NEW GENERATION OF ARTIFICIAL MICRORNAS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No.

61/947,732, filed March 4, 2014, entitled "New Generation of Artificial MicroRNAs, which is herein incorporated by reference. The present application also claims priority to U.S. Provisional Application No. 61/950,588, filed March 10, 2014, entitled "New Generation of Artificial MicroRNAs, which also is herein incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The development of this invention was partially funded by the government under grants from the National Science Foundation (MCB-0956526, MCB-1231726), National Institutes of Health (AI043288), National Institute of Food and Agriculture (MOW-2012- 01361). The government has certain rights in the invention.

FIELD

[0003] The field of the present disclosure relates generally to the field of molecular biology, more particularly relating to small RNA-directed regulation of gene expression. In particular, it relates to methods for down-regulating the expression of one or more target sequences in vivo. The disclosure also provides polynucleotide constructs and compositions useful in such methods, as well as cells, plants and seeds comprising the polynucleotides.

BACKGROUND

[0004] Reduction of the activity of specific genes (also known as gene silencing or gene suppression) is critical for normal cellular function in a variety of eukaryotes. Important to regulating gene expression, controlling integration of mobile genetic elements and defending against pathogens or pests, RNA-directed gene silencing is a conserved biological process that involves small RNA molecules. Small RNAs appear to function by base-pairing to complementary RNA or DNA target sequences. The consequence of these events, regardless of the specific mechanism, is that gene expression is modulated. In recent years, gene

1

RECTIFIED (RULE 91) - ISA/US silencing technology involving small RNAs has been used as an important tool to study and manipulate gene expression.

[0005] microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) are two distinct classes of plant small RNAs that act in post-transcriptional RNA silencing pathways to silence target RNA transcripts with sequence complementary (Chapman and Carrington, 2007; Martinez de Alba et al., 2013). Target repression can occur through direct

endonucleolytic cleavage, or through other mechanisms such as target destabilization or translational repression (Huntzinger and Izaurralde, 2011). MicroRNAs and tasiRNAs differ in their biogenesis pathway. While miRNAs originate from transcripts with imperfect self- complementary foldback structures that are usually processed by DICER-LIKE 1 (DCL1), tasiRNAs are formed through a refined RNA silencing pathway. TAS transcripts are initially targeted and sliced by a specific miRNA AGO complex, and one of the cleavage products is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE6 (RDR6). The resulting dsRNA is sequentially processed by DCL4 into 21-nt siRNA duplexes in register with the miR A-guided cleavage site (Allen et al., 2005; Dunoyer et al, 2005; Gasciolli et al., 2005; Xie et al., 2005; Yoshikawa et al., 2005; Axtell et al, 2006; Montgomery et al., 2008;

Montgomery et al., 2008). For both miRNA and tasiRNA intermediate duplexes, usually one strand is selectively sorted to an ARGONAUTE (AGO) protein according to the identity of the 5' nucleotide or to other sequence/structural elements of the small RNA or small RNA duplex (Mi et al, 2008; Montgomery et al, 2008; Takeda et al., 2008; Zhu et al, 2011).

[0006] Small RNA-directed gene silencing has been used extensively to selectively regulate plant gene expression. Artificial miRNA (amiRNA), synthetic tasiRNA (syn-tasiRNA), hairpin-based RNA interference (hpRNAi), virus-induced gene silencing (VIGS) or transcriptional silencing (TGS) methods have been developed (Ossowski et al., 2008; Baykal and Zhang, 2010). Since their initial application (Alvarez et al., 2006; Schwab et al., 2006), amiRNAs produced from different MIRNA precursors have been used to silence reporter genes (Parizotto et al., 2004), endogenous plant genes (Alvarez et al., 2006; Schwab et al., 2006), viruses (Niu et al., 2006) and non-coding RNAs (Eamens et al, 2011). Syn-tasiRNAs have been shown to target RNAs in Arabidopsis when produced from TASla (Felippes and Weigel, 2009), TASlc (de la Luz Gutierrez-Nava et al., 2008; Montgomery et al, 2008) and TAS3a (Montgomery et al, 2008; Felippes and Weigel, 2009) transcripts, or from gene fragments fused to an upstream miR173 target site (Felippes et al., 2012). Current methods to generate amiRNA or syn-tasiRNA constructs, however, can be tedious and cost- and time- ineffective for high-throughput applications.

[0007] Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are used for small RNA-based, specific gene silencing or knockdown in plants. Current methods to generate amiRNA or syn-tasiRNA constructs are not well adapted for cost-effective, large-scale production, or for multiplexing to specifically suppress multiple targets. Here we describe simple, fast and cost-effective methods with high-throughput capability to generate amiRNA and multiplexed syn-tasiRNA constructs for efficient gene silencing in Arabidopsis and other plant species. AmiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background BsaUccdB ('B/c')-based expression vector. B/c vectors for amiRNA and syn-tasiRNA cloning and expression contain a modified version of Arabidopsis MIR390a or TASlc precursors, respectively, in which a fragment of the endogenous sequence was substituted by a ccdB cassette. Several amiRNA and syn-tasiRNA sequences designed to target one or more endogenous genes were validated in transgenic plants that a) exhibited the expected phenotypes predicted by loss of target gene function, b) accumulated high levels of accurately processed amiRNAs or syn-tasiRNAs, and c) had reduced levels of the corresponding target RNAs.

[0008] However, current methods for generating small RNAs for targeting specific sequences are tedious and cost- and time-ineffective. Therefore, there is an unfulfilled need for efficient constructs and methods for inducing inhibition or suppression of one or more target genes or RNAs. It is to such constructs and methods, that this disclosure is drawn.

[0009] Further scope of the applicability of the present disclosure will become apparent from the detailed description and accompanying figures provided below. However, it should be understood that the detailed description and specific examples, while indicating several embodiments, are given by way of illustration only since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

SUMMARY [00010] The present disclosure relates to methods and constructs for modulating expression of one or more target sequences. Provided herein are methods for producing one or more sequence-specific microRNAs in vivo; also provided are constructs and compositions useful in the methods.

[00011] The methods and constructs provided in this disclosure are highly efficient methods for production of a new generation of plant MIR390a-basQd amiRNAs. The new methods and constructs use positive insert selection, and eliminate PCR steps, gel-based DNA purification, restriction digestions and sub-cloning of inserts between vectors, making them more suitable for high-throughput libraries.

[00012] Constructs and methods for producing specific small RNAs for inactivation or suppression of one or more target sequences or other entities, such as pathogens or pests (e.g. viruses, fungi, bacteria, nematodes, etc.) are also provided by this disclosure. Cells and organisms into which have been introduced a construct or a vector of this disclosure are also provided. Also provided are constructs and methods, where the small RNAs are produced in a tissue-specific, cell-specific or other regulated manner.

[00013] The present disclosure also relates to the production of plants with improved properties and traits using molecular techniques and genetic transformation. In particular, the invention relates to methods of modulating the expression of a target sequence in a cell using small RNAs. The disclosure also relates to cells or organisms obtained using such methods. Provided herein are plant cell and plants derived from such cells, as well as the progeny of such plants and to seeds derived from such plants. In such plant cells or plants, the modulation of the target sequence or expression of a particular gene is more effective, selective and more predictable than the modulation of the gene expression of a particular gene obtained using current methods known in the art.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[00014] The invention can be more fully understood form the following detailed description and the accompanying Sequence Listing, which form a part of this application. [00015] The sequence descriptions summarize the Sequence Listing attached hereto. The Sequence Listing contains standard symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. § 1 .822.

BRIEF DESCRIPTION OF THE FIGURES

[00016] The foregoing and other aspects, features, and advantages of the present disclosure will be better understood from the following detailed description taken in conjunction with the accompanying figures, all of which are given by way of illustration only, and are not limitative of the present specification, in which:

[00017] Figure 1. Arabidopsis thaliana MIR390a (AtMIR390d) is an accurately processed, conserved MIRNA foldback with a short distal stem-loop. A, AtMIR390a foldback processing diagram. miR390a and miR390a* nucleotides are highlighted in blue and green, respectively. Proportion of small RNA reads for the entire foldback are plotted as stacked bar graphs. Small RNAs are color-coded by size. B, Diagram of a canonical plant MIRNA foldback (adapted from Cuperus et al. 2011). miRNA guide and miRNA* strands are highlighted in blue and green, respectively. Distal stem-loop and basal stem regions are highlighted in black and grey. C, Distal stem-loop length of A. thaliana conserved MIRNA foldbacks. Box-plot showing the distal stem-loop length of A. thaliana conserved MIRNA foldbacks. The distal stem-loop length of AtMIR390a is highlighted with a red dot and indicated with an arrow. Outliers are represented with black dots. D, Distal stem-loop length of plant MIRNA foldbacks previously used for expressing amiRNAs. The Arabidopsis thaliana MIR3 '90a distal stem-loop length bar and name are highlighted in dark blue.

[00018] Figure 2. Direct cloning of amiRNAs in vectors containing a modified version of AtMIR390a that includes a ccdB cassette flanked by two Bsal sites BsaVccdB or 'B/c' vectors). A, Design of two overlapping oligonucleotides for amiRNA cloning. Sequences covered by the forward and the reverse oligonucleotides are represented with continuous or dotted lines, respectively. Nucleotides of AtMIR390a foldback, amiRNA guide strand and amiRNA* strand are in black, blue and green, respectively. Other AtMIR390a nucleotides that may be modified for preserving authentic AtMIR390a foldback secondary structure are in red. Rules for assigning identity to position 9 of the amiRNA* are indicated. B, Diagram of the steps for amiRNA cloning in AtMIR390a-B/c vectors. The amiRNA insert obtained after annealing the two overlapping oligonucleotides has 5'-TGTA and 5 -AATG overhangs, and is directly inserted in a directional manner into an AtMIR390a-B/c vector previously linearized with Bsal. Nucleotides of the Bsal sites and those arbitrarily chosen and used as spacers between the Bsal recognition sites and the AtMIR390a sequence are in purple and light brown, respectively. Other details are as described in panel A. C, Flowchart of steps from amiRNA construct generation to plant transformation.

[00019] Figure 3. Comparative analysis of the accumulation of several amiRNAs produced from AtMIR319a, AtMIR319a-21 or AtMIR390a foldbacks. A, Diagrams of AtMIR319a, AtMIR319a-21 and AtMIR390a foldbacks. Nucleotides corresponding to the miRNA guide strand are in blue, and nucleotides of the miRNA* strand are in green. Other nucleotides from the AtMIR319a, AtMIR319a-21 and AtMIR390a foldbacks are in light grey, dark grey, and black, respectively, except those nucleotides that were added in the AtMIR319a configuration are in light brown. Shapes of the AtMIR319a, AtMIR319a-21 d AtMIR390a foldbacks are in light grey, dark grey, and black, respectively. B, Accumulation of several amiRNAs expressed from the AtMIR319a, AtMIR319a-21 or AtMIR390a foldbacks in N. benthamiana leaves. Top, mean (n=3) relative amiRNA levels + s.d. when expressed from the AtMIR319a (light grey, amiRNA level =1.0), AtMIR319a-21 (dark grey, amiRNA level = 1) or

AtMIR390a (black) foldback. Only one blot from three biological replicates is shown. U6 RNA blot is shown as loading control.

[00020] Figure 4. Functionality of AtMIR39 Oa-based artificial miRNAs (amiRNAs) in Arabidopsis Coi-0 Tl transgenic plants. A, AtMIR390a-based foldbacks containing Lfy-, Ch42-, Ft- and Trich-amiRNAs. Nucleotides corresponding to the miRNA guide and miRNA* strands are in blue and green, respectively; nucleotides from the AtMIR390a foldback are in black except those that were modified to preserve authentic AtMIR390a foldback secondary structure that are in red. B, C, D and E, representative images of Arabidopsis Col-0 Tl transgenic plants expressing amiRNAs from the AtMIR390a foldback. B, Adult plants expressing 35S:GUS control (left) or 35S:AtMIR390a-Lfy with increased number of secondary shoots (top right) and leaf-like organs instead of flowers (bottom right). C _? Ten days-old seedlings expressing 35S:AtMIR390a-Ch42 and showing bleaching phenotypes. D, Adult control plant (35S:GUS) or plants expressing 35S:AtMlR390a-Ft plant with a delayed flowering phenotype. E, Fifteen days-old control seedling (35S:GUS), or seedling expressing 35S:AtMIR390a-Trich with increased number of trichomes. F,

Quantification of amiR A-induced phenotypes in plants expressing amiR-Lfy (top left), amiR-Ft (top right), and amiR-Ch42 (bottom). G, Accumulation of amiRNAs in Arabidopsis transgenic plants. One blot from three biological replicates is shown. Each biological replicate is a pool of at least 8 independent plants. U6 RNA blot is shown as a loading control. H, Mean relative level +/- s.e. of Arabidopsis LFY, CH42, FT, TRY, CPC and ETC2 mRNAs after normalization to ACT2, CPB20, SAND and UBQ10, as determined by quantitative real-time RT-PCR (35S:GUS = 1.0 in all comparisons).

[00021] Figure 5. Mapping of amiRNA reads from AtMIR390a-based foldbacks expressed in Arabidopsis Col-0 Tl transgenic plants. Analysis of amiRNA and amiRNA* reads in plants expressing amiR-Ft (top left), amiR-Lfy (top right), amiR-Ch42 (bottom left) and amiR-Trich (bottom right), respectively. amiRNA guide and amiRNA* strands are highlighted in blue and green, respectively. Nucleotides from the AtMlR390a foldback are in black except those that were modified to preserve authentic AtMIR390a foldback secondary structures that are in red. Proportion of small RNA reads are plotted as stacked bar graphs. Small RNAs are color- coded by size.

[00022] Figure 6. Direct cloning of syn-tasiRNAs in vectors containing a modified version of AtTASlc with a ccdB cassette flanked by two Bsal sites (BsaUccdB or 'B/c' vectors). A, Diagram of AtTASl c-based syn-tasiRNA constructs. tasiRNA production is initiated by miR173-guided cleavage of t e AtTASlc transcript. syn-tasiRNA-1 and syn-tasiRNA-2 are generated from positions 3'D3[+] and 3'D4[+] of the AtTASlc transcript, respectively. Nucleotides of AtTASlc, miR173, syn-tasiRNA- 1 and syn-tasiRNA-2 are in black, orange, blue and green, respectively. B, Design of two overlapping oligonucleotides for syn-tasiRNA cloning. Sequence covered by the forward and the reverse oligonucleotides are represented with continuous or dotted lines, respectively. C, Diagram of the steps for syn-tasiRNA cloning in AtTASl c-B/c vectors. The syn-tasiRNA insert obtained after annealing the two overlapping oligonucleotides has 5'-ATTA and 5'-CTTG overhangs, and is directly inserted into the i¾al-linearized AtTASl c-B/c vector. Nucleotides of the Bsal sites and arbitrary nucleotides used as spacers between the Bsal recognition site and the AtMIR390a sequence are in purple and light brown, respectively. Other details are as in panel A.

[00023] Figure 7. Functionality of AtTASl c-based syn-tasiRNAs in Arabidopsis Col-0 Tl transgenic plants. A, Organization of syn-tasiRNA constructs. Arrow indicates the miR173- guided cleavage site. tasiRNA positions 3'D1[+] to 3'D10[+] are indicated by brackets, with positions 3'D3[+] and 3'D4[+] highlighted in black. B, Representative images of Arabidopsis Col-0 transgenic lines expressing amiRNA or syn-tasiRNA constructs. C, Accumulation of amiRNAs and syn-tasiRNAs in Arabidopsis transgenic plants. Top, mean (n=3) relative Trich 21-mer (dark blue) and Ft 21-mer (light blue) levels + s.d. (35S:AtMIR390a-Trich and 35S:AtMIR390a-Ft lanes = 1.0 for Trich 21-mer and Ft 21-mer, respectively). One blot from three biological replicates is shown. Each biological replicate is a pool of at least 6 independent plants. U6 RNA blot is shown as a loading control. D, Syn-tasiRNA processing and phasing analyses in Arabidopsis Col-0 transgenic lines expressing syn-tasiRNAs (35S:AtTASlc-D3Trich-D4Ft and 35S:AtTASlc-D3Ft-D4Trich). Analyses of syn-tasiR- Trich, syn-tasiR-Ft and AtTASlc-derrved siRNA sequences by high-throughput sequencing. Pie charts, percentage of 19-24 nt reads; radar plots, percentages of 21-nt reads corresponding to each of the 21 registers from AtTASlc transcripts, with position 1 designated as immediately after the miR173-guided cleavage site. E, Mean relative level +/- s.e. of FT, TRY, CPC and ETC2 mRNAs after normalization to ACT2, CPB20, SAND and UBQ10, as determined by quantitative real-time RT-PCR (35S:GUS = 1.0).

[00024] Figure 8. AtMIR390a-B/c vectors for direct cloning of amiRNAs. A, Diagram of an AtMIR390a-B/c Gateway-compatible entry vector (pENTR-AtMIR390a-B/c) . B, Diagrams of AtMIR390a-B/c-based binary vectors for expression of amiRNAs in plants (pMDC32B- AtMIR390a-B/c, pMDC123SB-AtMIR390a-B/c and pFK210B-AtMIR390a-B/c). RB: right border; 35S: Cauliflower mosaic virus promoter; Bsal: Bsal recognition site, ccd : gene encoding the ccdB toxin; LB: left border; attLl and attL2: gateway recombination sites. Kan ^R : kanamycin resistance gene; Hyg ^R : hygromycin resistance gene; Basted: glufosinate resistance gene; Spec ^R : spectinomycin resistance gene. Undesired Bsal sites removed from the plasmid are crossed out.

[00025] Figure 9. Diagrams of AtMIR319a, AtMIR319a-21 and AtMIR390a foldbacks used to express several amiRNAs in N. benthamiana. Nucleotides corresponding to the miRNA guide and miRNA* are in blue and green, respectively. Other nucleotides from the

AtMIR319a, AtMIR319a-21 and AtMIR390a foldbacks are in light grey, dark grey, and black, respectively. Nucleotides that were added or modified that are in light brown and red, respectively. Shapes of the AtMIR319a, AtMIR319a-21 and AtMIR390a foldbacks are in light grey, dark grey, and black, respectively. [00026] Figure 10. Base-pairing of amiRNAs and target mRNAs. amiRNA and mRNA target nucleotides are in blue and brown, respectively.

[00027] Figure 11. AtTASlc-B/c vectors for direct cloning of syn-tasiRNAs. A, Diagram of an AtTASlc~B/c Gateway-compatible entry vector (pENTR-AtTASlc-B/c). B, Diagrams of AtTASlc-B/c binary vectors for expression of syn-tasiRNAs in plants (pMDC32B-AtTASlc- B/c, pMDC123SB-AtTASlc-B/c and pFK210B- AtTASlc-B/c). RB: right border; 35S:

Cauliflower mosaic virus promoter; Bsal: Bsal recognition site, ccdB: gene encoding the ccdB toxin; LB: left border; attLl and attL2: GATEWAY recombination sites. Kan ^R :

kanamycin resistance gene; Hyg ^R : hygromycin resistance gene; Basted: glufosinate resistance gene; Spec ^R ; spectinomycin resistance gene. Undesired Bsal sites removed from the plasmid are crossed out.

[00028] Figure 12. Organization of syn-tasiRNA constructs. Arrow indicates miR173-guided cleavage site. tasiRNA positions 3'D1(+) to 3'D10(+) are indicated by brackets, with positions 3'D3[+] and 3'D4[+] highlighted in black. The expected syn-tasiRNA-mRNA target interactions are represented. miR173, syn-tasiR-Trich and syn-tasiR-Ft sequences are in orange, dark blue and light blue, respectively. miR173 target site and syn-tasiRNA-mRNA target sequences are in light and dark brown, respectively.

[00029] Figure 13. Flowering time analysis of Arabidopsis Col-0 Tl transgenic plants expressing amiRNAs or syn-tasiRNAs. Mean (+ s.d.) days to flowering.

[00030] Figure 14. Processing analyses of syn-tasiRNAs expressed in Arabidopsis Col-0 Tl transgenic lines (35S:AtTASlc-D3Trich-D4Ft and 35S:AtTASlc-D3Ft-D4Trich). A, Small RNA size distribution of 19-24 nt siRNAs in both 3'D3[+] (up) and 3'D4[+] (bottom) positions in 35S:AtTASlc-D3Trich-D4Ft (left) and 35S:AtTASlc-D3Ft-D4Trich (right) transgenic plants. Correct syn-tasiR-Trich and syn-tasiR-Ft sequences are in dark and light blue, respectively. Other small RNA sequences are in grey. B, Distribution of small RNA reads (19-24 nt) having a 5' nucleotide within a -4/+4 region relative to the correct 5' nucleotide position of the syn-tasiRNA ('0' position). Other details as in panel A.

[00031] Figure 15. Processing and phasing analyses of endogenous AtTASJ c-tasiKNA in Arabidopsis Col-0 Tl transgenic lines expressing syn-tasiRNAs (35S:AtTASlc-D3Trich- D4Ft, 35S:AtTASlc-D3Ft-D4Trich and 35S:GUS control). Analyses of tasiR-3'D3[+] and tasiR-3'D4[+] (^/ ^S/oderived) siRNA sequences by high-throughput sequencing. Pie charts, percentage of 19-24 nt reads; radar plots, percentages of 21-nt reads corresponding to each register from AtTASlc transcripts, with position 1 designated as immediately after the miR173-guided cleavage site.

[00032] Figure 16. Processing analyses of endogenous AtTASlc-derrved siRNAs in

Arabidopsis Col-0 Tl transgenic plants expressing syn-tasiRNAs (35S:AtTASlc-D3Trich- D4Ft, 35S:AtTASlc-D3Ft-D4Trich and 35S:GUS control). A, Small RNA size distribution of 1 -24 nt siRNAs in both 3'D3[+] (up) and 3'D4[+] (bottom) positions in 35S:AtTASlc- D3Trich-D4Ft (left) and 35S:AtTASlc-D3Ft-D4Trich (right) transgenic plants. Correct tasiR- 3'D3[+] and tasiR-3'D4[+] sequences are in dark and light pink, respectively. Other small RNA sequences are in grey. B, Distribution of small RNA reads (19-24 nt) having a 5' nucleotide within a -4/+4 region relative to the correct 5' nucleotide position of the endogenous tasiRNA ('0' position). Other details are as in panel A.

[00033] Figure 17: Rice MIR390 foldback (OsMIR390) has a very short distal stem-loop that will make unexpensive the oligos necessary for cloning the amiRNAs.

[00034] Figure 18: A very high proportion of transgenic plants showed the expected amiRNA-induced phenotype, regardless of the MIRNA foldback (OsMIR390 or OsMIR390- AtL) from which the amiRNA was expressed.

[00035] Figure 19: A very high proportion of transgenic plants showed the expected amiRNA-induced phenotype, regardless of the MIRNA foldback (OsMIR390 or OsMIR390- AtL) from which the amiRNA was expressed.

[00036] Figure 20: A very high proportion of transgenic plants showed the expected amiRNA-induced phenotype, regardless of the MIRNA foldback (OsMIR390 or OsMIR390- AtL) from which the amiRNA was expressed.

[00037] Figure 21: A very high proportion of transgenic plants showed the expected amiRNA-induced phenotype, regardless of the MIRNA foldback (OsMIR390 or OsMIR390- AtL) from which the amiRNA was expressed. [00038] Figure 22: Artificial microRNA target mR As were significantly reduced in transgenic plants regardless the MIRNA foldback the amiRNA was expressed from (Figure 22).

[00039] Figure 23: Artificial microRNAs were processed more accurately when expressed from the chimeric (OsMIR390-AtL) compared to the wild-type foldback (OsMIR390; Figure 23).

[00040] Figure 24: Effects of amiRNA transfections in plants, (a) AtLMIR390a-based and OsMIR390-based amiRNA foldbacks; (b) miR390a and amiRNA accumulation in infiltrated Nicofiana leaves; (c) miR390a and amiRNA accumulation in transgenic Brachypodium colli.

[00041] Figure 25: Effects of amiRNA transfections in plants, (a) AtLMIR390-based amiRNA foldbacks; (b-c) photographs of wildtype and amiRNA-transfected plants;

quantification of amiRNA-indcued phenotype .

[00042] Figure 26: Design and annealing of overlapping oligonucleotides for direct amiRNA cloning.

[00043] Figure 27: OsMIR390-Bsai/ccdB-based (B/c) vectors for direct cloning of artificial miRNAs (amiRNAs). (a) Gateway-compatible entry clone; (b) plant binary vectors.

[00044] Figure 28: Oryza sativa MIR390 (OsMIR390) is an accurately processed, conserved MIRNA precursor with a particularly short distal stem-loop, (a) Diagram of a canonical plant MIRNA precursor (adapted from Cuperus et al. 2011). miRNA guide and miRNA* strands are highlighted in blue and green, respectively. Distal stem-loop and basal stem regions are highlighted in black and grey, respectively, (b) Distal stem-loop length of O. sativa conserved MIRNA precursors and of all plant catalogued MIR390 precursors. Box-plot showing the distal stem-loop length of O. sativa conserved MIRNA precursors and all catalogued MIR390 precursors. The distal stem-loop length of OsMIR390 is highlighted with an orange dot and indicated with an orange arrow. Outliers are represented with black dots, (c) OsMIR390 precursor processing diagram. miR390 and miR390* nucleotides are highlighted in blue and green, respectively. Proportion of small RNA reads for the entire OsMIR390 precursor are plotted as stacked bar graphs. Small RNAs are color-coded by size. [00045] Figure 29: Comparative analysis of accumulation and processing of several amiRNAs produced from AtMIR390a, AtMIR390a-OsL, OsMIR390 and OsMIR390-AtL precursors in Brachypodium transgenic calli. (a) Diagrams of AtMIR390a, AtMIR390a-OsL, OsMIR390 and OsMIR390-AtL precursors. Nucleotides corresponding to the miRNA guide strand are in blue, and nucleotides of the miRNA* strand are in green. Other nucleotides from AtMIR390a and OsMIR390 precursors are in black and grey, respectively. Shapes of AtMIR390a and OsMIR390 precursors are in black and grey, respectively, (b) Accumulation of miR390 (left) and of several 21 -nucleotide amiRNAs (right) expressed from the

AtMIR390a, AtMR390a-OsL, OsMIR390 or OsMIR390-AtL precursors in Brachypodium transgenic calli. Mean (n=3) relative amiRNA levels + s.d. when expressed from the

OsMIR390 (light grey, amiRNA level =1.0). Only one blot from three biological replicates is shown. 176 RNA blot is shown as loading control.

[00046] Figure 30: Functionality of amiRNAs produced from authentic OsMIR390~ or chimeric OsMIR390-AtL-based precursors in Brachypodium TO transgenic plants, (a) OsMIR390- and OsMIR390-AtL-based precursors containing Bril-, Cadi-, Cao and Spll l- amiRNAs. Nucleotides corresponding to the miRNA guide and miRNA* strands are in blue and green, respectively; nucleotides from AtMIR390a or OsMIR390 precursors are in black or grey, respectively, except those that were modified to preserve authentic AtMIR390a or OsMIR390 precursor secondary structures (red), (b-e) Representative images of plants expressing amiRNAs from OsMIR390-AtL or OsMIR390 precursors, or the control construct, (b) Adult control plant (left), or plants expressing 35S:OsMIR390-Bril (center) or

35S:OsMIR390-AtL-Bril (right), (c) Adult control plant (left), or plants expressing

35S:OsMIR390-Cad (center) or 35S:OsMIR390-AtL-Cadl (bottom), (d) Adult control plant (left), or plants expressing 35S:OsMIR390-Splll (center) or 35S:OsMIR390-AtL-Splll (right).

[00047] Figure 31: Target mRNA and amiRNA accumulation analysis in Brachypodium TO transgenic plants, (a) Mean relative level +/- s.e. of B. distachyon BdBRIl, BdCADl, BdCAO and BdSPLll mRNAs after normalization to BdSAMDC, BdUBC, BdUBI4 and BdUBIlO, as determined by quantitative real-time RT-PCR (35S:GUS = 1.0 in all comparisons), (b) Accumulation of amiRNAs in Brachypodium transgenic plants. In each blot the amiRNA accumulation of a single independent transgenic line per construct is analyzed. U6 RNA blot is shown as a loading control. [00048] Figure 32: Mapping of amiRNA reads from OsMIR390-AtL- or OsMR390-based precursors expressed in Brachypodium TO transgenic plants. Analysis of amiRNA and amiRNA* reads in plants expressing (a) amiR-BdBril, (b) amiR-BdCadl, (c) amiR-BdCao or (d) amiRBdSpll 1. amiRNA guide and amiRNA* strands are highlighted in blue and green, respectively. Nucleotides from the AtMIR390a or OsMIR390 precursors are in black and grey, respectively, except those that were modified to preserve the corresponding authentic precursor secondary structure (in red). Proportion of small RNA reads are plotted as stacked bar graphs. Small RNAs are colorcoded by size.

[00049] Figure 33: Transcriptome analysis of transgenic Brachypodium plants expressing amiRNAs from chimeric OsMIR390-AtL precursors. MA plots show log2 fold change versus mean expression of genes for each 35S:OsMIR390-AtL amiRNA line compared to the control lines (35S:GUS). Green, red and grey dots represent differentially underexpressed, differentially overexpressed or non-differentially expressed genes, respectively, in each amiRNA versus control comparison. The position of expected amiRNA targets is indicated with a circle.

[00050] Figure 34: Differential expression analysis of TargetFinder-predicted off-targets for each amiRNA versus control comparison. Histograms show the total number of genes (top panels) or the proportion of differentially underexpressed genes (bottom panels) in each target prediction score bin. Green, red and grey bars represent differentially underexpressed, differentially overexpressed or non-differentially expressed genes, respectively. In bottom panels, the name of the expected target gene is indicated when the target gene is the only gene differentially underexpressed in the corresponding bin.

[00051] Figure 35: 5' RLM-RACE mapping of target and potential off-target cleavage guided by amiRNAs in plants expressing (a) amiRBdBril, (b) amiR-BdCadl, (c) amiR- BdCao and (d) amiR-BdSpll 1. At the top of each panel, ethidium bromide-stained gels show 5'-RLM- RACE products corresponding to the 3' cleavage product from amiRNA-guided cleavage (top gel), and RT-PCR products corresponding to the gene of interest (middle gel) or control BdUBI4 gene (bottom gel). The position and size of the expected amiRNA-based 5' -RLM-RACE products are indicated. At the bottom of each panel, the predicted base- pairing between amiRNAs and prospective target RNAs is shown. The sequence and the name of authentic target rnRNAs are in blue. For each authentic or predicted target mRNA, the expected amiR A-based cleavage site is indicated by an orange arrow. Other sites are indicated with a black arrow. The proportion of cloned 5'-RLM-RACE products at the different cleavage sites is shown for amiRNA expressing lines, with that of control plants expressing 35S:GUS shown in brackets. TPS refers to 'Target Prediction Score'.

[00052] Figure 36: OsMIR390-B/c vectors for direct cloning of amiRNAs. (a) Diagram of an OsMIR390-B/c Gateway-compatible entry vector (pENTR-OsMIR390-B/c). (b) Diagrams of OsMIR390-B/c -based binary vectors for expression of amiRNAs in monocot species (pMDC32B-OsMIR390-B/c, _P MDC123SB-OsMIR390-B/c and pH7WG2B-OsMIR390~B/c). RB: right border; 35S: Cauliflower mosaic virus promoter; OsUbi: Oryza sativa ubiquitin 2 promoter; Bsal: Bsal recognition site, ccd : gene encoding the ccdQ toxin; LB: left border; attLl and attL2: gateway recombination sites. KanR: kanamycin resistance gene; HygR: hygromycin resistance gene; BastaR: glufosinate resistance gene; SpecR: spectinomycin resistance gene. Undesired Bsal sites removed from the plasmid are crossed out.

[00053] Figure 37: Generation of constructs to express amiRNAs from authentic OsMIR390 precursors, (a) Design of the two overlapping oligonucleotides required for amiRNA cloning into OsMIR39 O-based vectors. Sequences covered by the forward and reverse

oligonucleotides are represented with solid and dotted lines, respectively. Nucleotides of OsMIR390 precursor, amiRNA guide strand, and amiRNA* strand are in grey, blue, and green respectively. Other OsMIR390 nucleotides that may be modified for preserving authentic OsMIR390 precursor secondary structure are in red. Rules for assigning identity to positions 1 and 9 of amiRNA* are indicated, (b) Diagram of the steps for amiRNA cloning in OsMIR390 precursors. The amiRNA insert obtained after annealing the two overlapping oligonucleotides has 5'CTTG and 5'CATG overhangs and is directly inserted in a directional manner into an OsMIR390-B/c vector previously linearized with Bsal. Nucleotides of the Bsal sites and those arbitrarily chosen and used as spacers between the Bsal recognition sites and the OsMIR390 sequence are in purple and light brown, respectively. Other details are as described in A. C, flow chart of the steps from amiRNA construct generation to plant transformation.

[00054] Figure 38: Generation of constructs to express amiRNAs from chimeric OsMIR390- AtL precursors, (a) Design of the two overlapping oligonucleotides containing OsMIR390aa and AtMIR390a basal stem and distal stem loop sequences, respectively. Sequences covered by the forward and reverse oligonucleotides are represented with solid and dotted lines, respectively. Nucleotides of AtMIR390a and OsMIR390 precursors are in black and grey, respectively. Nucleotides of the amiRNA guide strand, and amiRNA* strand are in blue, and green respectively. Other OsMIR390 nucleotides that may be modified for preserving authentic OsMIR390 precursor secondary structure are in red. Rules for assigning identity to positions 1 and 9 of amiRNA* are indicated, (b) Diagram of the steps for generating constructs for expressing amiRNAs from chimeric OsMIR390-AtL precursors. The amiRNA insert obtained after annealing the two overlapping oligonucleotides has 5'CTTG and 5'CATG overhangs and is directly inserted in a directional manner into an OsMIR390-B/c vector previously linearized with Bsal. Nucleotides of the Bsal sites and those arbitrarily chosen and used as spacers between the Bsal recognition sites and the OsMIR390 sequence are in purple and light brown, respectively. Other details are as described in (a), (c) Flow chart of the steps from amiRNA construct generation to plant transformation.

[00055] Figure 39: Generation of constructs to express amiRNAs from chimeric AtMIR390a- OsL precursors, (a) Design of the two overlapping oligonucleotides containing AtMIR390a and OsMIR390 basal stem and distal stem loop sequences, respectively. Sequences covered by the forward and reverse oligonucleotides are represented with solid and dotted lines, respectively. Nucleotides of AtMIR390a and OsMIR390 precursors are in black and grey, respectively. Nucleotides of the amiRNA guide strand, and amiRNA* strand are in blue, and green respectively. Other AtMIR390a nucleotides that may be modified for preserving authentic AtMIR390a precursor secondary structure are in red. Rules for assigning identity to position 9 of amiRNA* are indicated, (b) Diagram of the steps for generating constructs for expressing amiRNAs from chimeric AtMIR390a-OsL precursors. The amiRNA insert obtained after annealing the two overlapping oligonucleotides has 5'TGTA and 5'AATG overhangs and is directly inserted in a directional manner into an AtMIR390a-B/c vector previously linearized with Bsal. Nucleotides of the Bsal sites and those arbitrarily chosen and used as spacers between the Bsal recognition sites and the AtMIR390a sequence are in purple and light brown, respectively. Other details are as described in (a), (c) Flow chart of the steps from miRNA construct generation to plant transformation.

[00056] Figure 40: Base-pairing of amiRNAs and Brachypodium target mRNAs. amiRNA and mRNA target nucleotides are in blue and brown, respectively. [00057] Figure 41: Plant height and seed length analyses in Brachypodium distachyon TO transgenic plants expressing amiR-BdBril from authentic OsMIR390 or chimeric OsMIR390- AtL precursors.

[00058] Figure 42: Quantification of amiR-BdCao-induced phenotype in Brachypodium distachyon 35S:OsMIR390-AtL-Cao, 35S:OsMIR390-Cao and 35S. GUS TO transgenic lines, (a) Quantification of chlorophyll a, chlorophyll b, chlorophyll a+b, chlorophyll a/b, and carotenoid content, (b) Absorbance spectra from 400 to 750 nm of leaves from

Brachypodium transgenic lines. Arrows indicate absorbance wavelengths of chlorophyll a (Chi a), chlorophyll b (Chi b), and carotenoids.

[00059] Figure 43: Comparative analysis of the accumulation and processing of several amiRNAs produced from AtMIR390a, AtMIR390a-OsL, OsMR390 and OsMIR390-AtL based precursors in Nicotiana benthamiana leaves, (a) Diagrams of AlMIR390a, AtMIR390a- OsL, OsMIR390 and OsMIR390a-AtL precursors. Nucleotides corresponding to the miRNA guide strand are in blue, and nucleotides of the miRNA* strand are in green. Other nucleotides from the AtMIR390a and OsMIR390 precursors are in black and grey, respectively. Shapes of he AtMIR390a and OsMIR390 precursors are in black and grey, respectively, (b) Accumulation of miR390 (left) and of several 21 -nucleotide amiRNAs (right) expressed from the AtMIR390a, AtMIR390a-OsL, OsMIR390 or OsMIR390-AtL precursors in N. benthamiana leaves. Mean (n=3) relative amiRNA levels + s.d. when expressed from the AtMIR390a (dark blue, amiRNA level =1.0). Only one blot from three biological replicates is shown. U6 RNA blot is shown as loading control.

[00060] Figure 44: Base-pairing of amiRNAs and Arabidopsis target mRNAs. amiRNA and mRNA target nucleotides are in blue and brown, respectively.

[00061] Figure 45: Functionality in Arabidopsis Tl transgenic plants of amiRNAs derived from AtMIR390a-based chimeric precursors containing Οιγζα sativa distal stem-loop sequences (AtMIR390a-OsL). (a) AtMIR390a- and AtMIR390a-OsL-based precursors containing Ft-, Ch42- and Trich-amiRNAs. Nucleotides corresponding to the miRNA guide and miRNA* strands are in blue and green, respectively; nucleotides from the AtMIR390a or OsMIR390 precursors are in black or grey, respectively, except those that were modified to preserve authentic AtMIR390a or OsMIR390 precursor secondary structures that are in red. (b-d) Representative images of plants expressing amiRNAs from AtMIR390a-OsL or AtMIR390a-OsL precursors, (b) Adult control plant (35S. GUS) or plants expressing

35S:AtMIR390a-Ft-OsL or 35S:AtMIR390a-Ft plant with a delayed flowering phenotype. (c) Ten days-old seedlings expressing 35S:AtMIR390a-OsL-Ch42 or 35S:AtMIR390a-Ch42 and showing bleaching phenotypes. (d) Fifteen days-old control seedling (35S. GUS), or seedling expressing 35S:AtMIR390a-OsL-Trich or 35S:AtMIR390a-Trich with increased number of trichomes. (e) Accumulation of amiRNAs in transgenic plants. One blot from three biological replicates is shown. Each biological replicate is a pool of at least 8 independent plants. U6 RNA blot is shown as a loading control, (f) Mean relative level +/- s.e. of A. thaliana FT, CH42, TRY, CPC and ETC2 mRNAs after normalization to ACT2, CPB20, SAND and UBQ10, as determined by quantitative real-time RT-PCR (35S:GUS = 1.0 in all

comparisons), (g) Mapping of amiRNA reads from AtMIR390a-OsL precursors expressed in transgenic plants. Analysis of amiRNA and amiRNA* reads in plants expressing amiR-AtFt (left), amiR~AtCh42 (center) and amiR-AtTrich (right), respectively. amiRNA guide and amiRNA* strands are highlighted in blue and green, respectively. Nucleotides from

AtMIR390a or OsMIR390 precursors are in black and grey, respectively, except those that were modified to preserve the corresponding authentic precursor secondary structure that are in red. Proportion of small RNA reads are plotted as stacked bar graphs. Small RNAs are color-coded by size.

[00062] Figure 46: Quantification of amiRNA-induced phenotypes in Arabidopsis transgenic plants expressing amiR-AtFt (left) and amiR-AtCh42 (right) from AtMIR390a or chimeric AtMIR390a~OsL precursors.

[00063] Figure 47: Target accumulation determined by RNA-Seq analysis in transgenic Brachypodium plants including 35S:OsMIR390- AtL-based or 35S:GUS constructs.

[00064] Figure 48: DNA sequence in FASTA format of all AtTASlc-based constructs used to express and analyze syn-tasiRNAs. Sequence corresponding to Syn-tasiRNA-1 (position 3'D3[+]) and syn-tasiRNA-2 (position 3'D4[+]) is highlighted in blue and green, respectively. Sequence corresponding to Arabidopsis tasiR-3'D[(+)]. tasiR-3'D4[+] is highlighted in dark and light pink respectively. All the other sequences from Arabiopsis TASic gene are high ^' ghted black.

[00065] Figure 49: DNA sequence in FASTA format of all MIRNA foldbacks used in this study to express and analyze amiRNAs. (A) atMTR319a foldbacks. Sequences unique to the 9

pri-miRNA, pre-miRNA, miRNA/amiRNA guide strand and miRNA*/amiR A* strand sequences are highlighted in grey, white, blue and gree, respectively. Bases of the pre- AtMIR319a that had to be modified to preserve the authentic AtMTR319a foldback structure are highlighted in red. Extra bases due to WMD2 design are highlighted in light brown. (B) AtMIR390a foldbacks. Sequence unique to the pre-AfMIR390a sequence is highlighted in black. Bases of the pre-AtMIR390a that had to be modified to preserve the authentic

AtMIR390a foldback structure are highlighted in red. Other details as in (A).

DETAILED DESCRIPTION

[00066] The following detailed description is provided to aid those skilled in the art. Even so, the following detailed description should not be construed to unduly limit, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present specification.

[00067] The contents of each of the publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control.

I. Terms

[00068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure pertains. Units, prefixes and symbols may be denoted in their SI accepted form. Provision, or lack of the provision, of a definition for a particular term or phrase is not meant to signify any particular importance, or lack thereof. Rather, and unless otherwise noted, terms used and the manufacture or laboratory procedures described herein are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references. The following definitions are provided to aid the reader in understanding the various aspects of the present disclosure.

[00069] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" includes a plurality of such plants, reference to "a cell" includes one or more cells and equivalents thereof known to those skilled in the art, and so forth. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Hence "comprising A or B" means including A, or B, or A and B. Furthermore, the use of the term "including", as well as other related forms, such as "includes" and "included", is not limiting.

[00070] Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5' to 3' direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as is known to one of ordinary skill in the art and is understood as included in embodiments where it would be appropriate. Nucleotides may be referred to by their commonly accepted single-letter codes. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxyl orientation, respectfully. Amino acids may be referred to herein by either their commonly loiown three letter symbols or by the one-letter symbols recommended by the IUPAC-IUM Biochemical Nomenclature Commission. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5 ^th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.

[00071] If ranges are disclosed, the endpoints of all ranges directed to the same component or property are inclusive and independently combinable (e.g., ranges of "up to about 25 wt.%, or, more specifically, about 5 wt.% to about 20 wt.%," is inclusive of the endpoints and all intermediate values of the ranges of "about 5 wt.% to about 25 wt.%," etc.). Numeric ranges recited with the specification are inclusive of the numbers defining the range and include each integer within the defined range.

[00072] The term "about" as used herein is a flexible word with a meaning similar to "approximately" or "nearly". The term "about" indicates that exactitude is not claimed, but rather a contemplated variation. Thus, as used herein, the term "about" means within 1 or 2 standard deviations from the specifically recited value, or ± a range of up to 20%, up to 15%, up to 10%), up to 5%, or up to 4%, 3%, 2%, or \% compared to the specifically recited value. [00073] As used herein, "altering level of production" or "altering level of expression" shall mean changing, either by increasing or decreasing, the level of production or expression of a nucleic acid sequence or an amino acid sequence (for example a polypeptide, an siRNA, a miRNA, an mRNA, a gene), as compared to a control level of production or expression.

[00074] By "amplification" when used in reference to a nucleic acid, this refers to techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re- annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of in vitro amplification can be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques. Methods of nucleic acid amplification can include, but are not limited to: polymerase chain reaction (PCR), strand

displacement amplification (SDA), for example multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), ligase chain reaction (LCR), irnmuno- amplification, and a variety of transcription-based amplification procedures, including transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), and rolling circle amplification. See, e.g., Mullis, "Process for Amplifying, Detecting, and/or Cloning Nucleic Acid Sequences," U.S. Pat. No. 4,683,195; Walker, "Strand Displacement Amplification," U.S. Pat. No.

5,455,166; Dean et al, "Multiple displacement amplification," U.S. Pat. No. 6,977,148;

Notomi et al, "Process for Synthesizing Nucleic Acid," U.S. Pat. No. 6,410,278; Landegren et al. U.S. Pat. No. 4,988,617 "Method of detecting a nucleotide change in nucleic acids";

Birkenmeyer, "Amplification of Target Nucleic Acids Using Gap Filling Ligase Chain Reaction," U.S. Pat. No. 5,427,930; Cashman, "Blocked-Polymerase Polynucleotide

Immunoassay Method and Kit," U.S. Pat. No. 5,849,478; Kacian et al,

"Nucleic Acid Sequence Amplification Methods," U.S. Pat. No. 5,399,491; Malek et al, "Enhanced Nucleic Acid Amplification Process," U.S. Pat. No. 5,130,238; Lizardi et al, BioTechnology, 6: 1197 (1988); Lizardi et al, U.S. Pat. No. 5,854,033 "Rolling circle replication reporter systems." In some embodiments, two or more of the

listed nucleic acid amplification methods are performed, for example sequentially.

[00075] "Antisense" and "Sense": DNA has two antiparallel strands, a 5'→ 3' strand, referred to as the plus strand, and a 3'→ 5' strand, referred to as the minus strand.

Because RNA polymerase adds nucleic acids in a 5'→ 3' direction, the minus strand of the DNA serves as the template for the RNA during transcription. Thus, an RNA transcript will have a sequence complementary to the minus strand, and identical to the plus strand (except that U is substituted for T). "Antisense" molecules are molecules that are hybridizable or sufficiently complementary to either RNA or the plus strand of DNA. "Sense" molecules are molecules that are hybridizable or sufficiently complementary to the minus strand of DNA.

[00076] As used herein "binds" or "binding" includes reference to an oligonucleotide that binds or stably binds to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding. Binding can be detected by either physical or functional properties of the target- oligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including both functional and physical binding assays. For instance, binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation and the like. Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures. The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (T _m ) at which 50% of the oligomer is melted from its target. A higher (T _m ) means a stronger or more stable complex relative to a complex with a lower (T _m ).

[00077] By "complementarity" refers to molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, or hybridize, to each other by forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when an oligonucleotide remains detectably bound to a target nucleic acid sequence under the required conditions. Complementarity is the degree to which bases in one nucleic acid strand base pair with (are complementary to) the bases in a second nucleic acid strand. Complementarity is conveniently described by the percentage, i.e., the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands. "Sufficient complementarity" means that a sufficient number of base pairs exist between the oligonucleotide and the target sequence to achieve detectable binding, and disrupt or reduce expression of the gene product(s) encoded by that target sequence. When expressed or measured by percentage of base pairs formed, the percentage complementarity that fulfills this goal can range from as little as about 50% complementarity to full (100%)

complementary. In some embodiments, sufficient complementarity is at least about 50%, about 75% complementarity, or at least about 90% or 95% complementarity. In particular embodiments, sufficient complementarity is 98% or 100% complementarity. Likewise, "complementary" means the base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.

[00078] As used herein "control" or "control level" means the level of a molecule, such as a polypeptide or nucleic acid, normally found in nature under a certain condition and/or in a specific genetic background. In certain embodiments, a control level of a molecule can be measured in a cell or specimen that has not been subjected, either directly or indirectly, to a treatment. A control level is also referred to as a wildtype or a basal level. These terms are understood by those of ordinary skill in the art. A control plant, i.e. a plant that does not contain a recombinant DNA that confers (for instance) an enhanced agronomic trait in a transgenic plant, is used as a baseline for comparison to identify an enhanced agronomic trait in the transgenic plant. A suitable control plant may be a non-transgenic plant of the parental line used to generate a transgenic plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant DNA, or does not contain all of the recombinant DNAs in the test plant.

[00079] As used herein, "encodes" or "encoding" refers to a DNA sequence which can be processed to generate an RNA and/or polypeptide. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[00080] As used herein, "expression" or "expressing" refers to production of a functional product, such as, the generation of an RNA transcript from an introduced construct, an endogenous DNA sequence, or a stably incorporated heterologous DNA sequence. A nucleotide encoding sequence may comprise intervening sequence (e.g. introns) or may lack such intervening non-translated sequences (e.g. as in cDNA). Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated (for example, siRNA, transfer RNA and ribosomai RNA). The term may also refer to a polypeptide produced from an mRNA generated from any of the above DNA precursors. Thus, expression of a nucleic acid fragment, such as a gene or a promoter region of a gene, may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/ or translation of RNA into a precursor or mature protein (polypeptide), or both.

[00081] The term "genome" as it applies to a plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found Within subcellular components (e.g., mitochondrial, plastid) of the cell.

[00082] As used herein, "heterologous" with respect to a sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/ or genomic locus. For example, with respect to a nucleic acid, it can be a nucleic acid that originates from a foreign species, or is synthetically designed, or, if from the same species, is substantially modified from its native form in composition and/ or genomic locus. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form.

[00083] By "host cell" or "cell" it is meant a cell which contains a vector and supports the replication and/or expression of the vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells. Alternatively, the host cells are monocotyledonous or dicotyledonous plant cells.

[00084] The term "hybridize" or "hybridization" as used herein means hydrogen bonding, which includes Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as base pairing. Complementary refers to the base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization, though waste times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed Green and Sambrook (2012) Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, herein incorporated by reference.

[00085] The term "introduced" means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, "introduced" in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct) into ac ell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[00086] As used here in "interfering" or "inhibiting" with respect to expression of a target sequence): This phrase refers to the ability of a small RNA, or other molecule, to measurably reduce the expression and/or stability of molecules carrying the target sequence. "Interfering" or "inhibiting" expression contemplates reduction of the end-product of the gene or sequence, e.g., the expression or function of the encoded protein or a protein, nucleic acid, other biomolecule, or biological function influenced by the target sequence, and thus includes reduction in the amount or longevity of the miRNA transcript or other target sequence. In some embodiments, the small RNA or other molecule guides chromatin modifications which inhibit the expression of a target sequence. It is understood that the phrase is relative, and does not require absolute inhibition (suppression) of the sequence. Thus, in certain embodiments, interfering with or inhibiting expression of a target sequence requires that, following application of the small RNA or other molecule (such as a vector or other construct encoding one or more small RNAs ), the target sequence is expressed at least 5% less than prior to application, at least 10% less, at least 15% less, at least 20% less, at least 25% less, or even more reduced. Thus, in some particular embodiments, application of a small RNA or other molecule reduces expression of the target sequence by about 30%, about 40%, about 50%, about 60%, or more. In specific examples, where the small RNA or other molecule is reduces expression of the target sequence by 70%, 80%, 85%, 90%, 95%, or even more.

[00087] The term "isolated" refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment; the isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material. Nucleic acids and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[00088] As used here "modulate" or "modulating" or "modulation" and the like are used interchangeably to denote either up-regulation or down-regulation of the expression of the product of a target sequence relative to its normal expression level in a wild type organism. Modulation includes expression that is increased or decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1 10%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150% , 155%, 160%, 165% or 170% or more relative to the wild type expression level.

[00089] As used herein, "microRNA" (also referred to herein interchangeable as "miRNA" or "miR") refers to an oligoribonucleic acid, which regulates the expression of a

polynucleotide comprising the target sequence transcript. Typically, microRNAs (miRNAs) are noncoding RNAs of approximately 21 nucleotides (nt) in length that have been identified in diverse organisms, including animals and plants (Lagos-Quintana et al., Science 294:853- 858 2001, Lagos-Quintana et al, Curr. Biol. 12:735-739 2002; Lau et al, Science 294:858- 862 2001 ; Lee and Ambros, Science 294:862-864 2001; Llave et al., Plant Cell 14: 1 605- 1619 2002; Mourelatos et al., Genes. Dev. 16:720-728 2002; Park et al., Curr. Biol. 12: 1484- 1495 2002; Reinhart et al, Genes. Dev. 16: 1616-1626 2002). Primary transcripts of miRNA genes form hairpin structures that are processed by the multidomain RNaseffl-like nuclease DICER and DROSHA (in animals) or DICER- LIKE1 (DCL1; in plants) to yield miRNA duplexes. As used herein "pre-microRNA" refers to these miRNA duplexes, wherein the foldback includes a "distal stem-loop" or "distal SL region" of partially complementary oligonucleotides, "mature miRNA" refers to the miRNA which is incorporated into RISC complexes after duplex unwinding. In one embodiment, the miRNA is the region comprising R] to R _n , wherein "n" corresponds to the number of nucleotides in the miRNA. In another embodiment, the miRNA is the region comprising R'i to R' _n , wherein "n" corresponds to the number of nucleotides in the miRNA. In one aspect, "n" is in the range of about from 15 to about 25 nucleotides, in another aspect, "n" is about 20 or about 21 nucleotides. The term miRNA is specifically intended to cover naturally occurring polynucleotides, as well as those that are recombinantly or synthetically or artificially produced, or amiRNAs.

[00090] As used herein "operably linked" refers to a functional arrangement of elements. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably Jinked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered "operably linked" to the coding sequence. In specific embodiments, operably linked nucleic acids as discussed herein are aligned in a linear concatamer capable of being cut into fragments, at least one of which is a small RNA molecule.

[00091] As used herein, "nucleic acid" means a polynucleotide (or oligonucleotide) and includes single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). Nucleic acids may also include fragments and modified nucleotides.

[00092] As used herein, "nucleic acid construct" or "construct" refers to an isolated polynucleotide which is introduced into a host cell. This construct may comprise any combination of deoxyribonucleotides, ribonucleotides, and/or modified nucleotides. The construct may be transcribed to form an RNA, wherein the RNA may be capable of forming a double-stranded RNA and/or hairpin structure. This construct may be expressed in the cell, or isolated or synthetically produced. The construct may further comprise a promoter, or other sequences which facilitate manipulation or expression of the construct.

[00093] The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and isolated plant cells, tissues and organs. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Also included with the term "plant" is algae and generally comprises all plants of economic importance. The term "plant" also includes plants which have been modified by breeding, mutagenesis or genetic engineering (transgenic and non- transgenic plants).

[00094] As used herein the phrase "plant cell" refers to plant cells which are derived and isolated from a plant or plant cell cultures.

[00095] As used herein the phrase "plant cell culture" refers to any type of native (naturally occurring) plant cells, plant cell lines and genetically modified plant cells, which are not assembled to form a complete plant, such that at least one biological structure of a plant is not present. Optionally, the plant cell culture of this aspect of the present invention may comprise a particular type of a plant cell or a plurality of different types of plant cells. It should be noted that optionally plant cultures featuring a particular type of plant cell may be originally derived from a plurality of different types of such plant cells.

[00096] The term "plant parts" includes differentiated and undifferentiated tissues including, but not limited to the following; roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture.

[00097] The term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant. [00098] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms

"polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

[00099] As used herein "promoter" includes reference to an array of nucleic acid control sequences which direct transcription of a nucleic acid. A "plant promoter" is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell.

Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as "tissue preferred". Promoters which initiate transcription only in certain tissue are referred to as "tissue specific". A "ceil type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" or "repressible" or "regulatable" promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, the presence of a specific molecule, such as C02, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters. Examples of inducible promoters include Cu-sensitive promoter, Gall promoter, Lac promoter, while Trp promoter, Nitl promoter and cytochrome c6 gene (Cyc6) promoter. A "constitutive" promoter is a promoter which is active under most environmental conditions. Examples of constitutive promoters include Ubiquitin promoter, actin promoter, PsaD promoter, RbcS2 promoter, heat shock protein (hsp) promoter variants, and the like. Representative examples of promoters that can be used in the present disclosure are described herein.

[000100] A skilled person appreciates a promoter sequence can be modified to provide for a range of expression levels of an operably linked heterologous nucleic acid molecule. Less than the entire promoter region can be utilized and the ability to drive expression retained. However, it is recognized that expression levels of mRNA can be decreased with deletions of portions of the promoter sequence. Thus, the promoter can be modified to be a weak or strong promoter. A promoter is classified as strong or weak according to its affinity for RNA polymerase (and/or sigma factor); this is related to how closely the promoter sequence resembles the ideal consensus sequence for the polymerase. Generally, by "weak promoter" is intended a promoter that drives expression of a coding sequence at a low level. By "low level" is intended levels of about 1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Conversely, a strong promoter drives expression of a coding sequence at a high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts.

[000101] As used herein "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed.

[000102] As used herein, a "recombinant construct", "expression construct", "chimeric construct", "construct" and "recombinant expression cassette" are used interchangeable herein. A recombinant construct comprises an artificial combination of nucleic acid fragments (e.g. regulatory and coding sequences) that are not found in nature. For example, a recombinant construct may comprise a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The recombinant construct can be incorporated into a plasmid, vector, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention. This construct may comprise any combination of deoxyribonucleotides, ribonucleotides, and/or modified nucleotides. The construct may be transcribed to form an R A, wherein the RNA may be capable of forming a double-stranded RNA and/or hairpin structure. This construct may be expressed in the cell, or isolated or synthetically produced. The construct may further comprise a promoter, or other sequences which facilitate manipulation or expression of the construct.

[000103] The term "residue" or "amino acid residue" or "amino acid" is used

interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively "protein"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[000104] As used herein, the phrase "sequence identity" or "sequence similarity" is the similarity between two (or more) nucleic acid sequences, or two (or more) amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity or sequence homology. Sequence identity is frequently measured as the percent of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions.

[000105] One of ordinary skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant similarity could be obtained that fall outside of the ranges provided. Nucleic acid sequences that do not show a high degree of identity can nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein. Means for making this adjustment are well-known to those of skill in the art. When percentage of sequence identity is used in reference to amino acid sequences it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.

[000106] Sequence identity (or similarity) can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Parti, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H, and Lipman, D., SUM J. Applied Math, 48: 1073 (1988).

Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection. See generally, (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402 (1997)).

[000107] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in (Altschul, S., et al., NCBI LM NIH Bethesda, Md. 20894; & Altschul, S., et al., J. Mol. Biol. 215: 403- 410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the

BLOSUM62 scoring matrix (see Henikoff & Henikoff(1989) Proc. Natl. Acad. Sci. USA 89: 10915).

[000108] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5877 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chern., 17: 149-163 (1993)) and XNU (Claverie and States, Comput. Chern., 17: 191-201 (1993)) low-complexity filters can be employed alone or in combination.

[000109] The term "silencing agent" or "silencing molecule" as used herein means a specific molecule, which can exert an influence on a cell in a sequence-specific manner to reduce or silence the expression or function of a target, such as a target gene or protein. Examples of silence agents include nucleic acid molecules such as naturally occurring or synthetically generated small interfering R As (siR As), naturally occurring or synthetically generated microR As (miRNAs), naturally occurring or synthetically generated dsRNAs, and antisense sequences (including antisense oligonucleotides, hairpin structures, and antisense expression vectors), as well as constructs that code for any one of such molecules.

[000110] A "small interfering RNA" or "siRNA" means RNA of approximately 21-25 nucleotides that is processed from a dsRNA by a DICER enzyme (in animals) or a DCL enzyme (in plants). The initial DICER or DCL products are double-stranded, in which the two strands are typically 21-25 nucleotides in length and contain two unpaired bases at each 3' end. The individual strands within the double stranded siRNA structure are separated, and typically one of the siRNAs then are associated with a multi-subunit complex, the RNAi- induced silencing complex (RISC). A typical function of the siRNA is to guide RISC to the target based on base-pair complementarity. The term siRNA is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

[000111] As used here "suppression" or "silencing" or "inhibition" are used interchangeably to denote the down-regulation of the expression of the product of a target sequence relative to its normal expression level in a wild type organism. Suppression includes expression that is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the wild type expression level.

[000112] As used herein, the phrases "target sequence" and "sequence of interest" are used interchangeably and encompass DNA, RNA (comprising pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, and may also refer to a polynucleotide comprising the target sequence. Target sequence is used to mean the nucleic acid sequence that is selected for suppression of expression, and is not limited to

polynucleotides encoding polypeptides. Target sequences may include coding regions and non-coding regions such as promoters, enhancers, terminators, introns and the like. The target sequence may be an endogenous sequence, or may be an introduced heterologous sequence, or transgene. The specific hybridization of an oligomeric compound with its target sequence interferes with the normal function of the nucleic acid. The target sequence comprises a sequence that is substantially or completely complementary between the oligomeric compound and the target sequence. This modulation of function of a target nucleic acid by compounds, which specifically hybridize to it, is generally referred to as "antisense".

[000113] The term "trans-acting siRNA" or "tasiRNA" or "ta-siRNA" refer to a subclass of siRNAs that function like miRNAs to repress expression of target genes, yet have unique biogenesis requirements. Trans-acting siRNAs form by transcription of tasiRNA-generating genes, cleavage of the transcript through a guided RISC mechanism, conversion of one of the cleavage products to dsRNA, and processing of the dsRNA by DCL enzymes. tasiRNAs are unlikely to be predicted by computational methods used to identify miRNA because they fail to form a stable foldback structure. A ta-siRNA precursor is any nucleic acid molecule, including single-stranded or double-stranded DNA or RNA, that can be transcribed and/or processed to release a tasiRNA. The term tasiRNA is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

[000114] II. Overview of Several Embodiments

[000115] In one embodiment, the invention relates to a heterologous or synthetic or artificial single-stranded ribonucleic acid (RNA) construct comprising: (i) a microRNA and a complement thereof, and (ii) a distal SL region operably linked in between the microRNA and the complement thereof, wherein the distal SL region consists of less than about 50 nucleotides.

[000116] In another embodiment, the invention relates to a heterologous or synthetic or artificial single-stranded ribonucleic acid (RNA) construct comprising: (i) a microRNA and a complement thereof, and (ii) a distal SL region operably linked in between the microRNA and the complement thereof wherein the distal SL region consists of less than about 45 nucleotides or less than about 44 nucleotides or less than about 43 nucleotides or less than about 42 nucleotides or less than about 41 nucleotides or less than about 40 nucleotides or less than about 39 nucleotides or less than about 38 nucleotides or less than about 37 nucleotides or less than about 36 nucleotides or less than about 35 nucleotides or less than about 34 nucleotides or less than about 33 nucleotides or less than about 32 nucleotides or less than about 31 nucleotides or less than about 30 nucleotides or less than about 29 nucleotides or less than about 28 nucleotides or less than about 27 nucleotides or less than about 26 nucleotides or less than about 25 nucleotides or less than about 24 nucleotides or less than about 23 nucleotides or less than about 22 nucleotides or less than about 21 nucleotides or less than about 20 nucleotides or less than about 19 nucleotides or less than about 18 nucleotides or less than about 17 nucleotides or less than about 16 nucleotides or less than about 15 nucleotides or less than about 14 nucleotides or less than about 13 nucleotides or less than about 12 nucleotides or less than about 11 nucleotides or less than about 10 nucleotides or less than about 9 nucleotides or less than about 8 nucleotides or less than about 7 nucleotides or less than about 6 nucleotides or less than about 5 nucleotides or less than about 4 nucleotides or less than about 3 nucleotides.

[000117] In another embodiment, the invention is a heterologous or synthetic or artificial single-stranded ribonucleic acid (RNA) comprising (i) a microRNA and a complement thereof, and (ii) a distal SL region in between the microRNA and the complement thereof, wherein the distal SL region consists of about 3 to about 40 nucleotides.

[000118] In accordance with another embodiment of the invention, the distal SL region can consists of between about 3 to about 50 nucleotides, between about 3 to about 45 nucleotides, between about 3 to about 40 nucleotides, between about 3 to about 35 nucleotides, between about 3 to about 30 nucleotides, between about 3 to about 20 nucleotides, between about 3 to about 15 nucleotides, between about 3 to about 10 nucleotides, between about 5 to about 50 nucleotides, between about 5 to about 50 nucleotides, between about 5 to about 45 nucleotides, between about 5 to about 40 nucleotides, between about 5 to about 35 nucleotides, between about 5 to about 30 nucleotides, between about 5 to about 20 nucleotides, between about 5 to about 15 nucleotides, between about 5 to about 10 nucleotides, between about 10 to about 50 nucleotides, between about 10 to about 45 nucleotides, between about 10 to about 40 nucleotides, between about 10 to about 35 nucleotides, between about 10 to about 30 nucleotides, between about 10 to about 20 nucleotides, between about 10 to about 15 nucleotides, between about 15 to about 50 nucleotides, between about 15 to about 45 nucleotides, between about 15 to about 40 nucleotides, between about 15 to about 35 nucleotides, between about 15 to about 30 nucleotides, between about 15 to about 20.

[000119] As used herein, the region that folds back between the micro-RNA and the complement thereof is referred to as the "distal stem-loop region" or "distal SL region." In an aspect of the invention, the region in between the microRNA and complement thereof could adopt a stem-loop structure or just a loop structure. In one embodiment of the invention, the region in between the micro RNA and the complement thereof is folded to form a symmetric stem-loop structure. In another embodiment, the region in between the micro RNA and the complement thereof is folded to form an asymmetric stem-loop structure.

[000120] In one embodiment of invention, the stem-loop is distal or downstream or 3' of the miRNA. In another embodiment, the stem-loop is proximal or upstream or 5' of the miRNA.

[000121] In another embodiment, the invention is a heterologous or synthetic or artificial single-stranded ribonucleic acid (RNA) comprising (i) a microRNA and a complement thereof, and (ii) a distal SL region in between the microRNA and the complement thereof, wherein the nucleotide sequence of the distal SL region is at least 75% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[000122] In accordance with another embodiment of the invention, the nucleotide sequence identity of the distal SL region is at least 70%, is at least 75%, is at least 80%, is at least 85%, is at least 90%, is at least 95%, is at least 97%, is at least 99%. In accordance with another embodiment of the invention, the nucleotide sequence identity of the distal SL region is identical or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[000123] In one embodiment of the invention, the RNA construct is operably linked between complementary nucleotide sequences. In another embodiment, the complementary nucleotide sequences are at least 75% identical to SEQ ID NO: 3 and SEQ ID NO: 4, or complements thereof. In another embodiment the complementary nucleotide sequences are at least 75% identical to SEQ ID NO: 5 and SEQ ID NO: 6, or complements thereof. In yet another embodiment the complementary nucleotide sequences are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical. In accordance with another embodiment of the invention, the complementary nucleotide sequences are identical or have 100% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4, or complements thereof; or the complementary nucleotide sequences are identical or have 100% sequence identity to SEQ ID NO: 5 and SEQ ID NO: 6, or complements thereof

[000124] In one embodiment of the invention, the RNA construct is a pre-microRNA that is processed into a microRNA, and wherein the microRNA modulates the expression of a target sequence. In another embodiment of the invention, the RNA is a pre-microRNA that is processed into a microRNA, and wherein the microRNA modulates or suppresses or reduces the expression of a target sequence. In accordance with another embodiment of the invention, the microRNA is an artificial microRNA. In yet another embodiment of the invention, the target sequence is a promoter, or an enhancer, or a terminator or an intron. In another embodiment, the target sequence is an endogenous sequence, in another embodiment the target sequence is a heterologous sequence. In one embodiment of the invention, the microRNA is substantially complementary to the target sequence. In another embodiment, the microRNA is sufficiently complementary to the target sequence. In another embodiment, the microRNA is completely complementary to the target sequence.

[000125] In one embodiment of the invention, the pre-microRNA has at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10; and wherein the region comprising Ri to R _n and the region comprising R'i to 'n represent the microRNA or the complement thereof; and wherein "n" corresponds to the number of nucleotides in the miRNA. In one aspect, "n" is in the range of from about 15 to about 25 nucleotides, in another aspect, "n" is from about 20, or "n" is from about 21 nucleotides.

[000126] In another embodiment of the invention, the pre-microRNA has a nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10. In accordance with another embodiment of the invention, the pre- microRNA has a nucleotide sequence is identical or has 100% sequence identity to SEQ ED NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10.

[000127] Also provided herein, is a heterologous or synthetic or an artificial deoxyribonucleic acid (DNA) comprising a polynucleotide or nucleotide sequence encoding an artificial or synthetic or heterologous single-stranded ribonucleic acid (RNA) comprising (i) a microRNA and a complement thereof, and (ii) a distal SL region in between the microRNA and the complement thereof.

[000128] In one embodiment, the invention relates to a vector comprising DNA encoding an artificial or synthetic or heterologous single-stranded ribonucleic acid (RNA) comprising (i) a microRNA and a complement thereof, and (ii) a distal SL region in between the microRNA and the complement thereof. In one embodiment, the vector further comprises a promoter or regulatory sequence. In another embodiment, the vector comprises a tissue-specific, cell- specific or other regulated manner. In another embodiment, the vector comprises a selectable marker or resistance gene. Typical markers and/or resistance genes are well known in the art and include antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin, the streptomycin phosphotransferase gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e. g., the bar gene), or other such genes known in the art.

[000129] In another embodiment of the invention, the vector comprises flanking nucleotide sequences; wherein the flanking nucleotide sequences are at least 75% identical to SEQ ID NO: 11 and SEQ ID NO: 12, or complements thereof; or wherein the flanking nucleotide sequences are at least 75% identical to SEQ ID NO: 13 and SEQ ID NO: 14, or complements thereof. In another embodiment, the vector comprises flanking nucleotide sequences; wherein the flanking nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to SEQ ID NO: 11 and SEQ ID NO: 12, or complements thereof; or wherein the flanking nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to SEQ ID NO: 13 and SEQ ID NO: 14, or complements thereof. In accordance with another embodiment of the invention, the vector comprises flanking nucleotide sequences; wherein the flanking nucleotide sequences are identical or 100% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 12, or complements thereof; or wherein the flanking nucleotide sequences are identical or 100% sequence identity to SEQ ID NO: 13 and SEQ ID NO: 14, or complements thereof.

[000130] In one embodiment, the invention relates to a cell expressing RNA or DNA, or complements thereof; or a vector encoding an artificial or synthetic or heterologous single- stranded ribonucleic acid (RNA) comprising (i) a microRNA and a complement thereof, and (ii) a distal SL region in between the microRNA and the complement thereof. In another embodiment the invention relates to a cell, wherein the cell expresses a RNA construct which is a pre-microRNA that is processed into a microRNA, and wherein the microRNA modulates the expression of a target sequence. In another embodiment of the invention, the RNA is a pre-microRNA that is processed into a microRNA, and wherein the microRNA modulates or suppresses or reduces the expression of a target sequence. Target sequences may include coding regions and non-coding regions such as promoters, enhancers, terminators, introns and the like. The target sequence may be an endogenous sequence, or may be an introduced heterologous sequence, or transgene. In one embodiment, the cell is a plant cell. In another aspect the plant cell is a monocotyledonous plant cell or a

dicotyledonous plant cell.

[000131] Provided herein, is a method of modulating expression of a target sequence, comprising: transforming a cell with a vector as described herein, or expressing a vector in a cell or applying or providing or introducing a microRNA to a cell. A method of modulating expression of a target sequence in a cell, comprising: transforming a cell with the vector as described herein, wherein the cell produces the microRNA, and wherein the microRNA modulates the expression of a target sequence in the cell.

[000132] In another embodiment, the invention relates to a method of modulating expression of a target sequence in cell, comprising providing, introducing, or applying the microRNA produced by the cell to a second cell, wherein the microRNA modulates the expression of a target sequence in the second cell. In one aspect the invention relates to passive provision of the microRNA to another cell; in another aspect the microRNA is actively provided to another cell. In one embodiment the second cell is from the same organism, in another embodiment the second cell is from a different organism. As a non-limiting example, passive provision of the microRNA to a cell in a different organism involves the uptake of the microRNA by a pathogen or pest, for example a virus, a bacterium, a fungus, an insect, etc.

III. Examples

[000133] The following examples are provided to illustrate various aspects of the present disclosure, and should not be construed as limiting the disclosure only to these particularly disclosed embodiments.

[000134] The materials and methods employed in the examples below are for illustrative purposes only, and are not intended to limit the practice of the present embodiments thereto. Any materials and methods similar or equivalent to those described herein as would be apparent to one of ordinary skill in the art can be used in the practice or testing of the present embodiments.

Example 1: Selection of Arabidopsis thaliana MIR390a precursor for direct cloning of artificial niiRNAs

[000135] Several properties of the AtMIR390a precursor make it attractive as a backbone to engineer a new generation of amiRNA vectors. First, small RNA library analyses indicate that the AtMIR390a precursor is processed accurately, as the majority of reads mapping to the AtMIR390a foldback correspond to the authentic 21 -nucleotide (nt) miR390a guide strand (Figure 1A). Second, as the MIR390 family is deeply conserved in plants (Axtell et al., 2006; Cuperus et al., 2011), AtMIR390a-based amiRNAs are likely to be produced accurately in different plant species. Third, the AtMIR390a precursor was used to express high levels of either 21 or 22-nt amiRNAs of the correct size inN. benthamiana leaves (Montgomery et al., 2008; Cuperus et al., 2010; Carbonell et al., 2012), demonstrating that the miR390 duplex sequence provides little or no specific information required for accurate processing. Fourth, the AtMIR390a foldback has a relatively short distal stem-loop (31 nt; Figure IB) compared to other conserved^, thaliana MIRNA foldbacks (Figure 1C), including those used previously for amiRNA expression in plants (Figure ID). A short distal stem-loop facilitates more cost-effective synthesis of partially complementary oligonucleotides (see next section) that span the entire foldback. And fifth, although authentic miR390a associates preferentially with AG07, association of AtMIR390a-based amiRNAs containing a 5'U or 5Ά can be directed to AGOl (Montgomery et al, 2008; Cuperus et al., 2010) or AG02 (Carbonell et al., 2012), respectively.

Example 2: Direct cloning of amiRNA sequences in AtMIR390a-based vectors

[000136] Details of the zero background cloning strategy to generate AtMIR390a-based amiRNA constructs are illustrated in Figure 2A. The amiRNA insert is derived by annealing of two overlapping and partially complementary 75-base oligonucleotides covering the amiRNA/ _^ 4t ZR390a-distal-loop/amiRNA* sequence (Figure 2A). Design of amiRNA oligonucleotides is described in detail in Supplemental Protocol SI. Forward and reverse oligonucleotides must have 5'-TGTA and 5'-AATG overhangs, respectively, for direct cloning into AtMIR390a-based vectors (see below). This strategy requires no oligonucleotide enzymatic modifications, PCR steps, restriction digestions, or DNA fragment isolation.

[000137] A series of AtMIR390a-based cloning vectors were developed and named iAtMIR390a-B/c' vectors (from AtMIR390a-BsaUccdB). They contain a truncated

AtMIR390a precursor sequence whose miRNA/distal stem-loop/amiRNA* region was replaced by a 1461 bp DNA cassette including the ccdB gene (Bernard and Couturier, 1992) flanked by two Bsal sites (Figure 2B, Table I, Fig. 9). foal restriction enzyme is a type lis endonuclease with non-palindromic recognition sites [GGTCTC(N]/Ns)] that are distal from the cleavage sites. Here, Bsal recognition sites are inserted in a configuration that allows both Bsal cleavage sites to be located outside the ccdB cassette (Figure 2B). After Bsal digestion, AtMIR390a-B/c vectors have 5'-TACA and 5'-CATT ends, which are incompatible. This prevents vector self-ligation and eliminates the need to modify the ends of insert

oligonucleotide sequences (Schwab et al., 2006; Molnar et al, 2009). The use of two Bsal sites in this configuration has been adapted from the Golden Gate cloning method (Engler et al., 2008), and was used in other amiRNA cloning methods (Chen et al., 2009; Zhou et al, 2013). Bsal digestion of the B/c vector and subsequent ligation of the amiRNA

oligonucleotide insert can be done in separate reactions, or combined in a single 5 min reaction. The amiRNA insert is ligated directionally into the foal-digested AtMIR390a-B/c vector and introduced into E. coli. Non-linearized plasmid molecules with no amiRNA insert fail to propagate in E. coli ccdB sensitive strains, such as DH5a or DH10B. In summary, compared to other amiRNA cloning methods (Schwab et al, 2006; Qu et al., 2007; Chen et al., 2009; Molnar et al., 2009; Wang et al, 2010; Eamens et al., 2011; Yan et al., 2011; Liang et al., 2012; Wang et al., 2012; Zhou et al., 2013), this method is relatively simple, fast, and cost-effective (Figure 2C).

[00Q138] pMDC32B-AtMIR390a-B/c,pMDCI23SB~AtA IR390a-B/c or pFK210B-AtMIR390a- B/c expression vectors were generated for direct cloning of amiRNAs and tested in different plant species (Table I, Fig. 8). Each vector contains a unique combination of bacterial and plant antibiotic resistance genes. The direct cloning of amiRNA inserts into plant expression vectors avoids the need for sub-cloning the amiRNA cassette from an intermediate plasmid to the expression vector (Schwab et al., 2006; Qu et al, 2007; Warthmann et al, 2008; Eamens et al., 2011; Yan et al, 2011). A pENTR-AtMIR390a-B/c GATEWAY-compatible entry vector was generated for direct cloning of the amiRNA insert and subsequent recombination into a preferred GATEWAY expression vector containing a promoter, terminator features of choice (Table I, Fig. 8).

Table I. Bsal/ccdB -based ('B/c') vectors for direct cloning of amiR As and syn- tasiRNAs.

Example 3: Comparison of amiRNA production from AtMIR390a and AtMIR319a precursors

[000139] To verify the accumulation in planta of AiMIR390a-denved amiRNAs, six different amiRNA sequences (amiR-1 to amiR-6) (Fig. 9) were directly cloned into pMDC32B- AtMIR390a-B/c (amiR-2 and amiR-3) or _P MDC123SB-AtMIR390a-B/c (amiR-1, amiR-4, amiR-5 and amiR6) and expressed transiently inN. benthamiana leaves. All AtMIR 90a- based amiRNAs had a U and C in 5'-to-3' positions 1 and 19, respectively, of the guide strand. They also contained G, A, C, and A in 5'-to-3' positions 1, 19, 20 and 21, respectively, of the amiRNA* strand (Figure 3A, Fig. 9). In addition, position 11 of the amiRNA guide strand was kept unpaired with position 9 of the amiRNA* to preserve the authentic AtMIR390a base-pairing structure (Figure 2A).

[000140] For comparative purposes, the same six amiRNA sequences were also expressed from AtMIR319a precursor, which has been most widely used to express amiRNAs in plants (Schwab et al, 2006). In this case, amiRNAs were cloned into pMDC32B-AtMIR319a-B/c (amiR-2 and amiR-3) or pMDC123SB-AtMIR319a-B/c (amiR-1, amiR-4, amiR-5 and amiR6; Figure 3 A, Supplemental Fig. S2), following the protocols used previously (Schwab et al., 2006). In the original ^£MZR3/9a-based cloning configuration, a 20 bp sequence in

AtMIR319a was replaced by a 21 bp sequence (Schwab et al, 2006) because it was initially thought that miR319a was only 20 bases long (Palatnik et al., 2003; Sunkar and Zhu, 2004). Later analyses, however, revealed that miR319a is predominantly a 21-mer, like the majority of plant miRNAs (Rajagopalan et al., 2006; Fahlgren et al., 2007). Consequently, the AtMIR319a foldbacks in the original AtMIR31 Pa-based configuration had a one base-pair elongated basal stem that did not seem to affect foldback processing (Schwab et al, 2006). Here, amiR-1, amiR-2 and amiR-3 were cloned in the original 20-mer configuration

(AtMIR319a) (Schwab et al., 2006), and amiR-4, amiR-5 and amiR-6 were cloned in the more recent 21-mer configuration (AiMIR319a~21) (wmd3.weigelworld.org) where the authentic 21 nt sequence of endogenous miR319a is replaced by the 21 nt sequence of the amiRNA, preserving the foldback structure of authentic AtMIR319a (Figure 3A, Fig. 9). All AtMIR319a- and AtMIR319a-21 -based amiRNAs had U and a C in positions 1 and 19, respectively, in the amiRNA guide, and A, U, U and C in positions 1, 19, 20 and 21, respectively, of the amiRNA*. Position 12 of the amiRNA* was kept unpaired with position 8 of the guide strand to preserve the authentic AtMIR319a base-pairing structure. Note that an extra A-U base pair is found in AtMIR319a-based foldbacks due to the AtMIR319a original 20-mer configuration (Figure 3 A, Fig. 9).

[000141 J In transient expression assays using N. bent amiana, each, of the six amiRNAs derived from the AtMIR390a foldbacks accumulated predominantly as 21 nt species, suggesting that the amiRNA foldbacks were likely processed accurately. In each case, the amiRNA from the AtMIR390a foldbacks accumulated to significantly higher levels than did the corresponding amiRNA from the AtMIR319a or AtMIR319a-21 foldbacks (P < 0.02 for all pairwise i-test comparisons; Figure 3B). The basis for differences in accumulation levels was not explored further. However, it is suggested that the more non-canonical loop-to-base processing mechanism for the AtMIR319a foldback (Addo-Quaye et al., 2009; Bologna et al., 2009; Bologna et al., 2013) may be relatively less efficient than the canonical base-to-loop processing pathway for AtMIR390a foldback.

Example 4: Functionality of AtMIR390a-based amiRNAs in Arabidopsis

[000142] To test the functionality of ^i R3P0a-based amiRNAs in repressing target transcripts, four different amiRNA constructs (Figure 4A) were introduced into in A. thaliana Col-0 plants. The small RNA sequences were shown previously to repress gene expression when expressed as amiRNAs from a AtMIR31 Pa-based foldback (Schwab et al., 2006; Liang et al., 2012) or from a syn-tasiRNA construct (Felippes and Weigel, 2009). In particular, amiR-Ft, amiR-Lfy and amiR-Ch42 each targeted a single gene transcript [LEAFY (LFY), CHLORINA 42 (CH42) and FLOWERING LOCUS T (FT) respectively], and amiR-Trich targeted three MYB transcripts [TRIPTYCHON (TRY), CAPRICE (CPC) and ENHANCER OF TRIPTYCHON AND CAPRICE2 (ETC2)] (Fig. 11). Plant phenotypes, amiRNA accumulation, mapping of amiRNA reads in the corresponding AtMIR390a foldback and target mRNA accumulation were measured in Arabidopsis Tl transgenic lines.

[000143] Twenty-three of 67 transgenic lines containing 35S:AtMIR390a-Lfy construct showed morphological defects like Ify mutants (Schultz and Haughn, 1991; Weigel et al., 1992; Schwab et al, 2006) (Supplemental Table SI), including obvious floral defects with leaf-like organs (Figure 4B) and significantly increased numbers of secondary inflorescence shoots (P < 0.01 two sample i-test, Figure 4F). Ninety-eight of 101 transgenic lines containing 35S:AtMIR390a-Ch42 construct were smaller than controls and had pale or bleached leaves and cotyledons (Figure 4C, Supplemental Table SI), as expected due to defective chlorophyll biosynthesis with a loss of Ch42 magnesium chelatase (Koncz et al., 1990; Felippes and Weigel, 2009). Sixty-three of these plants had a severe bleached phenoiype with a facie of visible true leaves at 14 days after plating (Figures 4C and 4F, Supplemental Table SI). Each of the 34 transformants containing 35S:AtMIR390ct-Ft was significantly delayed in flowering time compared to control plants not expressing the amiRNA (P < 0.01 two sample i-test, Figure 4D, Supplemental Table SI), as previously observed in small RNA knockdown lines (Schwab et al., 2006; Liang et al., 2012) and ft mutants (Koornneef et al., 1991). Finally, 52 out of 53 lines containing 35S:AtMIR390a- Trich had increased number of trichomes in rosette leaves; 15 lines had highly clustered trichomes on leaf blades like try cpc double mutants (Schellmann et al., 2002) or other amiR- Trich overexpressor transgenic lines (Schwab et al., 2006; Liang et al, 2012) (Figure 4E, Supplemental Table SI). Each of the MIR390a~based amiRNAs, therefore, conferred a high proportion of expected target-knockdown phenotypes in transgenic plants.

[000144] The accumulation of all four amiRNAs was confirmed by RNA blot analysis in Tl transgenic lines showing amiRNA-induced phenotypes (Figure 4G). In all cases, amiRNAs accumulated as a single species of 21 nt (Figure 4G), suggesting thatAtMIR390a-based amiRNAs were precisely processed. To more accurately assess processing and accumulation of the amiRNA populations, small RNA libraries from samples containing each of the

AtMIR390a-based constructs were prepared. In each case, the majority of reads from the AtMIR390a foldback corresponded to correctly processed, 21 nt amiRNA while reads from the amiRNA* strands were always relatively under-represented (Figure 5). It is possible that amiRNA* strands with an AGO-non-preferred 5' nucleotide (5'C for ami -Ft* and amiR- Trich*, and 5'G for amiR-Lfy* and amiRCh42*) were actually produced but were less stable. The library read data support the rational design strategy to place an AGO non-preferred 5' nucleotide (such as 5'G) at the 5' end of the amiRNA* to avoid competition with the amiRNA guide strand for AGO loading. Combined with previous data (Cuperus et al., 2010), AtMIR390a-based foldbacks can be rationally designed to produce accurately processed amiRNAs of 21 or 22 nts, the latter of which can be used to trigger tasiRNA biosynthesis.

[000145] Accumulation of amiRNA target mRNAs in A. thalianct transgenic lines was analyzed by quantitative RT-PCR assay. The expression of all target mRNAs was significantly reduced compared to control plants (P < 0.02 for all pairwise i-test comparisons, Figure 4H) when the specific amiRNA was expressed.

Example 5: Direct cloning of synthetic tasiRNAs in AtTASlc-based constructs

[000146] A new generation of functional syn-tasiRNA vectors based on a modified TASlc gene was produced with the potential to multiplex syn-tasiRNA sequences at DCL4- processing positions 3'D3[+]' and '3'D4[+] of AtTASlc transcript (see (Montgomery et al, 2008). The design of AtTASlc-bdseA syn-tasiRNA constructs expressing two syn-tasiRNAs is shown in Figure 6A.

[000147] Syn-tasiRNA vector construction is similar to that described for the amiRNA constructs (Figure 6C). Briefly, two overlapping and partially complementary

oligonucleotides containing syn-tasiRNA sequences are designed (for details see Figure 6B). Sequence of syn-tasiRNA- 1 can be identical or different to sequence of syn-tasiRNA-2. Theoretically, more than two syn-tasiRNA sequences can be introduced in the modified AtTASlc, with such design being more attractive if multiple and unrelated sequences have to be targeted from the same syn-tasiRNA construct. The syn-tasiRNA insert results from the annealing of two 46 nt-long oligonucleotides, and will have 5' -ATT A and 5'-GTTC overhangs. No PCR reaction, restriction enzyme digestion or gel purification steps are required to obtain the syn-tasiRNA insert. Several AtTASlc-b&sed cloning vectors were developed and named 'AtTASlc-B/c' vectors (from AtTASlc-BsaUccdB) (Table I, Fig. 11). These contain a truncated AtTASlc sequence with the 3'D3[+] -3'D4[+] region was replaced by the 1461 bp ccd cassette flanked by two Bsal sites in the orientation that allows both Bsal recognition sites to be located outside of the AtTASlc sequence (Figure 6C). Annealed oligonucleotides are directly ligated into the linearized AtTASlc-B/c expression vector in a directional manner (Figure 6C). Sub-cloning is only required if the syn-tasiRNA insert is inserted in the GATEWAY entry vector pENTR-AtTASlc-B/c that allows recombination with the y4/7¾S7c-syn-tasiRNA cassette to the GATEWAY expression vector of choice (Table I, Fig. 1 1). Compared to other syn-tasiRNA cloning methods (de la Luz Gutierrez-Nava et al., 2008; Montgomery et al, 2008; Felippes and Weigel, 2009), this method is relatively fast, efficient and cost-effective.

Example 6: Functionality of ^iZ^ c-based synthetic tasiRNAs in Arabidopsis

[000148] To test the functionality of single and multiplexed AtTASl c-based syn-tasiRNAs, and to compare to the efficacy of the syn-tasiRNAs with amiRNA, several syn-tasiRNA constructs were generated and introduced into Arabidopsis Col-0 plants (Figure 7). These constructs expressed either a syn-tasiRNA targeting i<T (syn-tasiR-Ft) and/or a syn-tasiRNA targeting TRY/CPC/ETC2 (syn-tasiR-Trich) in single (35S: AtTASl c-D3&D4Ft,

35S:AtTASlc-D3&D4Trich) or dual {35S:AtTASlc-D3Trich-D4Ft and 35S:AtTASlc-D3Ft- D4Trich) configurations (Figure 7A, Fig. 12). For comparative purposes, transgenic lines expressing 35S:AtMIR390a-Ft and 35S:AtMJR390a-Trich, as well as 35S. GUS control construct, were generated in parallel. The small RNAs produced in each pair of syn-tasiRNA and amiRNA vectors were identical. Plant phenotypes, syn-tasiRNA and amiRNA

accumulation, processing and phasing analyses of AtTASl c-based syn-tasiRNA, and target mRNA accumulation were analyzed in Arabidopsis Tl transgenic lines (Figure 7, Figs. 13-16 and Supplemental Table SII). Plant phenotypes were also analyzed in T2 transgenic lines to confirm the stability of expression (Supplemental Table SHI).

[0100] Seventy-three and 62% of the transformants expressing the dual configuration syn- tasiRNA constructs 35S:AtTASlc-D3Ft-D4Trich and 35S:AtTASlc-D3Trich-D4Ft, respectively, showed both Trich and Ft loss-of-function phenotypes (Supplemental Table SII), which were characterized by increased clustering of trichomes in rosette leaves and a delay in flowering time compared to the 35S:GUS transformants (Figure 7B). Plants expressing 35S:AtTASlc~D3&D4Trich or 35S:AtMIR390a-Trich constructs showed clear Trich phenotypes in 82% and 92% of lines, respectively. In contrast with amiR-Trich overexpressors, none of the syn-tasiRNA-Trich constructs triggered the double try cpc phenotype (Supplemental Table SII). Transformants expressing the 35S:AtTASlc-D3Ft- D4Trich and 35S:AtTASlc-D3Trich-D4Ft constructs had a significant delay in flowering time compared to control lines expressing the 35S:GUS, 35S:AtMIR390a-Trich or 35S:AtTASlc-D3&D4Trich constructs (P < 0.01 for all pairwise /-test comparison) although the 35S:AtMIR390a-Ft amiRNA lines showed the strongest delay in flowering (P < 0.001 two sample t-test) (Figure 7B, Fig. 13 and Supplemental Table SII). The trichome phenotypes were maintained in the Arabidopsis T2 progeny expressing 35S:AtMIR390a-Trich, 35S:AtTASlc-D3&D4-Trich, 35S:AtTASlc-D3Trich-D4Ft and 35S:AtTASlc-D3Ft-D4Trich constructs (Supplemental Table SIII).

[000149] Next, accumulation of syn-tasiR-Trich and syn-tasiR-Ft was compared to accumulation of amiR-Trich and amiR-Ft was analyzed by RNA blot assays using Tl transgenic plants showing obvious syn-tasiRNA- or amiRNA-induced phenotypes (Figure 7C). In all cases, syn-tasiRNA accumulated to high levels and as a single band at 21 nt (Figure 7C), suggesting that processing of AtTASlc-based constructs was accurate. When two copies of either syn-tasiR-Ft and syn-tasiR-Trich were expressed from a single construct, the corresponding RNAs accumulated to higher levels compared to when expressed in the dual syn-tasiRNA configuration containing only single copies of each RNA (Figure 7C).

Interestingly, amiR-Ft and amiR-Trich accumulated to higher levels than did any of the corresponding syn-tasiRNAs (Figure 7C). It is possible that one or more factors in the AtTASIc-dependent tasiRNA-generating pathway is (are) limiting relative to the ubiquitous miRNA biogenesis factors. It is also possible that RDR6-dependent 7¾.S7c-dsRNAs may be processed by DCL4 from both ends, resulting in the production of tasiRNAs in two registers (Rajeswaran et al., 2012) and limiting the accumulation of accurately processed syn- tasiRNAs from positions D3[+] and D4[+].

[0101] To further analyze processing and phasing of AtTASJc-ba&ed syn-tasiRNA expressed from the dual configuration constructs (35S:AtTASlc-D3Trich-D4Ft and 35S:AtTASlc-D3Ft- D4Trich), small RNA libraries were produced and analyzed. Analysis of 35S:AtTASlc- D3Trich-D4Ft small RNAs libraries confirmed that the syn-tasiRNA transcript yielded predominantly 21-nt syn-tasiR-Trich and syn-tasiR-Ft (51 and 67 % of the reads within ±4 nt of 3'D3[+] and 3'D4[+], respectively), and that the corresponding tasiRNAs were in phase with miR173 cleavage site (Figure 7D upper panel, Fig. 14 A and B left panels). Similarly, 35S:AtTASlc-D3Ft-D4Trich libraries revealed a high proportion of 21-nt syn-tasiR-Ft and syn-tasiR-Trich (45 and 65 % of the reads within ±4 nt of 3'D3[+] and 3'D4[+], respectively) and accurately phased tasiRNAs (Figure 7D lower panel, Fig. 14 A and B right panels). In both 35S:AtTASlc-D3Trich-D4Ft and 35S:AtTASlc-D3Ft-D4Trich libraries, relatively low levels of incorrectly processed siRNAs that overlap with the D3[+] and D4[+] positions were detected (Figure 14). While these small RNAs differ from the correctly processed forms by only one or a few terminal nucleotides, it is theoretically possible that these could have altered targeting properties. Additionally, analyses of endogenous small RNAs showed that expression of the syn-tasiRNA constructs, relative to expression of the 35S:GUS control construct, did not interfere with processing or accumulation of authentic AtTASlc tasiRNAs (Figs. 15 and 16).

[000150] Finally, accumulation of target mRNAs in the 35S:AtTASlc-D3Trich-D4Ft and 35S:AtTASlc-D3Ft-D4Trich transgenic lines was analyzed by quantitative RT-PCR assay (Figure 7E). The expression of all four target mRNAs (FT, TRY, CPC and ETC2) was significantly reduced in lines expressing both dual configuration syn-tasiRNA constructs compared to control plants expressing the 35S:GUS construct (P < 0.02 for all pairwise /-test comparison) (Figure 7E). However, target mRNA expression was reduced more in lines expressing the single configuration syn-tasiRNA constructs, and decreased even more in lines expressing the corresponding amiRNA (Figure 7E). Taken together with results presented above, the extent of target mRNA knockdown and resultant phenotypes correlates with amiRNA and syn-tasiRNA dosage.

[0102] Syn-tasiRNA technology was used before to repress single targets in Arabidopsis (de la Luz Gutierrez-Nava et al, 2008; Montgomery et al., 2008; Montgomery et al., 2008; Felippes and Weigel, 2009). Here, a single AtTASl c-based construct expressing multiple distinct syn-tasiRNAs triggered silencing of multiple target transcripts and resultant knockdown phenotypes. Theoretically, AtTASl c-based vectors could be designed to produce more than two syn-tasiRNAs to repress a larger number of unrelated targets. Therefore, the syn-tasiRNA approach may be preferred for applications involving specific knockdown of multiple targets.

Example 7: Plant materials and growth conditions [000151] Arabidopsis thaliana Col-0 mdNicotiana benthamiana plants were grown in a chamber under long day conditions (16/8 hr photoperiod at 200 μηιοΐ m ^"2 s ^"1 ) and 22°C constant temperature. Plants were transformed using the floral dip method with

Agrobacterium tumefaciens GV3101 strain (Clough and Bent, 1998). Transgenic plants were grown on plates containing Murashige and Skoog medium and Basra (50 mg/mi) or hygromycin (50 mg/ml) for 10 days before being transferred to soil. Plant photographs were taken with a Canon Rebel XT/EOS 350D digital camera and EF-S18-55mm #3.5-5.6 II or EF-lOOmm f/2.8 Macro USM lenses.

Example 8: DNA constructs

[000152] The cassette containing the AtMIR390a sequence lacking the distal stem-loop region, and including two Bsal sites, was generated as follows. A first round of PCR was done to amplify AtMIR390a-5' or AtMIR390a-V regions using primers AtMIR390a-F and Bsal- AtMIR390a-5'-R, or BsaI-AtMIR390a-3'-F and AtMIR390a-R, respectively. A second round of PCR was done using as template a mixture of the products of the first PCR round and primers AtMIR390a-F and AtMIR390a-R. The PCR product was cloned into pENTR-D- TOPO (Life Technologies) to generate pENTR-AtMIR390a-BsaI. A similar strategy was used to generate pENTR-AtTASlc-Bsal containing the AtTASlc cassette for syn-tasiRNA cloning: oligo pairs AtTASlc-F/BsaI-AtTASlc-5'-R and BsaI-AtTASlc-3'-F/AtTASlc-R were used for the first round of PCR, and oligo pair AtTASlc-F/AtTASlc-R was used for the second PCR.

[000153] A 2x35S promoter cassette including the Gateway attR sites of pMDC32 (Curtis and Grossniklaus, 2003) was transferred into pMDC123 (Curtis and Grossniklaus, 2003) to make pMDC123S. An undesired Bsal site contained in pMDC32,pMDC123S and pFK210 (de Felippes and Weigel, 2010) was disrupted to generate pMDC32B,pMDC123SB and pFK210B, respectively. pMDC32B-AtMIR390a-BsaI, pMDC123SB-AtMIR390BsaI and pFK210B-AtMIR390a-BsaI intermediate plasmids were obtained by LR recombination using pENTR-AtMIR390a-BsaI as the donor plasmid and pMDC32B,pMDC123SB and pFK210B as destination vectors, respectively. Similarly, pMDC32B-AtTASl c-Bsal and pMDC123SB- AtTASlc-Bsal intermediate plasmids were obtained by LR recombination using pENTR- AtTASlc-Bsal as the donor plasmid and pMDC32B and pMDC123SB as destination vectors, respectively. [000154] To generate zero background cloning vectors, a ccdB cassette was inserted in between the Bsal sites of plasmids containing the AtMIR390a-BsaI or AtTASlc-Bsal cassettes. ccdB cassettes flanked with Bsal sites and with AtMIR390a or AtTASlc specific sequences were amplified from pFK210 using primers AtMIR390a-B/c-F and AtMTR390a- B/c-R or AtTASlc-B/c-F and AtTASlc-Bc-R, respectively, with an overlapping PCR to disrupt an undesired Bsal site from the original ccdB sequence. These modified ccdB cassettes were then inserted between the Bsal sites into pENTR-AtMIR390a-BsaI, pENTR- AtTASlc-Bsal, pMDC32B-AtMIR390a-BsaI, pMDC32B-AtTASlc-BsaI, pMDC123SB- AtMIR390-BsaI, pMDC123SB-AtTASlc-BsaI and pFK210B-AtMIR390-BsaI\o generate pENTR-AtMIR390a-B/c, pENTR-AtTASl c-B/c, pMDC32B-AtMIR390a-B/c, pMDC32B- AtTASlc-B/c, pMDC123SB-AtMIR390a-B/c, pMDC123SB-AtTASlc-B/c and pFK210B- AtMIR390a-B/c, respectively.

[000155] ^ ZR3/9a-based amiRNA constructs pMDC32-AtMIR319a-amiR-l, pMDC32- AtMIR319a-amiR-2, pMDC32-AtMIR319a-amiR-3, pMDC32-AtMIR319a-21-amiR-4, pMDC32-AtMIR319a-21-amiR-5 and pMDC32-AtMIR319-21-amiR-6) were generated as previousiy described (Schwab et al, 2006) using the WMD3 tool (wmd3.weigelworld.org). The CACC sequence was added to the 5' end of the PCR fragments for pENTR-D-TOPO cloning (Life Technologies) and to allow LR recombination to pMDC32B or pMDC123SB. amiR-1, amiR-2 and amiR-3 were inserted in the AtMIR319a foldback, while amiR-4, amiR- 5, amiR-6, were inserted in the AtMIR319a-21 foldback.

[000156] The rest of the amiRNA and syn-tasiRNA constructs (pMDC32B-AtMIR390a-amiR- 1, pMDC32B-AtMIR390a-amiR-2, pMDC32B-AtMIR390a-amiR-3, pMDC32B-AtMIR390a- 21-amiRA, pMDC32B-AtMIR390a-21-amiR-5, pMDC32B-AtMIR390a-amiR-6, pMDC32B- AtMIR390a-Ft, pMDC32B-AtMIR390a-LJy, pMDC32B-AtMIR390a-Ch42, pMDC32B- AtMIR390a-Trich,pMDC32B-AtTASlc-D3&D4Ft, pMDC32B-AtTASlc-D3&D4Trich, pMDC32B-AtTASlc-D3Trich-D4Ft,pMDC32B-AtTASlc-D3Ft-D4Trich) were obtained as described in the next section. pMDC32-GUS construct was described previously

(Montgomery et al., 2008).

[000157] All oligonucleotides used for generating the constructs described above are listed in Supplemental Table SIV. The sequences and predicted targets for all the amiRNAs and syn- tasiRNAs used in this study are listed in Supplemental Table SV. The sequences of the amiRNA and syn-tasiRNA vectors are listed in the sections tht follow. The following amiRNA and syn-tasiRNA vectors are available from Addgene at www.addgene.org/:

pENTR-AtMIR390a-B/c (Addgene plasmid 51778), pMDC32B-AtMIR390a-B/c (Addgene plasmid 51776), pMDC123SB-AtMIR390a-B/c (Addgene plasmid 5177 '5), pFK210B- AtMIR390a~B/c (Addgene plasmid 51777), pENTR-AtTASlc-B/c (Addgene plasmid 1774), pMDC32B-AtTASlc-B/c (Addgene plasmid 51773) and pMDC123SB-AtTASlc-B/c (Addgene plasmid 51772).

Example 9: amiRNA and syn-tasiRNA oligo design and cloning

[000158] Detailed amiRNA and syn-tasiRNA oligo design and cloning protocols are given in Figures 2 and 6, and in the sections that follow. A web tool to design amiRNA and syn- tasiRNA sequences, together with the corresponding oligonucleotides for cloning into B/c vectors, will be available at website: p-sams.carringtonlab.org. All oligonucleotides used in this study for cloning amiRNA and syn-tasiRNA sequences are listed in Supplemental Table SIV.

[000159] For cloning amiRNA or syn-tasiRNA inserts into B/c vectors, 2 μ{ of each of the two overlapping oligonucleotides (100 μΜ stock) were annealed in 46 μΐ of Oligo Annealing Buffer (60 mM Tris-HCl pH7.5, 500 mM NaCl, 60 mM MgCl ₂ and 10 mM DTT) by heating the reaction for 5 min at 94°C and then cooling to 20°C (0.05°C/sec decrease). The annealed oligonucleotides were diluted in dH ₂ 0 to a final concentration of 0.30 μΜ. A 20 μΐ ligation reaction was incubated for lh at room temperature, and included 3 ul of the annealed and diluted oligonucleotides (0.30 μΜ) and 1 μΐ (75 ng/μΐ) of the corresponding B/c vector previously digested with Bsal. One-μΐ of the ligation reaction was used to transform and E. coli strain such as DH10B. or TOP10 that does not have ccdB resistance.

Example 10: Transient expression Assays

[000160] Transient expression assays in N. benthamiana leaves were done as described (Llave et al. 2002, Carbonell et al., 2012) using Agrobacterium tumefaciens GV3101 strain.

Example 11 : RNA blot Assays

[000161] Total RNA from A. thaliana or N. benthamiana was extracted using TRIzol reagent (Life Technologies) as described (Cuperus et al., 2010). RNA blot assays were done as described (Montgomery et al., 2008; Cuperus et al, 2010). Oligonucleotides used as probes for small RNA blots are listed in Supplemental Table SIV.

Example 12: Quantitative real-time RT-PCR (RT-qPCR)

[000162] RT-qPCR reactions were done using those RNA samples that were used for RNA blot and small RNA library analyses. Two micrograms of DNAsel-treated total RNA were used to produce first-strand cDNA using the Superscript III system (Life Technologies). RT- qPCR reactions were done in optical 96-well plates in a StepOnePlus™ Real-Time PCR System (Applied Biosystems) using the following program: 20 seconds at 95°C, followed by 40 cycles of 95°C for 3 seconds, 60°C for 30 seconds, and an additional melt curve stage consisting of 15 seconds at 95°C, 1 minute at 60°C and 15 seconds at 95°C. The 20 μΐ reaction mixture contained 10 μΐ of Fast SYBR ^® Green Master Mix (2X) (Applied

Biosystems), 2 μΐ diluted cDNA (1:5), and 300 nM of each gene-specific primer. Primers used for RT-qPCR are listed in Supplemental Table SIV. Target mRNA expression levels were calculated relative to 4 reference genes (AtACT2, AtCPB20, AtSAND and AtUBQlO) using the AACt comparative Ct method (Applied Biosystems) of the StepOne Software (Applied Biosystems, version 2.2.2). Three independent biological replicates were analyzed. For each biological replicate, two technical replicates were analyzed by RT-qPCR analysis.

Example 13: Preparation of small RNA libraries

[000163] Small RNA libraries were produced using the same RNA samples as used for RNA blots. Fifty- 100 μg of Arabidopsis total RNA were treated as described (Carbonell et al. 2012), but each small RNA library was barcoded at the amplicon PCR reaction step using an indexed 3 ' PCR primer (il, i3, i4, i5 or i9) and the standard 5'PCR primer (P5)

(Supplemental Table SVI). Libraries were multiplexed and submitted for sequencing using a HiSeq 2000 sequencer (Illumina).

Example 14: Small RNA sequencing analysis

[000164] Sequencing reads were parsed to identify library-specific barcodes and remove the 3' adaptor sequence, and were collapsed to a unique set with read counts. Unique sequences were aligned to a database containing the sequences of AtA4IR390a-based amiRNA, AtTASlc-based syn-tasiRNA and the control constructs using BOWTIE version 0.12.8 (Langmead et al., 2009) with settings that identified only perfect matches (-f -v 0 -a -S). Small RNA alignments were saved in Sequence Alignment/Map (SAM) format and were queried using SAMTOOLS version 0.1.19+ (Li et al., 2009). Processing of amiRNA foldbacks and syn-tasiRNA transcripts was assessed by quantifying the proportion of small RNA, by position and size, that mapped within ±4 nt of the 5' end of the miRNA and miRNA* or DCL4 processing position 3'D3[+] and 3'D4[+], respectively.

[000165] syn-tasiRNA constructs differ from endogenous AtTASlc at positions 3'D3 and 3'D4, but are otherwise the same. Therefore, reads for other syn-tasiRNA positions are indistinguishable from endogenous AtTASJc-deri ^' ved small RNAs. To assess the phasing of syn-tasiRNA constructs, small RNA reads from libraries generated from plants containing 35S.-GUS, 35S:AtTASlc-D3Trich-D4Ft or 35S:AtTASlc-D3Ft-D4Trich were first normalized to account for library size differences (reads per million total sample reads). Next, normalized reads for 21-nt small RNA that mapped to AtTASlc in the 35S:GUS plants were subtracted from the corresponding small RNA reads in plants containing syn-tasiRNA constructs to correct for endogenous background tasiRNA expression. Phasing register tables were constructed by calculating the proportion of reads in each register relative to the miR173 cleavage site for all 21-nt positions downstream of the cleavage site.

[000166] A summary of high-throughput small RNA sequencing libraries from Arabidopsis transgenic lines is provided in Supplemental Table SVI.

Example 15: Accession numbers

[000167] Arabidopsis gene and locus identifiers are as follows: CH42 (AT4G18480), CPC (AT2G46410), ETC2 (AT2G30420), Z 7(AT5G61850), r(ATlG65480), TRY

(AT5G53200). The miRBase (mirbase.org) locus identifiers of the conserved Arabidopsis MIRNA precursors (Figure IC) and of the plant MIRNA precursors used to express amiRNAs (Figure ID) are listed in Supplemental Table SVII and Supplemental Table SVIII, respectively.

[000168] High-throughput sequencing data from this article can be found in the Sequence Read Archive (ncbi.nim.nih.gov/sra) under accession number SRP036134. Example 16: Supplemental Tables SI through SVIII

Supplemental Table SI. Phenotypic penetrance of amiRNAs expressed in A. thaliana Col-0 Tl transgenic plants.

Supplemental Table SII. Phenotypic penetrance of amiRNAs or syn-tasiRNAs expressed in A. thaliana Col-0 Tl transgenic plants.

Supplemental Table SIII. Phenotypic penetrance of amiRNAs or syn-tasiRNAs expressed in A. thaliana Col-0 T2 transgenic plants.

Supplemental Table SIV. DNA oligonucleotides used in this study.

Supplemental Table SV. Sequences and predicted targets for all the amiRNAs and syn- tasiRNAs used in this study.

Supplemental Table SVI. Summary of high-throughput small RNA libraries from A. thaliana transgenic lines.

Supplemental Table SVII. miRBase Locus Identifiers of the Arabidopsis conserved MIRNA precursors used in this study.

Supplemental Table SVIII. miRBase Locus Identifiers of those plant MIRNA precursors previously used for expressing amiRNAs.

Example 17:

[000169] We generated Brachypodium distachyon transgenic plants expressing artificial miRNAs against Brachypodium distachyon BRIl, CAD, CAOl or SPLl l genes. In all cases, these artificial miRNAs were expressed them from two different foldbacks: OsMIR390 (the wild-iy/?e) and OsMIR390a (the chimeric foldback with rice OsMIR390 stem sequence but with Arabidopsis MIR390a distal stem-loop sequence).

[000170] Rice MIR390 foldback (OsMIR390) has a very short distal stem-loop, making expensive oligos unnecessary for cloning the amiRNAs (Figure 8), decreasing costs. A very high proportion of transgenic plants showed the expected amiRNA-induced phenotype, regardless of the MIRNA foldback (OsMIR390 or OsMIR390-AtL) from which the amiRNA was expressed (Figures 18-21).

[000171] Artificial microRNA target mRNAs were significantly reduced in transgenic plants regardless the MIRNA foldback the amiRNA was expressed from (Figure 22) However, artificial microRNAs were processed more accurately when expressed from the chimeric (OsMIR390~AtL) compared to the wild-type foldback (OsMIR390; Figure 23).

[000172] We suspect that because we are expressing the artificial microRNAs through an extremely potent promoter (called 35S, that leads to very high levels of artificial microRNA) we may be 'saturating' the system and that may explain why we do not see significant differences in phenotypes or in target mRNA accumulation in plants expressing the wild-type (OsMIR390) or the chimeric (OsMIR390-AtL) foldbacks.

[000173] However, we can predict that by expressing the artificial microRNAs to lower levels (without 'saturating' the system) we might see then a higher RNA silencing effect (stronger phenotypes, stronger reduction in target mRNAs) of artificial microRNAs expressed from the chimeric foldback compared to artificial microRNAs expressed from the wild-type foldback. This hypothesis is being tested by expressing the artificial microRNAs from a vector (pH7GW2) that contains a rice Ubiquitin promoter (called UBI) that is less strong than 35S.

[000174] We generated Arabidopsis thaliana transgenic plants expressing artificial microRNAs against Arabidopsis FT and CH42 gens. In both cases these artificial miRNAs were expressed from two different foldbacks: AtMFR390a (wild-type) and AtMIR390a-OsL (a MIRNA foldback with Arabidopsis MIR390a stem and shorter rice MTR390 distal stem- loop).

[000175] A very high proportion of transgenic plants showed the expected amiRNA-induced phenotype, regardless the MIRNA foldback (AtMIR390 or AtMTR390-OsL) the amiRNA was expressed from (Figure 24 & 25). Artificial microRNA target mRNAs were significantly reduced in transgenic plants regardless the MIRNA foldback the amiRNA was expressed from (Figure 24 & 25). Here, all artificial microRNAs were processed with similar accuracy regardless of the foldback (Figure 24 & 25).

[000176] Therefore, we can use the chimeric MIRNA foldback AtMTR390a-OsL to express efficient artificial microRNAs in Arabidopsis and saving money in the oligos needed for cloning (the length of the oligos for the AtMIR390a wild-type is 75 nt, and the length of the oligos for the chimeric AtMIR390a-OsL is 60 bp) (Figure 24 & 25).

Example 18: Designing and Cloning amiRNAs or syn-tasiRNAs

[000177] This example provides further information for designing and cloning amiRNAs or syn-tasiRNAs in BsaVccdB -based ('B/c') vectors containing AtMIR390a or AtTASlc precursors, respectively.

1. Selection of the amiRNA or syn-tasiRNA(s) sequence(s)

[000178] A link to a web tool for automated design of the amiRNA or syn-tasiRNA sequence(s) will be available at http.7/p-sams. carringtonlab. org/

2. Design of amiRNA or syn-tasiRNA oligonucleotides

[000179] A link to a web tool for automated design of the amiRNA or syn-tasiRNA oligonucleotide sequences will be available at http://p-sams.carringtonlab.org/

2.1 Design of amiRNA oligonucleotides 2.1.1 Sequence of the AtMIR390a cassette containing the amiRNA

[000180] The following FASTA sequence includes the amiRNA sequence inserted in the AtMTR390a precursor sequence:

>amiRNA in AtMlR390a precursor

[000181] TATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA ATATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC GAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCACT TCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

AACCCAAAAAAACAAAGTAGAGAAGAATCTjGTAXiX ₂ X3X4X5X6X7X8X9X _I oXiiXi2

X _l vX,.X,.X ,X _l 7X, X. X^ _i X _? ,ATGATGATCACATTCGTTATCTATTTTTTX,X _? X _l X _?

X ₃ X^ ₅ X<¾X ₈ X ₉ Xio ii i ₂ Xi ₃ Xi ₄ Xi ₅ Xi ₆ Xi _? i ₈ X ₁₉ CATTGGCTCTTCTTACTACAATG

AAAAAGGCCGAGGCAAAACGCCTAAAATCACTTGAGAATCAATTCTTTTTACTGT

CCATTTAAGCTATCT TTATAAACGTGTCTTATTTTCTATCTCTTTTGTTTAAACTA

AGAAACTATAGTATTTTGTCTAAAACAAAACATGAAAGAACAGATTAGATCTCA

TCTTTAGTCTC SEQ ID NO:368

[000182] Where: [000183] -X is a DNA base of the amiRNA sequence, and the subscript number is the base position in the amiRNA 21-mer

[000184] -X is a DNA base of the amiRNA* sequence, and the subscript number is the base position in the amiRNA* 21-mer

[000185] -X is a DNA base of the AtMIR390a foldback

[000186] -XJs a DNA base of the AtMIR390a foldback included in the oligonucleotides required to clone the amiRNA insert in B/c vectors

[000187] -X is a DNA base of the AtMIR390a foldback that may be modified to preserve the authentic AtMIR390a duplex structure

[000188] -X is a DNA base of the AtMIR390a precursor.

[000189] In the sequence above:

[000190] -Insert the amiRNA sequence where you see

XlX2X3 <X5 6X7X8 l0 ll l2Xl3 l l5 l6Xl7Xl8 l!> 20 21 SEQ ID NO:369

[000191] -Insert the amiRNA* sequence that has to verify the following base-pairing: [000192] X, X ₂ X ₃ X., X ₅ X _(l X ₇ X« X ₉ X10X11X12X13X14X1 X I (,X I ?X j sX i X X2 j SEQ ID

NO:370

[000193] ] I I I I I I I I I I I I I I 1 I I I I

[000194] icXisXn teXuXi.iXuXnXuXioX'. X* 7 X» Xs X4 X3 X2 X1 2 1 [000195] SEQ ID NO.-371

[000196] Note that:-In general, X,=T for amiRNA association with AGOl. SEQ ID NO:372 [000197] In this case, X _r =A SEQ ID NO:373

[000198] -Bases X _n and X ₉ DO NOT base-pair to preserve the central bulge of the authentic AtMIR390a duplex. The following base-pair rule applies:

[000199] -If Xir-G, then X _y =A SEQ ID NO:374 [000200] -If _u =C, then ₉ =T SEQ ID NO:375

[000201] -If u=A, then .,=G SEQ ID NO:376

[000202] -I f SEQ ID NO:377

2.1.2. Sequence of the amiRNA oligonucleotides [000203] The sequences of the two amiRNA oligonucleotides are: [000204] -Forward oligonucleotide (75 b),

[000205] ΤΟΤΑΧ,Χ2Χ ₃ Χ4Χ5 6Χ7Χ ₈ Χ9ΧΐθΧΐΐΧΐ2Χΐ ₃ Χΐ4Χΐ5Χΐ6Χΐ7Χΐ8Χΐ9 2 _θ Χ2ΐΑΤΟΑΤΟΑ TCACAT CGT ATCTATTrTTTX ₁ X ₂ X ₁ X ₂ X3X4X5X6X7X8X9 l0XllXl2Xl3Xl4Xl5Xl6Xl7 ΧΐδΧΐ9

[000206] SEQ ID NO:378

[000207] -Reverse oligonucleotide (75 b),

[000208] AATGY ₁₉ Y _l8 Y _l7 Y _l6 Y _l5 Y _l4 Y _l3 Y _l2 Y _{l l l0} Y ₉ Y ₈ Y ₇ Y ₆ Y ₅ Y ₄ Y ₃ Y ₂ Y _l Y ₂ Y _l AAAAAATG

ATAACGAATGTGATCATCATY21Y20Y19Y18Y17Y16Y15Y14Y13Y12Y11Y10Y 9Y8Y7Y6Y5Y4Y

[000209] Where:

[000210] -X1 2X3X4 5X6X7 8X9X10X11X12X13X14X15X16X17X18X19X20X21 =amiRNA sequence

[000211] SEQ ID NO:380

[000212] -ΧιΧ ₂ Χ ₃ Χ Χ5Χ6Χ7 8Χ9ΧιοΧιιΧΐ2Χΐ3Χΐ4Χΐ5Χΐ6Χΐ7Χΐ8Χΐ9 =partial amiRNA* sequence

[000213] SEQ ID NO:381

[000214] -Y ₂₁ Y ₂₀ Y ₁₉ Y ₁₈ Y ₁₇ Y ₁₆ Y ₁₅ Yi4Yi3Yi2YiiYioY9Y8Y7Y6Y5Y4Y ₃ Y2Yi =amiRNA reverse-complement sequence SEQ ID NO:382 [000215] -TGY _!9 Y ₁₈ Yi ₇ Yi6Yi5Yi4Yi3Yi2 iiYioY9Y8Y7Y6Y5Y4Y3Y ₂ Yi =amiRNA* reverse- complement sequence SEQ ID NO:383

[000216] -XiX ₂ = AtMlRJJ' Oa sequence that may be modified to preserve authentic

AtMIR390a duplex structure.

[000217] -Y ₂ Yi = reverse-complement of X)X ₂

Example 19.

[000218] The sequences of the two oligonucleotides to clone the amiRNA 'amiR-Trich' (TCCCATTCGATACTGCTCGCC) SEQ ID NO:384 are:

[000219] -Sense oligonucleotide (75 b),

[000220] TGTATCCCAT CGATACTGCTCGCCATGATGATCACATTCGTTATCTATTT

TTTGGCGAGCAGTCTCGAATGGGA SEQ ID NO:385

[000221] -Antisense oligonucleotide (75 b),

[000222] AATGTCCCATTCGAGACTGCTCGCCAAAAAATAGATAACGAATGTGATC ATCATGGCGAGCAGTATCGAATGGGA SEQ ID NO:386

[000223] Note: The 75 b long oligonucleotides can be ordered PAGE-purified, although oligonucleotides of 'Standard Desalting' quality worked well.

2.2 Design of syn-tasiRNA oligonucleotides

2.2.1 Sequence of the AtTASlc cassette containing the syntasiRNA(s)

[000224] The following FASTA sequence includes two syn-tasiRNA sequences inserted in the AtTASlc precursor sequence:

[000225] >syn-tasiRNA-l and syn-tasiRNA-2 in AtTASlc

[000226] AAACCTAAACCTAAACGGCTAAGCCCGACGTCAAATACCAAAAAGAGA AAAACAAGAGCGCCGTCAAGCTCTGCAAATACGATCTGTAAGTCCATCTTAACA CAAAAGTGAGATGGGTTCTTAGATCATGTTCCGCCGTTAGATCGAGTCATGGTCT TGTCTCATAGAAAGGTACTrrCGTTTACTTCTTTTGAGTATCGAGTAGAGCGTCGT CTATAGTTAGTTTGAGATTGCGTTTGTCAGAAGTTAGGTTCAATGTCCCGGTCCA ATTTTCACCAGCCATGTGTCAGTTTCGTTCCTTCCCGTCCTCTTCTTTGATTTCGTT GGGTTACGGATGTTTTCGAGATGAAACAGCATTGTTTTGTTGTGATTTTTCTCTAC AAGCGAA.TAGACCATTTATCGGTGGATCTTAGAAAATTAX1X2X3X4X5X6X7X8X9X1 θ ΐΐΧΐ2 ΐ3 ΐ ΐ5 ΐ<ίΧΐ7Χΐ8¾9Χ2θ 21¾¾Χ3Χ ^

sX^XzoXziGAACTAGAAAAGACATTGGACATATTCCAGGATATGCAAAAGAAAAC

AATGAATATTGTTTTGAATGTGTTCAAGTAAATGAGATTTTCAAGTCGTCTAAAG

AACAGTTGCTAATACAGTTACTTAT TCAATAAATAATTGGTTCTAATAATACAA

AACATATTCGAGGATATGCAGAAAAAAAGATGTTTGTTATTTTGAAAAGCTTGA

GTAGTTTCTCTCCGAGGTGTAGCGAAGAAGCATCATCTACTTTGTAATGTAATTT

TCTTTATGTTTTCACTTTGTAATTTTATTTGTGTTAATGTACCATGGCCGATATCG

GTTTTATTGAAAGAAAATTTATGTTACTTCTGTTTTGGCTTTGCAATCAGTTATGC

TAGTTTTTCTTATACCCTTTCGTAAGCTTCCTAAGGAATCGTTCATTGATTTCCACT

GCTTCATTGTATATTAAAACTTTACAACTGTATCGACCATCATATAATTCTGGGTC

AAGAGATGAAAATAGAACACCACATCGTAAAGTGAAAT

[000227] SEQ ID NO:387

[000228] Where:

[000229] -X is a DNA base of the syn-tasiRNA-1 sequence, and the subscript number is the base position in the syn-tasiRNA-1 21-mer

[000230] -X is a DNA base of the syn-tasiRNA-2 sequence, and the subscript number is the base position in the syn-tasiRNA-2 21-mer

[000231] -X is a DNA base of the AtTAS Ί c precursor included in the oligonucleotides required to clone the syn-tasiRNA insert in B/c vectors

[000232] -X is a DNA base of the AtTASlc precursor

[000233] Note that in general, X,--=T and X,=T for syn-tasiRNA association with AGOl. SEQ ID NO:388

[000234] In the sequence above, replace the sequences

i ₂ 3 4 _s 6 7 8 9 io ii i2 i3 i4 i5 i6 i7 ₁ 8 w 2o 2j SEQ ID NO:389 and X,X ₂ X3 XsX6 7¾¾XjoXi iXi2X.3Xi< i5Xi6Xi7Xi8Xi9X2oX2i SEQ ID NO:390 by the sequences of syn-tasiRNA_l and syn-tasiRNA_2, respectively.

2.2.2. Sequence of the syn-tasiRNA oligonucleotides

[000235] The sequences of the two syn-tasiRNA oligonucleotides are:

[000236] -Sense oligonucleotide (46 b):

[000237] ATTAX1X2X3X4X5X6X7X8X X10X11X12X13X14X15X16X17X18X19X20X21X1X2X3X4X5 6X7X8 9 10X11X1 X13X14X15 16X17 18 19X20X 1 SEQ ID NO:391

[000238] -Antisense oligonucleotide (46 b):

[000239] GTTCY ₂ ,Y3oYl9Yl8Yl7Yj6Yj5Yl4Yl3Yj2YuYloY9Y8Y7Y6Y5Y4Y3Y2YlY2l Y20Yl9Yl sYi7 YwYisYwYtaYnYii YioY9Y8Y7YiY5Y ₄ Y3Y2Yi SEQ ID NO:392 [000240] Where:

[000241] -X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X _{8 9 l0} X _ll X _l2 X _{l3 l4 l5 l6} X _{l7 l8} X _{l9 20} X ₂₁ =Syn-tasiRNA-l sequence SEQ ID NO:393

[000242] -XjX2X3X4X5X6X7X ₈ X9Xl0XnXl2Xl ₃ Xl4Xl5Xl6Xl7Xl ₈ Xl9X20X ₂ l=Syn-tasiR A-2 sequence SEQ ID NO:394

[000243] -Y ₂₁ Y20Yl9Yl8Yl7Yl6Yl ₅ Yl4Yl ₃ Yl2YllYl ₀ Y9Y8Y7Y6Y5Y4Y ₃ Y2Yl=Syn-tasiRNA-l reverse-complement sequence SEQ ID NO:395

[000244] -Y ₂₁ Y ₂₀ Yi ₉ Yi ₈ Yi ₇ Yi ₆ Yi ₅ Yi ₄ Yi ₃ Yi ₂ YnYioY ₉ Y ₈ Y ₇ Y ₆ Y ₅ Y ₄ Y ₃ Y ₂ Yi=syn-tasiRNA-2 reverse-complement sequence SEQ ID NO: 396

Example 20.

[000245] The sequences of the two oligonucleotides to clone syn-tasiRNAs 'syn-tasiR-Trich' (TCCCA TCGATACTGCTCGCC) SEQ ID NO:397 and 'syn-tasiR-Ft'

(TTGGTTATAAAGGAAGAGGCC) SEQ ID NO:398 in positions 3'D3[+] and 3'D4[+] of AtTASlc, respectively, are:

[000246] -Sense oligonucleotide (46 b): [000247] ATTATCCCATTCGATACTGCTCGCCWGGTTATAAAGGAAGAGGCC SEQ ID NO:399

[000248] -Antisense oligonucleotide (46 b):

[000249] GTTCGGCCTCTTCCTTTATAACCAAGGCGAGCAGTATCGAATGGGA [000250] SEQ ID NO:400

3. Cloning of the amiRNA/syn-tasiRNA sequences in BsaVccd (B/c) vectors

[000251] Notes:-Available B/c vectors are listed in Table I at the end of the section.

[000252] -At MIR390-B/C- and AtTASlc-B/c-based vectors must be propagated in a ccdB resistant E. coli strain such as DB3.1.

[000253] -Alternatively, Bsal digestion of the B/c vector and subsequent ligation of the amiRNA oligonucleotide insert can be done in separate reactions

3.1. Oligonucleotide annealing

[000254] -Dilute sense oligonucleotide and antisense oligonucleotide in sterile H ₂ 0 to a final concentration of 100 μΜ.

[000255] -Prepare Oligo Annealing Buffer:

[000256] 60 mM Tris-HCl (pH 7.5), 500 mM NaCl, 60 mM MgCl _2„ 10 mM DTT [000257] Note: Prepare 1 ml aliquots of Oligo Annealing Buffer and store at -20 ^° C. [000258] -Assemble the annealing reaction in a PCR tube as described below: [000259] Forward oligonucleotide (100 μΜ) 2 \xL [000260] Reverse oligonucleotide (100 uM) 2 μΐ,

[000261] Oligo Annealing Buffer 46 nL

[000262] Total volume 50 pL

[000263] The final concentration of each oligonucleotide is 4 μΜ. [000264] -Use a thermocycler to heat the annealing reaction 5 min at 94°C and then cool down (0.05°C/sec) to 20°C.

[000265] -Dilute the annealed oligonucleotides just prior to assembling the digestion-ligation reaction as described below:

[000266] Annealed oligonucleotides 3 μΐ,

[000267] di¾Q 37 μΙ _^

[000268] Total volume 40 uL

[000269] The final concentration of each oligonucleotide is 0.15 μΜ.

[000270] Note: Do not store the diluted oligonucleotides.

3.2. Digestion-ligation reaction

[000271] - Assemble the digestion-ligation reaction as described below: [000272] B/c vector (x ug/uL) Y yL (50 ng)

[000273] Diluted annealed oligonucleotides 1 \iL [000274] lOx T4 DNA ligase buffer 1 uL

[000275] T4 DNA ligase (400 U/pL) 1 uL

[000276] Bsal (10U/ μΤ, NEB) 1

[000277] dH _z O to 10 μΐ,

[000278] Total volume 10 μΐ,

[000279] Prepare a negative control reaction lacking Bsal.

[000280] -Mix the reactions by pipetting. Incubate the reactions at room temperature for 5 minutes at 37°C.

3.3. E.coli transformation and analysis of transformants [000281] -Transform 1-5 ul of the digestion-ligation reaction into an E. coli strain that doesn't have ccd resistance (e.g. DH10B, TOP 10, ...) to do counter-selection.

[000282] -Pick two colonies/construct, grow LB-Kan (100 mg/ml) cultures and purify plasmids.

[000283] -Sequence with appropriate primers: M13-F

(CCCAGTCACGACGTTGTAAAACGACGG) SEQ ID NO:401 and M13-R

(CAGAGCTGCCAGGAAACAGCTATGACC) SEQ ID NO:402 for pENTR-based vectors, attBl (ACAAGTTTGTACAAAAAAGCAGGCT) SEQ ID NO:403 and attB2

(ACCACTTTGTACAAGAAAGCTGGGT) SEQ ID NO:404 primers for pMDC32B-, pMDC123SB- or pFK210B-based vectors).

Figure 48 continued

STTGCTAATACAGTTACTTATTTCAATAAATAATTGGTTCTAATAATACAAAACA ATTCGAOGATAIOCA

GftAAJLAAAGATGTTTGTTATTTTGAAAAGCT'l'GAGTAGTTTCTCTCCGAGGTGTAG CGAAGililGCATCATC TACTTTGTAATGTAATTITCTTTATC-TTTTCACTTTGTAiiTTTTATTTGTGTTAATGT ACCATGGCCGATA TCGGTTTTATrGAMGAAAATT.TATGTTA.CTTCTGTTTTGC-OTTTGCAATCAGTTATG CTAGI'J'TTCTTAT ACCCTTTCGTAAGCTTGCTAAGGA J ai'leAlTGATTTCCAGTGCTTCATTOTATA TAAAaCTrj'ACAA CJGTATCGACCATCATATAATTeTGGGTGAAaA-aA'fGAAAATAGAACACOACATCGTA AAGTGAAATBBI

>AtTASlc-D3Ft-D4Trich

ACCTAAACCTAAACGGCTAAGCGCGACGTCAAATACCAAAAAGAGAAAAACAAGAGCGCC GTCAAGCTC TGCAAATACGATCTGTAAGTCCATCTTAACACAMHGTGAGATaGaTTCTTAGATCA GTTCCGCeGTTAG " TCQAGTCATGGTCTTC"fCTCATAGAAAGGTACTT.TCGTTTACTTGTTTTGAGTATCGA CTAGAaCGTCGT CTATAGTTAGTTTGAGA TaCGTTTCTGAGAAGTTAGGTTCAATGTCGCGGTCCAATTT'rCACCAG X¾1.'G! TGTCAGTTTeGTTCGTTCCCOTCGTGTTCTTTGATTTiZ'GTTaclGT ACGGATGTTTI'GGAaATGAAACAGC: ATTGTTTTGTTSTGATTWJ'CT^

31TCCTAATACftGTraCTTilTTTCSATMAlffiA'JTCGTTCTAATA4T»CAAAACA TATTCGAC-GATATGeA GAAAAAAAGATGTTTGTt'A TTTGAAAaGGTTGAGTAGTTTGTCTCCGAGGTGTAGCGAAGAAGCATCA'rc TAGTTTGTAATGrASJ TTCTTTATGTTITCAeTlTGTAATTTTATTTGTGTTTlATGTACCAT'GGGCGATA GGGTTTTAT GAJiAGAAMTTTftlGTTACTTCTGTITTGGCTTTGCAATCAGTTAT ^Q CTAGITTTCTTAT AeCCTTTCaTAACCTTCCraAGGAATCGTTCATTaATT CCACTGCTTCA TGTATATTAA7AJiCTTTACAA CTGTATCGACCATCATA AA'i' GTGGRTCAAGAGATGAAAATAGAACACCACATCGTAAAGTGAAAl

Figure 49

Figure 49 continued

Example 21.

DNA sequence of B/c vectors used for direct cloning of amiRNAs in zero- background vectors containing the OsMIR390 sequence.

Index:

> _P ENTR-OsMIR390-B/c >pMDC32B-OsMIR390-B/c

>pMDC123SB-OsMIR390-B/c

>pH7WG2B-OsMIR390-B/c

>pENT -OsMI 390-B/c (4122 bp)

[000284] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGA GTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG CGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA GCGCAACGCAATTAATACGCGTACCGCTAGCCAGGAAGAGTTTGTAGAAACGCA AAAAGGCCATCCGTCAGGATGGCCTTCTGCTTAGTTTGATGCCTGGCAGTTTATG GCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTGCTTCACAACGTTCAAATCCGC TCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAA ACGAAAGGCCCAGTCTTCCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTT CCCTACTCTCGCGTTAACGCTAGCATGGATGTTTTCCCAGTCACGACGTTGTAAA ACGACGGCCAGTCTTAAGCTCGGGCCCcaaataatgattttattttgactgatagtgacc tgttcgttgcaacaa attgatgagcaatgcttitftafaatgccaactrrgfacaaaaaagcaggciCCGCGGCC GCCCCCTTCACCGAGC TCGAGATGTTTTGAGGAAGGGTATGGAACAATCCTTGAGAGACCATTAGGCACC CCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGA GCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatggagaaaaaaatcactggatatac caccgtt gaiataicccaatggcatcgraaagaacatfftgaggeafftc

ggcGtttttaaagaccgtaaagaaaaataagGacaagttttatccggcctttattcacat tettgcccgcctgcitgaai

tccgtatggcaaTgaaagacggtgagciggtgatatgggatagtgttcacccttgit acaccgttttccatgagc

atcgctctggagtgaataceacgacgaW^

ttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagt ttcaccagttttgatttaaacgtggccaatatggaca acttcttcgceccc gttttcaccatgggc aaatattato

tttgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatg agtggcagggcggggcgtaaACGCGTG

GAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTG

CGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTAT

GCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTC

AAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATG AAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGG

CTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGG

CTGGTGAAATGCAGTTTAAGGTTl^CACCTATAAAAGAGAGAGC-CGTTATCGTCT

GTTTGTGGATGTACAGAGTGATATTATTGACACGCCCC3GCCGACGGATGGTGATC

CCCCTGGCCAGTGCACGTGTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGG

TGGTGCATATCGGGGATGAAAGCTGGCG ATGATGACCACCGATATGGCCAGTG

TGCCGGTlTCCGTTATCGGGGAACiAAGTGGCTGATCTCAGCCACCGCGAAAATG

ACATCAAAAA.CGCCATTAACCTGATGTTCTGGGGAA.TATAAATGTCAGGCTCCCT

TATACACAGCCAGTCTGCACCTCGACggtctcAcatggtttgttcttaccacacgac caattaaatcGAGC

TCAAGGGTGGGCGCGCCGacccagctttottgtacaaagttggcattataagaaagc ttttgcttatoaatttgttgcaac gaacaggtcactatcagtcaaaataaaatcattatttgCCATCCAGCTGATATCCCCTAT AGTGAGTCGT

ATTACATGGTCATAGCTGTTTCCTGGCAGCTCTGGCCCGTGTCTCAAAATCTCTG

ATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCT

TACATAAACAGTAATACAAGGGGTGTTatgagccataitcaacgggaaacgtcgagg cGgcgatiaaaftc caacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcagg tgcgacaatctaicgcttgtatgggaagcc cgatgcgccagagttgtttctgaaacatggcaaaggtagcgtt^^

aatttatgcctcttccgaccatcaagcattttatccgtactc^tgatgatgcatggt tactcaccactgcgatccccggaaaaacagcattc caggtattagaagaatatcctgattcaggtgaaa

igtccttttaacagcgatcgcgtatttcgtctcgcicaggcgcaatcacgaatgaat aacggttiggitgatgcgagtgattttgatga gcgtaatggctggcetgftgaaeaagictggaaagaa

cacttgataaeettaftrtigacgaggggaaatiaataggtt^^

catcctatggaaGtgcctcggtgagtttlctccttcattacagaaacggctttttca aaaatatggtattg

cagiticatttgatgctegatgagtft

CATTACGCTGACTTGACGGGACGGCGCAAGCTCATGACCAAAATCCCTTAACGT

GAGTTACGCGTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATC

TTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC

CGCTACCAGCGGTGGT TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAA

GGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG

TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC

TAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT

GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGG

GTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACC

TACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGAC AGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCC AGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTT GAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCC AGCAACGCGGCCTT TTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT

PURPLE/UPPERCASE: M13-forward binding site orange/lowercase: attl l BLUE/UPPERCASE: OsMIR390a 5' region RED/UPPERCASE: Bsal site magenta/lowercase: chloramphenicol resistance gene MAGENTA UPPERCASE: ccdB gene red/lowercase: inverted Bsal site blue/lowercase: OsMIR390a 3' region orange/iowerease/underlined: attL2

PURPLE/UPPERCASE/UNDERLINED: M13~reverse binding brown/lowercase: kajiamycin resistance gene

>pMDC32B-OsMIR390-B/c (11675 bp)

[000285] CCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCAC TCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAA GGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCT GCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGA TGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATA CTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTT AAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCT TCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAatggctaaaatg agaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaa ggaatgtctcctgctaaggtatataagct ggtgggagaaaatgaaaacctatatttaaaaatgacggacagccggtataaagggaccac ctatgatgtggaacgggaaaaggacat gatgctatggetggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatga tggctggagcaatctgctcatgagtgag gccgatggcgtcctttgctcggaagagtatgaagatgaacaaagccctgaaaagattatc gagctgtatgcggagtgcatcaggctctt tcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagccga attggattacttactgaataacgatctggcc gatgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcgagctgiat gattttttaaagacggaaaagcccgaag aggaacttgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatggca aagtaagtggctttattgatcttgggagaa gcggcagggcggacaagtggtatgaGattgGcttctgcgtccggtcgatcagggaggata tcggggaagaacagtatgtcgagctat tttttgacttactggggatcaagcctgattgggagaaaataaaatattatattttactgg atgaattgttttagTACCTAGAATGC ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTG CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG TCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC TTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG

AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC

ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGG

CTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT

CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAG

AGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCG

CCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGG

CCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGC

GGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTC

GGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTA

ATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGT

CACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGG

TTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAA

AGAGAACTTTTCGACCTTTTTCCCCTGGTAGGGCAATTTGCCCTAGCATCTGCTCC

GTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATG

ACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGT

CATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGC

GCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTG

CGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAA

AAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGC

GGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCT

CTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGG

CGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACC

GAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCA

GAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCC

CTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGT

ACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCT

ACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCT

TCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCA

CGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTT

CCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGA

TCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCC GCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGC

GCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTT

GGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGG

CCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGT

TCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTC

ATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAAT

GGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGC

AACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTG

CAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTT

GCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGC

CAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTT

GTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCA

AGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGC

GTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGA

CCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTA

AGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACAC

AGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCC

GATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAG

CGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGA

ATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGT

TGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTC

ATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACC

GCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCT

CGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACA

AACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGA

ACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGG

TTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTC

GGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCG

CCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGG

GTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCT

GGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGG

GGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTC

GATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCAT GCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCC

CGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCG

GGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTC

AAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAA

AACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCT

GGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGT

TCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAA

ACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAA

CTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAA

GCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGG

GGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCA

CGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTTAGGtit iaiccrgtcaAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAG

CTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA

GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA

CGGCCAGTGCCAAGCTTGGCGTGCCTGCAGGTCAACATGGTGGAGCACGACACA

CTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATT

GAGACTTI CAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAG

CTATCITiTC ACTTTATTGTGA A G ATAGTGGA A A AG G A AGGTG GCTCCT A C A A ATG

CCATCA1TGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGG CCCAAAGATXJGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCC

AACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACAC

AGTTGTCTACTCCAAAAATATCA AAGATACAGTCTCAGAAGACCAA AGGGCAAT

TGAGACTMCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCA

GCTATCTGTCACTTTATTGTGAAGAT GTGGAAAAGG AGGTGGCTCCTACAAAT

GCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTG

GTCGCAAAGATGGACCCCCACCCACGAGGAGCA ^' TCGTGGA AA AAGAAGACGTTC

CAACCACGTC ^' ITCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGG

ATGACGCACAATCCCACTATCCTTCGCAAGAC^CTTCCTCTATATAAGGAAGTTC

ATTTCATTTGGAGACXJACX CGACTCTAG-AGGATCCCCGGGTACCGGGCCCCCCC

TCGAGGCGCGCCAAGCTATCAAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGC

CGCCCCCTTCACCGAGCTCGAGATGTTTTGAGGAAGGGTATGGAACAATCCTTGA GAGACCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTG TGGATTTTGAGTTAGGAGCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatgga gaaaaiuiatcactggatataccaccgttgatatatcccaatggcatogtaaagaacatt ttgaggcatttcagtcagttgctc ataaccagaccgitcagctggatattacggccfttita^

cccgcctgatgaatgctcatccggagttccgtatggcaatgaaagacggtgagctgg tgatatgggatagtgltcacGcttgtta gttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttc cggcagtttctacacatatattcgcaagatgt ggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgttttt cgtcto

gttttgalttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgg gcaaatattatacgcaaggcgacaaggtgctgat gccgctggogattcaggitcatcatgccgtttgtgatggcttccatgtcggcagaatgct taatgaattacaacagta^ cagggcggggcgtaaACGCGTGGAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTA

TTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGT

ATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCG

ACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGC

ACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAA

AATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTG

CTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGA

GAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCG

GCCGA.CGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTC

CCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGAC

CACCGATATGGCCAGTGTGCCGGTTTCCGTl ^' ATCGGGGAAGAAGTGGCTGATCTC

AGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATA

TAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCACCTCGACggtctcAcatggtt tgttctt accacacgaccaattaaatcGAGCTCAAGGGTGGGCGCGCCG XCAGCT^

GTGOTTCGATAATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCCACCGCGGTGG

ATCCTGTTGCCGGTCTTGCGATG'ATTATCATATAATTTCTGTTGAATTACGTTAAG CATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGA

TTAGAOTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCG

CAAM £AGG £

ATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAAC ATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA CTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGT GCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTG GCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAA

TATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAG

GGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATC

AAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAA

AGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACC

CCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAA

GCAAGTGGATTGATGTGATAACatggtggagca^^

agaagaccaaagggciatigagacrttieaacaaagggtaatate

aaaaggacagtagaaaaggaaggi-ggcaccfacaiiatgccateatrgcgataaag gaaaggciatcgifcaapigcctcfgccgae agtggteccaaagatggacccccacccacgaggagcatogtggaaaaagaagacgttcca accacgtcttcaaagcaagtggaftg atgtgatatctccaetgacgteagggatgacgeacaaf ^^

gaggACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCTTTCG

CAGATCCCGGGGGGCAATGAGATATGAAAAAGCCTGAACTCACCGCGACGTCTG

TCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTC

GGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGT

CCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGG

CACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTA

GCGAGAGCCTGACCTATTGCATCTCCCGCCGTTCACAGGGTGTCACGTTGCAAGA

CCTGCCTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGAT

GCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCG

CAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATC

CCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGC

GCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCA

CCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATA

ACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTC

GCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCT

ACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACTCCGGGCGTATA

TGCTCCGCATTGGTCI GACCAACTCTATCAGAGCT GGTTGACGGCAATTTCGA

TGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGG

GACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGG

CTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAG

GGCAAAGAAATAGAGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGlttc

ATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCT AAGCGTCAATTTC-TTTACACCACAATATATCCTGCCA

brown/lowercase: kanaiiiycin resistance gene block vector ^' s .¾« ^■ // site cyaii/iowercase: T-DNA right border - GREEN/UPPERCASE: 2x35 S CaMV promoter ORANGE/UPPERCASE; at® I BLUE/UPPERCASE: OsMIR390 5' region RED/UPPERCASE: Bsal site magenta/lowercase: chloramphenicol resistance gene MAGENTA/UPPERCASE: cc B gene red/lowercase: inverted Bs l site blue/lowercase: OsMIR390 3' region ORANGE/UP PERCASE/UNDERLINEI): attB2 GREY/UPPERCASE/UNDERLINED: Nos terminator green/lowercase: CaMV promoter BROWN/UPPERCASE: hygromycin resistance gene gi-een/lowercase/uiidgrltnedi CaMV terminator CYAN/UPPERCASE: T-DNA left border >pMDC123SB-OsMIR390-B/c (11150 bp)

[000286] CCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCAC TCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAA GGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCT GCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGA TGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATA CTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTT AAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCT TCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAatggctaaaatg agaatatcaccggaattgaaaaaactgatcgaaaaataccgcrgcg†aaaa.g¾tac ggaaggaatgtctcctgctaaggtatataagci ggtgggagaaaatgaaaacctatatttaaaaatgacggacagcGggtataaagggaccac ctatgatgtggaacgggaaaaggacat gatgctatggctggaaggaaagctgcctgttcc iaaggtcctgcactttgaacggcatgatggctggagcaatctgctcatgagtgag gccgatggcgicctttgcicggaagag gaagatgaacaaagcc^^^

tcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagc cgaattggattacttactgaataacgatctggcc gatgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcgagctgtat gattttttaaagacggaaaagc aggaacitgtcitttcccacggcgacctgggagacagcaacatctttgtgaaagatggca aagtaagtggctttatigatcttgggagaa gcggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatca^

tttttgacttactggggatcaagcctgattgggagaaaataaaatattatattttac tggatgaattgttttagTACCTAGAATGC

ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG

AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTG

CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA

CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG

TCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC

TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG

TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG

TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC

ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA

GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG

CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT

CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC

TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC TTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG

AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC

ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGG

CTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT

CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAG

AGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCG

CCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGG

CCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGC

GGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTC

GGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTA

ATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGT

CACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGG

TTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAA

AGAGAACTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCC

GTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATG

ACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGT

CATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGC

GCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTG

CGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAA

AAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGC

GGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCT

CTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGG

CGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACC

GAATGCAGGTTTCTACCAGGTCGTC ITCTGCTTTCCGCCATCGGCTCGCCGGCA

GAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCC

CTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGT

ACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCT

ACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCT

TCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCA

CGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTT

CCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGA TCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCC

GCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGC

GCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTT

GGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGG

CCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGT

TCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTC

ATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAAT

GGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGC

AACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTG

CAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTT

GCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGC

CAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTT

GTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCA

AGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGC

GTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGA

CCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTA

AGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACAC

AGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCC

GATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAG

CGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGA

ATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGT

TGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTC

ATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACC

GCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCT

CGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACA

AACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGA

ACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGG

TTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTC

GGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCG

CCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGG

GTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCT

GGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGG

GGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTC GATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCAT

GCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCC

CGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCG

GGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTC

AAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAA

AACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCT

GGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGT

TCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAA

ACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAA

CTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAA

GCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGG

GGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCA

CGCCCTTITAAATATCGGTI ΑΎΊ ΛΑ ΛΛΛΓΧΚ ί TI ITGTG Π AGG

ix ^: !icctgicaAACACTGATAGTT AAACTGAAGGCGGGAAACGACAATCTGATCCAAG

CTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA

GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA

CGGCC AGTGCC AAGCTTGC ATGCCTGC AG GTC A AC ATGGTGGTGC ACGACACAC

TTGTCTACTCCAAAAATATCIITGATACAGTCTCAGAAGACCAAAGGGCAATTGA

GACTlTrcAACAAAGGGTAATATCCGGAAACCTCCTCGGAlTCCAWGCCCAGCT

ATCTGTCACTTIATrGTGAAGATAGTGGAAAAGGAAGOTGGCTCCTACAAATGCC

ATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTC

CCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTrCCAA

CCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACAC

TTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATrG

AGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGC

TATCTGTCACTrrATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGC

CATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGT

CCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCA

ACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATG

ACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATT

TCATITGGAGAGGACCTCGACTCTAGAGGATCCCCGGGTACCGGGCCCCCCCTCG

AGGCGCGCCAAGCTATCAAACAAGTTTG ^' IACAAAAAAGCAGGCTCCGCGGCCGC CCCCTTCACCGAGCTCGAGATGTTTTGAGGAAGGGTATGGAACAATCCTTGAGA GACCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTG GATTTTGAGTTAGGAGCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatggagaa aaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttga ggcatttcagtcagttgctcaatgtacctata acGagaccgitcagctggatattacggcctttttaaagaccgtaaagaaaaataagcaca agttttatccggctt

cgcctgatgaatgctcatccggagttccgta-tggcaatgaaagacggtgagctggt gatatgggatagtgttcacccttgttacaccgttt tccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggc agtttctacac

gtgttacggtgaaaacctggcctatttcccte^

gatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaa tattatacgcaaggcgacaaggtgctgat ctggcgattcaggttcatcalgccgtttgtgatggcttccatgtcggGagaatgcttaat gaattacaacagtactgcgatgagtggcag gcggggcgtaaACGCGTGGAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTT

GCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATG

TCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACA

GCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACA

ACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAAT

CAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTG

ACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAG

AGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATAITATTGACACGCCCGGCC

GACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCG

TGAACT1TACCCOGTGGTGCATATCGGGGATGAAAGCTGGCGCATGA.TGACCAC

CGATATGGCCAGTGTGCCGGTTTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGC

CACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAA

ATGTCAGGCTCCCTTATACACAGCCAGTCTGCACCTCGACggtctcAcatggtttgt tcttaccac acgaccaattaaatcGAGCTCAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAAAGTG

GTTCGATAATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCT

CGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCC

TGTTGCCGGTCTTGC-GATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATG

TAi'VTAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTT ^' ATGATTAG

AGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAA

CJAGGAIAAAi;™

ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACA ACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCT AACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTC GTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT

TGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAG

AATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAA

AGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCA

TCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATA

AAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGAC

CCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAA

AGCAAGTGGATTGATGTGATAAC ggtggagcacgacactctcgtctactccaagaatatcaaagatacagtct cagaagaccaaagggctaftgagacrtttoaacaaagggiaaiafcgggaaacctcotcg gattccattgcccagGtatcfgtcacitcat caaaaggacagtagaaaaggaaggtggcaceiacaaaigecaicaiigc^^

eagiggtCGcaaagatggacccceacceacgaggagcategtggaaaaag^^

gaigtgatatctccacigacgtaagggatgaegc^^

agaggACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGTCTAC

CATGAGCCCAGAACGACGCCCGGCCGACATCCGCCGTGCCACCGAGGCGGACAT

GCCGGCGGTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTT

CCGTACCGAGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCG

GGAGCGCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGC

CTACGCGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGAC

CGTGTACGTCTCCCCCC^

CACCTGCTGAAGreCCrGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATC

GGGCTGCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCC

CGCGCK^ATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGT

TTCTGGCAGCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCA

CCGAGATTTGACTCGAGtttote^^

¾5g;ttgagc §fe

ciaaagli^ cCCCCGAATTAA

ATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTrrACACCACAATATATCCTGCCA

brown/lowercase; kanamyein. resistance gene

QL ESEMQi BM MEQ C- A transversion to block vector's Bml sire cyan/lowercase; T-DNA right border GREEN/UPPERCASE: 2x35 S CaMV promoter

ORANGE/UPPERCASE: attB 1.

BLUE/UPPERCASE: OsMIR390 5' region

RED/UPPERCASE: Bsal site

magenta/lowercase: chloramphenicol resistance gene MAGENTA/UPPERCASE: cccB gene

red/lowercase: inverted Bsal site

blue/lowercase: OsMIR390 3' region

O ANGE ^' UPPERCASE/UNDERL-INED: attB2

GREY/UPPERCASETUNDERLINEP: Nos terminator green/lowercase: CaMV promoter

BROWN/UPPERCASE/UNDERLINED: BASTA resistance J^^ ^^l ^ ^- CaMV terminator

CYAN/UPPERCASE: T-DNA left border

> _P H7WG2B-OsMIR390-B/c (13122 bp)

[000287] TTTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACG AACGGATAAACCTTTTCACGCCCT ITAAATATCCGTTATTCTAATAAACGCTCTT

CGACAATCTGATCCAAGCTCAAGCTaagcitattcgg¾tcaag ^' gcggaagccagcg:cgccaccc acgtca gcaaatacgiaggcgcggggttgacggcgtcacccggtcctaacggcgaccaacaaacca gccagaagaaa^

aagtaaatigcactttgatccaectttiatte^

actaecatgaaeaactrttegteatg^

tgtgtttaaggtcgttgattgcacgagaaaaaaaaatccaaate^

ggtagtccaaagtaaaacttatagataataaaatgtggtccaaagcg^

gacaaacggcatcitetcgaaatitcccaaccg^

fcc¾¾gOagacggagaegteacgg

cctctcctcgcttcgtttcgattcgatttcgg

gagatgtttagggg tgtagatotgatggttgtgatttgggcacggttggttcgataggtggaatcgtggttagg tfttgggat¾ ggttctgatgattggggggaatttttacggttagatgaatfgftggatgattcgattggg gaaa

aaciagtcatgectgagtgaltggtgcgat^

attgagtttittggtgcggttggtgcaaacacaggctttaatatgttatatctgtttt

tggttcaattatgtagcttgtgcgtttcgatttga

gmgtcttgtcgctatatctgtca^

agtaatatcatgttaeaatctgtagtteatetata^

ttaiitctgaagticaggaiacgtgjgctgtt^

ggaatatgtttgctgtttgatccgttgttgtgte^

cccAAGCTTGACTAGTGATATCACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCC GCCCCCTTCACCGAGCTCGAGATGTTTTGAGGAAGGGTATGGAACAATCCTTGAG AGACCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGT GGATTTTGAGTTAGGAGCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAarggaga aaaaaatcactggataiaccaccgtigatatatcccaat^^

aaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataag cacaagttttatccggccttt^

ccgcctgatgaatgctcatGcggagttccgtatggcaatgaaagacgg gagctggtgatatgggatagtgttcaGccttgttacaccgt tttccatgagcaaacigaaacgttrtcatcgctctgga

cgtgttacggtgaaaacctggcGtatttccctaaagggtttaitgagaatatgtttt tcgt tgatttaaacgtggccaatatggacaacttctto

gctg¾cgattcaggttcatcatgccgtttgtgatggcttccatgtcggcagaatgc ttaatgaattacaacagtactgcgatgagtggcag ggcggggcgtaaACGCGTGGAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATT TGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTAT GTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGAC AGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCAC AACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAA TCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCT GACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGA GAGCCGlTATCGTCTGT ^' nOTGGATGTACAGAGTGATAlTATTGACACGCCCGGC CGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCC GTGAACTI ACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCA CCGATATGGCCAGTGTGCCGGTTTCCGTTATCGGGGAAGAAGTGGCTGATCTCAG CCACCGCGAAAATGACATCAAAAACGCCAT ^' rAACCTGATGTrCTGGGGAATATA AATGTCAGGCTCCCTTATACACAGCCAGTCTGCACCTCGACggtctcAcatggtttgttc ttacc acacgaccaattaaatcGAGCTCAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAAAG T GGTGATATCCCGcjigcca gciigagte

ig gtgagta _: gttoci¾tgate

aAftgfaaaatacttctetcaaiaaaattfctaaftcctaaaaccaaaalccagfga cctGCAGGCATGCGACGTCGGGC CCTCTAGAGGATCCCCGGGTACCGTGCAGCGTCGCGTCGGCX^CAAGCGAAGCAG

ACGGCACGGCATCTCIGTCGCTGCCrCTGGACCCCTCTCGAGAGTrCCGCTCCAC CGT1BGACT1IJCT€CGCTC1GGGCATC:CA.GAAA11IJCGTCGCGGAGCGGCAGAC

GTGAGCCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGCACCGGCAGCTACG GGGGATTCCTTTCCCACCGCTCCTTCCK:TTTCCCTTCCTCGCCCGCCGTAATAAAT

AGACACCCCCRCCACACCCTCTTTCCCCAACCTCGTG ^' NG'I ^' TCGGAGCGCACACA

CACACAACCAGATCTCCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAGGTACG

CCGCTCGTCCTCCCCCCCCCCCCCTCTCTACCTTCTCTAGATCGGCGITCCGGTCC

ATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTTG

TGI AGATCCGTGCTGCTAGCGTTCGTACACGGATGCGACCTGTACGTCAGACAC

GTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGCIAATCCTGGGATGGCTCTAG CCGTTCCGCAGACGGG GGATITCATGAllllTTllOll CGTrGCAI ^{^} AGGGTTr

GGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTCGGGTCATCT

mGATGCllTTTrTTGrCTrGGl GTGATGATGrGGTClGGlTGGGCGGFCGTrCT AGATCG<3AGTAGAAATCTGT rCAAACTACCTGGTGGAT TA rAATnTGGATC TGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATGATGGATGGAAA

TATCGATCTAGGATAGGTATACATGTTGATGCGGGT rrACTGATGCATATACAG

AGATGCTTTTTGTTCGCTTCJGTTGTGATGATGTGGTGTGGTTGGGCGGTCG

TCGl CTAGATCGGAGTAGAATACTG rTCAAACTACCTGGTGTAlTTATTAATTT

TGGAACTGTATGTGTGTGTCATACATCTTCATAGTTACGAGTTTAAGATGGATGG

AAATATCGATCTAGGATAGGTATACATGTTGATGTGGGTTTTACTGATGCATATA

CATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTTGAGTACCTATCTATT

ATAATAAACAAGTATGTTTTATAATTAT^

ATATGCAGCAGCTATATGTGOATTTTTXm

GCTTGGTACTGTITCTTTTGTC

GGTCGACTCTAGAGGATCCATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGA

GAAGTITCTGATCGAAAAGTrCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAG

GGCGAAGAATCTCGTGCTTTCAGC ^' ITCGAI'GTAGGAGGGCGTGGArA'rGTCCTGC

GGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTT

TGCATCGGCCGCGCTCCCGA ^' I CCGGAAGTGCTTGACA ^' ITGGGGAGTI AGCGAG

AGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGC

CTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGA

TCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAG

GAATCGGTCAATACACTACATGGCGTGAT'rTCATATGCGCGATTGCTGATCCCCA

TGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAG

GCTCTCGATGAGCTGATGCI TGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCG

TGCACGCGGA rrCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAG

CGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCA

ACATCITCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTT

CGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACTCCGGGCGTATATGCT

CCGCATTGGTCI GACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGAT

GCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACT

GTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGT

GTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCA

AAGAAATAGGAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAATTGTTATC

CGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGG

GTGCCTAATGAGTGAGCTAACTCACATTACTTAAGATTGAATCCTGTTGCCGGTC TTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAAC

ATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAAT

TATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAAT

TATCGCGCGCGGTGTCATCTATGTTACTAGATCGACCGGCATGCAAGCTGATAAT

TCAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTG

TCTAAGCGTCAATT ^' CCAGCCAGCCAACAG

CTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCA

GTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATG

TTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGA

TGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCTG

TGATCAATTCGggcacgaacccagtggacataagcctcgttcggttcgtaagctgta atgcaagtagcgtaactgccgtcac gc-aactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttc atggcttcttgitatgacatgittttttgggg tacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggto^

Gagggcagtcgccc-teaaacaaagttaaacatoatgggggaagcggtgatcgccgaagt atcgact aactatcagaggtagitggc gtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtg gatggcggcctgaagccacacagtgata ttgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatca acgaccttttggaaacttcggcttcccctgg ag gagGgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtg gcgttatccagc

aatttggagaatggcagcgcaatgacattettgcaggtatctte^

caagagaacatagcgtigccttggtaggtccagcggcggaggaactctttgatccgg ttcctgaacaggatctatttgaggcgctaaat gaaaecrtaacgciatggaactcgccgcccgactgggctgg¾

cagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcc tgccggcccagtatGagcccgtcatactt gaagctagacaggcttatc gacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacg tgaaag gcgagatcaccaaggtagtcggcaaataatgtctagctagaaattcgttcaagccgacgc cgcttcgccfgcgttaactcaagcgatt agatgcactaiigcacataattgctcacagccaaactatcaggtcaagtctgcttttatt atttttaagc

tgggagatatatcatgcatgacCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAG CGTCAGA

CCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATC

TGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATC

AAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACC

AAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA

GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTG

GCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG

CGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAA

CGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGC TTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACA

GGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCT

GTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG

GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTT

TTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA

ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGA

GCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCT

CCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATC

TGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTG

GGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGC

TTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC

ATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTG

ATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAG

ATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCG

ACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTG

CAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTA

AGAGT TTAATAAGTTTTAAAGAGTTrrAGGCGGAAAAATCGCCTTTTTTCTCTTT

TATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCA

ATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTAT

CCACAGGAAAGAGAACTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAG

CATCTGCTCCGTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGG

TAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACT

CCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAA

CTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCT

GCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGA

TCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTG

TGATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCC

GGTTTCGCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACC

GTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGG

TGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCT

CGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGC

ITGTCTCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGC

CATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGA AACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGG

TCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGG

GTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGG

GCGGCTTCCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCG

GATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATG

CGTTGCCGCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCA

GCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCT

CGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTA

CGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCC

TGGTTGTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCA

TTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTC

GGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCC

GCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCA

ACGTTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGGACTTAGCGTGTTTGT

GCTT TGCTCATTTTCTCTTTACCTCAT AACTCAAATGAGTTTTGATTTAATTTCA

GCGGCCAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGA

ACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGG

GACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCG

ATGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCAT

CCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATAT

GTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGC

GGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCG

CCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAAC

GGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGA

TCGGAATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGA

TGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAA

CCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAG

CGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCG

GCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCA

GACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGC

TCGAACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAA

ACGGTTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCAT

TCTCGGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCAC CGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTA

CAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCC

TTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGG

GCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGT

GCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAA

CACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACG

CAGGCCCGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGT

GCTGCGGGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAG

GTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTC

TCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCA

AGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGC

TCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGA

CTAAAACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCG

CGTAACTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGT

CAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTAC

t-yaii/lo ercase; T-DNA right border grey/lowercase: OslJbi promoter ORANGE/U PPERCASE I attBl BLUE/UPPERCASE: OsMIR390 5' region RED/UPPERCASE: Bsal site magenta/lowercase: chloramphenicol resistance gene MAGENTA/UPPERCASE: ccdB gene red/lowercase: inverted Bsal site blue/lowercase: OsMIR390 3' region ORANG]B UPPERCA$E UNDERLINED: attB2 CaMV termkaior GREY/UPPERCASE: ZmUbi promoter BROWN/UPPERCASE: ygroraycin resistance gene

brown/lowercase: spectiiiomycin resistance gene

Example 22.

[000288] DNA sequence of Bsal-ccdB -based (B/c) vectors used for direct cloning of amiRNAs or syn-tasiRNAs.

1. amiR A vectors

>pENTR-AtMIR390a-B/c (4491 bp)

[000289] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGA GTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG CGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA GCGCAACGCAATTAATACGCGTACCGCTAGCCAGGAAGAGTTTGTAGAAACGCA AAAAGGCCATCCGTCAGGATGGCCTTCTGCTTAGTTTGATGCCTGGCAGTTTATG GCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTGCTTCACAACGTTCAAATCCGC TCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAA ACGAAAGGCCCAGTCTTCCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTT CCCTACTCTCGCGTTAACGCTAGCATGGATGTTTTCCCAGTCACGACGTTGTAAA ACGACGGCCAGTCTTAAGCTCGGGCCCCAAATAATGATTTTATTTTGACTGATAG TGACCTGTTCGTTGCAACAAATTGATGAGCAATGCTTTTTTATAATGCCAACTTTG TACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCTATAGGGGGGAAAAAAAG GTAGTCATCAGATATATATTTTGGTAAGAAAATATAGAAATGAATAATTTCACGT TTAACGAAGAGGAGATGACGTGTGTTCCTTCGAACCCGAGTTTTGTTCGTCTATA AATAGCACCTTCTCTTCTCCTTCTTCCTCACTTCCATCTTTTTAGCTTCACTATCTC TCTATAATCGGTTTTATCTTTCTCTAAGTCACAACCCAAAAAAACAAAGTAGAGA AGAATCTGTAAGAGACCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCT CGTATAATGTGTGGATTTTGAGTTAGGAGCCGTCGAGATTTTCAGGAGCTAAGGA AGCTAAAatggagaaaaaaatcactggatatace^

cagttgctcaatgtacctataaccagaccgttcag^

gcGtttattcacattcttgcccgcctgatgaatgctcatccggagttccgtatggca atgaaagacggtgag

ttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctgga gtgaataccacgacgatttccggcagtttctacaca tataticgcaagatgtggcgtgtiacggtgaaaa

gggtgagtttcaccagttttgatttaaacgtggccaatatg^

gacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttc catgtcggcagaatgcttaatga^ actgcgatgagtggcagggcggggcgtaaACGCGTGGAGCCGGCTTACTAAAAGCCAGAT AACA

GTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTAT

ACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAG

TTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGT

CTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGG

AAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAAC

GGCTCTTTTGCTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTI ACACC

TATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTArrG

ACACGCCCGGCCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAG

ATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGC

GCATGATGACCACCGATATGGCCAGTGTGCCGGTTTCCGTTATCGGGGAAGAAG

TGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCA ^' JTTAACCTGATGTT

CTGGGGAATATAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCACCTCGACggt ctcAcattggctcttcttactacaatgaaaaaggccgaggcaaaacgcctaaaatcactt gagaatcaattctttttactgtccatttaagc tatcttttataaacgtgtcttattttctatctcttttgtttaaactaagaaactatagta ttttgtctaaaacaaaacatgaaagaa ctcatctttagtctcAAGGGTGGGCGCGCCGACC^

AAGAAAGCAT GCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAAT

AAAATCATTATTTGCCATCCAGCTGATATCCCCTATAGTGAGTCGTATTACATGG

TCATAGCTGTTTCCTGGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATT

GCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAAC

AGTAATACAAGGGGTGTTatgagccatattcaacgggaaacgtcgaggccgcgatta aattccaacatggatgctga itiataiggglaiaaatgggciegcgataaigtcgggcaata^

gtttctgaaacatggcaaaggfagcgttgccaatgat^

gaccatcaagcattttatcegtactectgatgatgcatg^

atatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggtt gcattcgattcctgtttgtaattgtcctttiaacagc gatcgcgiatttcgieicgcteaggcgcaato^ cctgttgaacaagtctggaaagaaatgcataaact^

atttttgacgaggggaaattaataggttgtattgatgttgg

gccicggtgagttttctccitcattacagaaacggctttttGaaaaatatggtattg ataatcGtgatatg

ctcgatgagtttttcTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATT ACGCT

GACTTGACGGGACGGCGCAAGCTCATGACCAAAATCCCTTAACGTGAGTTACGC

GTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGAT

CCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG

CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTGTTTTTCCGAAGGTAACTGG

CTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC

CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGT

TACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAG

ACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCAC

ACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA

GCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGT

AAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACG

CCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT

TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCC

TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT SEQ ID NO:405

PURPLE/UPPERCASE: M13-F binding site orange/lowercase: attLl BLUE/UPPERCASE: AtMIR390a 5' region RED/UPPERCASE: BsdL site magenta/lowercase: chloramphenicol resistance gene MAGENTA/UPPERCASE: cccB gene red/lowercase: inverted Bsal site blue/lowercase: AtMIR390a 3' region oran ge/Iowercase/i derliried : attL2

PURPLE/UPPERCASE/UNDERLINED: M13-Reverse binding site brown/lowercase: anamycin resistance gene

>pMDC32B-AtMIR390-B/c (12044 bp)

[000290] CCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCAC TCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAA GGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCT GCCGGGTTTGAAACACGGATGATCTGGCGGAGGGTAGCATGTTGATTGTAACGA TGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATA CTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTT AAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCT TCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAatggctaaaatg agaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaa ggaatgtctcctgctaaggtatataagct ggtgggagaaaatgaaaacctatatttaaaaatgacggacagccggtataaagggaccac ctatgatgtggaacgggaaaaggacat gatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatga tggctggagcaatctgctcatgagtgag gccgatggcgtcctttgctcggaagagtatgaagatgaacaaagccctgaaaagattatc gagctgtatgcggagtgcatcaggctctt tcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagccga attggattacttactgaataacgatctggcc gaigtggattgcgaaaactgggaagaagacactccatitaaagatccgcgcgagctgtat gattttttaaagacggaaaagcccgaag aggaacttgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatggca aagtaagtggctttattgatcttgggagaa gcggcagggcggacaagtggtatgacattgcettctgcgtccggtcgatcagggaggata tcggggaagaaeagtatgtcgagctat tttttgacttactggggatcaagcctgattgggagaaaataaaatattatattttactgg atgaattgttttagTACCTAGAATGC ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG AAAAGATCAAAGGATCTTCTTGAGATCCT TTTTTCTGCGCGTAATCTGCTGCTTG CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG TCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC TTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG

AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC

ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGG

CTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT

CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAG

AGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCG

CCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGG

CCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGC

GGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTC

GGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTA

ATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGT

CACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGG

TTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAA

AGAGAACTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCC

GTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATG

ACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGT

CATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGC

GCTGCCACTGCGrrCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTG

CGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAA

AAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGC

GGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCT

CTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGG

CGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACC

GAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCA

GAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCC

CTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGT

ACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCT

ACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCT

TCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCA

CGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTT

CCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGA TCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCC

GCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGC

GCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTT

GGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGG

CCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGT

TCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTC

ATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAAT

GGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGC

AACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTG

CAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACrrAGCGTGTTTGTGCTTTT

GCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGC

CAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTT

GTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCA

AGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGC

GTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGA

CCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTA

AGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACAC

AGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCC

GATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAG

CGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGA

ATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGT

TGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTC

ATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACC

GCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCT

CGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACA

AACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGA

ACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGG

TTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTC

GGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCG

CCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGG

GTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCT

GGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGG

GGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTC GATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCAT

GCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCC

CGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCG

GGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTGGCCAGGGCGTAGGTGGTC

AAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAA

AACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCT

GGTCGTCGGTGCTGACGCGGGCATAGCCCAGGAGGCCAGCGGCGGCGCTCTTGT

TCATGGCGTAATGTCTCCGGTTGTAGTCGCAAGTATTCTACTTTATGCGACTAAA

ACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAA

CTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAA

GCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGG

GGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCA

CGCCCT TTAAATATCCGT ATTCTAATAAACGCTC

f f!icci gica AAC ACTGATAGT TTAAACTGAAGGCGGGAAACGACAATCTGATCCAAG

CTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA

GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA

CGGCCAGTGCCAAGCTTGGCGTGCCTGCAGGTCAACATGGTGGAGCACGACACA

CTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATT

GAGACTTTTCAACAAAGGGTAATATGCGGAAACCTCCTCGGAT CCATTGCCCAG

CTATCTGTCACTITATTGTGAAGATAGTGGAAAAGGAAGGTGGC CCTACAAATG

CCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGG

TCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCC

A A.CC ACGTCTTC AA A GC A AGTGG ATTG ATGTG ATAAC ATGGTGG AGC ACQ AC A C

ACITGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAAT

TGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCAT GCCCA

GCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAAT

GCC ATCATTGCG ATAA AGG A A AGGCCATCGTTGAAGATGCCTCTGCCGACAGTG

GTCCCAAAGATGGACCCCCACCCACGAGGAGCA CGTGGAAAAAGAAGACGTrC

CAACCACGTCrrCAAAGCAAGTGGATTGATGTGATAtCTCCACTGACGTAAGGG

ATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTC

ATTTCATTTGGAGAGGACCTCGACTCTAGAGGATCCCCGGGTACCGGGCCCCCCC

TCGAGGCGCGCCAAGCTATCAAACAAGTITGTACAAAAAAGCAGGCTCCGCGGC CGCCCCCTTCACCTATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGG

TAAGAAAATATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGT

GTTCCTTCGAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTT

CCTCACTTCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCT

AAGTCACAACCCAAAAAAACAAAGTAGAGAAGAATCTGTAAGAGACCATTAGG

CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTT

AGGAGCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatggagaaaaaaatcactg gatatac caccgttgatatatcGcaatggcatcgtaaagaacattttgaggcatttcagtcagttgc tcaatgtacctataaccagaccgttcagctgg atattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggccttta ttcacattctt

cggagttcGgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcaGG Cttgttacaccgttttccatgagca cgttttcatGgctcfggagtgaataccacgacgatttccggcagtttctacacatatatt cgcaagatgtggcgtgttacggtgaaaacct ggcciatitccciaaagggittaiigagaa atgittttcgtcicagcca

atggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgac aaggtgctgatgccgctggcgattcaggttcat

GatgccgtitgtgatggGttccatgtcggcagaatgcttaatgaattacaacagtac tgGgaigagiggcagggcggggcgtaaACG

CGTGGAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATT

TTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAG

GTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTT

GCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGA

ATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGA

TGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAG

GGGCTGGTGAAATGCAGTrrAAGGTTTACACCTATAAAAGAGAGAGCCGTTATC

GTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGCCGACGGATGGT

GATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTAC

CCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCC

AGTGTGCCGGTI CCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAA.

AATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGC

TCCCTTATACACAGCCAGTCTGCACCTCGACggtctcAcattggctcttcttactac aatgaaaaaggccg aggcaaaacgcctaaaatcacttgagaatcaattctttttactgtccatttaagctatct tttataaacgtgtcttattttctatctct^ ctaagaaactatagtattttgtctaaaacaaaacatgaaagaacagattagatctcatct ttagtctcAAGGGTGGGCGCGC

CGM CAGCTTIOT

AGCGGCCGCCCACCGCGGTGGAGCTCGAA ^' RTTCCCCGATCGTTCAAACATTTGGC

AAJ AAGTIT^

ATTTCTCTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTA TTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGC-GA

TAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCAT

CTATGTTACTGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTAT

CCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGG

GGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTT

TCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG

GGAGAGGCGGTTTGCGTATTGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGA

CACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGC

TATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGC

CCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTAC

AAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGAC

AGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGA

CGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACafggtggagcacga cactc tegtetaetccaagaatatcaaagat^^

icggatfccattgcccagctatcigfcacttcatcaaaaggacagtagaaa ggaaggtggcacciacaaatgccatcattgcgataaa ggaaaggctatcgttcaagatgcctctgecgacagtggtcccaaagatggacccccaccc acgaggagcatcgiggaaaaagaaga cgttccciaccacgtcttcaaagcaagtggatteatgigatatctocactgacgfaaggg atgacgcacaalcccactatccttcgcaaga ccttcctc atataaggaagttcatttcamggagaggACACGCTGAAATCACCAGTCTCTCTCTACAAA

TCTATCTCTCTCGAGCTTTCGCAGATCCCGGGGGGCAATGAGATATGAAAAAGCC

TGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTC

TCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATG

TAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACA

AAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGT

GCTTGACATTGGGGAGTTTAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCA

CAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTACAAC

CGGTCGCGGAGGCTATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCG

GGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTT

CATATGCGCGATTGCTGATCeCCATGTGTATCACTGGCAAACTGTGATGGACGAC

ACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGG

ACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCT

GACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGG

GGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGT

ATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCG CCACGACTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCT

TGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAA

TCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCG

CGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGAC

GCCCCAGCACTCGTCCGAGGGCAAAGAAATAGAGTAGATGCCGACCGGATCTGT

CGATCGACAAGCTCGAGtttotccataataatgt^ actaaaatccagatcCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGC A ATGTGTTATT AAGTTGTCT AAGCGTC AATTTG ΤΪΤΑ C ACC A C A ATAT A TCCTGCC A SEQ ID NO:406 brown/lowercase: kanamycin resistance gene

£Ύ£Ν/ϋ ^' ΕΕΕΕ Ε υ^ C~>A traiisversioii to block vector ^' s Bsa! site cyan/lowercase: T-DNA right border

GREEN/UPPERCASE: 2x35S CaMV promoter

ORANGE/UPPERCASE: attBl

BLUE/UPPERCASE: AtMIR390a 5' region

RED/IJPPERCASE: Aral site magenta/lowercase: chloramphenicol resistance gene

MAGENTA/UPPERCASE: ccd gene red/lowercase: inverted Bsal site blue/lowercase: AtMIR390a 3' region

ORANGE/UPPERCASE/UN DERLI ED: attB2

GREY/UPPERCASE/UNDERLINED: Nos terminator green/lowercase: CaMV promoter

BROWN/UPPERCASE: hygromycin resistance gene green/lowere-ase/underliiied: CaMV terminator

>pMDC123SB-AtMIR390a-B/c (11519 bp)

[000291] CCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCAC TCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAA GGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCT GCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGA TGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATA CTATGT ATACGCCAACTT GAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTT AAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCT TCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAatggctaaaatg agaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaa ggaatgtctcctgctaaggtatataagct ggtgggagaaaatgaaaacctatatttaaaaatgacggacagccggtataaagggaccac ctatgatgtggaacgggaaaaggacat gatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatga tggctggagcaatctgctcatgagtgag gccgatggcgtcctttgctcggaagagtatgaagatgaacaaagccctgaaaagattatc gagctgtatgcggagtgcatcaggctctt tcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagccga attggattacttactgaataacgatctggcc gatgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcgagctgtat gattttttaaagacggaaaagcccgaag aggaacttgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatggca aagtaagtggctttattgatcttgggagaa gcggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggata tcggggaagaacagtatgtcgagctat ttttl ^' gacttaetggggatca^gcctgattgggagaaaa

ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG

AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTG

CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA

CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG

TCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC

TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG

TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG

TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC

ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA

GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG

CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT

CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC

TTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG

AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC

ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGG

CTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT

CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAG

AGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCG

CCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGG

CCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGC

GGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTC

GGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTA

ATAAGT TTAAAGAGTTTTAGGCGGAAAAATCGCCTTTri CTCTTTTATATCAGT

CACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGG

TTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAA

AGAGAACT TTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCC

GTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATG

ACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGT

CATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGC

GCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTG

CGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAA

AAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGC

GGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCT

CTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGG

CGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACC

GAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCA

GAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCC

CTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGT

ACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCT

ACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCT

TCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCA

CGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTT CCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGA

TCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCC

GCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGC

GCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTT

GGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGG

CCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGT

TCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTC

ATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAAT

GGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGC

AACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTG

CAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTT

GCTCATTTTCTCTTTACCTCAT AACTCAAATGAGTTTTGATTTAATTTCAGCGGC

CAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTT

GTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCA

AGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGC

GTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGA

CCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTA

AGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACAC

AGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCC

GATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAG

CGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGA

ATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGT

TGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTC

ATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACC

GCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCT

CGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACA

AACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGA

ACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGG

TTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTC

GGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCG

CCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGG

GTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCT

GGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGG GGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTC

GATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCAT

GCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCC

CGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCG

GGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTC

AAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAA

AACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCT

GGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGT

TCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAA

ACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAA

CTTAGGAGTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAA

GCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGG

GGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCA

tafcctgtoaAACACTGAmGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAG

CTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA

GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA

CGGCCAGTGCCAAGCTTGCATGCCTGCAGGTCAA.CATGGTGGTGCACGACACAC

TTGTCTACTCCAAAAATATCTTTGATACAGTC CAGAAGACCAAAGGGCAATTGA

GACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCT'

ATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCC

ATCATrGCGATAAAGGAAAGGCCATCGTrGAAGATGCCTCTGCCGACAGTGGTC

CCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAA

CCACGTCTrCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACAC

TTOTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTG

AGACTTT C A A C A A AGGGTA ATATCCGG A A A CCTCCTCGG A TTCC ATTGCCC AGC

TATCTGTCACTTTATTGTGAAGATACJTCJGAAAAGGAAGGTGGCTCCTACAAATGC

CATCAlTGCGATAAAGGAAAGGCCATCGTTGAAGAIYirCCTCTGCCGACAGTGGT

CCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCA

ACCAeGTC TCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGA G

ACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATr

TCATTTGGAGAGGACCTCGACTCTAGAGGATCCCCGGGTACCGGGCCCCCCCTCG AGGCGCGCCAAGCTATC AAAC A A GTTTGT AC A AAAAAGCA GGC I CCGCGGCCGC

CCCCTTCACCTATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAA

GAAAATATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTT

CCTTCGAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCT

CACTTCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAG

TCACAACCCAAAAAAACAAAGTAGAGAAGAATCTGTAAGAGACCATTAGGCACC

CCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGA

GCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatggagaaaaaaatcactggata taccaccgtt gatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgt acctataaccagaccgttca ggcctttttaaagaecgiaaagaaaaataagcacaagttttatccggccittattcacat ictigcccgcctgatgaatgcicatGcggagt ^' tccgtatggcaatgaaagacggtgagetggtgatatgggata^

atcgctctggagigaataccacgacgatitccggcagittetacaca!atattegca aga gt

tfccctaaagggrtfattgagaatatgtttttcg^

acttettcgcccecgttitcac^

tttgtgatggcttccatgtcggcagaaigcttaatgaattacaacagtactgcgatg agtggcagggcggggcgtaaACGCGTG

GAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTG

CGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTAT

GCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTC

AAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATG

AAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGG

CTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGG

CTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCT

GTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGCCGACGGATGGTGATC

CCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGG

TGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTG

TGCCGGTTTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATG

ACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCT

TATACACAGCCAGTCTGCACCTCGACggtctcACATTGGCTCTTCTTACTACAATGA A

AAAGGCCGAGGCAAAACGCCTAAAATCACTTGAGAATCAATTCTTTTTACTGTCC

ATT AAGCTATCTTTTATAAACGTGTCTTATTTTCTATCTCTTTTGTTTAAACTAAG

AAACTATAGTATTTTGTCTAAAACAAAACATGAAAGAACAGATTAGATCTCATCT

T AGTCTCAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAAAGTGGTTCGATA

ATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCGAA I I I C GTCTTGj^ AAJI ACAT^

AATTATCGCGCGCGG7\?fTCATCTATG rACTAGATCGGGAATTCGTAATCATGGT

CATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACG

AGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCAC

ATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAG

CTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAG

AGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCA

AAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAA

TATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAG

GACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAA

AGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACC

CACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGT

GGATTGATGTGATAACatggtggagcacgacactotc tctactocaagaatafcaaagatacagtctcagaagacca aagggefattgagactittcaacaaagggtaaiateg

gtagaaaaggaaggiggcaccaaeaaatgcc tcaftgeg

aagafggaeececacceaeeaggagcategigg^^

cactgacgtaaggptgacgcacaateccactaiccttcgcaagaccftcctctatai aaggaagttcatttcatttggagaggACAC

GCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGTCTACCATGAGC

CCAGAACGACGCCCGGCCGACATCCGCCGTCCCACCGAGGCGGACATGCCGGCG

GTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACrrCCGTACCG

AGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCGGGAGCGCT

ATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGCCTACGCGG

GCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGTACG

TCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACCCACCTGCT

GAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATCGGGCTGCC

CAACGACCCGAGCGTGCGCA.TGCACGAGGCGCTCGGATA.TGCCCCCCGCGGCAT

GCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTGGCA

GCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCACCGAGATT

TGACTCGAGttit3cci3taata _; atg¾

iataagaaacccttagtaigtattfgtatttgtaaaate^ cCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTA TTAAGTTGTCTAAGCGTCAATT I ( TTT A C ACCAC A AT ATA.TCCTGCCA SEQ ID NO:407 brown/lowercase: kanamycin resistance gene

C Niii M C">A transversion to block vector ^' s Bsal site eyan/!owercase: ^'

GREEN/UPPERCASE: 2x.35S CaMV promoter ORANGE/UPPERCASE : attB i BLUE/UPPERCASE: AtMIR390a 5' region RED/UPPERCASE: Bsal site magenta lowercase: chloramphenicol resistance gene MAGENTA/UPPERCASE: ccdB gene red/lowercase: inverted Bsal site blue/lowercase: AtMIR390a 3' region green/lowercase: CaMV promoter

BROWN/UPPERCASE/UNDERLINED: BASTA resistance

CYAN/UPPERCASE: T-DNA left border

>pFK210B-AtMIR390-B/c (7916 bp)

[000292] GTTATCAGCTTGCATGCCGGTCGATC

TAGT ACATAGA O T GT TTCTATCGCGTATTA AACAG AMT^

AAACTllATroCCAAA.TG1TTGAACGA ^r rCTGCTTGACTCTAGGGGTCATCAGArr

TCGGTGACGGGCAGGACCGGACG ^' GGGCGGCACCGGCAGGCTGAAGTCCAGCTGC

CAGAAACCCACGTCATGCCAGTTCCCGTGCTTGAAGCCGGCCGCCCGCAGCATG

CCGCGGGGGGCATATCCGAGCGCCTCGTGCATGCGCACGCTCGGGTCGTTGGGC

AGCCCGATGACAGCGACCACGCTCTTGAAGCCCTGTGCCTCCAGGGACTTCAGC

AGGTGGGTGTAGAGCGTGGAGCCCAGTCCCGTCCGCTGGTGGCGGGGGGAGACG

TACACGGTGGACTCGGCCGTCCAGTCGTAGGCGTTGCGTGCCTTCCAGGGACCCG

CGTAGGCGATGCCGGCGACCTCGCCGTCCACCTCGGCGACGAGCCAGGGATAGC

GCTCCCGCAGACGGACGAGGTCGTCCGTCCACTCCTGCGGTTCCTGCGGCTCGGT

ACGGAAGTTGACCGTGCTIOTCTCGATGTAGTGGTTGACGATGGTGCAGACCGCC

GGCATGTCCGCCTCGGTGGCACGGCGGATGTCGGCCGGGCGTCGTTCTGGGCTCA

TGGTAGATCCCCTCGATCGAGTTGAGAGTOAATATGAGACTCTAATTGGATACCG

AGGGGAATTTATGGAACGTCAGTGGAGCATTTTTGACAAGAAATATTTGCTAGCT

GAIAGTGACCTTAGGCGACTTTlOAACGCGCAA'rAATGGTITC ^' rGACGTA'rG ^' rGC

TTAGCTCATTAA.ACTCCAGAAACCCGCGGCTCAGTGGCTCCTTCAACGTTGCGGT

TCTGTCAGTTCCAAACGTAAAACGGCTTGTCCCGCGTCATCGGCGGGGGTCATAA

CGTGACTCCCTTAATTCTCCGCTCATGTATCGATAACATTAACGTTTACAATTTCG

CGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCC

TCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTT

GGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGC

GCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGG

TCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCATTCG

GTCCCCAGATTAGCCTTTTCAATTTCAGAAAGAATGCTAACCCACAGATGGTTAG

AGAGGCTTACGCAGCAGGTTTCATCAAGACGATCTACCCGAGCAATAATCTCCA

GGAAATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAAGATTCAGGAC

TAACTGCATCAAGAACACAGAGAAAGATATATTTCTCAAGATCAGAAGTACTAT

TCCAGTATGGACGATTCAAGGCTTGCTTCACAAACCAAGGCAAGTAATAGAGAT

TGGAGTCTCTAAAAAGGTAGTTCCCACTGAATCAAAGGCCATGGAGTCAAAGAT

TCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACA

GAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCaacatggtggagcacga cacact igteiaeiccaaaaaiatcaaagaracagfcicag^^ cggaftccattgcccageiaretgleactM^

aaaggccafcgttgaaptgcctctgccgacagtggfcccaaagafggacccccaccc acgaggagcatogtggaaaaagaagac gficimaecaegicttcaaagcaagtggatfgalgigat

ccftcctctatataaggaagttcattlcatttggagagAACACGGGGGACGAGCTTC TAGAGGATCACAA

GTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCTATAGGGGGGAAA

AAAAGGTAGTCATCAGATATATATTTTGGTAAGAAAATATAGAAATGAATAATT

TCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTCGAACCCGAGTTTTGTTCG

TCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCACTTCCATCTTTTTAGCTTCAC

TATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCACAACCCAAAAAAACAAAG

TAGAGAAGAATCTGTAAGAGACCATTAGGCACCCCAGGCTTTACACTTTATGCTT

CCGGCTCGTATAATGTGTGGATTTTGAGTTAGGAGCCGTCGAGATTTTCAGGAGC

TAAGGAAGCTAAAaiggagaaaaaaatcactggataiaccaccg gaiaiatoccaaiggcatcgiaaagaacaiittga ggcattteagtcagttgctcaatgtacctataaccagaccgttcagrt^

aagttttatccggcctttatteacattcttgGCCgcctgatgaatgctcatccggag ttccgiaiggcaatg

atgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttc atcgctctggagtgaataccacgacgatttccggc agtttctacacatatattcgcaagatgtggcgigttacggtgaaaacctggcctatttcc ctaaagggtttattgagaatatgttttte agccaatcccigggtgagtttcaccagttttgatttaaacgtggccaatatggacaacti cttcgcccccgttttcaccatgggcaaatatt atacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtg atggctte^

aatfacaacagtacigcgatgagtggcagggcggggcgtaaACGCGTGGAGCCGGCT TACTAAAAGCCA

GATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGA

TATGTATACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACA

GTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATAT

CTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGA

ACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGA

AATGAACGGCTCTTTTGCTGACGAGAACAGGGGCTGGTGAAATGCAGTITAAGG

TTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGA

TATTATTGACACGCCCGGCCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTG

CTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAA

GCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTTTCCGTTATCGGGGA

AGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCT

GATGTI TGGGGAATATAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCACCT

CGACggtctcAcattggctcttcttactacaatgaaaaaggccgaggcaaaacgcct aaaatcacttgagaatcaattctttttactgt ccatttaagctatcttttataaacgtgtcttattttctatcte^ agattagatctcatctttagtctcAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAAA GTGGT

GATCCTAGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCA

TCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATT

GGGAAAACTG-TTTTTCTTGTACCATTTGTTGTGCTTGTAAT TACTGTGTTTT

TTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTT ^'

AATGAATGATATGGTCCTl TGTTCATTCTCAAATTAATATTATTrGTT TT

CTCTTATTTGTTGTGTGT GAATTTGAAATTATAAGAGATATGCAAACATTTT

GTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATAT

GAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGCAACAA

ATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTC

CTCTTGTGTITrAGACATTTATGAAC I CCrrTATGTAATTTTCCAGAATCC

TTGTCAGATTCTAATCATTGCTTTATAATTATAGTTATACTCATGGA TTGTA

GITGAGTATGAAAATATTTTTTAATGCAT TTATGACTTGCCAATTGATTGAC

AACATGCATCAATCGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTCCG

AGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCAC

AATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTA

ATGAGTGAGCTAACTGACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG

GGAAACGTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGC

GGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT

CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCC

ACAGAATCAGGGGATAACGCAGGAAAGAACATGAAGGCCT .· · .·· , , . aaCTAAGTCGCTGTATGTGTTTGTTTGAGATCTCATGTGAGCAAAAGGCCAGCAA

AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGC

CCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCG

ACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC

CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGC

GTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC

GCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTT

ATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG

GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA

GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTA

TCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAGAAGAGTTGGTAGCTCTTGATC

CGGCAAACAAACCACCGCTGGTAGCGGTGGT TTTTTGTTTGCAAGCAGCAGATT ACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTG ACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTA AAGTATATATGTGTAACATTGgtctagtgattatttgccgactaccttggtgatctcgcc tttcacgtagtgaacaaat tcttccaactgatctgcgcgcgaggccaagcgatcttcttgtcGaagataagcctgccta gcttcaagtatgacgg

cggcaggcgctccattgcccagtcggcagcgacatccttcggcgcgattttgccggt tactgcgctgtaccaaatgcgggacaacgta agcaetacatttcgcieatcgccagcccag

ggaaccggatcaaagagttcctccgccgctggacctaccaaggcaacgctatgttct cttgcttttgtca

gtcgatcgtggctggctcgaagatacctgcaagaatgtcattgcgctgccatictcc aaattgcagttcgcgcti

cggaatgatgtegtcgigeaeaacaatggtgacticiacagcgcgg

tcgttgatcaaagctcgccgcgttgtttcatcaagcctte^

ccactgcggagccgtacaaatgtac.ggccagcaacgt^ggttcgagatggcgctcg atgacgccaactaGCtctgatagttgagtcg atacttGggcgatcaccgcttccctcatAACACCCCTTGTATTACTGTTTATGTAAGCAG ACAGTTT

TATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAG

ACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAT

CACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAAT

CACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTC

TGGCTGGATGATGGGGCGATTCAGGCGATCCCCATCCAACAGCCCGCCGTCGAG

CGGGCTTTTTTATCCCCGGAAGCCTGTGGATAGAGGGTAGTTATCCACGTGAAAC

CGCTAATGCCCCGCAAAGCCTTGATTCACGGGGCTTTCCGGCCCGCTCCAAAAAC

TATCCACGTGAAATCGCTAATCAGGGTACGTGAAATCGCTAATCGGAGTACGTG

AAATCGCTAATAAGGTCACGTGAAATCGCTAATCAAAAAGGCACGTGAGAACGC

TAATAGCCCTTTCAGATCAACAGCTTGCAAACACCCCTCGCTCCGGCAAGTAGTT

ACAGCAAGTAGTATGTTCAAT AGCTTTTCAATTATGAATATATATATCAATTATT

GGTCGCCCTTGGCTTGTGGACAATGCGCTACGCGCACCGGCTCCGCCCGTGGACA

ACCGCAAGCGGTTGCCCACCGTCGAGCGCCAGCGCCTTTGCCCACAACCCGGCG

GCCGGCCGCAACAGATCGTTTTATAAATTTTTnrTTTTTGAAAAAGAAAAAGCCCG

AAAGGCGGCAACCTCTCGGGCTTCTGGATTTCCGATCCCCGGAATTAGAGATCT

SEQ ID NO:408 brown/lowercase; spectinoniycin resistance gene CYAN/UPPERCASE: T-DNA left border GREY/UPPERCASE/UNDERLINED: Nos terminator BROWN/U PPERC AS E/ UNDERLINED : BASTA resistance gene GREY/UPPERCASE: Nos promoter

CYAN/UPPEB£ASP^^ C->T transversioii to block vector's Bsal site

GREEN/UPPERCASE: 35S promoter

ORANGE/UPPERCASE: attBl

BLUE/UPPERCASE: AtMIR390a 5' region

RED/UPPERCASE: Bsal site magenta/lowercase: chloramphenicol resistance gene

MAGENTA/UPPERCASE: cccB gene red/lowercase: inverted Bsal site blue/lowercase: AtMIR390a 3' region

ORANG E/U PPERC ASE/UNDERL INED : attB2

GREY/UPPERCASE/BOLD: Pea rbes terminator cyiiii/Io ercase; T-DNA right border

2. syn-tasiRNA vectors

>pENTR-AtTASlc-B/c (4989 bp)

[000293] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGA GTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG CGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA GCGCAACGCAATTAATACGCGTACCGCTAGCCAGGAAGAGTTTGTAGAAACGCA AAAAGGCCATCCGTCAGGATGGCCTTCTGCTTAGTTTGATGCCTGGCAGTTTATG GCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTGCTTCACAACGTTCAAATCCGC TCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAA ACGAAAGGCCCAGTCTTCCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTT CCCTACTCTCGCGTTAACGCTAGCATGGATGTTTTCCCAGTCACGACGTTGTAAA ACGACGGCCAGTCTTAAGCTCGGGCCCCAAATAATGA^

TGACCTGTTCGTTGCAACAAATC^

TACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCAAACCTAAACCTAAACGG

CTAAGCCCGACGTCAAATACCAAAAAGAGAAAAACAAGAGCGCCGTCAAGCTCT

GCAAATACGATCTGTAAGTCCATCTTAACACAAAAGTGAGATGGGTTCrrAGATC

ATGTTCCGCCGTTAGATCGAGTCATGGTCTTGTCTCATAGAAAGGTACTTTCGTTT

ACTTCTTTTGAGTATCGAGTAGAGCGTCGTCTATAGTTAGTTTGAGATTGCGTTTG

TCAGAAGTTAGGTTCAATGTCCCGGTCCAATTTTCACCAGCCATGTGTCAGTTTC

GTTCCTTCCCGTCCTCTTCTTTGATTTCGTTGGGTTACGGATGTTTTCGAGATGAA

ACAGCATTGTTTTGTTGTGATTTTTCTCTACAAGCGAATAGACCATTTATCGGTGG

ATCTTAGAAAATTAAGAGACCATTAGGCACCCCAGGCXTTACACTTTATGCTTCC

GGCTCGTATAATGTGTGGATTTTGAGTTAGGAGCCGTCGAGATTTTCAGGAGCTA

AGGAAGCTAAAatggagaaaaaaatcactggatataccaccgttgatatatcGcaat ggcatcgtaaagaacattttgaggc atttcagtcagltgctcaatgtacctataaccagaccgttc^

ttiatccggccttiattcacattcttgcccgccfgaigaatgctcaiccggagftcc gtatggcaatgaaagacggtgagctggtgatatgg gatagtgttcacccttgttacaccgttttccatga

ctacacatatattcgcaagatgtggcgtgttacggtg^

aatccctgggtgagtttcacGagttttgatttaaacgtggccaatatggacaacttc ttcgcccccg t

caaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgat ggcttccatgtcggcagaatgcttaatgaatta caacagtactgcgatgagtggcagggcggggcgtaaACGCGTGGAGCCGGCTTACTAAAA GCCAGAT

AACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATAT

GTATACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTG

ACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTC

CGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACG

CTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAAT

GAACGGCTCTTTTGCTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTT

ACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATAT

TATTGACACGCCCGGCCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTG

TCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCT GGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTTTCCGTTATCGGGGAAG

AAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGA

TGITCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCACCTCG

ACggtctcAgaactagaaaagacattggacatattccaggatatgcaaaagaaaaca atgaatattgttttgaatgtgttcaagtaaat gagattttcaagtcgtctaaagaacagttgctaatacagttacttatttcaataaataat tggttctaataatacaaaacatattcgaggatat gcagaaaaaaagatgtttgttattttgaaaagcttgagtagtttctctccgaggtgtagc gaagaagcatcatctactttgtaatgtaattttc

Watgttttcactttgtaattttatttgtgttaatgtaccatggccgatatcggtttt attgaaagaaaattt

cagttatgctagttttcttataccctttcgtaagcttcrt

cgaccatcatataattctgggtcaagagatgaaaatagaacaccacatcgtaaagtg aaatAAGGGTGGGCGCGCCGA CCCAGCHTTCTTGTACAAAGTTGGCATM

GCAACGAACAG(JTCACTATCAGTCA AAATAAAATCATTATTTGCCATCCAGCTGA TATCCCCTATAGTGAGTCGTATTACATGGTCATAGCTGTTTCCTGGCAGCTCTGGC CCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATG AACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTatgagccatattca acgggaaacgtcgaggccgcgattaaattccaacatggafgctgatttatatgggtataa atgggctcgcgataatgtcgggc-aatcag gtgcgacaatctatcgcttgiat^ggaagcccgatgcgccaga

gatgagatggtcag ctaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcct gatgatgcatggttact caccactgegatccceggaaaaacagcatrcca^

cctgcgccggttgeattegattccfgtt¾^

cggitlggttgatgcgagtgattttgatgacgagcgtaat ^' ggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccat caccggattcagtcgtcactcatggtgatttGtcacttgataaccttatttttgacgagg ggaaaitaataggttg

cggaatcgcagaccgataccaggatcttgccatcc ggaactgcctcggtgagttttctccttcattacag

atggtattgataatcctgatatgaataaattgcagtttcattt

GTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGCAAGCTCATG

ACCAAAATCCCTTAACGTGAGTTACGCGTCGTTCCACTGAGCGTCAGACCCCGTA

GAAAAGATCAAAGGATCTTCT GAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTT

GCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT

ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACT

GTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGC

CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA

GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCG

GTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTA

CACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGA AGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGC GCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTT TCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGC CTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGC CTTTTGCTCACATGTT SEQ ID NO:409

PURPLE/UPPERCASE: M13-F binding site orange/lowercase: attt 1 BLUE/UPPERCASE: AtTASlc 5 ' region RED/UPPERCASE: Bsal site red/lowercase: inverted Bsal site magenta/lowercase: Chloramphenicol resistance gene MAGENTA/UPPERCASE: ccdB gene blue/lowercase: AtTASlc 3' region orange/iowercase/underlined: attL2

PURPLE/UPPERCASE/UNDERLINED : Ml 3-R binding site brown/lowercase: Kanamycin resistance gene

> _P MDC32B-AtTASlc-B/c (12550 bp)

[000294] CCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCAC TCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAA GGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCT GCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGA TGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATA CTATGT ATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTT TCTGGTATTT AAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCT TCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAatggctaaaatg agaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaa ggaatgtctcctgetaaggtatataagct ggtgggagaaaatgaaaacctatatttaaaaatgacggacagccggtataaagggaccac ctatgatgtggaacgggaaaaggacat gatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatga tggctggagcaatctgctcatgagtgag gccgatggcgtcctttgctcggaagagtatgaagatgaacaaagccctgaaaagattatc gagctgtatgcggagtgcatcaggctctt tcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagccga attggattacttactgaataacgatctggcc gatgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcgagctgtat gattttttaaagacggaaaagcccgaag aggaacttgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatggca aagtaagtggctttattgatcttgggagaa gcggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggata tcggggaagaacagtatgtcgagctat tttttgacttactggggatGaagcctgattgggagaaaataaaatattatattttactgg atgaattgttttagTACCTAGAATGC

ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG

AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTG

CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA

CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG

TCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC

TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG

TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG

TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC

ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA

GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG

CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT

CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC

TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC

TT TGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG

AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC

ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGG

CTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT

CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAG

AGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCG

CCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGG

CCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGC

GGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTC

GGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTA

ATAAGTTTrAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGT CACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGG

TTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAA

AGAGAACTTTOGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCC

GTAGATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATG

ACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGT

CATTTGACCCGATCAGCTTGCGCAGGGTGAAACAGAACTTCTTGAACTCTCCGGC

GCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTG

CGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAA

AAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGC

GGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCT

CTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGG

CGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACC

GAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCA

GAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCC

CTTCCC1TCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGT

ACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCT

ACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCT

TCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCA

CGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTT

CCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGA

TCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCC

GCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGC

GCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTT

GGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGG

CCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGT

TCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTC

ATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAAT

GGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGC

AACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTG

CAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTT

GCTCAT TTCTCTTTACCTCAT AACTCAAATGAGTTTTGATTTAATTTCAGCGGC

CAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTT

GTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCA AGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGC

GTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGA

CCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTA

AGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACAC

AGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCC

GATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAG

CGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGA

ATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGT

TGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTC

ATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACC

GCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCT

CGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACA

AACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGA

ACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGG

TTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTC

GGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCG

CCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGG

GTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCT

GGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGG

GGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTC

GATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCAT

GCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCC

CGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCG

GGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTC

AAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAA

AACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCT

GGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGT

TCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAA

ACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAA

CTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAA

GCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGG

GGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCA

CGCCCTT TAAATATCCGTTATTCTAATAAACGCTCTlTTCTCT AGGttm AACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAG CTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA CGGCCAGTGCCAAGCTTGGCGTGCCTGCAGGTCAACATGCJTGGAGCACGACACA CTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAG-AAGACCAAAGGGCAATT ^' GAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAG CTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATG CCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGG TCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCC AACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACAC ACTTGTCTACTCCAAAAATATCAAAGATACAG CTCAGAAGACCAAAGGGCAAT TGAGACTTTTCAACAAAGGGTA AT TCCGGAAACCTCCTCGGATTCCATTGCCCA GC ATCTGTCACTTTATrGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAAT GCCATCATTGCGATAAAGGAAAGGCCATCG rGAAGATGCCTCTGCCGACAGTG GTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTC CAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGG ATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTC ATTTCATTTGGAGAGGACCTCGACTCTAGAGGATCCCCGGGTACCGGGCCCCCCC TCGAGGCGCGCCAAGCTATCAAACAAGTTTGTA.CAAAAAAGCAGGCTCCGCGGC CGCCCCCTTCACCCCTTCACCAAACCTAAACCTAAACGGCTAAGCCCGACGTCAA ATACCAAAAAGAGAAAAACAAGAGCGCCGTCAAGCTCTGCAAATACGATCTGTA AGTCCATCTTAACACAAAAGTGAGATGGGTTCTTAGATCATGTTCCGCCGTTAGA TCGAGTCATGGTCTTGTCTCATAGAAAGGTACTTTCGTTTACTTCTTTTGAGTATC GAGTAGAGCGTCGTCTATAGTTAGTTTGAGATTGCGTTTGTCAGAAGTTAGGTTC AATGTCCCGGTCCAATTTTCACCAGCCATGTGTCAGTTTCGTTCCTTCCCGTCCTC TTCTTTGATTTCGTTGGGTTACGGATGTTTTCGAGATGAAACAGCATTGTTTTGTT GTGATTTTTCTCTACAAGCGAATAGACCATTTATCGGTGGATCTTAGAAAATTAA GAGACCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTG TGGATTTTGAGTTAGGAGCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatgga gaaaaaaatcactggatataGcaccgttgatatatcccaatggcatcgfaaagaacatft tgaggcatttcagtcagtt

ataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaata agcacaagttttatccggcctttattcacattcttg cccgcctgatgaatgctcatccggagttGcgtatggcaatgaaagacggtgagctggtga tatgggatagtgttcacccttgt^ gttttccatgagcaaactgaaacgttftcatcg^^

ggcgtgttacggtgaaaacctggcctatttccctaaagg^

gftttgatttaaacgtggccaatatggaGaacttcttcgcccccgttttcaccatgg gcaaaiattatacgGaaggcgacaa gccgctggcgattcaggttcatcatgccgtttgtgatggcttcca-tgtcggcagaatgc ttaatgaattacaacagtactgcgatgagtgg cagggcggggcgtaaACGCGTGGAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTA

TTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGT

ATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCG

ACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGC

ACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAA

AATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTG

CTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGA

GAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCG

GCCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTC

CCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGAC

CACCGATATGGCCAGTGTGCCGGTTTCCGTTATCGGGGAAGAAGTGGCTGATCTC

AGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATA

TAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCACCTCGACggtctcAgaactag aaaa gacattggacatattccaggatatgcaaaagaaaacaatgaatattgtittgaatgtgtt caagtaaatgagattttcaagtcgtctaaaga acagttgctaatacagttacttatttcaataaataattggttctaataatacaaaacata ttcgaggatatgcagaaaaaaagatgtttgttatt ttgaaaagcttgagtagtttctctccgaggtgtagcgaagaagcatcatctactttgtaa tgtaattttctttatgttttcacttt gtgttaatgtaccatggccgatatcggttttattgaaagaaaatttatgttacttctgtt ttggctttgcaatcagttatgctagttttcttataccc tttcgtaagcttcctaaggaatcgttcattgatttccactgcttcattgtatattaaaac tttacaactgtatcgaccatcatalaattctgg aagagatgaaaatagaacaccacatcgtaaagtgaaatAAGGGTGGGCGCGCCGACCCAG CTTTCITGT

ACAAAGTGGTTCGATAATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCCACCGC

GGTGGAGCTCGAATTTCCCCGATCGTTCAAACATITGGCAATAAAGTTTCTrAAG

ATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACG

TTAAGCATGTAATAATTAACATGTAATGCATGACGTTA ^{^} n ATGAGATGGGT TT

TATGAT AGAGTCCCGCAATTATACATTTAATACGCGATAGAAA CAAAATATA

GCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTC

TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACA

CAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAG

CTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTG

TCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT ATTGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAA

GAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACA

AAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTC

ATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGAT

AAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGA

CCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCA

AAGCAAGTGGATTGATGTGATAACatggtggxigea^

ctcagaagaccaaagggctatigagacttttcaacaaagggtaatalcgggaaacct cctcggattccattgcccagctatatgtcacttc atcaaaaggacagtagaaaaggaaggtggcacctecaaafgccatcattgcgafaaagga aaggctaregitcaagatgccfctgcc gacagtggtcccaEagaiggaaccGcacccacgaggagcarcgfggaaaaagaagacgft ccaaccacgtcttoaaagcaagtgga ttgatgtgatatctccaetgacgtaag^^

gagaggACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCTTT CGCAGATCCCGGGGGGGAATGAGATATGAAAAAGCCTGAACTCACCGCGACGTC

TGTCGAGAAGTTrCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTC TCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATAT

GlCClOCGGGTAAAlAGClOCGCCGAlOGTl ^{^} rCTACAAAGA!rGIlATGTTTATC

GGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTT

TAGCGAGAGCC rGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTrGCAA

GACCTGCCTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATG

GATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGITCGGCCCATTCGGA

CCGCAAGGAATCGGI AATACACTACATGGCGTGAI ^v rrCATATGCGCGATTGCTG

ATCCCCATGTGTATCACTX3GCAAACTGTGATOGACGACACCGTCAGTGCGTCCGT

CGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCG

GCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGC

ATAACAGCGGTCATTGACTGGAGCGAG GATGTTCGGGGAT.rCCCAATACGAG

GTCGCCAACATCTTCTTCTGrGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGC

GCTACiTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACrCCGGGCGT

ATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTT

CGATGATGCAGCT GGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGC

CGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGA

TGGCTGTGT GAAGTACTCGCCGATAG ^' FGGAAACCGACGCCCCAGCACTCGTCC

GAGGGCAAAGAAATAGAGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGA tagtatetatt gt¾^

TTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTG TCTAAGCGTCAATTTG ^' ΓΤΤ AC A CC AC A A T AT ATCCTC1CC A SEQ ID NO:410

brown/lowercase: kanamycin resistance gene

Εΐ /ίΜΙΕΕ^ C->A transversion to ioek vector ^' s Bsal site cyan/lowercase: T-KNA lig t border

G EEN/IJ PPERCASE: 2x35S CaMV promoter

ORANGE/UPPERCASE: aitB l

BLUE/UPPERCASE: AtTASlc 5' region

RED/UPPERCASE: Bsal site magenta/lowercase: chloramphenicol resistance gene

MAGENTA/UPPERCASE: ccdB gene red/lowercase: inverted Bsal site blue/lowercase: AtTASlc 3' region

ORANGE/LTPERCASE UNDERLiNED: attB2

GREY/UPPERCASE UNDERL.I ED: Nos terminator green/lowercase: CaMV promoter

BROWN/UPPERCASE: hygromycin. resistance gene

CYAN/UPPERCASE: T-DNA left border

>pMDC123SB-AtTASlc-B/c (12017 bp) [000295] CCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCAC TCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAA GGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCT GCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGA TGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATA CTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATrr AAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCT TCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAatggctaaaatg agaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaa ggaatgtctcctgctaaggtatataagct ggtgggagaaaatgaaaacctatatttaaaaatgacggacagccggtataaagggaccac ctatgatgtggaacgggaaaaggacat gatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatga tggctggagcaatctgctcatgagtgag gccgatggcgtcctttgctcggaagagtatgaagatgaacaaagccctgaaaagattatc gagctgtatgcggagtgcatcaggctctt tcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagccga attggattacttactgaataacgatctggcc galgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcgagctgtat gattttttaaagacggaaaagcccgaag aggaacttgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatggca aagtaagtggctttattgatcttgggagaa gcggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggata tcggggaagaacagtatgtcgagctat ttttfgacttactggggatcaagcctgaitgggagaaaataaaatattataftttactgg atgaattgttttagTACCTAGAATGC ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTG CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG TCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC TTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGG

CTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT

CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAG

AGGTTT CACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCG

CCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGG

CCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGC

GGCGGGGCGTAGGGAGCGGAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTC

GGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTA

ATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGT

CACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGG

TTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAA

AGAGAACTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCC

GTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATG

ACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGT

CATTTGAGCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGC

GCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTG

CGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAA

AAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGC

GGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCT

CTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGG

CGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACC

GAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCA

GAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCC

CTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGT

ACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCT

ACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCT

TCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCA

CGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTT

CCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGA

TCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCC

GCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGC

GCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTT GGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGG

CCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGT

TCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTC

ATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAAT

GGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGC

AACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTG

CAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTT

GCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGC

CAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTT

GTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCA

AGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGC

GTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGA

CCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTA

AGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACAC

AGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCC

GATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAG

CGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGA

ATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGT

TGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTC

ATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACC

GCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCT

CGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACA

AACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGA

ACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGG

TTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTC

GGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCG

CCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGG

GTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCT

GGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGG

GGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTC

GATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCAT

GCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCC

CGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCG GGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTC

AAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAA

AACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCT

GGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGT

TCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAA

ACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAA

CTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAA

GCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGG

GGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCA

CGCCCTTI AAATATCCGTTATTCT

tafcctgtcaAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAA G

CTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA

GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA

CGGCCAGTGCCAAGCTTGCATGCCTGCAGGTCAACATGGTGGTGCACGACACA.C

TTGTCTACTCCAAAAATATCTI GATACAGTCTCAGAAGACCAAAGGGCAATTGA

GACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCT

ATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAA GCC

ATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTC

CCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGT CCAA

CCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACAC

TTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTG

AGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGC

TATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGC

CATCATRGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGT

CCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCA

ACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATG

ACGCAC:AATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATT

TCAL RGGAGAGGACCTCGACTCTAGAGGATCCCCGGGTACCGGGCCCCCCCTCG

AGGCGCGCCAAGCTATCAAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGC

CCCCTTCACCAAACCTAAACCTAAACGGCTAAGCCCGACGTCAAATACCAAAAA

GAGAAAAACAAGAGCGCCGTCAAGCTCTGCAAATACGATCTGTAAGTCCATCTT

AACACAAAAGTGAGATGGGTTCTTAGATCATGTTCCGCCGTTAGATCGAGTCATG GTCTTGTCTCATAGAAAGGTACTTTCGTTTACTTCTTTTGAGTATCGAGTAGAGCG

TCGTCTATAGTTAGTTTGAGATTGCGTTTGTCAGAAGTTAGGTTCAATGTCCCGGT

CCAATT TCACCAGCCATGTGTCAGTTTCGTTCCTTCCCGTCCTCTTCTTTGATTTC

GTTGGGTTACGGATGTTTTCGAGATGAAACAGCATTGTTTTGTTGTGATTTTTCTC

TACAAGCGAATAGACCATTTATCGGTGGATCTTAGAAAATTAAGAGACCATTAG

GCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGT

TAGGAGCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatggagaaaaaaatcact ggatata ccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttg ctcaatgtacctataaccagaccgttcagctg gatattaeggccrttttaaagaccgtaaagaaaaataagcacaa

c€-ggagttccgtatggcaatgaaagacggtgagctggtgaiatgggatagtgtt cacccttgttac ccgttttccatgagcaaactgaa acgttitcatcgctctggagtgaataccacgacgatttccggcagt tctacacatatattcgcaagatgtggcgtgiiacggtgaaaacc ggcctatttccctaaagggittatigagaata^

atggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgac aaggtgctgatgccgctggcgattcaggttcat catgccgtttgtgatggcttccatgtcggcagaatgcttaatgaattacaacag actgcgatgagtggcagggcggggcgtaaACG

CGTGGAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATT

TTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAG

GTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTT

GCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGA

ATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGA

TGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAG

GGGCTGGTGAAATGCAGTITAAGGTTrACAC-CTATAAAAGAGAGAGCCGTTATC

GTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGCCGACGGATGGT

GATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCGGTGAACTTTAC

CCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCC

AGTGTGCCGGTTTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAA

AATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGC

TCCCTTATACACAGCCAGTCTGCACCTCGACggtctcAGAACTAGAAAAGACATTGG

ACATATTCCAGGATATGCAAAAGAAAACAATGAATATTGTTTTGAATGTGTTCAA

GTAAATGAGATTTTCAAGTCGTCTAAAGAACAGTTGCTAATACAGTTACTTATTT

CAATAAATAATTGGTTCTAATAATACAAAACATATTCGAGGATATGCAGAAAAA

AAGATGTTTGTTATTTTGAAAAGCTTGAGTAGTTTCTCTCCGAGGTGTAGCGAAG

AAGCATCATCTACTTTGTAATGTAATTTTCTTTATGTTTTCACTTTGTAATTTTATT

TGTGTTAATGTACCATGGCCGATATCGGTTTTATTGAAAGAAAATTTATGTTACTT CTG1TTTGGCTTTGCAATCAGTTATGCTAGTTTTCTTATACCCTTTCGTAAGCTTCC TAAGGAATCGTTCATTGATTTCCACTGCTTCATTGTATATTAAAACTTTACAACTG TATCGACCATCATATAATTCTGGGTCAAGAGATGAAAATAGAACACCACATCGT

AAAGTGAAATAAGGGTGGGCGCGCCGACCCAGCTT Cl ^' TGTACAAAGTGG'ITCG ATAATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCGAA r

ITCClCCGATO

CC TCTTCffiA™

COC IIAT LAT™

ATAAA ATCGCGCGCGGTGTCATCTATCNI ^' ACTAGATCGGGAATTCGTAATCAT

GGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACAT

ACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACT

CACATTAATTGCGTTGCGCTCACTGCCCGCTTTGCAGTCGGGAAACCTGTCGTGC

CAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGC

TAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATAT

CAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGT

AATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAA

AGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGG

AAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCC

ACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCA

AGTGGATTGATGTGATAACatggtggagciicgacactctegtctactccaagaata tcaaagatacagtctcagaag a xaaagggciattgagaet†ttcaaeaaagggta af¾

gacagtagaaaaggaaggtggcacctacaaatgccatcattgcgaiaaaggaaaggc tatcgttcaagatgcctctgccgacagtgg fcccaaagatggacccccaccc8Cgifggagcatcgtggaaaaagaagacgttccaacca cg(:cficaaagcaagiggaii¾atgtga tatctccacigacgtaagggatgacgcaGaatcccact tc(;ttcgcaagaccifcctctatataaggaagttcaitiGatttggagaggA

CACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGTCTACCATG

AGCCCAGAACGACGCCCGGCCGACATCCGCCG ^' rGCCACCGAGGCGGACATGCCG

GCGGTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTTCCGTA

CCGAGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCGGGAGC

GCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGCCTACG

CGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGT

ACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACCCACCT

GC?TGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATCGGGCT GCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCGCGG

CATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTG

GCAGCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCACCGAG

ATTTGACTCGAGtifct^ataaiaaigigt c^atoCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTG

TTATTAAGTTGTCTAAGCGTCAATTTCiTTlAC'ACC ACA ATA.TATCCTGCCA SEQ ID NO:411 brown/lowercase: kanam cia resistance gene

ΩΥ ΰΡΕΕ8£Δ^ΰΜΰΕ Ι _ί Ο^·· C->A teansverskm to block vector's Bsal site cyari/lo ercase: T-DN.A right border

GREEN/UPPERCASE: 2x358 CaMV promoter

ORANGE/UPPERCASE: at© I.

BLUE/UPPERCASE: AtTASlc 5 ' region

RED/UPPERCASE: Bsal site magenta/lowercase: chloramphenicol resistance gene

MAGENTA/UPPERCASE: ccdB gene red/lowercase: inverted Bsal site blue/lowercase: AtTASlc 3' region

O ^' RANGE/UPPERC A SE/UNPERLINED : attB2

GREY/UPPERCASE/UNDERLINED: Nos terminator green/lowercase: CaMV promoter

B RO WN/UPPERC ASE/UNDERLINED : BASTA resistance gene greeiylo ercase/tmclerljiied: CaMV terminator CYAN/UPPERCASE: T-DNA left border Table 1 : P enotypic penetrance of artificial m!RNAs expressed in A. thaliana

arniRNA MIRNA T1 Phenotypic"

Foldback analyzed penetrance amiR-Ft AUVHR390 64 100% amiR-Ft AtMfR390-OsL 44 100% amiR-Ch42 AtMIR39Q 406 100%

3% weak · 28% intermediate 69% severe ami -Ch42 AtW!IR390-OsL 267 98%

3% weak

33% intermediate

64% severe

* Atransformant shows the Ft phenotype when its 'days to flowering' value is higher than the 'days of flowering' average of the 35$:GUS control set

Ch42 phenotype is scored in 10 days-old seedling and Is considered 'weak ¹ , 'intermediate' or 'severe' If seedlings have >2 leaves, exactly 2 [eaves or no leaves (only Z cotyledons), respectively.

A) Sequences of OsMXRJSO-baaed amiRNA precursors

3G'I.5TSSA&CA&TCC

llGGTTOSTTi

>OsMIR.330-Bril-AtI* _„.,„:

>OsHTR380 ^OaJ ^ _' ^ _{' *"} '4Η½*¾-#Ρ¾Γί1 " . _ »J|

- ^> OaHIIt390-Caol-v? ^ ^ 1, _, : _{j. i' j} |

>OaMIR39ii-Oftol _^ AfcI. _^

1 _ -.i -Gft¾q>¾¾CC G}¾> » ' ".·.. ^{■ ■} - .¾¾! J!! ^i' ': ! rGATGATOAC¾FTC

Figure 50 >0SHIR390-Pds-V2

TCGAAAT CAAACT&G OeMIE3S0-P<3s -AtL

.TGATGATCACATTC GTTATCTATTJ'TTTGj

>OsMlR390-Splll-v2

kCTlOTACAAGGGe SSgjTCG A A A I'CAAACTAG

>OsMIK390 - elF4A- l-v2

^S¾¾¾1I§^S1GGTATGG ACAATGCTTG[ TACCftACACAAGT'J CC CCA TCGAAATCAAACTAH

>OSMXR390 - elF4A- 1-AtL ^^m^m a _t - — ^ _B m

>OsiaiR390 - dlF4A- 2 ~ v2

^^^^^^^^^^^^^gGG¾¾C¾¾TCCTTG^^^^^^^^^^^^¾TCGA TC ¾ CT ¾ >0SBIR3 a <0 ~ elF4A-2 -Atli

Figure 50 continued

Figure 51

B) Sequences of AfcJMXR3 ^" 9Pa-based amiRNA precursore

>AtMIR390a-173-21

AtMIR390a- 472 - 2-.-OsI.-v2

TATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAAA ATAGAAATGAATAATTTCAG GTTTAACGAAGAGGAGATGACGTGTGTTCCTTCGAACCCGAGTTTTGTTCGTCTATAAAT AGCACCTTCTe TTCTCCTTCTTCCTCACTTCCATCTTTTTAGGTTCACTATCTCTCTATAATCGGTTTJAT C'CTTCTCTAAG iTAJGGG.CGaC<¾AGGAAAftAOAroiw Bi¾HTO

ATCACTTaAGATiTCAAT'TCTTTTTACTGTCeATT.TAAGCTATCTTTTATAAACG TGTCTTATTTTCTATCT GTTTTGTTTAAAC AAGAAACTATAGTATTITGTGTAAAi CAftAfiGATGAAAGAACAGATTAGATCTGATC TTTAGTCTC

>AtMIR390a- 828 -21-Os3_-v2

TATAGGGGGGAAAAAAAGGTAGTCATGAGATATATATTTTGGTAAGAAAATATAGAAATG fiATAATTTGAC GTTTAACGAAGAGGAGATGACGTGTGTTeCTTCGAACCCGAGTT'TTGTTCGTCTATAAA TAGCACCTTCTC TTCTCCTTCTTCCTCACT'ICCATCTTTTTAGCTTCACTATCTCTGTATAATCGGTTTTA TCTTTGTGTAAG ^AG¾APrraA¾¾¾¾AF'A¾kf«.^ jB^^ jtgTC TGCTT¾AAyG GTA TCC

kTACTC BTgaAGCa GAC'AJwSBSB^TOMra

ATCACTTGAGAATC¾A½C½TT¾CTGT^^

CTTTTGTTTAAACTAAGAAACTATAG aTTTTGTC AAAACAfiAAeATGaAAGAACAGATTAGATCTGATC TTTAGTCTC

>AtMIR390a- oh42 -OsI.-v2

TATAGGGGGGAAAAAAAGGTAGTCSTCAGATATATATTtTGGimGAMATATAGAAATGAA TAJiTmeA' GTTTAACGAAGAGGAGATGACGTGTGTTCCT CGAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTC^ TTCTCCTTCTTCCTCACTTCCATGTTTyTAGGTTCAGTATCTeTCTATAATCGGTTTTAT G^TTCTeiAAG ¹ : GACA¾GGG¾fiAA¾¾AGAAfSy^^¾^¾|^^C^M ^^!g^STl' G GTG GGG TCCG'I'i

TsGA^TTCCTIG¾ ¾eiTA¾C¾l^

TCACTTG GAATC

CTTTTGTTTAAAC'TAAGAAAGTATAGTATTTTGTCTSRSACAAR&CATGAAA GAACAGATTAGATCTGAl^C TTTAGTCTC

>&tMIR390aa- !?fc

;ATAaGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAAATATAGAAATG AATAATTTGA GTTTAACGAAGAGGAGATG&GGTGTGTTCC TCGAAGCCGAGTTTTGTTCGTCTATAAATAGCACGTirCTC'! TTC CCTTCTTCCTCACTTCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTT CTCTAAG;

CAAAACGCCTAJiAATCAClVGAGAATCAAT ^^

TCTTATTTTCTATCTCTTTTGTTTAAAGT&RGAAACTATAGTA1¾ ^' TTG'5CTAAAACAAAA ^' GATGAAAGAAC] GATTAGATCTCATCT'J'TAGTCTCl

Example 23. High Through-put Cloning and High Expression of amiRNAs in Monocots

[000296] Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to generate amiR A constructs for silencing transcripts in monocot species are not well adapted for simple, cost-effective and large-scale production. Here, a new series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-basQd vectors were validated in transgenic

Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-basQd precursors that include distal stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants exhibited the expected phenotypes predicted by loss of target gene function, and accumulated high levels of amiRNAs and reduced levels of the corresponding target RNAs. Genome-wide transcriptome profiling combined with 5'-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific.

[000297] A new generation of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost-effective and large-scale production of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390~based precursors including Arabidopsis thaliana MIR390a distal stem-loop sequences accumulated elevated levels of highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.

[000298] MicroRNAs (miRNAs) are a class of -21 nt long endogenous small RNAs that posttranscriptionally regulate gene expression in eukaryotes (Bartel, 2004). In plants, DICER-LIKE 1 processes MIRNA precursors with imperfect self-complementary foldback structures into miRNA/miRNA* duplexes (Bologna and Voinnet, 2014). Typically, one strand of the miRNA duplex is sorted into an ARGONAUTE (AGO) protein according to the identity of the 5'-terminal nucleotide (nt) of the miRNA (Mi et al, 2008; Montgomery et al, 2008; Takeda et al, 2008) and/or to other sequence or structural properties of the miRNA duplex (Zhu et al, 2011; Endo et al, 2013; Zhang et al, 2014). Plant miRNAs target transcripts with highly complementary sequence through direct AGO-mediated

endonucleolytic cleavage, or through other cleavage-independent mechanisms such as target destabilization or translational repression (Axtell, 2013).

[000299] Artificial miRNAs (amiRNAs) can be produced accurately by modifying the miRNA/miRNA* sequence within a functional MIRNA precursor (Alvarez et ah, 2006; Schwab et ah, 2006). AmiRNAs have been used in plants to selectively and effectively knockdown reporter and endogenous genes, non-coding RNAs and viruses (Ossowski et ah, 2008; Tiwari et ah, 2014). Recently, cost- and time-effective methods to generate large numbers of amiRNA constructs were developed and validated for eudicot species (Carbonell et ah, 2014). These included a new generation of eudicot amiRNA vectors based on

Arabidopsis thaliana MIR390a (AtMIR390a) precursor, whose relatively short distal stem- loop allows the cost-effective synthesis and cloning of the amiRNA inserts into "B/c" expression vectors (Carbonell et ah, 2014). In monocots, OsMIR528 precursor has been used successfully to express amiRNAs for silencing endogenous genes in rice ( arthmann et ah, 2008; Butardo et ah, 2011; Chen et ah, 2012a; Chen et ah, 2012b). However, OsMIR528- based cloning methods have not been optimized for efficient generation of monocot amiRNA constructs.

[000300] A new series of amiRNA expression vectors for high-throughput cloning and high- level expression in monocot species are described and tested. The new vectors contain a truncated sequence from Oryza sativa MIR390 (OsMIR390) precursor in a configuration that allows the direct cloning of amiRNAs. OsMIR390-b< ^r xsed amiRNAs were generally more accurately processed and accumulated to higher levels in transgenic Brachypodium distachyon (Brachypodium) when processed from chimeric precursors {OsI IR390-AtL) containing Arabidopsis thaliana (Arabidopsis) MIR390a AtMIR390a) distal stem-loop sequences. Functionality of OsMIR390-AtL-b&SQd amiRNAs was confirmed in

Brachypodium transgenic plants that exhibited the phenotypes expected from loss of target gene function, accumulated high levels of amiRNAs and reduced levels of the corresponding target RNAs. Moreover, genome-wide transcriptome profiling in combination with 5'-RLM RACE analysis confirmed that the amiRNAs were highly specific. We also describe a cost- optimized alternative to generate amiRNA constructs for eudicots, as amiRNAs produced from chimeric AtMIR390a-based precursors including AtMIR390a basal stem and OsMIR390 short distal stem-loop sequences are highly expressed, accurately processed, and effective in target gene knockdown in A. thaliana.

AmiR A vectors based on the OsMIR390 precursor

[000301] Previously, the short AtMIR390a precursor was selected as the backbone for high- throughput cloning of amiRNAs in a new generation of vectors for eudicot species (Carbonell et al, 2014). These vectors allow a zero-background, oligonucleotide cloning strategy that requires no enzymatic modifications, PCR steps, restriction digestions, or DNA fragment isolation (Carbonell et al, 2014). The short distal stem-loop (Figure la) of AtMIR390a precursor provides a cost-advantage by reducing the length of synthetic oligonucleotides corresponding to the amiRNA precursor sequence. To develop a comparable system for monocot species, a search for conserved, short Oryza sativa (rice) MIRNA (OsMIRNA) precursors that could be adapted for amiRNA vectors was done. Rice MIRNA precursors were analyzed as they have been subjected to extensive prior analysis (Arikit et al., 2013). The distal stem-loop length of 142 OsMIRNA precursor sequences (median length=54 nt, Figure lb) from 23 conserved miRNA families (Table SI) revealed that the OsMIR390 precursor was one of the shortest (16 nt). Moreover, OsMIR390 contains the shortest distal stem-loop of all 51 sequenced MIR390 precursors from 36 species (median length=47 nt, Figure lb, Table S2), including those from maize (ZmaMIR390a and ZmaMIR390b), sorghum (SbiMIR390a) and B. distachyon (BdiMIR390) with lengths of 137, 148, 134 and 107 nt respectively. The MIR390 family is among the most deeply conserved miRNA families in plants (Axtell et al, 2006; Cuperus et al, 2011).

[000302] Publicly available small RNA data sets from rice (Heisel et al, 2008; Zhu et al, 2008; Johnson et al, 2009; Zhou et al, 2009; He et al, 2010) were analyzed to assess the OsMIR390 precursor processing accuracy. Approximately 70% of reads mapping to the OsMIR390 foldback correspond to the authentic 21-nt miR390 guide strand (Figure lc). Given the short distal stem-loop sequence and relatively accurate precursor processing characteristics, OsMIR390 was selected as the backbone for amiRNA vector development.

[000303] A series of OsMIR390-based cloning vectors named OsMIR390-B/c ^' ' (from

OsMIR390~Bsa lccdQ) were developed for direct cloning of amiRNAs (Figure SI, Table I). OsMIR390-B/c vectors contain a truncated OsMIR390 precursor sequence whose miRNA/distal stem-loop/amiRNA* region was replaced by a DNA cassette containing the counter-selectable ccdB gene (Bernard and Couturier, 1992) flanked by two Bsal sites.

AmiRNA inserts corresponding to amiRNA/Oi KJ ^-distal-stem-loop/amiRNA* sequences are synthesized using two overlapping and partially complementary 60-base oligonucleotides (Figure S2). Forward and reverse oligonucleotides must have 5'-CTTG and 5'-CATG overhangs, respectively, for direct cloning into OsMIR390-based vectors (Figure S2).

[000304] OsMIR390-B/c vectors include _P MDC32B-OsMIR390-B/c,pMDC123SB- OsMIR390-B/c and pH7WG2B-OsMIR390-B/c plant expression vectors, each of which contains a unique combination of bacterial and plant antibiotic resistance genes and regulatory sequences (Figure SI, Table I). Additionally, a pENTR-OsMIR390-B/c

GATE AY-compatible entry vector was generated for direct cloning of the amiRNA insert and subsequent recombination into a preferred GATEWAY expression vector containing a promoter, terminator or other features of choice (Figure SI, Table I).

High accumulation of amiRNAs derived from chimeric precursors in Brachypod im calli

[000305] To test amiRNA expression from OsMIR390 precursors, transformed B. distachyon calli containing amiRNA constructs expressing miR390 or modified versions of several miRNAs from Arabidopsis (amiR173-21, amiR472-21 or amiR828-21) (Cuperus et al, 2010) were analyzed (Figure 2a). In addition, the same amiRNAs were expressed from a chimeric precursor (OsMIR390-AtL) composed of the OsMIR390 basal stem mdAtMIR390a distal stem-loop (Figure 2a, Figure S3). Each amiRNA was also expressed from the reciprocal chimeric precursors (AtMIR390a-OsL) containing the AtMIR390a basal stem and OsMIR390 distal stem-loop (Figure 2a, Figure S4). A 35S:GUS construct expressing the β-glucuronidase transcript was used as negative control.

[000306] Surprisingly, miR390 accumulated to highest levels when expressed from the chimeric OsMIR390-AtL precursor compared to each of the other three precursors (P< 0.001 for all pairwise t-test comparisons; Figure 2b). Moreover, each amiRNA expressed from OsMIR390-AtL chimeric precursors also accumulated to significantly higher levels when compared to the other precursors (P< 0.026 for all pairwise i-test comparisons; Figure 2b). miR390 and each amiRNA derived from authentic AtMIR390a or chimeric AtMIR390a-OsL precursors accumulated to low or non-detectable levels, indicating that the AtMIR390a stem is suboptimal for the accumulation and/or processing of amiRNAs in Brachypodium.

[000307] To assess the accuracy of precursor processing, small RNA libraries from samples expressing OsMIR390-AtL-based amiRNAs were prepared and sequenced (Figure 2c). For comparative purposes, small RNA libraries from samples containing amiRNAs produced from authentic OsMIR390 precursors were also analyzed. In each case, the majority of reads mapping to the chimeric OsMIR390-AtL precursors corresponded to correctly processed 21 nt amiRNAs (Figure 2c). In contrast, processing of authentic OsMIR390 precursors including amiRNA sequences was less accurate, as revealed in each case by a lower proportion of reads corresponding to correctly processed sequences (Figure 2c).

Gene silencing in Brachypodium and Arabidopsis by amiRNAs derived from chimeric precursors

[000308] To test the functionality of OsMIR390-AtL-derrved amiRNAs in repressing target transcripts in Brachypodium, BRASSINOSTEROID-INSENSITIVE 1 (BdBRIl), CINNAMYL ALCOHOL DEHYDROGENASE 1 (BdCADl), CHLOROPHYLLIDE A OXYGENASE (BdCAO) and SPOTTED LEAF 11 (BdSPLll) gene transcripts were targeted by amiRNAs expressed from the chimeric OsMIR390-AtL and from authentic OsMIR390 precursors (Figure 3a). The sequences for amiR-BdBril, amiR-BdCadl, amiR-BdCao and amiR- BdSpll 1 (Figure S5) were designed using the "P-SAMS amiRNA Designer" tool (http://p- sains.ca.tTmgtonlab.org,, Fahlgren et al. in preparation). Plants expressing 35S:GUS were used as negative controls. Plant phenotypes, amiRNA accumulation, amiRNA reads from sequencing data, and target mRNA accumulation were measured in Brachypodium TO transgenic lines.

[000309] Sixteen out of 20 and 11 out of 17 transgenic lines containing 35S:OsMIR390-AtL- Bril or 35S:OsMIR390-Bril, respectively, which were predicted to have brassinosteroid signaling defects, had reduced height and altered architecture (Figure 3b, Figure S6, Table S3). Most organs, particularly leaves, exhibited a contorted phenotype from the earliest stages of development (Figure 3b). Inflorescences had reduced size (Figure 3b), and contained smaller seeds compared to control lines (Figure S6). AmiR-BdBril -induced phenotypes were similar to those described for the Brachypodium bril T-DNA mutants from the BrachyTAG collection (Thole et al, 2012). These phenotypes are consistent with the expectation of plants with brassinosteroid signaling defects (Zhu et al, 2013). All 27 transgenic lines containing 35S:OsMIR390-AtL-Cadl, and 52 out of 55 lines including 35S:OsMIR390-Cadl, exhibited reddish coloration of lignified tissues such as tillers, internodes and nodes (Figure 3c, Table S3), as expected from Cadi knockdown and loss of function mutant analyses (Bouvier d'Yvoire et al, 2013; Trabucco et al, 2013).

[000310] Each of 27 35S:OsMIR390-AtL-Cao-expressing plants, and 12 of 12 of

35S:OsMIR390-Cao-expressmg plants exhibited light green color compared to control plants (Figure 3d, Table S3), as expected due to reduction in chlorophyllide a to b conversion during chlorophyll b synthesis (Tanaka et al, 1998; Oster et al, 2000; Philippar et al, 2007).

Biochemical analysis of chlorophyll content in transgenic lines confirmed that chlorophyll b content in 35S:OsMIR390-AtL-Cao and 35S:OsMIR390-Cao lines was reduced to approximately 57% and 67%, respectively, compared to levels measured in control plants (Figure S7). Carotenoid content was also notably reduced (to almost 50%) in lines expressing amiR-BdCao from chimeric or authentic precursors (Figure S7), as observed before in Arabidopsis cao mutants (Philippar et al, 2007). Finally, 39 of 43 transgenic lines containing 35S:OsMIR390-AtL-Splll, and 22 of 24 35S:OsMIR390-Splll -expressing plants displayed a spontaneous cell death phenotype characterized by the development of necrotic lesions in leaves (Figure 3e). This was consistent with expectations based on phenotypes of SPL11- knockdown amiRNA rice lines (Zeng et al, 2004). Phenotypes induced by all four sets of amiPvNAs were heritable in self-pollinated Tl plants expressing OsMIR390- or OsMIR390- AZ-based amiRNA precursors from pMC32B vectors containing 35S regulatory sequences (Table S4).

[000311] Accumulation of amiRNA target mRNAs in Brachypodium transgenic lines expressing OsMIR390-AtL- or OsMIR390-based amiRNAs was analyzed by quantitative real time RT-PCR (RT-qPCR) assay. The expression of all target mRNAs was significantly reduced compared to control plants (P < 0.005 for all pairwise i-test comparisons, Figure 4a) when the specific amiRNA was expressed. No significant differences were observed in target mRNA levels between lines expressing OsMIR390-AtL- or OsMIR390-based amiRNAs. [000312] AmiR-BdBril, amiR-BdCao and amiR-BdSplll produced from chimeric

OsMIR390-AtL precursors were also expressed using pH7WG2B-based constructs that contain the rice ubiquitin (UBI) regulatory sequences. Each of the three UBI promoter-driven amiRNAs induced the expected phenotypes in a relatively high proportion of Brachypodium TO lines (Table S3), and in the one case tested (amiR-BdSpIl 1), phenotypes were heritable in the Tl generation (Table S4).

[000313] Finally, we tested if the reciprocal chimeric AtMIR390a-OsL precursor could be used to express amiRNAs efficiently in eudicots. The synthesis of AtMIR390a-OsL-based constructs requires shorter oligonucleotides than the generation ofAtMIR390 -based constructs, and therefore would be a further cost-optimized alternative. As shown in

Nicotiana benthamiana and Arabidopsis assays, AtMIR390-OsL precursors are accurately processed (Appendix SI, Figures S8-S10). Indeed, amiRNAs produced from chimeric AtMIR390a-OsL precursors are highly expressed, accurately processed and highly effective in target gene knockdown in Tl Arabidopsis transgenic plants (Appendix SI, Figures S9- Sl 1, Table S5). Moreover, amiRNA induced phenotypes were still obvious in T2 plants confirming the heritability of the effects (Table S6). Therefore, the use of AtMIR390a-OsL precursors may be an attractive alternative to express effective amiRNAs in eudicots in a cost-optimized manner.

Accuracy of processing of OsMIR390 and OsMIR390-AtL chimeric precursors in Brachypodium

[000314] The accumulation of each amiRNA from chimeric and OsMIR390 precursors was analyzed by RNA blot analysis in TO transgenic lines showing amiRNA-induced phenotypes (Figure 4b). i most cases, OsMIR390~AtL-derived amiRNAs accumulated to higher levels and as more uniform RNA species (Figure 4b). AmiRNAs from the OsMIR390 precursor accumulated to rather low levels (except in transgenic lines containing 35S:OsMIR390-Cao) and generally as multiple species (Figure 4b).

[000315] To more accurately assess processing and accumulation of the amiRNA populations, small RNA libraries from transgenic lines expressing amiRNAs from chimeric OsMIR390- AtL or authentic OsMIR390 precursors were prepared (Figure 5). Three of the four amiRNAs produced from chimeric OsMIR390-AtL precursors accumulated predominantly as 20-nt species (Figure 5a, c and d); only amiR-BdCadl accumulated mainly as a 21 nt RNA (Figure 5b). Processing of authentic OsMIR390 precursors generally resulted in a high proportion of small RNAs of diverse sizes, except for OsMIR390-Cadl precursors (Figure 5).

[000316] The reasons explaining the accumulation of OsMIR390a-AtL-based amiRNAs that are 1 nt-shorter than expected are not clear. AmiRNAs shorter than expected and differing on their 3' end were also described using AtMIR319a precursors in Arabidopsis (Schwab et al, 2006). Importantly, a recent study has shown that amiRNA efficacy is not affected by the loss of the base-pairing at the 5' end of the target site (Liu et al, 2014). Regardless, the inaccurate processing of an amiRNA precursor leading to the accumulation of diverse small RNA populations could conceivably induce undesired off-target effects. This potential complication argues against using authentic OsMIR390 precursors to express amiRNAs in Brachypodium and possibly other monocot species.

[000317] Reads from the amiRNA* strands from each of the OsMIR390 and OsMIR390-AtL- derived precursors were under-represented, relative to the amiRNA strands (Figure 5). The rational P-SAMS design tool uniformly specifies an amiRNA* strand containing an AGO- non preferred 5'G residue, which likely promotes amiRNA* degradation.

High specificity of amiRNA derived from chimeric precursors in Brachypodium

[000318] To assess amiRNA target specificity at a genome-wide level, transcript libraries from control (35S:GUS) and amiRNA-expressing lines were generated and analyzed. Only lines expressing amiRNAs from the more accurately processed OsMIR390-AtL precursors were analyzed. Differential gene expression analyses were done by comparing, in each case, the transcript libraries obtained from four independent control lines with those obtained from four independent amiRNA-expressing lines exhibiting the expected phenotypes. Four hundred and ninety four, 1847 and 818 genes were differentially expressed in plants expressing amiR-BdBril, amiR-BdCao and amiR-BdSpll 1, respectively (Figure 6, Data SI). In contrast, only 21 genes were differentially expressed in plants expressing amiR-BdCadl (Figure 6, Data SI). The high number of differentially expressed genes in amiR-BdBril-, amiR-BdCao- and amiR-BdSpll 1 -expressing lines may reflect the complexity of the corresponding targeted gene pathways involving hormone signaling, photosynthesis and cell death/pathogen resistance respectively. As expected, BdCADl, BdCAO and BdSPLll were differentially underexpressed in plants expressing amiR-BdCadl, amiR-BdCao and amiR- BdSpll 1, respectively (q<0.01, Wald test) (Figure 6, Data SI). However, BdBRIl was not called as differentially expressed (q=0.42, Wald test) (Figure 6, Data SI) despite being notably downregulated in 35S:OsMIR390-AtL-Bril plants as shown by RT-qPCR analysis (Figure 4a). Because the power of statistical tests involving count data decreases with lower count numbers (Rapaport et al, 2013), this result could be explained by the low accumulation of BdBRIl even in control plants (Figure S12, Data S2). Therefore, the differential expression analysis on R A-Seq data approach may not be appropriate to evaluate the differential expression of genes with genuine low expression and/or low coverage, as suggested before (Rapaport et al. , 2013).

[000319] To assess potential off-target effects of the amiRNAs, TargetFinder (Fahlgren and Carrington, 2010) was used to generate a genome-wide list of potential candidate targets that share relatively high sequence complementarity with each amiRNA. TargetFinder ranks the potential amiRNA targets based on a Target Prediction Score (TPS) assigned to each amiRNA-target interaction. Scores range from 1 to 11, that is, from highest to lowest levels of sequence complementarity between the small RNA and putative target RNA. Indeed, when designing amiRNAs with the "P-SAMS amiRNA Designer" tool, "optimal" amiRNAs are selected when i) their interaction with the desired target has a TPS=1, and ii) no other amiRNA-target interactions have a TPS<4 (Fahlgren et al., in preparation). Therefore, direct off-target effects with amiRNAs described here can only occur through amiRNA-target RNA interactions with a TPS in the [4, 11] interval. It was hypothesized that off-target effects, if due to base-pairing between amiRNAs and the affected transcripts, would be reflected by the presence of differentially underexpressed genes corresponding to target RNAs with lower TPS scores in the [4, 11] interval. Therefore, we next analyzed for all TargetFinder-predicted targets for each amiRNA if their corresponding genes were differentially underexpressed in amiRNA-expressing lines versus controls.

[000320] As expected from P-SAMS design, BdCadl, BdCao and BdSplll were the only genes differentially underexpressed in the [1,4[ TPS interval in plants expressing amiR- BdCadl, amiR-BdCao and amiR-BdSpll 1, respectively (Figure 7, Data S3). On the other hand, 2958, 1290, 1528 and 1533 genes corresponded to target RNAs with calculated TPS scores in the [4, 11] interval in TargetFinder analyses including amiR-BdBril, amiR- BdCadl, amiR-BdCao and amiR-BdSpll 1, respectively (Figure 7). In all cases, the number of differentially underexpressed genes corresponding to predicted targets with a TPS in the [4, 11] interval was low (Figure 7, upper panels). Moreover, in each of the four cases the proportion of differentially underexpressed genes among TargetFinder-predicted targets was also low in the [4, 11] TPS interval (Figure 7, bottom panels). Indeed, in this same interval, 0.84%, 1.31% and 0.78% of the genes were differentially underexpressed in amiR-BdBril-, amiR-BdCao-, and amiR-BdSpl 11 -expressing lines, respectively. In each case, this percentage was lower than the percentage of differentially underexpressed genes from transcripts with a TPS not included in the [4, 11] interval in the same samples (1.12%, 3.74% and 1.55% respectively). In amiR-BdCad-expressing lines, although the percentage of genes differentially expressed in the [4, 11] interval (0.07%) was higher compared to the percentage of genes differentially underexpressed in the ]4, 11[ interval (0.04%), this difference was not statistically significant (P=0.45, Fisher test). Together, these results indicate that globally TargetFinder-predicted targets were not preferentially downregulated in the amiRNA- expressing lines.

[000321] Next, we used 5'-RLM-RACE to test for amiRNA-directed off-target cleavage of undenepresmted transcripts. This analysis detects 3' cleavage products expected from small RNA-guided cleavage events. Only TargetFinder predicted targets with a TPS<7 were included in the analysis, as targets with higher score are not considered likely to be cleaved, according to previous studies (Addo-Quaye et al, 2008). For all specific targets, 3' cleavage products of the expected size were detected in samples expressing the corresponding amiRNA, but not in control samples expressing 35S. GUS (Figure 8). Sequencing analysis confirmed that the majority of sequences comprising these products, in each case, contained a canonical 5' end position predicted for small RNA-guided cleavage (Figure 8). In contrast, for all potential off-target transcripts, no obvious amiRNA-guided cleavage products were detected in either amiRNA-expressing or 35S:GUS lines (Figure 8). Additionally, sequencing analysis failed to detect even low-level amiRNA-guided cleavage products among potential off-targets (Figure 8).

[000322] High amiRNA specificity was previously indicated for AtMIR319a-derived amiRNAs in Arabidopsis based on genome-wide expression profiling (Schwab et al, 2006). However, a recent and systematic processing analysis of AtMIR319a-basQd amiRNA precursors in petunia (Guo et al, 2014) showed that multiple small RNA variants are generated from different regions of the precursor, and that many of these small RNAs meet the required criteria for amiRNA design (Schwab et al, 2006). Here, the fact that chimeric OsMIR390-AtL precursors produce high levels of accurately processed amiRNAs not only in Brachypodium (Figures 2, 4 and 5) but also in a eudicot species such as N. benthamiana (Figure S8), strongly suggests that these precursors will be functional in a wide range of species.

[000323] We have developed and validated a new generation of expression vectors based on the OsMIR390 precursor for high-throughput cloning and high expression of amiRNAs in monocots. OsMiR390-B/c-based vectors allow the direct cloning of amiRNAs in a zero- background strategy that requires no oligonucleotide enzymatic modifications, PCR steps, restriction digestions, or DNA fragment isolation. Thus, OsMIR390-B/c-basQd vectors are particularly attractive for generating large-scale amiRNA construct libraries for silencing genes in monocots.

"P-SAMS amiRNA Designer" tool was used to design four different amiRNAs, each of which was aimed to target specifically one Brachypodium gene transcript. We show that chimeric OsMIR390-AtL precursors including OsMIR390 basal stem and AtMIR390a distal stem-loop were processed more accurately, and the resulting amiRNAs generally accumulated to higher levels than amiRNAs derived from authentic OsMIR390 precursors in Brachypodium transgenic plants. Each P-SAMS-designed amiRNA induced the expected phenotypes predicted by loss of target gene function, and specifically decreased expression of the expected target gene. Chimeric OsMIR390-AtL precursors designed using P-SAMS, therefore, are likely to be highly effective and specific in silencing genes in monocot species.

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

[000324] Arabidopsis thaliana Col-0 and N. benthamiana plants were grown as described (Carbonell et al, 2014). Brachypodium distachyon 21-3 plants were grown in a chamber under long day conditions (16/8 hr photoperiod at 200 μηιοΐ nf ² s ^"1 ) and 24°C/18°C temperature cycle.

[000325] Arabidopsis thaliana plants were transformed using the floral dip method with Agrobacterium tumefaciens GV3101 strain (Clough and Bent, 1998). A. thaliana transgenic plants were grown on plates containing Murashige and Skoog medium hygromycin (50 mg/ml) for 10 days before being transferred to soil. Embryogenic calli from B. distachyon 21-3 plants were transformed as described (Vogel and Hill, 2008). Photographs of plants were taken as described (Carbonell et al, 2014).

DNA constructs

[000326] pENTR-OsMIR390-BsaI construct was generated by ligating into pENTR (Life Technologies) the DNA insert resulting from the annealing of oligonucleotides Bsal- OsMIR390 -F and BsaI-OsMIR390-R. Rice ubiquitin 2 promoter and maize ubiquitin promoter-hygromycin cassettes were transferred into the GATEWAY binary destination vector pH7WG2 (Karimi et al 2002) to generate pH7WG2-OsUbi. pH7WG2-OsMIR390-BsaI, PMDC123SB-OsMIR390-BsaI and pMDC32-OsMIR390-BsaIwere obtained by LR recombination using pENTR-OsMIR390-BsaI as the donor plasmid and pH7WG2-OsUbi, pMDC32B (Carbonell et al, 2014) and pMDC123SB (Carbonell et al, 2014) as destination vectors, respectively. A modified ccdB cassette (Carbonell et al, 2014) was inserted between the Bsal sites of _P ENTR-OsMIR390-BsaI, pMDCl 23SB-OsMIR390-BsaI,pMDC32B- OsMIR390-Bsal and pH7WG2-OsMIR390-Bsal To generate pENTR-OsMIR390-B/c, pMDC123SB-OsMIR390-B/c, pMDC32B-OsMlR390-B/c and pH7WG2-OsMIR390-B/c, respectively. Finally, an undesired Bsal site was disrupted in pH7WG2-OsMIR390-B/c to generate pH7WG2B-OsMIR390-B/c. The sequences of the OsMIR390-B/c-based amiRNA vectors are listed in Appendix S2. The following amiRNA vectors for monocots are available from Addgene (http://www.addgene.org/): pENTR-OsMIR390-B/c (Addgene plasmid 61468), pMDC32B-OsMIR390-B/c (Addgene plasmid 61467) _P MDC123SB-OsMIR390-B/c (Addgene plasmid 61466) and pH7WG2B-OsMIR390-B/c (Addgene plasmid 61465). pMDC32B- AtMIR390a-B/c (Addgene plasmid 51776) was described before (Carbonell et al, 2014).

[000327] The rest of the amiRNA constructs (pMDC32B-AtMIR390a-OsL-l 73-21, pMDC32B-AtMIR390a-OsL-472-21, pMDC32B-AtMIR390a-OsL-828-21, pMDC32B- AtMIR390a-OsL-Ch42,pMDC32B-AtMIR390a-OsL-Ft,pMDC32B-AtMIR390 a-OsL-Trich, pMDC32B-OsMIR390, _P MDC32B-OsMIR390-AtL, pMDC32B-OsMIR390-l 73-21,

PMDC32B-OsMIR390-l 73-21 -AtL, pMDC32B-OsMIR390-472-21, pMDC32B-OsMIR390- AtL-472-21, _P MDC32B-OsMIR390-828-21, _P MDC32B-OsMIR390-AtL-828-21, pMDC32B- OsMIR390-Bril, pMDC32B-OsMIR390-AtL-Bril, pMDC32B-OsMIR390-Cao, pMDC32B- OsMIR390-AtL-Cao, pMDC32B-OsMIR390-Cadl, pMDC32B-OsMIR390-AtL-Cadl, pMDC32B-OsMIR390-Splll, pMDC32B-OsMIR390-AtL-Splll, pH7WG2B-OsMIR390- Bril-AtL, pH7WG2B-OsMIR390-Cao-AtL, and pH7WG2B-OsMR390-Spll 1-AtL) were obtained as described in the next section. Control construct pH7WG2-GUS was obtained by LR recombination using pENTR-GUS (Life technologies) as the donor plasmid and pH7GW2-OsUbi as the destination vector. pMDC32-GUS construct was described previously (Montgomery et al, 2008). The sequence of all amiRNA precursors used in this study are listed in Appendix S3. All oligonucleotides used for generating the constructs described above are listed in Table S7.

amiRNA oligonucleotide design and cloning

[000328] Sequences of the amiRNAs expressed in A. thaliana were described previously (Schwab et al, 2006; Felippes and Weigel, 2009; Liang et al, 2012; Carbonell et al, 2014). Sequences of the amiRNAs expressed in Brachypodium, and their corresponding

oligonucleotides for cloning in OsMIR390~B/c vectors, were designed with the "P-SAMS amiRNA Designer" tool (http://p-sams.carringtonlab.org) (Fahlgren et al, in preparation). The sequences and predicted targets for all the amiRNAs used in this study are listed in Table S8.

The generation of constructs to express amiRNAs from authentic AtMIR390a precursors was described before (Carbonell et al, 2014). Detailed oligonucleotide design for amiRNA cloning in OsMIR390, OsMIR390-AtL and AtMIR390a-OsL precursors is given in Figures S2, S3 and S4, respectively. The amiRNA cloning procedure is described in Appendix S4. All oligonucleotides used in this study for cloning amiRNA sequences are listed in Table S7.

Transient expression assays in N. benthamiana

[000329] Transient expression assays in N. benthamiana leaves were done as described (Carbonell et al, 2014) with tumefaciens GV3101 strain.

RNA-blot assays

[000330] Total RNA from Arabidopsis, Brachypodium or N. benthamiana was extracted using TRlzol® reagent (Life Technologies) as described (Cuperus et al, 2010). RNA blot assays were done as described (Cuperus et al, 2010). Oligonucleotides used as probes for small RNA blots are listed in Table S7.

Quantitative real-time RT-qPCR

[000331] RT-qPCR reactions and analyses were done as described (Carbonell et al, 2014). Primers used for RT-qPCR are listed in Table S7 (and are named with the prefix 'q'). Target mRNA expression levels were calculated relative to four thaliana (AtACT2, AtCPB20, At SAND and AtUBQIO) or B. distachyon (BdSAMDC, BdUBC18, BdUBU and BdUBIlO) reference genes as described (Carbonell et al, 2014).

5'-RLM-RACE

[000332] 5' RNA ligase-mediated rapid amplification of cDNA ends (5'-RLM-RACE) was done using the GeneRacer™ kit (Life Technologies) but omitting the dephosphorylation and decapping steps. Total RNA (2 μg) was ligated to the GeneRacer RNA Oligo Adapter. The GeneRacer Oligo dT primer was then used to prime first strand cDNA synthesis in reverse transcription reaction. An initial PCR was done by using the GeneRacer 5' and 3' primers. The 5' end of cDNA specific to each mRNA was amplified with the GeneRacer 5' Nested primer and a gene specific reverse primer. For each gene, control PCR reactions were done using gene specific forward and reverse primers. Oligonucleotides used are listed in Table S7. 5'-RLM-RACE products were gel purified using MinElute gel extraction kit (Qiagen), cloned using the Zero Blunt® TOPO® PCR cloning kit (Life Technologies), introduced into Escherichia coli DH10B, screened for inserts, and sequenced.

Chlorophyll and carotenoid extraction and analysis

[000333] Pigments from Brachypodium leaf tissue (40 mg of fresh weight) were extracted with 5 ml 80% (v/v) acetone in the dark at room temperature for 24 hours, and centrifuged at 4000 rpm during two minutes. One hundred μΐ of supernatant was diluted 1:2 with 80% (v/v) acetone and loaded to flat bottom 96-well plates. Absorbance was measured from 400 to 750 nm wavelengths in a SpectrMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) using the software SoftMax Pro 5 (Molecular Devices, Sunnyvale, CA). Content in chlorophyll a, chlorophyll b, and carotenoids was calculated with the following formulas: Chlorophyll a (mg L in extract)=12.21*Absorbance663 _nm -2.81*Absorbance6 _47nra ; Chlorophyll b (mg L in extract)=20.13*Absorbance64 _7nm ~5.03*Absorbance663nm; Carotenoid (mg L in extract)=[1000*Absorbance ₄₇ onm -3.27*Chlorophyll a (mg/L) - 104*Chlorophyll b

(mg/L)]/227.

Preparation of small RNA libraries

[000334] Fifty to 100 g of Arabidopsis, Brachypodium or Nicotiana total RNA were treated as described (Carbonell et al, 2012; Gilbert et ah, 2014), but each small RNA library was barcoded at the amplicon PCR reaction step using an indexed 3' PCR primer (il-i8, ilO or il 1) and the standard 5'PCR primer (P5) (Table S7). Libraries were multiplexed and subjected to sequencing analysis using a HiSeq 2000 sequencer (Illumina).

Small RNA sequencing analysis

[000335] Small RNA sequencing analysis was done as described (Carbonell et al, 2014). Custom scripts to process small RNA data sets are available at

https://github.coffl/carringtoniab/srtools. A summary of high-throughput small RNA sequencing libraries from transgenic Arabidopsis inflorescences and Brachypodium calli or leaves, and from N. benthamiana agroinfiltrated leaves, is provided in Table S9. O. sativa small RNA data sets used in the processing analysis of authentic OsMIR390 presented in Figure lb were described previously (Cuperus et al, 2010).

Preparation of strand-specific transcript libraries

[000336] Ten μg of total RNA extracted from four independent lines per construct were treated with TURBO DNAse I DNA-free (Life Technologies). Samples were depleted of ribosomal RNAs by treatment with Ribo-Zero Magnetic Kit "Plant Leaf (Epicentre) according to manufacturer's instructions. cDNA synthesis and strand-specific transcript libraries were made as described (Wang et al, 2011 ; Carbonell et al, 2012), with the following modifications. Ribo-Zero treated RNAs were fragmented with metal ions during 4 minutes at 95°C prior to library construction, and 14 cycles were used in the linear PCR reaction. DNA adaptors 1 and 2 were annealed to generate the Y-shape adaptors, and PE-F oligonucleotide was combined with one indexed oligonucleotide (PE-R-N701 to PE-R-N710) in the linear PCR (see Table S7). DNA amplicons were analyzed with a Bioanalyzer (DNA HS kit; Agilent), quantified using the Qubit HS Assay Kit (Invitrogen), and sequenced on a HiSeq 2000 sequencer (Illumina).

Transcriptome analysis

[000337] FASTQ files were de-multiplexed with the parseFastq.pl perl script

(https://github.com/carringtonlab/srtools). Sequencing reads from each de-multiplexed transcript library were mapped to B. distachyon transcriptome (v2.1, Phytozome 10) using Butter (Axtell, 2014) and allowing one mismatch. Differential gene expression analysis was done using DESeq2 (Love et al, 2014) with a false discovery rate of 1%. For each 35S:GUS versus 35S:OsMIR390-AtL pairwise comparison, genes having no expression (0 gene counts) in at least five of the eight samples were removed from the analysis. Differential gene expression analysis results are shown in Data SI.

[000338] TargetFinder vin (https ://github . com/carrin gtonlab/Tar getFinder) (Fahlgren and Carrington, 2010) was used to obtain a ranked list of potential off-targets for each amiRNA.

[000339] A summary of high-throughput RNA-Seq libraries from transgenic Brachypodium leaves is provided in Table S10.

Accession numbers

[000340] A. thaliana gene and locus identifiers are as follows: AtACT2 (AT3G18780), AtCBP20 (AT5G44200), AtCH42 (AT4G 18480), AtCPC (AT2G46410), AtETC2

(AT2G30420), _4iPT (AT1G65480), AtSAND (AT2G28390), _4i7R7(AT5G53200) and AtUBQIO (AT4G05320). B. distachyon gene and locus identifiers are as follows: BdBRIl (Bradi2g48280), BdCADl (Bradi3g06480), BdCAO (Bradi2g61500), BdSAMDC

(Bradi5gl4640), BdSPLll (Bradi4g04270), BdUBC18 (Bradi4g00660), BdUBI4

(Bradi3g04730) and BdUBIJO (Bradilg32860). The miRBase Chttp://mirbase.org (Kozomara and Griffiths- Jones, 2014) locus identifiers of the conserved rice MIRNA precursors and plant MIR390 precursors (Figure lb) are listed in Table SI and Table S2, respectively.

[000341] High-throughput sequencing data from this article can be found in the Sequence Read Archive (http://www.Dcbi.nlm.nih.gov/sra) under accession number SRP052754. Table 1: OsMR390-3saI/ccdB ('B c' ) Yecfors for direct cloning of aniiRRAs.

Vector Bacterial Plant GATEWAY Backbon Promote Terminato Plant antibiotic antibiotic use e I r species resistance resistance tested pENTR- Kanamycin - pENTR - - -

OsMIRSaO- Done

S/c I

pMDC123S Kauamycin BASTA - CaMV l!OS Nicoiiana

3- pMDCI2 2x35S bentimmiema

OsMJR390- 3

S/c

pMDC32 - Kanamyctti - CaMV nos Nicotiana

OsMK.390- Hygrouiyciii Hygrornyci pMDC32 2x35S benthamiana

B/c II Brachypodium distach on pH7WG2B- - Os CaMV Smchypodimn OsMIR390- Spectiflomyci Hygrornyci pH7W02 UbiquitSn distachyon

Btc 11 11

Table SI. trtiRbase Locus Identifiers of the Oryza saliva conserved MTRNA precursors used in this study.

Locus

MIRNA precursor

Identifier osa-MIR156a 10000653 osa~MIR156b ΜΓ0000654 osa-MIR156c 10000655 osa- IR156d I0000G56 osa-MIR156e M100006S7 osa-MIR156f ΜΓΟ0ΟΟ658 osa-MIR156g MI0000659 osa-MIR156h ,viroooo660 osa-MIR156i M10000661 osa-MlR156j MI0000662 osa-MIR156k M1000I090 osa-MIR1561 I000109I osa-MIR159a.l MI AT0001022 osa-MIR159b M 10001093 osa-MlR159c MI000J094 osa-MIR159d 1000I095 osa-MIR159e Γ00ΟΙΟ96 osa-MIR159f ΜΪ00 1097 osa~MlRi60a MI0000663 osa-M!R160b MI0000664 osa-MIR160c I000066S osa-MIR160d MI0000666 osa-MIR160e MiOOOllOO os»-MIR160f MtOOOI 101 osa-MIRt62a M10000667 osa-MIR162b MtOOOI 1 2 osa-MI l64a M10000668 osa-MIR164b M 10000669 osa-MIRl64c ΜΓ0001103 asa~MnU64d MI000I JG4 osa-MIRiu4c ΜΓ00ΟΠΟ5 osa-MlRI64f MI000U59 osa-MIR166a M 10000670 osa-MIR166b MI000O671 osa- IR166c MI0000672 osa-MIR166d ΜΊ0000673 osa- IR166e I00J )674 osa-MER166f · M 10000675 osa-MDU66g MtOOOl 142 osa-MIRI66h ΜΓ0ΟΟΠ43 osa-MlRl 66i iOOOi 144 osa-MH 66j Mrooonsi osa-MHU66k MIOOOI 107 osa- IR1661 MIOOOI 108 osa-MIR166m MIOOOI 157 osa- IR166n MI AT0001088 osa-MIR167a 10000676 osa-MIR167b 10000677 osa-MIR167e M10000678 osa-MIR.I67d MIOOO I 109 osa-MIR167e MIOOOI 1 f O osa- IR ^' 167f MIOOOI 11 1 osa-MIR167g MIOOOI 112 osa- IR167h IOOOI 1 13 osa-MIR167i MIOOOI 1 14 osa-MIR167j MIOOOI 156 oea-MIR168a ΜΙ0ΟΟΠ 15 osa-MlR169a MI0000679 osa-MIR169b MIOOOI 1 17 osa-MIR169c MIOOOI 118 osa-MlR16Pd M IOOOI 1 19 osa-MIR169e 1 MIOOOI 120 osa-MIR169f MIOOOI 121 osa-MIR169g MIOOOI 122 osa-M!Riea. MIOOOI 123 osa-MIR16M MIOOOI 124 osa-MIR169j MIOOOI 125 osa-MlR169k MIOO I 126 osa-MIR1691 MIOOOI 127 osa-MIR169m MIOOOI 128 osa-MIR169n MIOOOI 129 osa-MJR169a ΜΓ000Π 0 osa-MIR169p MIOOOI 131 osa-MlR169q MIOOOI 132 osa-MIR171a MI0000680 osa-MJR171b MIOOO I 133 osa-MIR171c MIOOOI 134 osa-MJR171d. MIOOOI 135 osa-MIR171e MIOOOI 136 osa-MIR171f M [000 It 37 osa- I 17Ig ΓΟΟ0Π38 osa-MIR17ih M [0001147 osa~MIR171i IVirOOO! Ϊ55 osa-MIR172a IQ00H39 osa-MIR172b M 10001.140 osa-MI 172c MI0001141 osa-MIR172d MI000I154 osa-MIK.319a 10QO 1.098 osa-MIK3!9b M1000I099 osa-MIR390 10001690 osa-MIR.3 3 MLQ OI! osa-MIR393b M 10001148 osa- IR394 Ml 0001027 osa-MIR395a Ml 0001042 osa-MI 3 5b MI0001028 osa-MIR395e M 1000104! osa-MIR395d MT0001029 osa- IR395e MI0001030 osa- IR395f MI0001043 osa- IR395g M 100(110 1 osa-MIR395h ^' T0001032 os -MIR i MJO0OJO33 osa~MlR395j M 10001034 osa-MIR395k M 10001035 osa-MIR395I iv! 10001036 osa-MIK395m MT0005084 osa-MIR395n 100050S5 osa-MJR395o Mi00050S(5 osa- IR395p M10005087 osa- IR395q M10005088 osa-MIR395r ΜΪ0005092 osa-MIR395s M 10001037 osa-MI 395t 1.0001038 osa-MIR395u i0001044 osa-MIR395v M10005090 osa-MIR395w 0005091 osa-M!R396a M 10001046 osa-MIR396b M [0001047 osa- I 396c _. M10001048 asa- IR396d iOO 13049 osa«MIR396e MI0001703 osa-MIR396f I0010563 osa-M3R396h I001304S osa ^» IR3 7a MI0001049 osa-MIR397b I0001050 osa-MIR398a 1QOOI051 osa-MIR398b ΜΪ0001052 osa-MI 399a MI0001053 osa-MlR399b M1000105 asa~MIR3 9c M 1.0001055 osa~MIR399d M10001056 osa-MIR399e f000i057 osa-MIR399f Mrooo¾05¾ osa-MI 399g MI0001059 osa-MIR399-i I000106O osa- IR399i MIO0O1O61 osa-MIR399j IOOOI062

0sa~MIR399k MI000I063 osa- IR408 MI0001149 osa~MIRS28 Μ|ΰ003201 osa-MIR827 10 10490

Table S2. miRbaso Locus Identifiers of plant MR390 precursors used in this study.

MIRNA Locus precursor Identifier aly- IR390a Mi00H5fi9 aIy-MIR390b M100I4570 afli-MIR390a 10001000 ath-MIR390b MWOOIOOl bna-MIR390a M10006447 bna-M!R390b MI0006448 bna»MIR390c MI0006449 cca- IR390 MI002I077 cme-MIR3 Qa 10023238 cme-MiR390b mnm cme- IR390c M1Q023239 cme-MIR390d M10023237

CSI-MIR390 M1O013317 ghr- IR390a MI0005647 ghv-MIR390b 10G0564S ghr-MIR390c M1O005649 gma-MIR39Qa MI00Q7214 gma-MIR390b MI00072I5 gma-MIR390c M100I7845 gma~MIR390d ΜΪ002Ι700 gma-MlR 0e MI0021701 gma-MTR390f M.I0021 02 gma-MIR390g M 1-0021703 hax-MIR390a C0022249

Uex- IR390b M 10022250 mdm- IR390a M 10023073 mdm-MIR390b M10023074 mdm-MIR390c M 10023075 mdm-MIR390d M 10023076 mdm-MUG90, ΜΪ002377 mdm-MTR390f M 10023078 mtr-MIR390 M10005586 nta-MIR390a MK502I391 nta-MIR390b MT002I392 nta-MI.R390c MT002I393 pde-MIR390 M 10022095 pta- R390 M10005787 ptc-MlR390a Mt()002305 ptc- IR39Qb M10002306 ptc-MIR390c MIQO023O7 pte~MIR390d M 10002308 rco-MIE.390a M 10013410 reo-MlR390b M l li tcc-MIR390a MI00J7503 tec- JR3 0b M10017504 vvi-MIR390 M|pJ06552

Table S3: Thenotypic penetrance of aniiRNAs expressed in

Brachypodium TO transgenic plants

Construct TO analyzed Phettofypte penetrance'

3$S:OsMtR390~Bnl 11 64%

35S:OsMIR39Q-AtL~Bril 20 80%

UBJ:O$MIR390~AtL~Bril 22 32%

35S:OsMIR390-Cadl 52 94%

358:OsMIR39()-AtL-Cadl 27 100%

3SS:OsMIR390~C o 12 100%

35S;O$MR390-AtZ~Cao 27 100%

UBI:OsMIR39Q~AtL~Cao 32 53%

35S;O$MIR390~Sp!ll 22 95%

35S:OsMR390~AtL-Sptt 1 43 91%

UBl:OsMIR39Q~AiL~SpllI 13 61%

f ^l The Bril phenotype was defined as a shorter height and presence of splradly leaves in amiR-Bril ti¾nsformants when compared to transfermaiits of the 35S: GUS control set.

The Cadi phenotype was defined as the presence of brown to red colorations in stems and nodes in amiR-Cad transformants.

The Cao phenotype was defined as a lighter green color amiR-Caol transfonnants when compared to transformants of the 35S;GUS control set.

The Spll ί phenotype was defined as the presence of necrotic areas m leaves from amiR-Spl i 1 transformants,

Table S4: Pheiiotypic penetrance of amiRNAs expressed in

Brachypodium Tl transgenic plants

Construct Tl analyzed Phenotypic penetrance ⁰

35S:OsMIR39Q-Bril 1 100%

35S:OsMlR39Q-AtL-Bril 2 50%

35S:OsMlR390-AtL-Cadl 6 100%

3SS:OsMR390-AtL-Cao 2 100%

35S:OsMlR390-AtL~Splll 4 100%

UBl:OsMR390-A(L-Spfll 4 100%

"The Bril phenotype was defined as a shorter height and presence of splindly leaves in amiR-Bri 1 transformants when compared to transformants of the 35S:GUS control set.

The Caol phenotype was defined as a lighter greed color amiR-Caol ITansfoitnants when compared to transformants of the 35S:GUS control set

The Cad phenotype was defined as the presence of brown to red colorations in stems and nodes in amiR-Cad transformants.

The Spll 1 phenotype was defined as the presence of necrotic areas in leaves from aroiR-SpH .1 transformants.

Table S5: Phenotypio penetrance of amiRNAs

expressed in Arabidopsis Tl transgenic plants

Construct Tl analyzed Phenotypie penetrance"

35S:AtMR390a-Ft 64 100%

35S;AtMIR390a~OsL~Ft 44 100%

35S;AtMlR390a-Ch42 406 100%

3% weak

2S% intermediate 69% severe

35S:AiMIR390a-OsL~Ch42 267 98%

3% weak

33% intermediate 64% severe

35S AtMlR390a-Trich 45 93%

12% try cpc type

35S:AtMR390a-OsL-Trkh 69 99%

9% try cpc type ^a The Ft phetiotype was defined as a higher 'days to flowering' value when compared to the average 'days to flowering' value of the

35S:GUS control set

The Ch42 phenotype was scored in 10 days-old seedling and was considered 'weak', 'intermediate' or 'severe' if seedlings have >2 leaves, exactly 2 leaves or no leaves (only 2 cotyledons), respectively. The Trich phenotype was defined as a higher number of trichomes when compared to traiisformants of the 35S:GUS control set. Plants with a Trich phenotype were considered 'tiy Ret pe ⁸ if they resembled the Arabidopsis try cpc double mutant.

Table S6: Phenotypic penetrance of amiRNAs expressed in

Arabidopsis T2 transgenic plants

Construct Ϊ2 analyzed Phenotypic penetrance*

3$S;AtMIR390a~Ft 5 100%

35S:AtMIR390a-OsL-Ft 5 100%

35S:AtMM39Qa~Trich 10 90%

35S:AtMIR390a~OsL~Trich 10 90%

"The Ft plienotype was defined as a higher 'days to flowering' value when compared to the average 'days to flowering' valne of the 35S:GUS control set.

The Trich phenotype was defined as a higher number of tnchomes when compared to ttansformants of the 3SS;GUS control set.

TGTAl rrrCCTACrCCGCCCATACTCGAAATCAAACTAGTATGGGCGGCGTAGGAAAAA

amiR472-21 UUUUUCCUACUCCGCCCAUAC RFU, RPS5, CC-NBS- Arabidopsis thaiiana Cuperus er a/., 2010 inn, s

amiR828-21 UCUUGCUUAAAUGAGUAUUCC MTBU3, MYB82, TAS4 Arabidopsis thaiiana Cupcms et a!., 2010 araiR-AtCh42 UUAAGUGUCACGGAAAUCCCU CH42 Arabidopsis thaiiana Felippes and Wcigel, 2009

Carbonel 'i ai, 2014 amiR-AtFt UUGGl JUAUA A AGGAAG AGG CC FT Arabidopsis thaiiana Scbwabb cr (j/., 2006

CarboneU et ai, 2014 amiR-AtTrich U CCCAUUCG UACUGCUCG CC TRY* CP ETC2 Arabidopsis thaiiana Schwabb er al, 2006

Carbonell et ai, 2014 amiR-BdBri I UCGCAAUCUUCCGCCUUGCUC BRXl Brachypodium dtstadiyon This work amiR-BdCadl UCGAUCUGAGAAGUAAGCCCA CADI Brachypodium distachyon This work araiR-BdCao UCUGCAUGGAUUGUAAACCCA CAO Brachypodium distachyon This work amiR UUAGCAACACUACAAGGGCAC SPUl Brachypodium distachyon This work

Table S9. Summary of high-throughput small KNA libraries from Arabidopsis, Brachypodium oiNicoliana benthamiana plants, , „

Sample Construct Species Tissue 3'PCR Barcode Adaptor- ID primer Sequence parsed reads

1 35S:AlMR390a-173-21 K benthamiana Leaf il CGATGT 25,652,072

2 35S:AtMR390a-472~21 N, benthamiana Leaf i3 CAGATG 23,512,059

3 3SS:AtUIR39()a-82$-21 H, benthamiana Leaf ^' iS TTACCA 26,746,930

4 35S:AtMR390a-OsL-l 73-21 N. benthamiana Leaf iJ CGATGT 42,522,405

5 35S:AtMIR390a-OsL-472-21 N, benthamiana Leaf i2 GATCAC 47,332,026

6 3SS:AtMIR390a-OsL-S28-21 N, benthamiana Leaf 13 CAGATG 52,048,606

7 S5S:OsMR390-173-2l B. distac yon Callus il CGATGT 14,756,652

8 3>S:OsMlR390~472-2l B. distachyon Callus 13 CAGATG 69,380,781

9 3SS;OsMR390-S28-21 B. distachyon Callus i5 TTACCA 60,437,057 to 3SS OsMlR390~Atl-l 73-21 B. distachyon Callus Ϊ2 GATCAC 17,972,261

11 . 3SS:OsMR390a-Atl-472-21 B. distachyon Callus TACGTT 25,830.535

12 3SS:OsM -AtL-m-2l B, distachyon Callus «6 ACTGTA 25,129,002

13 35S:AtMR390a-Osl-AtCh42 A, thaliana Inflorescence 110 TGCTAG 10,429,854

14 35S:AiMIR390a-OsL-AtFt A, thaliana Inflorescence il l CTTGTA 32,295,61.7

15 35$:AlMIR390a-OsL-AtTrich A, thaliana Inflorescence i4 TACGTT 51,516,926

16 35$:OsMIR390-BdBril B, distachyon Leaf il CGATGT 19,319,670

17 B. di'stacityon Leaf i2 GATCAC 20,856,916

18 3SS:OsMlR390-BdCadl B, distachyon Leaf i5 TTACCA 21,308,138

19 3SS;OsMIR39»-Atl~BdCadl B, distachyon Leaf 16 ACTGTA 22,929,175

20 3SS:OsMIR390-BdCao B, distachyon Leaf i3 CAGATG 21,930,111

21 35S:OsMR390-AtL-BdC o- B, distachyon Leaf 14 TACOTT 22,199,088

22 35S:OsMIR390-BdSplU B, distachyon Leaf i7 ATCACG 21.231,525

23 35S:OsMR390~AtL-BdSplll B, distachyon Leaf i8 ACTTGT 24,735,881

Table SIO, Summary of Wgh-t roughput sirand-specifio transcript RNA libraries from independent Biachypodium TO transgenic lines _{ir ui m}

Sample Construct PE Pritner-R Index Adaptor- ID Index Sequence parsed reads

1 35&GUS N707 GTAGAGA 16,779,027

2 35S.-GVS N708 CCTCTCT 20,182,946

3 35&GUS N7G9 AGCGTAG 19,472,243

4 358;GUS N710 CAGCCTC 19,128,516

5 35S:0sMlR39Q~AtL-BdBril Ν7ΘΙ TAAGGCG 17,265,195

6 35S:OsMlR390-A.tL-BdBril N702 CGTACTA 16,300,58s

7 3SS:OsMR390~AtL«3dBril ^" N703 AGGCAGA 15,724,668

8 35S:OsMIR39Q~>AiL-BdBril N704 CCTGAG 18,807,736

9 35S:O$MR390-AtL~BdCadI N709 AGCGTAG 22,853,726

10 3SS;OsMR590-AtL-BdCadl N7 0 CAGCCTC 22,562,039

11 35S:OsMR390-AtL-BdCadl N701 TAAGGCG 16,877,134

12 35S:QsMlR39Q-Atl-BdCadt N702 CGTACTA 17,142,684

13 35S;OsMR390-AtL-BdCao N705 AGGAGTC 18,778,386

14 3SS:OsMlR390-AtL~BdCao N706 CATGCCT 19,333,658

15 35S:OsMIR390-AlL-BdCao N707 GTAGAGA 19,648,254

16 35S:OsMR39Q-AlL-BdC(to N708 CCTCTCT 20,379,073

17 3$S:OsMlR39Q-A&~BdSpU I Ή703 AGGCAGA 16,234,590

18 35S;OsMlR390-AtL-BdSplU N704 TCCTGAG 15,407,203

19 35S OsMR39Q-AiL~BdSplll N705 AGGAGTC 21,167,509

20 35S:OsMR390-AlL-BdSpUl N7G6 CATGCCT 19,068,045

Characterization of AtMIR390a-OsL~based amiRNAs in eudicots

Accumulation and processing of amiRNAs produced from AiMJR390a- or OsMIR390- based precursors in Nicotiana benthamiana

[000342] A key feature of the At R390a-B/c-based cloning system to produce amiRNA constructs for eudicots is that the amiRNA insert can be synthesized by annealing two relatively short 75 bases-long oligonucleotides (Carbonell et al., 2014). Because the oligonucleotides containing OsMIR390 distal stem-loop sequences are even shorter (60 bases), we first tested if amiRNAs derived from precursors including OsMfR390 distal stem- loop sequences could be expressed efficiently in eudicot species. This would reduce the synthesis cost of the oligonucleotides required for generating AtMIR390a-based amiRNA constructs, and benefit the generation of large amiRNA construct libraries for gene

knockdown in eudicots such as those reported recently (Hauser et al., 2013; JoverGil et al, 2014).

[000343] To test the functionality of authentic OsMIR390 precursors to produce high levels of accurately processed small RNAs, miR390 and three different amiRNA sequences

(amiR173-21, amiR472-21 and amiR828-21) (Cuperus et al., 2010) were directly cloned into pMDC32B-OsMIR390-B/e (Figure SI, Table I) and expressed transiently in N. benthamiana leaves (Figure S5). The same small RNA sequences were also expressed from the chimeric AtMIR390a-OsL precursor including AtMIR390a basal stem and OsMIR390 distal stem-loop sequences (Figure S4, Figure S8a). For comparative purposes, the same small RNA

sequences were expressed from the authentic AtMIR390a precursor or from a chimeric precursor including OsMIR390 basal stem and AtMTR390a stem-loop sequences (0sMIR390- AtL) (Figure S3, Figure S8a). Samples expressing the B-glucuronidase transcript from the 35S: GUS construct were used as negative controls.

[000344] MiR390 accumulated to similar levels when expressed from each of the different precursors (Figure S8b). In each case, amiRNAs expressed from AtMIR390a-OsL precursors did not accumulate to significantly different levels than did the corresponding amiRNAs produced from authentic AtMIR390a precursors (P> 0.11 for all pairwise t-test comparisons) (Figure S8b). AtMIR390a-OsL-derived amiRNAs accumulated predominantly to 21 nt species, suggesting that the chimeric amiRNA precursors were likely processed accurately (Figure S8b). Finally, amiRNAs produced from either authentic OsMIR390 or chimeric OsMIR390-Ath precursors did not always accumulated as 21 nt species (e g miR828-21 and amiR472-21 from OsMIR390 or OsMTR390-AtL precursors, respectively) (Figure S8b). Therefore, further analyses focused on characterizing AtMlR390a-OsL-based amiRNAs.

[000345] To more accurately assess processing of the amiRNA populations produced from AtMIR390a-OsL precursors, small RNA libraries were prepared and sequenced. For comparative purposes, small RNA libraries from samples containing AtMIR390a-derived amiRNAs were also analyzed. In each case, the majority of reads from either the chimeric AtMIR390a-OsL or authentic AtMIR390a precursors corresponded to correctly processed, 21 nt amiRNA (Figure S8c).

Gene Silencing in Arabidopsis by amiRNAs derived from chimeric precursors

[000346] To test the functionality of AtMIR390a-OsL based amiRNAs in repressing target transcripts, three different amiRNA constructs were introduced into A. thaliana Col-Oplants. For comparative purposes, the same three amiRNA sequences were also expressed from authentic AtMTR390a precursors as reported before (Carbonell et aL, 2014). In particular, amiR-AtFt, and amiR-AiCh42 each targeted a single gene transcript [FLOWERING LOCUS T (FT) and CHLORINA 42 (CH42), respectively], and amiRAtTrich targeted three MYB transcripts [TRIPTYCHON (TRY), CAPRICE (CPC) and ENHANCER OF TRIPTYCHON AND CAPRICE2 (ETC2)] (Figure S9). Plants including 35S: GUS were used as negative controls. Plant phenotypes, amiRNA accumulation, mapping of amiRNA reads in

AtMIR390a-OsL precursors and target mRNA accumulation were measured in Arabidopsis Ti transgenic lines.

[000347] Each of the 44 transformants containing 35S:AtMIR390a-OsL-Ft was significantly delayed in flowering time compared to control plants not expressing the amiRNA (P < 0.01 two sample t-test, Figure S 1 Ob, Figure SI 1, Table S5), as previously observed in amiRNA knockdown lines (Schwab et al., 2006; Liang et al, 2012; Carbonell et al., 2014) and ft mutants (Koornneef et aL, 1991). Two hundred and sixty-six out of 267 transgenic lines containing 355:AtMIR390a-OsL-Ch42 were smaller than controls and had bleached leaves and cotyledons (Figure SlOc, Figure SI 1, Table S5), as consequence of defective chlorophyll biosynthesis and loss of Ch42 magnesium chelatase (Koncz et al., 1990; Felippes and Weigel, 2009). One hundred and seventy of these plants had a severe bleached phenotype with a lack of visible true leaves at 14 days after plating (Figure S 10c, Figure SI 1, Table S5). Finally, 68 out of 69 lines containing 35S:AtMIR390a-OsL-Trick had increased number of trichomes in rosette leaves; six lines had highly clustered trichomes on leaf blades like try cpc double mutants (Schellmann et al, 2002) or other amiR-Trich overexpressor transgenic lines (Schwab et al., 2006; Liang et al., 2012; Carbonell et al., 2014) (Figure SlOd, Table S5). The delayed flowering and trichome phenotypes were maintained in the Arabidopsis T2 progeny expressing amiR-Ft and amiR-Trich, respectively, from chimeric AtMIR390a-OsL precursors (Table S6). No obvious phenotypic differences were observed between plants expressing the amiRNAs from the AtMIR390a-OsL or AtMIR390a precursors in either Tl or T2 generations (Figure S lOb-d, Figure Sll, Tables S5 and S6). In summary, AtMIR390- OsL-based amiRNAs conferred a high proportion of expected and heritable target-knockdown phenotypes in transgenic plants.

[000348] The accumulation of all three amiRNAs produced from chimeric AtilllR390-0sL or authentic AtlllIR390a precursors was confirmed by RNA blot analysis in Tl transgenic lines showing amiRNA-induced phenotypes (Figure SlOe). In ail cases, AtM[R390-0sLand AtMIR390a-derived amiRNAs accumulated to similarly high levels and as a single species of 21 nt (Figure SlOe), suggesting that AtMIR390a-OsL-based amiRNAs were as accurately processed as AtMIR390a-based amiRNAs. To more precisely assess processing and accumulation of the AtMIR390a-OsL-based amiRNA populations, small RNA libraries from samples containing each of the AtMIR390a-OsL-based constructs were prepared. In each case, the majority of reads from AtMIR390a-OsL precursors corresponded to correctly processed, 21 nt amiRNA while reads from the amiRNA* strands were always relatively under-represented (Figure SlOg) as observed before with the same amiRNAs expressed from AtMIR390a precursors (Carbonell et l., 2014).

[000349] Finally, accumulation of target mRNAs in A. thaliana transgenic lines expressing AtMIR390a-OsL- or AtMTR390a-based amiRNAs was analyzed by quantitative real time RT-PCR assay. The expression of all target mRNAs was significantly reduced comparedto control plants (P < 0.023 for all pairwise t-test comparisons, Figure SI Of) when the specific amiRNA was expressed. No significant differences were observed in target mRNA expression between lines expressing AtMIR390a-OsL- or AtilllR390a-based amiRNAs.

[000350] Collectively, all these results indicate that amiRNAs produced from chimeric AtIVER390a-OsL precursors are highly expressed, accurately processed and highly effective in target gene knockdown. Therefore, the use of chimeric AtMlR390a-OsL precursors is an attractive alternative to express effective amiRNAs in eudicots in a cost-optimized manner.

DNA sequence of B/c vectors used for direct cloning of amiRNAs in zero- background vectors containing the OsMIR390 sequence.

>pENTR-OsMIR390-B/c (4122 bp)

[000351] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGA GTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG CGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA GCGCAACGCAATTAATACGCGTACCGCTAGCCAGGAAGAGTTTGTAGAAACGCA AAAAGGCCATCCGTCAGGATGGCCTTCTGCTTAGTTTGATGCCTGGCAGTTTATG GCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTGCTTCACAACGTTCAAATCCGC TCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAA ACGAAAGGCCCAGTCTTCCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTT CCCTACTCTCGCGTTAACGCTAGCATGGATGTTTTCCCAGTCACGACGTTGTAAA ACGACCKJCCAGTCTTAAGCTC^

attgafgagcaatgctttttiataatgccaactttgtacaaaaaagcaggcfCCGCGGCC GCCCCCTTCACCGAGC

TCGAGATGTTTTGAGGAAGGGTATGGAACAATCCTTGAGAGACCATTAGGCACC

CCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGA

GCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatggagaaaaaaatcactggata taccaccgtt gatatatcccaatggcatcgtaaagaacattttgaggcatttcagtc^

ggccrttttaaagaccgluaagaaaaataagcacaagttltatcc

tccgtatggcaatgaaagacggtgagctggtgatatgggatagigttcacccttgttaca ccgttttccatgagcaaactgaaacgttttc atcgctotggagtgaataccacgacgatttccggcagtttctacacatataitcgcaaga tgtggcgtgttacggtgaaaacctggccte ttcectaaagggfttattgagaatatgttrttcgtctcagcra^

acttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctga tgccgctggcgattcaggttcatcatgccg tttgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagt ggcagggcggggcgtaaACGCGTG

GAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTG

CGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTAT

GCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTC AAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATG

AAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGG

CTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGG

CTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCT

GTTTGTGGATGTACAGAGTGATArrATTGACACGCCCGGCCGACGGATGGTGATC

CCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGG

TGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTG

TGCCGGTTTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATG

ACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCT

TATACACAGCCAGTCTGCACCTCGACggtctcAcatggtttgttcttaccacacgac caattaaatcGAGC

TCAAGGGTGGGCGCGCCGaeccagctitottgtacaaa^

gajtcajretcac^^

ATTACATGGTCATAGCTGTTTCCTGGCAGCTCTGGCCCGTGTCTCAAAATCTCTG ATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCT

TACATAAACAGTAATACAAGGGGTGTTatgagccataftcaaGgggaaacgtcgagg ccgcgattaaattc caacatggatgctgatttatatgggtataaatgggcicgcgataatgtOgggc atcaggtgcgacaatctatcgcttgtatgggaagcc cgatgc-gccagagitgtttctgaaacatggcaaa^

aatttatgcctettecgaccatcaagcat^^

caggtattagaagaatatcctgattcaggtgaaaatattgttgatgc^^^

tgtccttttaacagcgatcgcgtatttegtctcgctcaggcgcaatcacgaatgaat

gcgtaatggctggcctgttgaacaagtctggaaagaaatgcat^^

cacttgataaccttatttttgacgaggggaaattaatuggtt^

catcctatggaactgcctcggtgagttttctccttc^

cagtttcatttgatgctcgatgagtttttcTAATCAGAATTGGTTAATTGGTTGTAA CACTGGCAGAG

CATTACGCTGACTTGACGGGACGGCGCAAGCTCATGACCAAAATCCCTTAACGT

GAGTTACGCGTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATC

TTCTTGAGATCCTT TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC

CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAA

GGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG

TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC

TAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT

GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGG

GTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACC TACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGAC AGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCC AGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTT GAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCC AGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT

SEQ ID NO.:416

PURPLE/UPPERCASE: M 3-forward binding site orange/lowercase: atiL 1 BLUE/UPPERCASE: OsMIR390a 5' region RED/UPPERCASE: Bsal site magenta/lowercase: chloramphenicol resistance gene MAGENTA/UPPERCASE: ccd gene red/lowercase: inverted Bsal site blue/lowercase: OsMIR390a 3' region orange/lowercase/underlined; attL2

PURPLE/UPPERCASE/U DERLINED: M13-reverse binding site brown/lowercase: kanamycin resistance gene

>pMDC32B-OsMIR390-B/c (11675 bp)

[000352] CCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCAC TCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAA GGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCT GCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGA TGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATA CTATGT ATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTrTTCTGGTATTT AAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCT TCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAatggctaaaatg agaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaa ggaatgtctcctgctaaggtatataagct ggtgggagaaaatgaaaacctatatttaaaaatgacggacagccggtataaagggaccac ctatgatgiggaacgggaaaaggacat gatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatga tggctggagcaatctgctcatgagtgag gccgatggcgtcctttgctcggaagagtatgaagatgaacaaagccctgaaaagattatc gagctgtatgcggagtgcatcaggctctt tcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagccga attggattacttactgaataacgatctggcc gatgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcgagctgtat gattttttaaagacggaaaagcccgaag aggaacttgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatggca aagtaagtggctttattgatcttgggagaa gcggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggata tcggggaagaacagtatgtcgagctat tttttgacttactggggatcaagcctgattgggagaaaataaaatattatattttactgg atgaattgitttagTACCTAGAATGC

ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG

AAAAGATCAAAGGATCTTCTTGAGATCCTTrrTTTCTGCGCGTAATCTGCTGCTTG

CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA

CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG

TCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC

TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG

TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG

TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC

ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA

GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG

CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT

CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC

TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC

TTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG

AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC

ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGG

CTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT

CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAG

AGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCG

CCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGG

CCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGC GGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTC

GGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTA

ATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGT

CACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGG

TTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAA

AGAGAACTTTTCGACCTT TTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCC

GTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATG

ACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGT

CATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGC

GCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTG

CGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAA

AAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGC

GGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCT

CTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGG

CGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACC

GAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCA

GAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCC

CTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGT

ACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCT

ACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCT

TCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCA

CGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTT

CCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGA

TCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCC

GCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGC

GCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTT

GGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGG

CCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGT

TCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTC

ATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAAT

GGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGC

AACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTG

CAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTT GCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGC

CAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTT

GTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCA

AGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGC

GTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGA

CCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTA

AGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACAC

AGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCC

GATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAG

CGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGA

ATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGT

TGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTC

ATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACC

GCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCT

CGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACA

AACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGA

ACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGG

TTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTC

GGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCG

CCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGG

GTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCT

GGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGG

GGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTC

GATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCAT

GCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCC

CGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCG

GGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTC

AAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAA

AACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCT

GGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGT

TCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAA

ACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAA

CTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAA GCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGG GGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCA CGCCCTTTTAAATATCCGTTATTCTAATAAACGC

ia iiCtgtoaAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAG

CTCAAGCTGCTCTAGCATTCGCCAT CAGGCTGCGCAACTGTTGGGAAGGGCGAT

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA

GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA

CGGCCAGTGCCAAGCTTGGCGTGCCTGCAGGTCAACATGGTGGAGCACGACACA

CTTGTCTACTCCAAA ATATCAAAGATAC.AGTCTCAGAAGACCAAAGGGCAATT

GAGACTmCAACAAAGGGTAATATCCGGAAACCTCCTCGGATOCATTGCCCAG

CTATCTGT ACTTTATTGTGAAGATAGTGGAAA AGGAAGGTGGCTCCTACAAATG

CCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGG

TCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAA.AGAAGACGTTCC

AACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACAC

ACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAAT

TGAGACTTITCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCA

K^TATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAAT

GGCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTG

GTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTC

CAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGG

ATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGITC

ATTTCATTTGGAGAGGACCTCGACTCTAGAGGATCCCCGGGTACCGGGCCCCCCC

TCGAGGCGCGCCAAGCTATCAAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGC

CGCCCCCTTCACCGAGCTCGAGATGTTTTGAGGAAGGGTATGGAACAATCCTTGA

GAGACCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTG

TGGATTTTGAGTTAGGAGCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatgga gaaaaaaatcactggatataccacGgttgatatatcccaatggcatcgtaaagaacattt tgaggcatttcagtcagttgctcaat ataaccagaccgttcagctggatattacggCGtMaaagaccgtaaagaaaaataagcaca agttttatccggccttiattcacattcttg cccgcctgatgaatgctcatccggagttccgtatggcaatgaaagaeggtgagctggiga tatgggatagtgttcacccttgttaGac gttttccatgagcaaactgaaacgttttcatcgctctgg^

ggcgtgttacggtgaaaacctggcctatttccctaaagggtitattgagaatatgtt tttcgte

gttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgg gcaaatattatacgcaaggcgacaaggtgctgat gccgciggcgalicaggticatcalgccgltlgtgatggctf ^' cca cagggcggggcgtaaACGCGTGGAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTA

TTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGT

ATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCG

ACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGC

ACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAA

AATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTG

CTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGA

GAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCG

GCCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTC

CCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGAC

CACCGATATGGCCAGTGTGCCGGTTTCCGTTATCGGGGAAGAAGTGGCTGATCTC

AGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATA

TAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCACCTCGACggtctcAcatggtt tgttctt accacacgaccaattaaatcGAGCTCAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACA AA

GTGGTTCGATAATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCCACCGCGGTGG

AGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAG ^r lTTCTTAAGATTGA

Al TCTGIlXiCCGCT irAi illCCCGCAATIM

CA CTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTGAATTCGTAATC

ATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAAC

ATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA

CTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGT

GCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTG

GCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAA

TATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAG

GGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATC

AAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAA

AGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACC

CCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAA

GCAAGTGGATTGATGTGATAACatggtggagcaGgacactctcgtctactecaagaa tafcaaagatacagtclc agaagaceaaagggciaiigagaettitoaaeaaagggia aiegggaaaeei^

aaaaggacagtagaaaaggaaggtggcacctacaaatgccalx^attgcgataaagg aaaggcfaicgrteaagatgccfctgccgac agtggtcccaaagatggaccGCcaGccacgaggagcatcgiggaaaaagaagacgttCGa accacgtcttcaaagcaaglggattg atgtgatarctccactgaogtaagggatgacgcacaatcccastarccttcgcaagacct tcctciatataaggaagttcatttcafffgga. gaggACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCTTTCG

CAGATCCCGGGGGGCAATGAGATATGAAAAAGCCTGAACT ^' CACCGCGACGTCTG

TCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTC

GGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGT

CCTGCGGGTAAATAGC GCGCCGATGGTTTCTAGAAAGATCGTTATGTTTATCGG

CACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTA

GCGAGAGCCTGACCTATTGCATCTCCCGCCGTTCACAGGGTGTCACGTTGCAAGA

CCTGCCTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGAT

GCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCG

CAAGGAATCGGTCAATACAGTACAIXJGCGTGATTTCATATGCGCGATTGCTGATC

CCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGC

GCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCA

CCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATA

ACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTC

GCCAACATCT CTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCT

ACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACTCCGGGCGTATA

TGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGA

TGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGG

GACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGG

CTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAG

GGCAAAGAAATAGAGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGtttc tccafaataafgtgtgagtagttc cagataagggaatfagggttoctafagggf tcg

gtattt¾tatttgiaaaatactt^

ATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCT AAGK GTCAATTTGTTTACACCAC:AATATATCCTG CA

SEQ ID NO.:417 brown/lowercase; kanamycin. resistance gene C~>A transversion to block vector's B l site cyan/lowercase: T-DNA right: border GREEN/UPPERCASE: 2x358 CaMV promoter ORANGE/UPPERCASE: attB 1 BLUE/UPPERCASE: OsMR390 5' region RED/UPPERCASE: Bsal site magenta/lowercase : chloramphenicol resistance gene MAGENTA UPPERCASE: ccd gene red/lowercase: inverted Bsal site blue/lowercase: OsMIR390 3' region ORANGE UPPERCASE/UNDERLINED: attB2 GREY UPPERCASE/UNDERLINED: Nos terminator Rreen/lowercase: CaMV i

BROWN/UPPERCASE: hygromycin resistance gene green lowercase/underlined: CaMV terminator CYAN/UPPERCASE: T ^' -DNA left border

>pMDC123SB-OsMIR390-B/c (11150 bp)

[000353] CCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCAC TCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAA GGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCT GCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGA TGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATA CTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTT AAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCT TCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAatggctaaaatg agaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaa ggaatgtctcctgctaaggtatataagct ggtgggagaaaatgaaaacctatatttaaaaatgacggacagcc^

gatgctatggctggaaggaaagctgcctgttc-caaaggtc-ctgcactttgaacgg catgatggctggagcaatctgctcatgagtgag gcc atggcgtcetttgctcggaagagtatgaagatgaacaa

tcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagc cgaattggattacttactgaataacgatctggcc gatgtggattgcgaaaactgggaagaagacactocatttaaagatccg^

aggaacttgtcttttcccacggegacctgg¾agac

gcggcagggcggacaagtggtatgacattgccttetgcgte^

tttttgacttactggggatcaagcctgattgggagaaaataaaatattatattttac tggatgaattgttttagTACCTAGAATGC

ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG

AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTG

CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA

CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG

TCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC

TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG

TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG

TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC

ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA

GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG

CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT

CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC

TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC

TTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG

AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC

ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGG

CTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT

CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAG

AGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCG

CCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGG

CCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGC

GGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTC

GGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTA ATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGT

CACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGG

TTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAA

AGAGAACTTTTCGACCTTI TTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCC

GTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATG

ACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGT

CATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGC

GCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTG

CGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAA

AAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGC

GGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCT

CTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGG

CGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACC

GAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCA

GAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCC

CTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGT

ACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCT

ACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCT

TCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCA

CGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTT

CCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGA

TCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCC

GCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGC

GCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTT

GGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGG

CCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGT

TCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTC

ATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAAT

GGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGC

AACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTG

CAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTT

GCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGC

CAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTT GTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCA

AGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGC

GTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGA

CCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTA

AGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACAC

AGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCC

GATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAG

CGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGA

ATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGT

TGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTC

ATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACC

GCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCT

CGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACA

AACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGA

ACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGG

TTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTC

GGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCG

CCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGG

GTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCT

GGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGG

GGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTC

GATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCAT

GCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCC

CGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCG

GGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTC

AAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAA

AACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCT

GGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGT

TCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAA

ACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAA

CTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAA

GCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGG

GGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCA CGCCCTT TAAA A CCGTTATTCTAATAAACGCTCTTTTCTCTTAGGfttacccgecaa taicctgrcaAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAG

CTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA

GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA

CGGCCAGTGCCAAGCTTGCATGCCTGCAGGTCAACATGGTGGTGCACGACACAC

TTGTCTACTCCAAAAATATCTTTGATACAGTCTCAGAAGACCAAAGGGCAATTGA

GACrmCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCT

ATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCC

ATCATTCCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTC

CCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAA

CCACGTCtTCAAAGCAAGTGGATTGATGTGATA-^CATGGTGGAGCACGACACAC .

TTGTCT ACTCC A A A A A T ATC A A A G A T AC A G TCTC A G A A G A CC A A AG G GC A ATTG

AGACTTl CAACAAAGGGTAATATCCGGAAACCTCCTCGGATrcCATTGCCCAGC

TATCTGTCACITTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGC

CATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGT

CCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCA

ACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCrcCACTGACGTAAGGGATG ΑΟΟΑ€ΑΑΊ ^' Α€ΟΑεΐΑ;Γ€εΊ1ϊΑ3€ΑΑΟΑ€Χ Ί ^' Ί εΤ€Τ Ι 1ΑΑΟΟ Α(3Τ1ΙΛΊΑ ^·'

TCATTTGGAGAGGACCTGGACTCTAGAGGATCCCCGGGTACCGGGCCCCCCCTCG

AGGCGCGCCAAGCTATCAAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGC

CCCCTTCACCGAGCTCGAGATGTTTTGAGGAAGGGTATGGAACAATCCTTGAGA

GACCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTG

GATTTTGAGTTAGGAGCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatggagaa aaaaatcactggatataccaccgttgatatatccea^^

accagaccgrtcagctggatattacggcctt^

cgcctgatgaatgctcatccggagttccgiatggcaatgaaagacggtgagctggtg atatgggatagtgttcacccttgttacaccgttt tecatgagcaaactgaaacgttrtcatcg^

gtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgttttt cgtctca

gatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaa tattatacgcaaggcgaGaaggtgctgatgccg ciggcgattcaggttcatcatgccgrtto

gcggggcgtaaACGCGTGGAGCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATT T GCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATG TCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACA

GCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACA

ACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAAT

CAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTG

ACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAG

AGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGCC

GACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCG

TGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCAC

CGATATGGCCAGTGTGCCGGTTTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGC

CACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAA

ATGTCAGGCTCCCTTATACACAGCCAGTCTGCACCTCGACggtctcAcatggtttgt tcttaccac acgaccaattaaatcGAGCTCAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTA.CAAA.G TG

GTTCGATAATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCT

C ¹ > ' , , ^f ! ' M v I > ' ^> , · , I ! _ ^■ ·

ISTJOCTO

AGTCCCGCAAlTATAm

CTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGTA

ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACA

ACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCT

AACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTC

GTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT

TGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAG

AATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAA

AGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCA

TCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATA

AAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGAC

CCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAA

AGCAAGTGGATTGATGTGATAACatggtggageacgaca^^^

cagaagaccaaagggctattgagacttttcaacaaagggtaatatcgggaaacctcc tcggattccattgcccagciatct ¾ca.cttcat caaaaggscagtagaaaaggaaggtggcacctacaaatgccatcattgcgataaaggaaa ggctatcgtf ^' GaagatgctTcfgccga cagtggtcccaaagafggacccccacccacgaggagcategfggaaaaagaagacgtfcc aaccacgfctteaaagcaagtggatt gatgigatatctccactgacglaagggatgaegeacaai^^ agaggACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGTCTAC

CATGAGCCCAGAACGACGCCCGGCCGACATCCGCCGTGCCACCGAGGCGGACAT

GCCCiGCGGTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTF

CCGTACCGAGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCG

GGAGCGCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGC

CTACGCGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGAC

CGTGTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACC

CACCTGCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATC

GGGCTGC'CCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCC

CGCGGCATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGT

TTCTGGCAGCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCA

CCGAGATTTGACTCGAGftietcca aataatgtgtgagtagte

catg!g!itgagci!faiaa

cta atccagatcCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCA

ATGTGTTAT AAGT GTCTAAGCGTCAATTTGTI ACACCACAATATATCCTGCCA

SEQ ID NO..-418 brown/lowercase: kanamycin resistance gene

- s Bsal site cyan/lowercase: T-TXN . right border GREEN/UPPERCASE: 2x35S Ca V promoter ORANGE/UPPERCASE; attB l BLUE/UPPERCASE: OsMIR390 5' region RED/UPPERCASE: Bsal site magenta/lowercase: chloramphenicol resistance gene MAGENTA/UPPERCASE: ccd gene red/lowercase: inverted Bsal site blue/lowercase: OsMIR390 3' region ORANGE UPPERCASE/UNDERLINED: a«B2 GREY/UPPERCASE/UNDERLINED: Nos terminator green/lowercase: CaMV promoter

BROWN/UFPERCASE/UNDERLINED: BASTA resistance gene CaMV terminator CYAN/UPPERCASE: T-DMA left border

>pH7WG2B-OsMIR390-B/c (13122 bp)

[000354] TTTGA TCCCGAGGGGAA CCCTGTGGTTGGCA TGCA CA TA CAM TGGA CGAA C GGATAAACCTTTTCACGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTT A GGtttacccgccaatatatcctgtcaAA CA C TGA TA GTTTAAA CTGAA GGCGGGAAA CGA CAA TCTG ATCCAAGCTCAAGCTaagcttattcgggtcaaggcggaagccagcgcgccaccccacgtc agcaaatacggaggcg cggggttgacggcgtcacccggtcctaacggcgaccaacaaaccagccagaagaaattac agtaaaaaaaaagtaaattgca ctttgatccaccttttaUacctaagtctcaatttggatcacccttaaacctatcttttca at gggccgggttgtgg

aacaacttttcgtcatgtctaacttccctttcagcaaacatatgaaccatatatagagga gatcggccgtatactagagctgatgtgtt taaggtcgttgattgcacgagaaaaaaaaatccaaatcgcaacaatagcaaatttatctg gttcaaagtgaaaagatatgtttaaa ggtagtccaaagtaaaacttatagataataaaatgtggtccaaagcgtaattcactcaaa aaaaatcaacgagacgtgtaccaa acggagacaaacggcatcttctcgaaatttcccaaccgctcgctcgcccgcctcgtcttc ccggaaaccgcggtggtttcagcgtg gcggattctccaagcagacggagacgtcacggcacgggactcctcccaccacccaaccgc cat aataccagccccctcatctc ctctcctcgcatcagctccacccccgaaaaatttctccccaatctcgcgaggctctcgtc gtcgaatcgaatcctctcgcgtcctcaa ggtacgctgcttctcctctcctcgcttcgtttcgattcgatttcggacgggtgaggttgt tttgttgctagatccgattggtggttaggg tcgatgtgattatcgtgagatgtttaggggttgtagatctgatggttgtgatttgggcac ggttggttcgataggtggaa^ gttttgggattggatgttggttctgatgattggggggaatttttacggttagatgaattg ttggatgattcgattgggg

atctgttggggaattgtggaactagtcatgcctgagtgattggtgcgatttgtagcgtgt tccatcttgtaggccttgttgcgagcatgtt cagatctactgttccgctcttgattgagttattggtgcggttggtgcaaacacaggcttt aatatgttataM^

tctgtagggtagttcttcttagacatggttcaattatgtagcttgtgcgtttcgatttga tttcatatgttcacagattagataatgatgaac tcttttaattaattgtcaatggtaaataggaagtcttgtcgctatatctgtcataatgat ctcatgttactatctgccagtaatttatgctaa gaactatattagaatatcatgttacaatctgtagtaatatcatgttacaatctgtagttc atctatataatctattgtggtaatttcW atctgtgtgaagattattgccactagttcattctacttatttctgaagttcaggatacgt gtgctgttac^^ atgtgcctgttactatctttttgaatacatgtatgttctgttggaatatgtttgctgttt gatccgttgttgtgtccttaat^

ccctatctgtttggtgattatttcttgcagattcagatcgggccc GCTTGACTAGTGATATCACAAGlTTGTAC

AAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCGAGCTCGAGATGTTTTGAGGAAGG

GTATGGAACAATCCTTGAGAGACCATTAGGCACCCCAGGCTTTACACTTTATGCTTC CG

GCTCGTATAATGTGTGGATTTTGAGTTAGGAGCCGTCGAGATTTTCAGGAGCTAAGG AA

GCTAAAatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcat cgtaaagaacattttgaggcatttcag tcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaag accgtaaagaaaaataagcacaagtttt atccggcctttattcacattcttgcccgcctgatgaatgctcatccggagttccgtatgg caatgaaagacggtgagctggtgatatg ggatagtgttcacccttgttacaccgttttccatgagcaaactg acgttttcatcgctctggagtgaataccacgacgatttccggc agtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcct tttccctaaagggtttattgagaatatgtttitcgt ctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaa cttcttcgcccccgttttcaccatgggca aatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccg tttgtgatggcttccatgtcggcagaa tgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaaACGCGTGGAG CCGGCTTACTAAA

AGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATAC TG

ATATGTATACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGT GA

CAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGG TC

TGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAG

CGGAAAA TCAGGAA GGGA TGGCTGA GGTCGCCCGGTTTA TTGAAA TGAACGGCTCTTT

TGCTGA CGA GAA CA GGGGCTGGTGAAA TGCA GTTTAA GGTTTA CA CCTATAAAA GA GA

GAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGCCG A

CGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAA C

TTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGG C

CAGTGTGCCGGTTTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAA T

GACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTT AT

ACACAGCCAGTCTGCACCTCGACggtctcAcatggtttgttcttaccacacgaccaa ttaaatcGAGCTCAA

GGGTGGGCGCGCCGA CCCAGCTTTCTTGTA CAAA GTGGTGA TA TCCCGcssccatgctagag tccgcaaaaatcaccagtctctctctacaaatctatctctctctatttttctccagaata atgtgtgagtagttcccagataagg agggttcttatagggtttcgctcatgtgttgagcatataagaaacccttagtatgtattt gtatttgtaaaatacttctatcaataaaattt ctaattcctaaaaccaaaatccagtsacctGCAGGCATGCGACGTCGGGCCCTCTAGAGG ATCCCCG

GGTACCGTGCAGCGTCGCGTCGGGCCAAGCGAAGCAGACGGCACGGCATCTCTGTC

GCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTC G

GCATCCAGAAATTGCGTGGCGGAGCGGCAGACGTGAGCCGGCACGGCAGGCGGCCT

CCTCCTCCTCTCACGGCACCGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCG C TTTCCCTTCCTCGCCCGCCGTAATAAATAGACACCCCCTCCACACCCTCTTTCCCCAAC

CTCGTGTTGTTCGGAGCGCACACACACACAACCAGATCTCCCCCAAATCCACCCGTC G

GCACCTCCGCTTCAAGGTACGCCGCTCGTCCTCCCCCCCCCCCCCTCTCTACCTTCT C

TAGATCGGCGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTT GT

GTTA GA TCCGTGTTTGTGTTA GA TCC GTGCTGCTA GC GTTCGTA CA CGGA TGCGA CCT

GTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGGAATCCTG GG

A TGGCTCTA GCCGTTCCGCA GA CGGGA TCGA TTTCA TGA TTTTTTTTGTTTCGTTGCA TA

GGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTCGGG TCA

TCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGGGCGGTCGT TCT

AGATCGGAGTAGAAATCTGTTTCAAACTACCTGGTGGATTTATTAATTTTGGATCTG TAT

GTGTG TGCCA TA CA TA TTCA TA G TTA CGAA TTGAA GA TGA TGGA TGGAAA TA TCGA TCTA

GGATA GGTA TA CA TGTTGA TGCGGGTTTTA CTGA TGCA TA TA CA GA GA TGC TTTTTGTTC

GCTTGGTTGTGA TGA TGTGGTGTGGTTGGGCGGTCGTTCA TTCGTTCTAGA TCGGA GT

AGAATACTGTTTCAAACTACCTGGTGTATTTATTAATTTTGGAACTGTATGTGTGTG TCAT

ACATCTTCATAGTTACGAGTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATA CAT

GTTGATGTGGGTTTTACTGATGCATATACATGATGGCATATGCAGCATCTATTCATA TGC

TCTAACCTTGAGTACCTATCTA ATAATAAACAAGTATGTTTTATAATTATTT

ATATACTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCT TCA

TACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTGG TGT

TACTTCTGCAGGTCGACTCTAGAGGATCCATGAAAAAGCCTGAACTCACCGCGACGT C

TGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTC G

GAGGGCGAA GAA TCTCGTGCTTTCAGC TTCGA TGTA GGAGGGCGTGGA TA TGTCCTGC

GGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTG CA

TCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTAGCGAGAGCCTG

ACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACC G

AACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGATCGCTGCGGCCG

ATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACA

CTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAA CT

GTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTT

TGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAAC

AATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATG

TTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCT T

GTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGC CACGACTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGT

TGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCG

ATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCT

GGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTC

GTCCGAGGGCAAAGAAATAGGAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAA AT

TGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCC TG

GGGTGCCTAA TGA GTGA GCTAA CTCA CA TTA CTTAA GA TTGAA TCCTGTTGCCGGTCTT

GCGA TGA TTA TCA TA TAA TTTCTGTTGAA TTA C GTTAA GCA TGTAA TAA TTAA CA TGTAA T

GCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTT AAT

ACGCGATA GAAAA CAAAA TA TA GCGCGCAAA CTAGGA TAA A TTA TCGCGCGCGGTGTC

ATCTATGTTACTAGATCGACCGGCATGCAAGCTGATAATTCAATTCGGCGTTAATTC AG

TACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACA CCA

CAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAA T

CACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTT

GTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAG C

TGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATG A

CAGAGCGTTGCTGCCTGTGATCAATTCGggcacgaacccagtggacataagcctcgt tcggttcgtaagctgt aatgcaagtagcgtaactgccgtcacgcaactggtccagaaccttgaccgaacgcagcgg tggtaacggcgcagtggcggtttt catggcttcttgttatgacatgtttttttggggtacagtctatgcctcgggcatccaagc agcaagcgcgttacgccgtgggtcgatgtt tgatgttatggagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaa acatcatgggggaagcggtgatcg ccgaagtatcgactcaactatcagaggtagttggcgtcatcgagcgccatctcgaaccga cgttgctggccgtacatttgtacggct ccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccg taaggcttgatgaaacaacgcggcg agctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccg cgctgtagaagtcaccattgttgtgcac gacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcag cgcaatgacattcttgcaggtatcttc gagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagc gttgccttggtaggtccagcggcgg aggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaa cgctatggaactcgccgcccgactgg gctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaacc ggcaaaatcgcgccgaaggatgtcg ctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagcta gacaggcttatcttggacaagaag aagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcg agatcaccaaggtagtcggcaaat aatgtctagctagaaattcgttcaagccgacgccgcttcgccggcgttaactcaagcgat tagatgcactaagcacataattgctca cagccaaactatcaggtcaagtctgcttttattatitttaagcgtgcataataagcccta cacaaattgggagatatatcatgcatgac

CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAT CA

AAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAA AAC CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAA

GGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTA G

TTA GGCCA CCA CTTCAA GAA CTCTGTA GCA CCGCCTA CA TA CCTCGCTCTGCTAA TCCT

GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAG A

CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA

GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATG A

GAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG

GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTA

TAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTC AG

GGGGGCGGA GCCTATGGAAAAA CGCCA GCAA CGCGGCCTTTTTA CGGTTCCTGGCCT

TTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA ACC

GTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCA

GCGA GTCAGTGA GCGA GGAA GCGGAA GA GCGCCTGA TGCGGTA TTTTCTCCTTA CGC

ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATG CC

GCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGC C

CCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATC

CGCTTA CA GA CAA GCTGTGACCGTCTCCGGGA GCTGCA TGTGTCA GA GGTTTTCA CCG

TCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTG

GCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTT

TTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCG

CAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGGCCAGACAG

TTA TGCA CA GGCCAGGCGGGTTTTAA GA GTTTTAA TAA GTTTTAAA GA GTTTTA GGCGG

AAAAATCGCCTTTTTTCTCTTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAA TGT

ACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCC C

AATGTACGTGCTATCCACAGGAAAGAGAACTTTTCGACCTTTTTCCCCTGCTAGGGC AA

TTTGCCCTAGCATCTGCTCCGTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCA G

GTTGCGGTAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATC

GTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTT G

AACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCT G

CCTTGCCTGCGGCGCGGCGTGCCAGGCGGTA GAGAAAA CGGCCGA TGCCGGGA TCG

ATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATC T

CGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGC T

CTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCG G CCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGC

AGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGA G

TACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCTTCCCTTCCCG

GTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATC C

CACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGC

TCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGGAAAACGGCC

ACGTCCATGATGCTGCGACTATCGCGGGTGCCCACGTCATAGAGCATCGGAACGAAA A

AA TC TGGTTGC TCGTCGCCCTTGGGCGGCTTCCTAA TCGA CGGCGCA CCGGCTGCCG

GCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGG

GGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCTTCAACTTCT

CCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGAC

CGTCA CGCCGA TTCCTCGGGCTTGGGGGTTCCA GTGCCA TTGCA GGGCCGGCA GA CA

ACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACG

GCGTCGGTGCCTGGTTGTTCTTGA TTTTCCA TGCCGCCTCCTTTA GCCGCTAAAA TTCA

TCTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATA GCA

GCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCT C

CGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAA

CGTTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCT

TTTGCTCA TTTTCTCTTTA CCTCA TTAA CTCAAA TGA GTTTTGA TTTAA TTTCA GCGGCCA

GCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGC

CGGCGGCGGCA GTGCCTGGGTA GCTCA CGCGCTGCGTGA TACGGGACTCAAGAA TG

GGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTGCCTTTG

ATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGC T

GCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCA

CCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCAAGTCCGCCGCCT

GGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGG

TCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCC

GCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCG

GCGAGTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCT

TTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTA CTC

ATCGCATCATATACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATC AC

CTTTTTAGACGGCGGCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCC

AGCTTGGCATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGC GCGGGCGGCTCGAACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTA

ATGAAAAACGGTTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGC G

TTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGC

ACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTA

CAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTC

CTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGG

GGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTCG

ATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCATGCGG

CCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCC

GGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGC

GGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGG

CCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAA ACAGCTTGGTGC

AGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGA

CGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGTTCATGGCGTAATGTCTCC

GGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAAACACGCGACAAGAAAACGCC AG

GAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTT T

CAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTT

GGATCAAAGTAC

SEQ ID NO.:419

grey/lowercase: OsUbi promoter ORANGE/UPPERCASE: attBl BLUE/UPPERCASE: OsMIR390 5' region RED/UPPERCASE: Bsal site magenta/lowercase: chloramphenicol resistance gene MAGENTA/UPPERCASE: ccdB gene red/lowercase: inverted Bsal site blue/lowercase: OsMIR390 3' region ORANGE/UPPERCASE/UNDERLINED: attB2 green/lowercase/urtderlirsed: CaMV terminator GREY/UPPERCASE: ZmUbi promoter BROWN/UPPERCASE: hygromycin resistance gene CY AN/UPPERCASE: T-DN A left border brown/lowercase: spectinomycin resistance gene

· , ,. ^' · L to block vector' Bsol site

[000355] DNA sequence in FASTA format of all the MIRNA precursors used in this study to express and analyze amiRNAs.

(a) Sequences of OsMIR390-based amiRNA precursors

[000356] Sequences unique to the pri-miRNA, pre-miRNA, miRNA/amiRNA guide strand and miRNA*/amiRNA* strand sequences are highlighted in grey, white, blue and green, respectively. Bases of the pre-OsMIR390 that had to be modified to preserve the authentic OsMIR390 precursor structure are highlighted in red.

>OsMIR390

[000357] 3TATGGAACAATCCTTGnBaDBBffifflBBD

SEQ ID NO.:420

>OsMIR390-AtL

SEQ ID NO.:421

>OsMIR390-l 73-21

[000359] GA GA TGTTTTGAGGAA GGGTA TGGAA CAA TCC TTC

i ^' ^TCGAAATCAAACTAMGA ITGGTTTGTTCTTA CCA CACG ACCAATTAAATC

SEQ ID NO.:422

>OsMIR390-AtL-173-21

SEQ ID NO.:423

OsMIR390-472-21

STGGTTTGTTCTT

SEQ ID NO..-424

>OsMIR390-AtL-472-21

SEQ ID NO.:425

>OsMIR390-828-21 SEQID O.:426

>OsM I Κ39Θ-Α tL-828-21

SEQ ID NO.:427 >OsMIR390-Bril

[000365] _ jGTATGGAACAATCCTTG]

rGGTTTGTTCT

SEQ IDNO.:428

>OsMIR390-AtL-Bril

SEQIDNO..-429

>OsMIR390-Cadl

SEQIDNO.:430

>OsMIR390-AtL-Cadl SEQ ID NO.:431

>OsMIR390-Cao

[000369] _^ ^" · . ^■ ^ ^{A A r'} '" ^A —— ^B

SEQ ID NO.:432

>OsMIR390-AtL-Cao

SEQ ID NO.:433

>OsMIR390-Splll

SEQ ID NO.:434

>OsMIR390-AtL-SpIl 1

^} TATGGAACAATCCTTG||nHBHnEMH

^TGATGATCACATTCGTTATCTATTTTTTC

SEQ ID NO.:435

(b) Sequences of AtMIR390a-based amiRNA precursors [000372] Sequence unique to the φ\-ΑίΜΙΚ3>90α sequence is highlighted in black. Bases of the pre-AtMIR390a that had to be modified to preserve the authentic AtMIR390a precursor structure are highlighted in red. Other details as in (a).

>AtMIR390a

[000373] TATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA

AACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCAC 3 rCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

GCTCTTCTTACTf CAATGAAAAAGGCCGAGGCAAAACGCCTAAAATCACTTGAG

ATCAATTCTTTTTACTGTCCATTTAAGCTATCTTTTATAAACGTGTCTTATTTTCT

5 .TCTCTTTTGTTTAAACTAAGAAACTATAGTATTTTGTGTAAAACAAAACATGAA

AGAACAGATTAGATCTCATCTTTAGTCTC

SEQ ID NO.:436

>AtMIR390a-OsL

[000374] ^{^} ATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA

1 TATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

GAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCAC

CCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

CCCAAAAAAACAA jAGTAGAGAAGAATCTGT^

rCGAAATCAAACTAGGS GGCTCTTCTTACH

TGAAAAAGGCCGAGGCAAAACGCC 1 AAAA 1 CAC ^' l 1 AGAA TCAA I 1 CT I 1 ΤΓΑ

TGTCCATTTAAGCTATCTTTTATAAACGTGTCTTATTTTCTATCTCTTTTGTTTAA

CTAAGAAACTATAGTATTTTGTCTAAAAGAAAACATGAAAGAAGAGATTAGAT

TCATCTTTAGTCTC

SEQ ID NO.:437

>AtMIR390a-l 73-21 [000375] ^' ATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA

GAACCCGAGTTTTGTTCGTCTATAAATAGCAGCTTCTCTTCTCCTTGTTCCTCAG

TCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

CTCTTCTTACT ACAATGAAAAAGGCCGAGGC AA \ACGCCTAAAATCAC TTGAGA

G TCAATTCTTTTTACTGTCCATTTAAGCTATCTTTTATAAACGTGTCTTATTTTCTA

TCTCTTTTGTTTAAACTAAGAAACTATAGTATTTTGTGTAAAACAAAACATGAAA

AACAGATTAGATCTCATCTTTAGTCTC

SEQ ID NO.:438

>AtMIR390a-OsL-173-21

[000376] TATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAG

TATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

GAACCCGAGTTTTGTTCGTGTATAAATAGCACGTTCTCTTCTCCTTCTTCCTCAC I

TCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

E ACCCAAAAAAACAA AGTAGAGAAGAATCTG17

TGAAAAAGGCCGAGGCAAAAGGCCTAAAATCAGTTGAGAATCAATTCTTTTTA

TGTCCATTTAAGCTATCTTTTATAAACGTGTCTTATTTTCTATCTCTTTTGTTTAA

GTAAGAAACTATAGTATTTTGTCTAAAACAAAACATGAAAGAACAGATTAGAT

TCATCTTTAGTCTC

SEQ ID NO.:439

AtMIR390a-472-21

[000377] ATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA

1 TATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

GAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTGTTCCTCAC I

TCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC GCTCTTCTTACTQ

ATCAATTCTTTTTACTGTCCATTTAAGCTATCTTTTATAAACGTGTCTTATTTTCT CTCTTTTGTTTAAACTAAGAAACTATAGTATTTTGTCTAAAACAAAACATGAA

GAACAGATTAGATCTCATCTTTAGTCTC

SEQ ID NO.:4440

AtMIR390a-OsL-472-21

[000378] I V\1\A(jG(KXi(jAA \ :\GG ^' ] ^" AG ^' )X ^, A I ^' CACJA ^' I ^' A Γ.Λ ^' 1 ^' ATTTTGGTAAGAAA

TATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

GAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCAC 1

CCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

«M¾¾^^MC¾ AGTAC _T AGAAGAATCTGT7

CGAAATCAAACTAGL . ITGGC I CTrCTTACTHSS

TGAAAAAGG G.ACiGC ^' AAAACGCC Ι ΑΛΛΑ R ^' AC I rGAG.VATCAATTGTTTTTA

CTGTCCATTTAAGCTATCTT ^' ITATAAACGTG ^' I CTT TTTTC rATCTCTTTTGTTTAA

ACTAAGAAAC I A I AG 1 ΑΊ ^" n ^" I Ci ^" l ^" C TAAAACAAAACA ^" l ^" GA,\AGAACAGATTAGAT

CTCATCTTTAGTCTC

SEQ ID NO.:441

>AtMER390a-828-21

[000379] TATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA

TATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

AACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCAC J

TCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

GCTCTTCTTACT ACAATGAAAAAGGCCGAGGCAAAACGCCTAAAATCACTTGAG

m TCAATTCTTTTTACTGTCCATTTAAGCTATCTTTTATAAACGTGTCTTATTTTCT

1 TCTCTTTTGTTTAAACTAAGAAACTATAGTATTTTGTCTAAAACAAAACATGAA

GAACAGATTAGATCTCATCTTTAGTCTC

SEQ ID NO.:442 >AtMI 390a-OsL-828-21

[000380] A AGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA

TATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

GAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCAC I

TCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

CCCAAAAAAACAA AGTAGAGAAGAATCTGTV

TCGAAATCAAACTAC rGGCTCTTCTTACTSSS XiAAAAAGUC riAGGCA.-\.-\.-\CG CTA.\A.-VrC.\CTI ^' G.-\GA/\TCA.\ ^' l ^" TC ^" r rT ^" ri ^' .\

TG I CC ATI TAAGCTA T T Π I A TAAACGTG IX I ΪΑ ΊΊ ΓΊΓΤΑ ΚΊ C I ^" I 1 ^" Γ(.ί ΓΓΑΑ

CTAAGAAACTATAG ^' rATITI ^' GTCTAAAA AAAAC.ATCjA.-VACiAACAGATTAG I ^"

CTCATCTTTAGTCTC

SEQ ID NO.:443

>AtMffi390a-Ch42

[000381] ^' ATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA ATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

GAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCAC I

TCCATCTTTTTAGCTTCAC ATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

m CCCAAAAAAACAA jAGTAGAGAAGAATCTGTA^^^^^^^^^^¾

ATGATGATCACATTCGTTATCTATTTTTT G( TTGG CTCTTCTTACTA!! AATGAAAAAGG( ( ( . U■( i( \ \ \ ( >( l I \ \ \ \ I ⁽ \c I I c■ \( . \

ATCAATTCTTTTTACTGTCCATTTAAGCTATCTTTTATAAACGTGTCTTATTTTCTA

CTCTTTTGTTTAAACTAAGAAACTATAGTATTTTGTCTAAAACAAAACATGAAA

GAACAGATTAGATCTCATCTTTAGTCTC

SEQ ID NO.:444

>AtMIR390a-OsL-Ch42

[000382] TATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA

ATATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

GAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCAC ^R

TCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC ^■ XAC CAAAAAAACAA AGTAGAGAAGAATCTGTAl I \ ⁽ . K \· ⁽ .( . \ \ \ m i I

TTGGCTCTTCTTACTi

^TGAAAAAGGCCGAGGCAAAACGCCTAAAATCACTTGAGAATCAATTCTTTTTA

:TGTCCATTTAAGCTATCTTTTATAAACGTGTCTTATTTTCTATCTCTTTTGTTTAA CTAAGAAACTATAGTATTTTGTCTAAAACAAAACATGAAAGAACAGATTAGAT

CTCATCTTTAGTCTC

SEQ ID NO.:445 >AtMIR390a-Ft

[000383] rATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA TATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

AACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCACl

TCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC ACCCAAAAAAACAA jAGTAGAGAAGAATCTGTAi I K id I I \ I \ ¾ id \ \( . M idi

|ATGATGATCACATTCGTTATCTATTTTTTGG ( K I I I I i n , \ l \ \ \( \ GCTCTTCTTACT! \CAATGAAAAAGGCCGAGGGAAAACGCG 1 AAAA ^' l CAC 1 1 GAC

GAACAGATTAGATCTCATCTTTAGTCTC

SEQ ID NO.:446 >AtMIR390a-OsL-Ft

[000384] TATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA TATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

GAACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCACT

rCCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

AACCCAAAAAAACAA JA AGGT TAAGGA AG GA AA AG GA AA AT ^' I C CTTGGTTA Al| I K id I 1 \ l \ \ \C it I \ \ ⁽ , U iLl

ITCGAAATCAAACTAGC PTGGCTCTTCTTAC1¾

AATGAAAAAGGCCGAGGCAAAACGCCTAAAATCACTTGAGAATCAATTCTTTTT

ACTGTCCATTTAAGCTATCTTTTATAAACGTGTCTTATTTTCTATCTCTTTTGTTTA

AACTAAGAAACTATAGTATTTTGTCTAAAACAAAACATGAAAGAACAGATTAGA

rCTCATCTTTAGTCTC SEQ ID NO.:447

>A f M I R390a -Tri ch

[000385] TATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA

TATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

AACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCAC 1

^' CCATCTTTTTAGCTTCACTATCT TCTATA ΑΊ CGG I ^' Γ Γ Ι ΑΊ C 1 Ύ I CTC I AAG I CAC

GCTCTTCTTACTE CAATGAAA.-XAGGCCGACiCiCAAAACGCC ΑΑΑΑ IX, ^" AC I I GAG

1 ATCAATTCTTTTTACTGTCC AT rTAAG ATCT I ^' TTATAAACGTG ΊΊ ΑΤΪ Γ 1X

E TCTCTTTTGTTTAAACTAAGAAACTATAGTATTTTGTCTAAAACAAAACATGAA

GAACAGATTAGATCTCATCTTTAGTCTC

SEQ ID NO.:448

>AtMIR390a-OsL-Trich

[000386] TATAGGGGGGAAAAAAAGGTAGTCATCAGATATATATTTTGGTAAGAAA

TATAGAAATGAATAATTTCACGTTTAACGAAGAGGAGATGACGTGTGTTCCTTC

AACCCGAGTTTTGTTCGTCTATAAATAGCACCTTCTCTTCTCCTTCTTCCTCAC I

CCATCTTTTTAGCTTCACTATCTCTCTATAATCGGTTTTATCTTTCTCTAAGTCAC

AACCCAAAAAAACAA [AGTAGAGAAGAATCTGIV

TGAAAAAGGCCGAGGCA,\AACGCCT.\AAATCAC r rCiAC]AATCAATTCTTTTTA

CTGTCCATTTAAGCTATCTTTTATAAACGTGTCTTATTTTCTATCTCTTTTGTTTAA

CTAAGAAACTATAGTATTTTGTCTAAAACAAAACATGAAAGAACAGATTAGAT

CTCATCTTTAGTCTC

SEQ ID NO.:449

[000387] Protocol to clone amiRNAs in Bsal/ccdB-b&sed ('B/c') vectors containing the OsMIR390 precursor.

[000388] Notes .-Available OsMIR390 B/c vectors are listed in Table I at the end of this protocol. [000389] -OsMJR390-B/c~based vectors must be propagated in a ccdB resistant E. coli strain such as DB3.1.

[000390] -Alternatively, Bsa/ digestion of the B/c vector and subsequent ligation of the amiRNA oligonucleotide insert can be done in separate reactions

3.1. Oligonucleotide annealing

[000391] -Dilute sense oligonucleotide and antisense oligonucleotide in sterile H20 to a final concentration of 100 μΜ.

[000392] -Prepare Oligo Annealing Buffer: [000393] 60 mM Tris-HCl (pH 7.5) [000394] 500 mM NaCl [000395] 60 mM MgCl ₂ [000396] 10 mM DTT

[000397] Note: Prepare 1 ml aliquots of Oligo Annealing Buffer and store at -20° C. [000398] -Assemble the annealing reaction in a PCR tube as described below: [000399] Forward oligonucleotide (100 μΜ) 2 μΐ, [000400] Reverse oligonucleotide (100 μΜ) 2 μΐ.

[000401] Oligo Annealing Buffer 46 \i

[000402] Total volume 50 \iL

[000403] The final concentration of each oligonucleotide is 4 μΜ. [000404] -Use a thermocycler to heat the annealing reaction 5 min at 94°C and then cool down (0.05°C/sec) to 20°C.

[000405] -Dilute the annealed oligonucleotides just prior to assembling the digestion-ligation reaction as described below:

[000406] Annealed oligonucleotides 3 uL

[000407] dBbO 37 μΐ,

[000408] Total volume 40 μΐ,

[000409] The final concentration of each oligonucleotide is 0.15 μΜ.

[000410] Note: Do not store the diluted oligonucleotides.

3.2. Digestion-ligation reaction

[000411] - Assemble the digestion-ligation reaction as described below:

[000412] B/c vector (x ug/uL) Y μΐ, (50 ng)

[000413] Diluted annealed oligonucleotides 1 μΐ, [000414] lOx T4 DNA ligase buffer 1 μΐ,

[000415] T4 DNA ligase (400 U/pL) 1 μΐ,

[000416] Bsal (10U/ xL, NEB) 1 uL

[000417] dHaO to 10 yL

[000418] Total volume 10 pL

[000419] Prepare a negative control reaction lacking Bsal.

[000420] -Mix the reactions by pipetting. Incubate the reactions for 5 minutes at 37°C.

3.3. E.coli transformation and analysis of transformants [000421] -Transform 1-5 ul of the digestion-ligation reaction into an E. coli strain that doesn't have ccdB resistance (e.g. DH10B, TOP10, ...) to do counter-selection.

[000422] -Pick two colonies/construct, grow LB-Kan (100 mg/ml) cultures and purify plasmids.

[000423] -Sequence with appropriate primers: M13-F

(CCCAGTCACGACGTTGTAAAACGACGG) SEQ ID NO.:450 and M13-R

(CAGAGCTGCCAGGAAACAGCTATGACC) SEQ ID NO.:451 for pENTR-based vectors; attBl (ACAAGTTTGTACAAAAAAGCAGGCT) SEQ ID NO.:452 and attB2

(ACCACTTTGTACAAGAAAGCTGGGT) SEQ ID NO.:453 primers for pMDC32B-, pMDC123SB- or pH7WG2B-based vectors).

[000424] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure specifically described herein. Such equivalents are intended to be encompassed within the scope of the following claims.

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