BACKGROUND OF THE INVENTION Multicellular organisms need to control proliferation of pluripotent stem cells, also referred to as meristematic cells in the apices, cambium, and pericycle of flowering plants. Because organ differentiation of plants is predominantly postembryonic and does not involve cell migration, plant stem cells need to be controlled by short-and long-distance signals to achieve equilibrium between cell proliferation and differentiation. The role of short- distance signaling in plant development has been extensively researched, and some of the key genes involved have been identified (Fletcher, 2002, Annu.

Rev. Plant Biol. 53 45; Becraft, 2002, Annu. Rev. Cell Dev. Biol. 18 163; Takayama and Sakagami, 2002, Curr. Opin. Plant Biol. 5 382; Clark et al, 1997, Cell 89,575 ; Trotochaud et al, 2000, Science 289 613; Spaink, 2000, Annual Review of Microbiology 54 257; Endre et a/., 2002, Nature 417 962; Stracke et

a/., 2002 Nature 417 959). Legume nodulation involves regulation via short and long distance signals and provides a useful model for investigating plant cell proliferation and differentiation.

Legume nodulation is important in supplying nitrogen to ecological and agricultural systems. Modern agriculture methods use nitrogen fertilizer to improve crop yield. However, nitrogen fertilisers account for a substantial cost of crop production. Moreover large-scale production of artificial nitrogen fertilizer may negatively impact upon the local environment as well as the global nitrogen cycle. Producing nitrogen fertilizer requires the consumption of considerable amounts of fossil fuels and the generation of greenhouse gases.

Also, while nitrogen fertilizer increase crop yields, crops do not capture all of the applied fertilizer and the remainder runs off to contaminate rivers and other waterways. Accordingly, genetically engineering nodulation and nitrogen fixation in plants including commercial crops such as wheat, rice and maize and further modifying nodulation and nitrogen fixation in legumes could have great benefits for the environment on a global scale.

Nodule meristems form in response to mitogenic signals from symbiotic bacteria called rhizobia (Spaink, 2000, Annual Review of Microbiology 54 257), but nodule proliferation is restricted by autoregulation of nodulation (AON) (Pierce and Bauer, 1983, Plant Physio. 73 286; Gresshoff and Delves, 1986, Plant Gene Research 3 159; Carroll et al, 1985a, Proc. Natl.

Acad. Sci. USA 82 4162; Carroll et al, 1985b, Plant Physiol. 78 34). Phenotypic mutations affecting nodule meristems have been identified, including mutations that confer supernodulation as a result of a defect in AON. Allelic

supernodulating (nts) mutants of soybean were first isolated by EMS (ethylmethane sulfonate) mutagenesis (Carroll et al, 1985a, Proc. Natl. Acad.

Sci. USA 82 4162; Carroll et a/, 1985b, Plant Physiol. 78 34; Delves et al, 1988, Journal of Genetics 67 1). These mutants altered at the NTS-1 locus also develop more lateral roots, leading to a bushy root system in the absence of nodulation, and reduced root growth in the presence of prolific nodulation.

Subsequently, additional mutations in the NTS-1 locus were induced chemically or by fast neutrons (Gremaud and Harper, 1989, Plant Physiol 89169; Akao and Kouchi, 1992, Soil Sci Plant Nutr 38 183; Men et al., 2002, Genome Letters 3 147). Isolation of mutants in other legumes confirmed the generality of AON (Postma et al, 1988, J. Plant Physiol. 132 424; Park and Buttery, 1988, Can. J.

Plant Sci. 68 199; Wopereis et al., Plant Journal 23 97), and reciprocal grafting of supernodulating and wild-type genotypes showed that long-distance signaling was involved and that the leaf genotype controlled proliferation of nodule primordia (Delves et al, 1992, Plant, Cell and Environm. 15 249; Francisco and Harper, 1995, Plant Sci 107 167; Delves et al., 1986, Plant Physio. 82 588).

Although phenotypic supernodulating plants are known, the molecular or biochemical structure and function of NTS-1 remained elusive.

Accordingly, a gene (s) and protein (s) involved in long distance cellular regulation and supernodulation was unknown prior to the present invention.

SUMMARY OF THE INVENTION It is an object of the invention to provide an alternative or improvement to the abovementioned background art.

The inventors have surprisingly found that autoregulation of nodulation (AON) is controlled at least in part by the receptor-like protein kinase GmNARK (Glycine max nodule autoregulation receptor kinase). GmNARK expression in the leaf unexpectedly has a major role in long-distance communication with nodule and lateral root primordia. GmNARK mutants described herein are characterised by changes in root development, for example increased lateral root formation and supernodulation. This finding has led to new possibilities of producing transgenic plants with potential for inducing nodulation in plants that do not normally form nodules, eg. wheat, rice and other cereal grains, increasing nodulation in plants that normally form nodules, eg legumes, and controlling root architecture by regulating lateral root formation and tap root development. Also, the identification of the role of GmNARK, and mutants thereof, in regulating root development, eg. lateral root formation and supernodulation (improved nitrogen fixation), has led to methods for identifying plants with these phenotypes. Further, the isolated GmNARK promoter allows for a means for controlling nucleic acid expression in plants, in particular in cases wherein expression is desired in tissues other than shoot apical meristem or nodules.

In a first aspect, the invention provides an isolated GmNARK mutant protein kinase comprising at least one mutation of at least one amino acid residue of a GmNARK protein kinase.

Preferably, the GmNARK protein kinase comprises an amino acid sequence as set forth in SEQ ID NO: 1.

In one form, the isolated GmNARK mutant protein kinase

comprises at least one amino acid deletion.

The at least one amino acid deletion may comprise a truncation.

The isolated at least one amino acid deletion may be within a kinase domain of the GmNARK protein kinase.

Alternatively, or in addition, the at least one amino acid deletion may be located within an extracellular domain of the GmNARK protein kinase.

Preferably, the at least one amino acid deletion is selected from the group consisting of: a non-sense mutation at amino acid residue Q106, a non-sense mutation at amino acid residue K115, a non-sense mutation at amino acid residue K606, a non-sense mutation at amino acid residue Q920 and an amino acid substitution at amino acid residue 837.

In another form, the isolated GmNARK mutant protein kinase comprises at least one amino acid substitution.

The amino acid substitution may replace a valine at amino acid residue 837 with a different amino acid.

Preferably, the different amino acid is alanine.

In a second aspect, the invention provides an isolated fragment or variant of the isolated GmNARK mutant protein kinase of the first aspect.

The isolated GmNARK mutant protein kinase of the first and second aspects may be characterised by a reduction or elimination of kinase activity when compared with the GmNARK protein.

In a third aspect, the invention provides an isolated protein comprising at least 70% amino acid sequence identity with a GmNARK protein kinase comprising an amino acid sequence as set forth in SEQ ID NO: 1,

wherein said isolated protein is characterised by at least one analogous mutation as described above.

Preferably, the isolated protein is characterised by a reduction or elimination of kinase activity when compared with a corresponding non-mutated isolated protein.

In a fourth aspect, the invention provides an isolated protein comprising an amino acid sequence selected from the group consisting of: (i) amino acid sequence as set forth in SEQ ID NO. 6; (ii) amino acid sequence as set forth in SEQ ID NO. 7; (iii) amino acid sequence as set forth in SEQ ID NO. 8; (iv) amino acid sequence as set forth in SEQ ID NO. 9; and (v) amino acid sequence as set forth in SEQ ID NO. 10.

In a fifth aspect, the invention provides an isolated protein comprising an amino acid sequence at least 70% identical with the abovementioned isolated protein of the fourth aspect.

In a sixth aspect, the invention provides an isolated nucleic acid encoding an isolated GmNARK mutant protein kinase according to the first and second aspects.

In a seventh aspect, the invention provides an isolated nucleic acid encoding an isolated protein according to the third, fourth or fifth aspects.

The invention also relates to an isolated nucleic acid fragment of the isolated nucleic acid of the preceding aspects.

In an eighth aspect, the invention provides an isolated nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NOS: 11 and 12.

In a ninth aspect, the invention provides an isolated nucleic acid comprising a nucleotide sequence at least 70% identical with the isolated nucleic acid of the eighth aspect.

In one form, the invention provides an isolated nucleic acid fragment of the isolated nucleic acid of the eighth and ninth aspects, wherein said isolated nucleic acid fragment comprises at least one regulatory element.

In a tenth aspect, the invention provides a genetic construct comprising at least one of the isolated nucleic acids of any one of the preceding aspects.

The genetic construct may comprise a vector.

The vector may comprise an expression vector.

The genetic construct in another form may comprise a chimeric gene wherein the isolated nucleic acid is the isolated nucleic acid according to the eighth or ninth aspects operably linked to a transcribable nucleic acid.

In one form, the transcribable nucleic acid is any one or more of the isolated nucleic acids according to the preceding aspects.

In an eleventh aspect, the invention provides a host cell or tissue comprising the isolated nucleic acid of any one of the sixth to ninth aspects.

In a twelfth aspect, the invention provides a host cell or tissue comprising a genetic construct according to the tenth aspect.

Preferably, the host cell or tissue is a plant cell or tissue.

Preferably, the plant cell or tissue is transformed with the isolated nucleic acid or genetic construct.

The plant cell or plant tissue may be obtained from a dicotyledon.

Preferably, the plant cell or tissue is a legume.

More preferably, the legume is Glycine max, Glycine soja, Lotus japonicus or Lotus comiculatus.

The plant cell or tissue may be obtained from a monocotyledon.

Preferably, the monocotyledon is a wheat, rice, barley or maize.

In a thirteenth aspect, the invention provides a transgenic plant comprising an isolated nucleic acid according to any one of the sixth to ninth aspects.

In one form, the transgenic plant comprises a genetic construct according to the tenth aspect.

The genetic construct may be an expression vector.

The transgenic plant may be a dicotyledon.

Preferably, the dicotyledon is a legume.

More preferably, the legume is Glycine max, Glycine soja, Lotus japonicus or Lotus comiculatus.

The transgenic plant may be a monocotyledon.

Preferably, the monocotyledon is a wheat, rice, barley or maize.

In a fourteenth aspect, the invention provides a plant cell, fruit, leaf, root, shoot, flower, seed, cutting and other reproductive material useful in sexual or asexual propagation, progeny plants inclusive of F1 hybrids, male- sterile plants and all other plants and plant products obtainable from a transgenic plant according to the thirteenth aspect.

In a fifteenth aspect, the invention provides a method of producing a transgenic plant including the step of transforming a plant cell or tissue with

an isolated nucleic acid according to any one of the sixth to ninth aspects.

Preferably, the isolated nucleic acid is an expression construct according to the tenth aspect.

Preferably, the transgenic plant is a dicotyledon.

More preferably, the dicotyledon is a legume.

Even more preferably, the legume is Glycine max, Glycine soja, Lotus japonicus or Lotus comiculatus.

The transgenic plant may be a monocytyledon.

In a sixteenth aspect, the invention provides a method for detecting GmNARK protein in a biological sample suspected of comprising said GmNARK protein, said method including the steps of:- (a) isolating the biological sample; (b) combining with the isolated biological sample at least one antibody or antibody fragment capable of binding with the GmNARK protein; and (c) detecting antibody or antibody fragment bound to the GmNARK protein, which indicates the presence of GmNARK.

The GmNARK protein may comprise a GmNARK mutant and antibody binding thereto indicates presence of said GmNARK mutant in said biological sample.

In one form, the antibody is capable of binding a kinase domain of GmNARK.

In one form, lack of antibody binding to the kinase domain of

GmNARK indicate presents of a GmNARK mutant.

In a seventeenth aspect, the invention provides a method for detecting GmNARK nucleic acid in a biological sample suspected of comprising said GmNARK nucleic acid, said method including the steps of:- (I) isolating the biological sample ; (II) combining with the isolated biological sample at least one isolated GmNARK nucleic acid of any one the sixth to ninth aspects; and (III) detecting a sample nucleic acid hybridised to said at least one isolated GmNARK nucleic acid, which indicates the presence of GmNARK nucleic acid.

In one form, the GmNARK nucleic acid is a nucleic acid encoding a GmNARK mutant and hybridisation thereto by said sample nucleic acid indicates presence of said GmNARK mutant in said biological sample.

In one form, the isolated GmNARK nucleic is capable of hybridising with a nucleic acid comprising a nucleotide sequence encoding a kinase domain of GmNARK.

In an eighteen aspect, the invention provides a method for detecting GmNARK protein kinase enzymatic activity in a biological sample suspected of comprising said GmNARK protein, said method including the steps of:- (a) isolating the biological sample; (b) combining with the isolated biological sample a substrate for the GmNARK protein kinase; and

(c) detecting kinase activity, which indicates the presence of kinase active GmNARK.

In one form, kinase activity less than that of wild type GmNARK kinase activity indicates presence of a GmNARK mutant in said biological sample.

In a nineteenth aspect, the invention provides a method for identifying a plant defective in autoregulation of nodulation, including the steps of detecting a GmNARK mutant according to any one of the sixteenth, seventeeth or eighteenth aspects.

Preferably, the plant is characterised by supernodulation.

More preferably, the plant is characterised by increased nitrogen fixation.

Preferably, the biological sample is selected from the group consisting of: leaf, stem, root, root tip, apical meristem, seed, nodule and flower.

In a twentieth aspect, the invention provies a kit for detecting GmNARK or GmNARK mutant in a biological sample comprising one or more isolated proteins according to the abovementioned aspects.

In a twenty first aspect, the invention provides a kit for detecting GmNARK or GmNARK mutant in a biological sample comprising one or more isolated nucleic acids according to the abovementioned aspects.

In a twenty second aspect, the invention provides a kit for detecting GmNARK or GmNARK mutant in a biological sample comprising one or more reagents for detecting kinase activity.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES In order that the invention may be readily understood and put into practical effect, preferred embodiments will now be described by way of example with reference to the accompanying figures and tables wherein like reference numerals refer to like parts and wherein: FIG. 1A. Photograph of wild-type and supernodulation (AON- defective) phenotypes.

FIG. 1B. Genetic (G. max nts246 x G. soja CPI 100070 mapping population) and physical maps of soybean NTS-1 region. Deletion in FN37 mutant is shown in light shading. Flanking markers pUTG132a and UQC-IS1 were used as anchor points to isolate BAC clones and build contigs. Dashed lines show microsynteny to Arabidopsis chromosomes 2 and 4.

FIG. 1C Putative protein structure of GmNARK : SP = signal peptide. LRR = leucine-rich repeats (circles); TM = transmembrane domain.

Mutations resulting in either nonsense or amino acid change, the location of the original AFLP product (UQC-IS5) derived from BAC75M10 and the intron (arrow) are shown.

FIG. 1D. GmNARK (GmClv1B) and GmClv1A are orthologues of CLAVATA1. The phylogenetic tree was constructed for a selection of related proteins using ClustalX version 1.81 and Bootstrap analysis. All of these predicted proteins are from Arabidopsis except where otherwise noted. The three most closely-related receptor-like kinases to CLAVATA1 in Arabidopsis, designated RLK-A (GI : 15239123), RLK-B (GI : 15229189) and RLK-C (GI : 15235366), fall into a phylogenetic clade separate from that containing

CLAVATA1, GmNARK and GmCLV1A. Two receptor-like kinases designated RLK-D (At2g21480 in B) and RLK-E (At4g39110 in B) corresponding to the Arabidopsis regions syntenic with the soybean GmNARK region (B), were surprisingly more distantly related to GmNARK. Other distantly related proteins included in the phylogenetic analysis were CLAVATA2 (G1 : 6049563), the brassinosteroid receptor BR) 1 (Gi : 2392895), the auxin response protein PINOID (Gl : 7208442), the tomato disease resistance proteins CF-2 (G) : 1184075) and HCR2-5D (Gt : 3894393), and the Medicago sativa NORK (G) : 21698781), the M. truncatula NORK (G) : 21698783) and the Lotus japonicus SYMRK (G1 : 21622628) proteins required for root nodule formation. The scale bar is an indicator of genetic distance based on branch length.

FIG. 2A: Genomic Southern analysis using GmNARK probe on G. soja CP1100070 (CPI), G. max PS55, G. soja P1468. 397 (P1468), and mutant FN37. Arrows indicate missing fragments in the deletion mutant. The arrow head indicates duplicated fragment in CPI, PS55 and P1468, but only a single fragment in FN37.

FIG. 2B. Genomic PCR of several soybean genotypes, BAC75M10 and BAC112J23 with GmNARK-specific and GmCLV1A-specific oligonucleotide primers.

FIG. 2C. Non-quantitative RT-PCR analysis of expression of GmNARK in G. soja (WT) and FN37 with GmNARK-specific 3'UTR and actin primers. PCR template was prepared with (+) and without (-) reverse transcriptase.

FIG. 2D. Quantitative RT-PCR analysis of expression with

GmNARK-specific 3'UTR primers in Bragg, nts1007 and nts382 leaves and shoot apical meristem (SAM) regions from nodulated plants. Values are fold increase ratios from two PCR replicates, normalization against actin actin2/5, relative to wild-type meristems. Average SE was less than 1 %.

FIG. 2E. SAM regions of Bragg and nts1007 plants showing similar morphology of the tunica and central zone as well as alternating leaf primordia. Bar= 65 microns.

FIG. 3. Soybean mutant FN37 obtained after fast neutron mutagenesis confers shoot-controlled supernodulating phenotype. Grafting analysis of supernodulation in FN37. Average nodule number for ungrafted and grafted combinations was determined from at least four plants. Statistical analysis was done by ANOVA using version 8.0 of Statistical Analysis Software (SAS Institute Inc., Cary, NC, USA). Means represented by the same letter are not significantly different at P = 0005. WT = wild-type G. soja P1468. 397 (ungrafted control) ; WT/\NT = wildtyp scion over wild-type stock; WT/m = wild- type scion over FN37 stock; m = FN37 (ungrafted control); m/m = FN37 scion over FN37 stock; FN/WT = FN37 scion over wild-type stock.

FIG. 4 Genetic and physical maps around the NTS-1 locus of soybean. Originally developed RFLP/PCR marker pUTG132a and an AFLP marker UQC-IS1 were used to start the walk from the north and south of the gene. Eighteen BACs were identified using pUTG132a as a probe. Three BACs spanning the"south"-side contig have been orientated relative to the NTS-1 locus with the deletion mutant FN37 and F2 recombinants between pUTG132a and NTS-1. Similarly, 15 BACs were identified using UQC-IS1 as a probe, and

3 clones constitute the"north"-side contig. The genetic map spans NTS-1, with UQG-AS1 and UQC-IS3 mapped 0.9 cM south and 0.6 cM north of the gene, respectively (UQnts mapping population). A putative location of the deletion is shown on the right, and probes located outside the deletion are indicated with an"X"on the FN37 chromosome.

FIG. 5. Example of the BAC fingerprinting. Clones were digested with Hindi) and separated in 1% agarose overnight. The gel was stained with SYBRe Gold. PBeloBAC11 vector band as well as unique fingerprint fragments in lanes 3 and 5 are shown by arrows. The most informative BACs (lanes 3 and 5 in this experiment) then were chosen and assembled into overlapping contigs.

FIG. 6A. PCR analysis of deletion mutant FN37 with probes mapped close to NTS-1. Schematic representation of four PCR probes used for the analysis. The T7 probe was derived from the"south"end of the BAC156F11 ; ST7 is the"south"end of BAC17107. The order of the probes relative to NTS-1 has been established by previous mapping (Men and Gresshoff, 2001, Plant J. Physiol. 158 999).

FIG. 6B. Agarose gel analysis of amplified probes. DNA in lanes : m = FN37; WT D wild-type G. soja P1468. 397; EMS = nts382 mutant ;-= negative (no DNA) control.

FIG. 7A. Southern blot analysis of FN37 and wild-type soybean DNA. Two radiolabeled probes were used simultaneously. One probe was deleted from the FN37 DNA based on a PCR test (UQG-AS2). The other was PCR positive on FN37 DNA (ST7 end of BAC17107). The absence of hybridization signal in FN37 DNA after Xbal digestion is probably due to the

small molecular size of the corresponding fragment. DNA in lanes : WT = G. soja P1468. 397; m = FN37.

FIG. 7B. Southern blot analysis of FN37 and wild-type soybean DNA. A radiolabeled UQC-IS4 probe (homologous to soybean ATPase, accession number AW756248) was used on genomic DNA digested with several restriction endonucleases. Arrows indicate altered restriction fragments caused by the deletion break point.

FIG. 8. Protein phosphorylation activities of the GmNARK kinase domain produced by heterologous prokaryotic system. Gel (A) is an SDS- PAGE showing protein migration pattern stained with coomassie blue. Gel (B) is a phosphorimager scan of gel (A) showing protein phosphorylation via incorporation of [Y-32P] ATP. Gel (C) is a Western blot of a replicate gel of gel (A), reacted with anti-His antibodies. Lane: 1 = E. coli crude protein extract (negative control), 2 = chromatography fixation aliquot not comprising the GmNARK kinase domain (negative control), 3 = crude protein extract of E. coli expressing GmNARK kinase domain, 4 = chromatography fraction comprising the GmNARK kinase domain, 5 = histidine-tag containing protein (Western blot positive control) and 6 = molecular weight marker proteins.

FIG. 9: amino acid sequence alignment of GmNARK (SEQ ID NO: 1), GmCLV1A (SEQ ID NO: 2), HAR1 (SEQ ID NO: 3), CLV1 (SEQ ID NO: 4) and consensus sequence. SP = Signal peptide; PC = Paired cysteines; TM = Transmembrane domain. Roman numerals refer to domains of a protein kinase.

FIG. 10: Nucleotide acid sequence of GmNARK promoter (SEQ

ID NO: 11) with predicted cis-regulatory elements on GmNARK. The translational start site (TSS) is indicated as white text on black background.

Numbers indicate the positions relative to the TSS. Three putative TATA boxes are underlined in bold and labeled as TATA1 (-43 to-49), TATA2 (-114 to-121), and TATA3 (-159 to-163) respectively. The putative negative regulatory region (NRR;-301 to-308) found both in GmNARK and LjHAR1 promoter is written on grey background. Six motifs of GmNARK promoter in silico identified by MEME are indicated as white text on grey background and labeled as Motif1 (-94 to- 106), Motif2 (-548 to-558), Motif3 (-712 to-730), Motif4 (-966 to-1001), Motif5 (- 1642 to-1679) and Motif6 (-1713 to-1762).

FIG. 11. Nucleotide sequence of a GmNARK promoter fragment of the nucleic acid shown in FIG. 10 (SEQ ID NO: 12) with regulatory regions indicated.

FIG. 12. Differential tissue expression of GmNARK RNA at different times; 16 = 16 days after inoculation and planting ; 28 = 28 days. FTF = first trifoliate leaf, NOD = nodule, RT = root tip region, SAM = Shoot apical meristem, STF = second trifoliate leaf, TTF= third trifoliate leaf and UF = Unifoliate leaf.

FIG. 13. Expression levels of ribosomal RNA (rRNA) in the same tissues and times as shown in FIG. 12.

FIG. 14. Expression levels of Actin 1 RNA in the same tissues and times as shown in FIG. 12.

FIG. 15. Quantitation of GmNARK RNA in the indicated plant samples relative to RT16 (root tip at day 16) normalised to actin as shown in

FIGS. 10 and 12.16 = 16 days after inoculation and planting ; 28 = 28 days; NOD = nodule ; YNOD = young nodule ; RT = root tip region; UF = Unifoliate leaf ; FTF = first trifoliate leaf ; STF = second trifoliate leaf and TTF= third trifoliate leaf ; SAM = Shoot apical meristem.

FIG. 16. Quantitation of GmNARK RNA in the indicated plant samples relative to RT16 (root tip at day 16) normalised to rRNA as shown in FIGS. 10 and 11.

FIG. 17. Diagram of 5'deletions of the GmNARK promoter. ATG indicates the first codon. The number is indicated in base pairs relative to the translation start site (+1). All GmNARK promoter constructs are fused to a GUS gene containing intron. M1, M2, M3, M4, M5 and M6 represent potential Motif1, Motif2, Motif3, Motif4, Motif5 and Motif6 respectively. NRR represents putative negative regulatory region TABLE 1: Mutant alleles of GmNARK, 1 nts382, nts1007, nts246, nts1116 and en6500 are allelic as F1 hybrids show increased nodulation compared to wild type (Delves et al, 1998, Journal of Genetics 67,1 ; Akao and Kouchi, 1992, Soil Sci Plant Nutr 38,183). PS55 is an Australian soybean variety into which nts1007 mutant allele was introgressed. 2 first description of allele. 3 supernodulating F2 plants derived from G. max nts246 x G. soja CPI 100070. * = nonsense termination mutation.

TABLE 2. Quantitative Real Time PCR analysis of GmNARK expression. R1 = replicate 1; R2 = replicate 2; Ave = mean average between R1 and R2; Ct = cycle threshold number; ACt = difference between GmNARK and actin values ; AACt = difference between each individual sample and the

reference value (Bragg SAM). Ratios are calculated using formula 2-AACt. For each sample, a no RT enzyme reaction was performed also in two replicates Table 3).

TABLE 3. Ct values for the non-reverse transcriptase control. R1 = replicate 1; R2 = replicate 2.

TABLE 4. GmNARK promoter elements ; symbols in table are those used in the PLACE Database; R = A or G, W = A or T.

TABLE 5. GmNARK promoter element groupings.

TABLE 6. Values for quantitation of GmNARK RNA used in FIGS. 11-13.

TABLE 7. Values for quantitation of GmNARK RNA in the indicated plant samples relative to RT16 (root tip at day 16) normalised to rRNA ; data used in FIG. 14.

TABLE 8. Values for quantitation of GmNARK RNA in the indicated plant samples relative to RT16 (root tip at day 16) normalised to actin; data used in FIG. 15.

DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all technical and scientific terms used herein have a meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any method and material similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.

For the purpose of the present invention, the following terms are defined below.

Regulation of Cellular Proliferation and Induction of Supernodulation

Proliferation of legume nodule primordia is controlled by shoot- root signaling known as autoregulation of nodulation (AON). Mutants defective in AON show supernodulation and increased numbers of lateral roots. As described herein, it has been discovered that AON is controlled by the receptor- like protein kinase GmNARK (Glycine max nodule autoregulation receptor kinase). GmNARK is similar to Arabidopsis CLAVATA1 (CLV1), which functions in a protein complex controlling stem cell proliferation by short- distance signaling in shoot apices. However, surprisingly GmNARK expression in the leaf has a major role in long-distance communication with nodule and lateral root primordia.

The discovery that GmNARK, a receptor-like protein kinase, is part of the signaling circuit for AON creates the possibility of characterizing associated long distance signals in plant development as exemplified by control of root development and nodulation.

GmNARK and GmNARK mutants The present invention provides a"GmNARK mutanY', which comprises a modification to GmNARK, GmNARK related protein or respective fragments thereof. A GmNARK mutant may comprise one or more mutations as described herein, including deletion of one or more amino acids (inclusive of a truncation at the N-terminus and/or C-terminus; and/or one or more internal in-frame deletions), amino acid substitution and other modifications to a GmNARK protein, related protein or fragment thereof. The modification may occur at any part of the protein, including the extracellular domain, transmembrane domain and/or kinase domain.

In one form, the invention provides isolated peptides comprising respective amino acid sequences as set forth in SEQ ID NOS : 6-10, and shown in FIG. 1C, which are examples of GmNARK mutants of the invention. These examples of GmNARK mutants are characterised as either non-sense mutations or amino acid substitutions. Non-sense mutations include stop codons at residues: Q106, K115, K606 and Q920. An amino acid substitution mutant is exemplified by V837A.

Non-sense mutations are examples of an amino acid deletion.

GmNARK mutants Q106*, K115* and K606* are examples of GmNARK mutants comprising a deletion in the extracellular domain of GmNARK.

GmNARK mutants V837A and Q920 are examples of GmNARK mutants comprising respective amino acid substitution and deletion in the kinase domain.

Amino acid sequences for respective examples of GmNARK mutants comprise the following peptides: (i) peptide comprising amino acids 1-105 (SEQ ID NO: 6); (ii) peptide comprising amino acids 1-114 (SEQ ID NO: 7); (iii) peptide comprising amino acids 1-605 (SEQ ID NO: 8); (iv) peptide comprising amino acids 1-919 (SEQ ID NO: 9); and (v) peptide comprising amino acids 1-987 wherein valine at residue 837 is substituted with alanine (SEQ ID NO: 10); wherein amino acid reside numbers are based on wild type GmNARK amino acid sequence as set forth in SEQ ID NO: 1 and shown in FIG. 9.

It will be appreciated that the invention is not limited to these

exemplified mutants and other GmNARK mutants are contemplated. Other GmNARK mutants may comprise mutations that are known in the art including those mutations described herein. Preferably, the GmNARK mutant reduces or eliminates kinase activity of GmNARK. Such mutants may delete a part of, or all of, or substitute one or more amino acids of the kinase domain with another amino acid. GmNARK mutants, such as mutant V837A, substitute an amino acid within the kinase domain. Mutations affecting kinase activity include amino acid substitutions outside of the kinase domain that may affect the folding of the protein to thereby affect the kinase activity. Mutations not located within the kinase domain may affect GmNARK binding with other proteins. For example, a mutation in the extracellular domain of GmNARK, and analogous mutation in other Leucine Rich Region-Receptor-like kinases (LRR-RLK), may prevent normal receptor dimerisation. Prevention of dimerisation may prevent proper biological activity, including proper kinase activity.

Mutations may also comprise deletions of one or more amino acids wherein the reading frame of the protein is preserved. Such deletions may be internal to the protein. A deletion or substitution of an amino acid that this phosphorylated may also influence the biological activity of the protein.

The abovementioned non-sense mutations, and other mutations, may delete part of, or the entire, kinase domain. The identified mutants result in a supernodulation phenotype with a loss or reduction in kinase activity.

For the purposes of this invention, by"isolated"is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free

from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material includes material in native and recombinant form. For example, GmNARK nucleic acid and encoded proteins, inclusive of mutants thereof, have been respectively isolated from a plant, namely soybean.

By"endogenous"nucleic acid or protein is meant a nucleic acid or protein that may be found in a native cell, tissue or plant in isolation or otherwise. An endogenous nucleic acid or protein may be a"wild type"nucleic acid or protein that is characterized by a nucleotide sequence, amino acid sequence or phenotype that is naturally-occurring, normal or non-mutated for a majority of a population.

Wild type GmNARK encodes a receptor-like protein kinase (RLK) comprising a 24 amino acid N-terminal signal peptide (MRSCVCYTLLLFIFFIWLRVATCS ; SEQ ID NO: 13), an extracellular domain comprising 19 tandem copies of a 24 amino acid leucine-rich repeat (LRR), a transmembrane domain (TRVIVIVIALGTAALLVAVTVYM ; SEQ ID NO: 14) and a C-terminal cytoplasmic kinase domain. A 49 amino acid region interrupts the LRR domain between repeats 11 and 12. Overall, the protein comprises 15 potential N-glycosylation sites.

By"protein"is also meant'polypeptide","peptide"or fragments thereof, referring to an amino acid polymer, comprising natural and/or non- natural amino acids, including L-and D-isomeric forms as are well understood in the art. For example, GmNARK may be referred to as both a protein or

polypeptide.

A"peptide"is a protein having no more than fifty (50) amino acids.

In one embodiment, a"fragmenf'includes an amino acid sequence which constitutes less than 100%, but at least 20%, preferably at least 30%, more preferably at least 80% or even more preferably at least 90% of said polypeptide.

The fragment may also include a"biologically active fragment" which retains biological activity of a given polypeptide or peptide. For example, a biologically active fragment of GmNARK comprises a fragment that is capable of regulating root development, nodulation, binding a substrate and/or comprising kinase activity. The biologically active fragment constitutes at least greater than 1% of the biological activity of the entire polypeptide or peptide, preferably at least greater than 10% biological activity, more preferably at least greater than 25% biological activity and even more preferably at least greater than 50% biological activity. As described herein, biological activity of a GmNARK mutant may be equal to or great than that of wild type GmNARK, for example as determined by a reduction or loss in autoregulation of nodulation that may be measured by number of nodules, relative percentage of nodules per root length. A biologically active fragment may also comprise one or more mutations of GmNARK that result in modified biological activity, for example increased nodulation in number and/or percentage (also referred to in some cases as"supernodulation"and"hypernodulation"). For example, supernodulation encompasses nodulation greater that of wild type, including 2 fold greater, 5 fold greater, 10 fold greater, 20 fold greater, even 30 fold greater

than wild type nodulation. Supernodulation generally refers to a loss or reduction in autoregulation of nodulation characterised by an increase in total nodule number and/or larger percentage of roots comprising nodules when a root is inoculated with an organism such as rhizobia. Preferably, a plant characterized by supernodulation comprises nodules on more than 10% of the roots, more than 30%, more than 50%, more than 75%, more than 85%, more than 90% and even more than 95% of the roots. It will be appreciated that the number of nodules and percent of nodulation depends in part on a stage of plant development that the inoculation by the organism occurs. In general, in the absence of nodulation the number of lateral roots increases leading to a bushy root system and in the presence of prolific nodulation, root growth is reduced. Generally, nodules of a supernodulating plant remain in a juvenile developmental stage and do not senesce are quickly as wild type nodules.

Accordingly, a supernodulating plant may retain nodules for a longer period of time. Further, supernodulation results in a phenotype wherein nodules may be formed even at nitrate levels that would normally inhibit nodule formation.

Accordingly, supernodulating GmNARK mutants are no longer under external regulation by nitrate levels.

A"protein kinases a term known in the art and encompasses a protein, or fragment thereof, capable of self or autophosphorlyation and/or phosphorylation of another protein, which may be a same type of protein or different type of protein. The protein kinase may be a single protein, two or more proteins (for example a dimer) and/or a complex of subunit proteins, such as insulin receptor. The protein kinase may be membrane bound, for

example, an LRR-RLK, or soluble, for example, protein kinase C. The protein kinase may be an activator or inhibitor of another protein that interacts with the protein kinase.

In another embodiment, a"fragment"is a small peptide, for example of at least 6, preferably at least 10 and more preferably at least 20 amino acids in length, which comprises one or more antigenic determinants or epitopes. Larger fragments comprising more than one peptide are also contemplated, and may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled"Peptide Synthesis"by Atherton and Shephard, which is included in a publication entitled"Synthetic Vaccines"edited by Nicholson and published by Blackwell Scientific Publications. Alternatively, peptides can be produced by digestion of a polypeptide of the invention with a suitable proteinases. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques.

As used herein,"variant"protein is a protein of the invention in which one or more amino acids have been replaced by different amino acids. A GmNARK mutant is a variant of GmNARK. A variant comprises a mutation of a protein when compared with a starting protein. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions).

Substantial changes in function are made by selecting substitutions that are less conservative. Other replacements would be non- conservative substitutions and relatively fewer of these may be tolerated.

Generally, the substitutions which are likely to produce the greatest changes in a polypeptide's properties are those in which (a) a hydrophilic residue (e. g. , Ser or Thr) is substituted for, or by, a hydrophobic residue (e. g. Leu, lie, Phe or Val) ; (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (e. g. , Arg, His or Lys) is substituted for, or by, an electronegative residue (e. g., Glu or Asp) or (d) a residue having a bulky side chain (e. g. , Phe or Trp) is substituted for, or by, one having a smaller side chain (e. g., Ala, Ser) or no side chain (e. g. , Gly).

Protein and Nucleic Acid Sequence Comparison Terms used herein to describe sequence relationships between respective nucleic acids and polypeptides include"comparison window", "sequence identity", "percentage of sequence identity"and"substantial identity".

Because respective nucleic acids/polypeptides may each comprise (1) only one or more portions of a complete nucleic acid/polypeptide sequence that are shared by the nucleic acids/polypeptides, and (2) one or more portions which are divergent between the nucleic acids/polypeptides, sequence comparisons are typically performed by comparing sequences over a"comparison window" to identify and compare local regions of sequence similarity. A"comparison window'refers to a conceptual segment of typically at least 6 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i. e., gaps) of about 20% or less as

compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (for example ECLUSTALW and BESTFIT provided by WebAngis GCG, 2D Angis, GCG and GeneDoc programs, incorporated herein by reference) or by inspection and the best alignment (i. e. , resulting in the highest percentage similarity or identity over the comparison window) generated by any of the various methods selected.

Search for potential cis-element motifs of the GmNARK promoter was performed using the homology search tool, SIGNAL SCAN program, available at the PLACE Web site (http ://www. dna. affrc. go. jp/htdocs/PLACE), incorporated herein by reference.

The ECLUSTALW program can be used to align multiple sequences. This program calculates a multiple alignment of nucleotide or amino acid sequences according to a method by Thompson, J. D. , Higgins, D. G. and Gibson, T. J. (1994). This is part of the original ClustalW distribution, modified for inclusion in EGCG. The BESTFIT program aligns forward and reverse sequences and sequence repeats. This program makes an optimal alignment of a best segment of similarity between two sequences. Optimal alignments are determined by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman.

ECLUSTALW and BESTFIT alignment packages are offered in WebANGIS GCG (The Australian Genomic Information Centre, Building J03, The University of Sydney, N. S. W 2006, Australia).

Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference.

A detailed discussion of sequence analysis can be found in Chapter 19.3 of Ausubel et al, supra.

The term"sequence identity"'is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity"is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e. g. , A, T, C, G, U) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i. e. , the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example,"sequence identity"may be understood to mean the"match percentage"calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co. , Ltd. , South San Francisco, California, USA).

A protein may share amino acid sequence similarity or identity with a same or different protein. For example, a mutation of GmNARK may substitute a different amino acid compared with the original or wild type amino acid of GmNARK. Such a mutation results in the mutant protein comprising a different amino acid sequence when compared with the original or wild type

protein. For example, in relation to one GmNARK mutant described herein, amino acid substitution at residue 837 replaces a valine with an alanine. The GmNARK mutant is no longer identical with the wild type GmNARK, but shares amino acid sequence similarity thereto.

Alternatively, a different protein may share amino acid sequence identity or similarity with GmNARK, such as CLV1A, HAR1, CLV1 and others.

It is contemplated that analogous mutations described herein for GmNARK may be introduced into other proteins having a similar amino acid sequence, such as CLV1A, HAR1 and CLV1. For example, an analogous mutation of the V837A substitution mutation in GmNARK is V831A for GmCLV1A, V836A for HAR1 and V834A for CLV1. Analogous non-sense mutation at Q106* of GmNARK is Q106* for GmCLV1A, M107 for HAR1 and A103 for CLV1 ; analogous non- sense mutation at K115* of GmNARK is K115 of GmCLV1 a, S116 of HAR1 and L112 of CLV1; analogous non-sense mutation at K606* of GmNARK is K606 of GmCLV1a, K608 of HAR1 and T604 of CLV1; and analogous non-sense mutation at Q920* of GmNARK is Q914 of GmCLV1A, Q921 of HAR1 and Q916of CLV1.

Similar mutations as described herein for GmNARK may likewise function analogously in other proteins having a similar amino acid sequence.

For example, protein kinases, in particular serine/theonine protein kinases such as membrane bound leucine-rich repeat (LRR) class of receptor-like kinases (RLKs). Such LRR-RLK comprise: SR160/CURL3, BRI1, BAK1, CLV1, HAR1/SYM78, FLS2, EXS EMS1, HAESA, ER, SERK1, SYMRK, SYM19 and NORK and others, for example as described in Dievart and Clark, 2003,

Current Opinion in Plant Biology 6 507, incorporated herein by reference. It is preferred that the proteins share at least 70%, preferably at least 90% and more preferably at least 95% sequence identity with the amino acid sequences of polypeptides of the invention, including fragments thereof.

Proteins having similar amino acid sequence, both the same protein that has been modified or mutated and a different protein, may also be referred to as a"homolog"or"orthologs", which are functionally-related polypeptides and their encoding nucleic acids, isolated from other organisms.

For example CLV1A protein isolated from Arabidopsis is an otholog of GmNARK.

With regard to protein and polypeptide variants, these can be created by mutagenising a polypeptide or by mutagenising an encoding nucleic acid, such as by random mutagenesis or site-directed mutagenesis. Examples of nucleic acid mutagenesis methods are provided in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al., supra which is incorporated herein by reference.

It will be appreciated by the skilled person that site-directed mutagenesis is best performed where knowledge of the amino acid residues that contribute to biological activity is available. For example, amino acid residues located within the kinase domain, in particular amino acids that are phosphorylated or bind substrate.

In cases where this information is not available, or can only be inferred by molecular modeling approximations, for example, random mutagenesis is contemplated. Random mutagenesis methods include chemical

modification of proteins by hydroxylamine (Ruan et al., 1997, Gene 188 35), incorporation of dNTP analogs into nucleic acids (Zaccolo et a/., 1996, J. Mol.

Biol. 255 589) and PCR-based random mutagenesis such as described in Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91 10747 or Shafikhani et a/., 1997, Biotechniques 23 304, each of which references is incorporated herein. It is also noted that PCR-based random mutagenesis kits are commercially available, such as the DiversifyT" kit (Clontech). Mutagenesis may also be induced by chemical means, such as ethyl methane sulphonate (EMS) and/or irradiation means, such as fast neutron irradiation of seeds as is common in the art and described herein.

As used herein,"derivative"polypeptides are polypeptides of the invention that have been altered, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. Such derivatives include amino acid deletions and/or additions to polypeptides of the invention, or variants thereof.

"Additions"of amino acids may include fusion of the peptide or polypeptides or variants thereof with other peptides or polypeptides. Particular examples of such peptides include amino (N) and carboxyl (C) terminal amino acids added for use as"tags". Use of an N-terminal 6X-His tag for isolating an expressed fusion polypeptide is described herein.

N-terminal and C-terminal tags include known amino acid sequences which bind a specific substrate, or bind known antibodies, preferably monoclonal antibodies. pRSET B vector (ProBondTM ; Invitrogen Corp. ) is an example of a vector comprising an N-terminal 6X-His-tag which binds

ProBond resin.

Other derivatives contemplated by the invention include, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide or polypeptide synthesis and the use of cross linkers and other methods which impose conformational constraints on the polypeptides, fragments and variants of the invention. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate ; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4; reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; and trinitrobenzylation of amino groups with 2,4, 6-trinitrobenzene sulphonic acid (TNBS).

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, by way of example, to a corresponding amide.

The guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3- butanedione, phenylglyoxal and glyoxal.

Sulphydryl groups may be modified by methods such as performic acid oxidation to cysteic acid; formation of mercurial derivatives using 4- chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate ; 2-chloromercuri- 4-nitrophenol, phenylmercury chloride, and other mercurials; formation of a

mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide ; carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides or by oxidation with N-bromosuccinimide.

Tyrosine residues may be modified by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

The imidazole ring of a histidine residue may be modified by N- carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, use of 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6- methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids.

Proteins in relation to the invention such as those exemplified in FIG. 9 and GmNARK mutants comprising an amino acid sequence as set forth in SEQ ID NOS: 6-10 (inclusive of fragments, variants, derivatives and homologs in general) may be prepared by any suitable procedure known to those of skill in the art.

For example, the polypeptide may be prepared by a procedure including the steps of: (i) preparing an expression construct which comprises a

recombinant nucleic acid of the invention, operably linked to one or more regulatory nucleotide sequences, for example a T7 promoter; (ii) transfecting or transforming the expression construct into a suitable host cell, for example E. coli ; and (iii) expressing the polypeptide in said host cell.

Preferably, the recombinant nucleic acid of the invention encodes a protein comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 1-10, or fragment thereof, more preferably as set forth in SEQ ID NOS: 6- 10.

Recombinant proteins may be conveniently expressed and purified by a person skilled in the art using commercially available kits, for example"ProBondT""Purification System"available from Invitrogen Corporation, Carlsbad, CA, USA, herein incorporated by reference. Alternatively, standard molecular biology protocols may be used, as for example described in Sambrook, et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), incorporated herein by reference, in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. 1995-1999), incorporated herein by reference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. 1995-1999) which is incorporated by reference herein, in particular Chapters 1,5, 6 and 7. Such recombinant expressed proteins may be useful as antigens for preparation of an antibody capable of binding wild type GmNARK and/or mutated forms

thereof as described hereinafter.

Nucleic Acids The term"nucleic acid"as used herein designates single or double stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA. A nucleic acid may be native or recombinant and may comprise one or more artificial nucleotides, e. g. nucleotides not normally found in nature. Nucleic acid encompasses modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and methylcytosine).

The term"isolated nucleic acid"as used herein refers to a nucleic acid subjected to in vitro manipulation into a form not normally found in nature.

Isolated nucleic acid includes both native and recombinant (non-native) nucleic acids. For example, a nucleic acid isolated from human or mouse.

The terms"mRNA","RNA"and"transcripf'are used interchangeably when referring to a transcribed copy of a transcribable nucleic acid.

In the context of transgenic plants, a"transgene"comprises any nucleic acid transferred or transformed into a cell of a plant. A transgene may encode a protein for example GmNARK (SEQ ID NO: 1) or GmNARK mutant.

Preferably, the GmNARK mutant comprises an amino acid sequence as set forth in SEQ ID NOS: 6-10, inclusive of fragments thereof. Alternatively, a transgene may not encode for a protein, for example in relation to a promoter or regulatory element such as the GmNARK promoter, for example the GmNARK promoter comprising nucleotide sequences as set forth in SEQ ID NOS: 11 and

12, inclusive of fragments thereof. The transgene need not be transcribed, for example in the case of homologous recombination and gene knockouts. In one embodiment, the transgene comprises a transcribable nucleic acid, such as GmNARK and mutants thereof. The transgene may modify the plant cell, and plant comprising said cell, by modifying a characteristic or phenotype of the cell or plant. For example, the transgene may induce cell division and/or supernodulation.

A"polynucleotide"is a nucleic acid having eighty (80) or more contiguous nucleotides, while an"oligonucleotide"has less than eighty (80) contiguous nucleotides.

In one embodiment, a nucleic acid"fragmenY'comprises a nucleotide sequence that constitutes less than 100% of a nucleic acid of the invention. A fragment includes a polynucleotide, oligonucleotide, probe, primer and an amplification product, eg. a PCR product. Examples of fragments are primers set forth in SEQ ID NOS: 15-17.

A"probe"may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern blotting, Southern blotting or microarray analysis, for example.

A"primef'is usually a single-stranded oligonucleotide, preferably having 20-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid"template"and being extended in a template- dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase. For example,

the following primers were used to amplify Genomic GmNARK and GmCLV1A nucleic acids.

As used herein, the term"varianf', in relation to a coding nucleic acid, means a nucleotide sequence thereof that has been mutagenized or otherwise altered so as to encode a substantially same, or modified, protein. In relation to a regulatory nucleic acid such as a regulatory element, the term "varianf'means a nucleic acid that has been mutagenized or otherwise altered so as to result in a substantially same, or modified, regulatory activity. Such changes may be trivial, for example in cases where more convenient restriction endonuclease cleavage and/or recognition sites are introduced without substantially affecting the encoded protein or regulatory activity. Other nucleotide sequence alterations may be introduced so as to modify the biological activity or promoter activity. These alterations may include deletion or addition of one or more nucleotide bases, or involve an alternation resulting in a non-conservative amino acid substitution. Such alterations can have profound effects upon biological activity of an encoded protein (for a coding nucleic acid) or regulatory activity (for a coding or non-coding nucleic acid such as a promoter). The alteration may either increase or decrease activity as required.

In this regard, nucleic acid mutagenesis may be performed in a random fashion

or by site-directed mutagenesis in a more"rational"manner. Standard mutagenesis techniques are well known in the art, and examples are provided in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds Ausubel et al. (John Wiley & Sons NY, 1995), which is incorporated herein by reference. Mutagenesis also includes mutagenesis using chemical and/or irradiation methods such as EMS and fast neutron mutagenesis of plant seeds.

It will be appreciated that nucleic acid variants may result in protein variants as described herein.

A"genetic construcf'encompasses a nucleic acid, chimeric gene and a nucleic acid vector. The genetic construct may comprise a recombinant nucleic acid, such as GmNARK or GmNARK mutant, and/or the GmNARK promoter or fragments. Such nucleic acids include those encoding a protein comprising an amino acid sequences as set forth in SEQ ID NOS: 1-10. A nucleic acid vector may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome. The vector may comprise a recombinant nucleic acid that is not transcribed. The nucleic acid vector may be useful in homologous recombination, knocking-in a nucleic acid, knocking out a nucleic acid and other molecular methods of modifying an endogenous nucleic acid as are common in the art. Such modification of an endogenous nucleic acid may reduce or eliminate expression of said endogenous nucleic acid and and/or encoded protein. For example, knocking out endogenous GmNARK or knocking out the kinase domain of GmNARK by homologous recombination may result in a supernodulation phenotype. The nucleic acid vector in one embodiment is an expression vector as described

below. The nucleic acid vector may comprise viral and pathogen nucleic acids, such as plant pathogen nucleic acids. A plant pathogen nucleic acid includes T DNA plasmid, modified (including for example a recombinant nucleic acid) or otherwise, from Agrobacterium.

The host cell may be plant, yeast, animal (including insect) or bacterial. The vector may be suitable for expression of the operably linked recombinant nucleic acid. Of particular use is a recombinant T-DNA plasmid that may be transferred into the bacteria, Agrobacterium tumefaciens, for transfer into a plant via Agrobacterium tumefaciens infection.

An"expression vector'is one embodiment of a vector may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome. An example of an expression vector is pRSET B (Invitrogen Corp. ) and derivations thereof.

By"operably linked"is meant that said regulatory nucleotide sequence (s) is/are positioned relative to the recombinant nucleic acid of the invention to initiate, regulate or otherwise control transcription. A nucleic acid comprising a regulatory sequence and any transcribable nucleic acid is referred to as a"chimeric gene". The regulatory sequence is commonly 5'of the transcribable nucleic acid, but this need not always be the case. For the purposes of host cell expression, the recombinant nucleic acid may be operably linked to one or more regulatory sequences.

Regulator nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of

host cells. The nucleic acid shown in FIGS. 9 and 10 as set forth as SEQ ID NOS: 11 and 12 are an examples of regulatory nucleotide sequences that control expression of endogenous GmNARK. The term"promoteK'used herein is understood to refer to a regulatory nucleic acid, inclusive of fragments thereof. Such fragments may comprise one or more regulatory elements, for example those elements shown in tables 4-7.

Typically, said one or more regulatory nucleotide sequences may include, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.

Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. For example, the lac promoter is inducible by IPTG.

The expression vector may further comprise a selectable marker nucleic acid to allow the selection of transformed host cells. Selectable marker nucleic acid are well known in the art and will vary with the host cell used. For example, an ampicillin resistance gene for selection of positively transformed host cells when grown in a medium comprising ampicillin.

The expression vector may also include a fusion partner (typically provided by the expression vector) so that the recombinant polypeptide of the invention is expressed as a fusion polypeptide with the fusion partner. An advantage of fusion partners is that they assist identification and/or purification of the fusion polypeptide. Identification and/or purification may include using a

monoclonal antibody or substrate specific for the fusion partner, for example a 6X-His tag or GST. A fusion partner may also comprise a leader sequence for directing secretion of a recombinant polypeptide, for example an alpha-factor leader sequence.

Well known examples of fusion partners include hexahistidine (6X-HIS)-tag, N-Flag, Fc portion of human IgG, glutathione-S-transferase (GST) and maltose binding protein (MBP), which are particularly useful for isolation of the fusion polypeptide by affinity chromatography. For the purposes of fusion polypeptide purification by affinity chromatography, relevant matrices for affinity chromatography may include nickel-conjugated or cobalt-conjugated resins, fusion polypeptide specific antibodies, glutathione-conjugated resins, and amylose-conjugated resins respectively. Some matrices are available in"kit" form, such as the ProBondT" Purification System (Invitrogene Corp.) which incorporates a 6X-His fusion vector and purification using ProBondT" resin.

In order to express the fusion polypeptide, it is necessary to ligate a nucleic acid according to the invention into the expression vector so that the translational reading frames of the fusion partner and the nucleotide sequence of the invention coincide.

The fusion partners may also have protease cleavage sites, for example enterokinase (available from Invitrogen Corp. as EnterokinaseMax), Factor Xa or Thrombin, which allow the relevant protease to digest the fusion polypeptide of the invention and thereby liberate the recombinant polypeptide of the invention therefrom. The liberated polypeptide can then be isolated from the fusion partner by subsequent chromatographic separation.

Fusion partners may also include within their scope"epitope tags", which are usually short peptide sequences for which a specific antibody is available.

As hereinbefore, polypeptides of the invention may be produced by culturing a host cell transformed with an expression construct comprising a nucleic acid encoding a protein, protein fragment, variant or polypeptide homolog, of the invention. The conditions appropriate for polypeptide expression will vary with the choice of expression vector and the host cell. For example, a nucleotide sequence of the invention may be modified for successful or improved polypeptide expression in a given host cell.

Modifications include altering nucleotides depending on preferred codon usage of the host cell, for example codon usage in plants, in particular legumes.

Alternatively, or in addition, a nucleotide sequence of the invention may be modified to accommodate host specific splice sites or lack thereof. These modifications may be ascertained by one skilled in the art.

A host cell for nucleic acid and/or protein expression may be prokaryotic or eukaryotic.

Useful prokaryotic host cells are bacteria.

A typical bacteria host cell is a strain of E. coli.

Agrobacterium tumefaciens is a bacteria useful for transforming plant cells, in particular when said bacteria comprises a recombinant T-DNA plasmid.

Useful eukaryotic cells include yeast and SF9 cells, which may be used with a baculovirus expression system, and mammalian cells.

The host cell may be a cell from a plant or plant part, including a stem, root, leaf, seed or nodule.

The plant may be any suitable plant, including for example, a monocotyledon or dicotyledon.

The plant may be, for example, wheat, maize, rice, tobacco, Arabidopsis, legumes such as soybean, Glycine max, Glycine soja L. , pea, cowpea, broadbean, Lotus japonicus, Lotus comiculatus, and Medicago truncatula.

The recombinant polypeptide may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), incorporated herein by reference, in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et a/., (John Wiley & Sons, Inc. 1995-1999), incorporated herein by reference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et a/., (John Wiley & Sons, Inc. 1995-1999) which is incorporated by reference herein, in particular Chapters 1,5 and 6.

In one embodiment, nucleic acid homologs encode polypeptide homologs of the invention, inclusive of variants, fragments and derivatives thereof.

In yet another embodiment, nucleic acid homologs are nucleic acids having one or more codon sequences altered by taking advantage of codon sequence redundancy.

A particular example of this embodiment is optimization of a

nucleic acid sequence according to codon usage as is well known in the art.

This can effectively"tailor"a nucleic acid for optimal expression in a particular organism, or cells thereof, where preferential codon usage has been established. In this regard, specific codons used in plants may be used. In one embodiment legume specific codons are used for expression in legumes.

In another embodiment, a nucleic acid of the invention shares at least 60%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% nucleotide sequence identity with GmNARK, GmNARK mutants and GmNARK promoter, including respective fragments thereof. When referring to nucleotide sequence identity or similarity, it will be appreciated that in one embodiment an original or starting nucleic acid (for example a wild type nucleic acid) may be mutated or modified such that the mutated nucleic acid has a different nucleotide sequence when compared with the original nucleic acid. For example, the nucleotide sequence of the GmNARK mutant encoding the V837A substitution has a different nucleotide sequence than the wild type nucleotide sequence.

In another embodiment, different nucleic acids or genes may have a similar nucleotide sequence or share nucleotide sequence identity. For example, a GmNARK nucleic acid has a similar nucleotide sequence as GmCLV1A, HAR1 and CLV1. Analogous mutations of the GmNARK nucleic acid may likewise be made in nucleic acids having a similar nucleotide sequence, for example GmCLV1A, HAR1 and CLV1. Accordingly, GmCLV1A, HAR1 and CLV1 may be mutated or modified to make analogous mutations as described herein in relation to GmNARK. Nucleic acids that share nucleotide

sequence identity may also be referred to as"homologs"and"orthologs". A "paralog"may also have a same or similar nucleotide sequence when compared with the nucleic acids of the invention.

In yet another embodiment, similar nucleic acids or nucleic acid homologs hybridise to nucleic acids of the invention under at least low stringency conditions, preferably under at least medium stringency conditions and more preferably under high stringency conditions.

"Hybridise and Hybridisation"is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-DNA, RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementary nucleotide sequences occur through base-pairing.

In DNA, complementary bases are: (i) A and T; and (ii) C and G.

In RNA, complementary bases are: (i) A and U; and (ii) C and G.

In RNA-DNA hybrids, complementary bases are: (i) A and U; (ii) A and T; and (iii) G and C.

Modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and methylcytosine) may also engage in base pairing.

"Stringency"as used herein, refers to temperature and ionic strength conditions, and presence or absence of certain organic solvents and/or detergents during hybridisation. The higher the stringency, the higher will be the required level of complementarity between hybridizing nucleotide sequences.

"Stringent conditions"designates those conditions under which only nucleic acid having a high frequency of complementary bases will hybridize.

Reference herein to low stringency conditions includes and encompasses:- (i) from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridisation at 42°C, and at least about 1 M to at least about 2 M salt for washing at 42°C ; and (ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2xSSC, 0. 1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at room temperature.

Medium stringency conditions include and encompass:- (i) from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridisation at 42°C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42°C ; and (ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M

NaHP04 (pH 7.2), 7% SDS for hybridization at 65°C and (a) 2 x SSC, 0. 1% SDS; or (b) 0.5% BSA, 1 mM EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at 42°C.

High stringency conditions include and encompass:- (i) from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridisation at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 420C ; (ii) 1% BSA, 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65°C, and (a) 0.1 x SSC, 0. 1% SDS ; or (b) 0.5% BSA, 1mM EDTA, 40 mM NaHP04 (pH 7.2), 1% SDS for washing at a temperature in excess of 65°C for about one hour; and (iii) 0.2 x SSC, 0. 1% SDS for washing at or above 68°C for about 20 minutes.

In general, the Tm of a duplex DNA decreases by about 1°C with every increase of 1 % in the number of mismatched bases.

Notwithstanding the above, stringent conditions are well known in the art, such as described in Chapters 2.9 and 2.10 of Ausubel et al., supra, which are herein incorporated by reference. A skilled addressee will also recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization.

Typically, complementary nucleotide sequences are identified by

techniques that include a step whereby nucleotides are immobilized on a matrix (such as a synthetic membrane, eg nitrocellulose, for blotting methods, a surface of a chip for use with microarray methods, or a bead or other substrate), a hybridization step, and a detection step. Southern blotting is used to identify a complementary DNA sequence ; Northern blotting is used to identify a complementary RNA sequence. Dot blotting and slot blotting can be used to identify complementary DNA/DNA, DNA/RNA or RNA/RNA polynucleotide sequences. Such techniques are well known by those skilled in the art, and have been described in Ausubel et al., supra, at pages 2.9. 1 through 2.9. 20, herein incorporated by reference.

According to such methods, Southern blotting involves separating DNA molecules according to size by gel electrophoresis, transferring the size- separated DNA to a synthetic membrane, and hybridizing the membrane bound DNA to a complementary nucleotide sequence.

In dot blotting and slot blotting, DNA samples are directly applied to a synthetic membrane prior to hybridization as above.

An alternative blotting step is used when identifying complementary nucleic acids in a cDNA or genomic DNA library, such as through the process of plaque or colony hybridisation. Other typical examples of this procedure are described in Chapters 8-12 of Sambrook et al., supra which are herein incorporated by reference.

Typically, the following general procedure can be used to determine hybridisation conditions. Nucleic acids are blotted/transferred to a synthetic membrane, as described above. A nucleotide sequence of the

invention is labeled as described above, and the ability of this labeled nucleic acid to hybridise with an immobilized nucleotide sequence analysed.

A skilled addressee will recognise that a number of factors influence hybridisation. The specific activity of radioactively labeled polynucleotide sequence should typically be greater than or equal to about 108 dpm/, ug to provide a detectable signal. A radiolabeled nucleotide sequence of specific activity 108 to 109 dpm/g can detect approximately 0.5 pg of DNA. It is well known in the art that sufficient DNA must be immobilised on the membrane to permit detection. It is desirable to have excess immobilised DNA, usually 10 pg. Adding an inert polymer such as 10% (w/v) dextran sulfate (MW 500,000) or polyethylene glycol 6000 during hybridisation can also increase the sensitivity of hybridisation (see Ausubel et a/., supra at 2.10. 10).

To achieve meaningful results from hybridisation between a nucleic acid immobilised on a membrane and a labeled nucleic acid, a sufficient amount of the labeled nucleic acid must be hybridised to the immobilised nucleic acid following washing. Washing ensures that the labeled nucleic acid is hybridised only to the immobilised nucleic acid with a desired degree of complementarity to the labeled nucleic acid.

Methods for detecting labeled nucleic acids hybridised to an immobilised nucleic acid are well known to practitioners in the art. Such methods include autoradiography, chemiluminescent, fluorescent and colourimetric detection.

A nucleic acid array, e. g. a microarray, uses hybridization-based technology that, for example, may allow detection and/or isolation of a nucleic

acid by way of hybridization of complementary nucleic acids. A microarray provides a method of high throughput screening for a nucleic acid in a sample that may be tested against several nucleic acids attached to a surface of a matrix or chip. In this regard, a skilled person is referred to Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al.

John Wiley & Sons NY, 2000).

Each nucleic acid occupies a known location on an array. A nucleic acid target sample probe is hybridised with the array of nucleic acids and an amount or relative abundance of target nucleic acid hybridised to each probe in the array is determined.

High-density arrays are useful for monitoring gene expression and presence of allelic markers that may be associated with a phenotype, such as supernodulation. Fabrication and use of high density arrays in monitoring gene expression have been previously described, for example in WO 97/10365, WO 92/10588 and US Patent No. 5,677, 195, all incorporated herein by reference.

In some embodiments, high-density oligonucleotide arrays are synthesised using methods such as the Very Large Scale Immobilised Polymer Synthesis (VLSIPS) described in US Patent No. 5,445, 934, incorporated herein by reference.

A nucleic acid having a similar nucleotide sequence as that of the invention may be prepared according to the following procedure: (i) obtaining a nucleic acid extract from a suitable host, for example a plant species; (ii) creating primers which are optionally degenerate wherein

each comprises a portion of a nucleotide sequence of the invention; and (iii) using said primers to amplify, via nucleic acid amplification techniques, one or more amplification products from said nucleic acid extract.

Plant transformation and transgenic plants Other aspects of the present invention relate to transgenic plants and a method of producing transgenic plants.

The identification and cloning of GmNARK and GmNARK mutants, opens up a possibility of beneficial manipulating plant root systems.

Plants, including crops, forests and garden plants, are completely dependent on a healthy root system for absorption of water and nutrients from soil. It is now possible that transgenic expression of GmNARK and mutants thereof may improve an ability of a plant to absorb water and nutrients from soil. Such transgenic plants may have increased water and nutrient absorption thereby improving crop yields.

Supernodulation can increase nitrogen fixation. Transgenic plants made in accordance with the present invention may be engineered to increase nodulation and nitrogen fixation in crops like wheat, rice and maize thereby decreasing a requirement for nitrogen fertilizers. Further, transgenic plants expressing GmNARK, or mutants thereof, may also express other nucleic acids that may further improve nodulation. Expressing additional nucleic acids in conjunction with GmNARK mutants could result in crops like wheat, rice and maize, being grown without the need for nitrogen fertilizer.

Transgenic plants expressing wild type or mutant forms of GmNARK may be used to control root development in plants. With the identification of GmNARKs regulating root development, wild type GmNARK may be expressed in suitable abundance to suppress lateral root formation to thereby favour deep tap root growth. This may be useful in situations where water supply is scare and deep roots are essential for plant survival.

Alternatively, expression of mutated GmNARK and/or knocking out or inhibiting endogenous wild type GmNARK may be useful in situations where a deep tap root is undesirable, or example in hydroponics, potted plants, or where a deep tap root may damage subterranean structures such as pipes.

Supernodulation mutants may also be useful for increasing nodulation when using nodules as bio-factories to produce a desired compound, such as a bio-active compound or biologically active protein for use in a pharmaceutical composition. Increasing the number and/or frequency of nodules may improve yield and ease of harvesting of the bio-active compound, that may be recombinantly expressed or endogenous to the nodule and/or symbiotic organism of the nodule.

In one embodiment, the method of producing a transgenic plant includes the steps of: (i) transforming a plant cell or tissue with a genetic construct comprising an isolated nucleic acid encoding for a protein comprising an amino acid sequence as set forth in SEQ ID NOS. 1-10; and (ii) selectively propagating a transgenic plant from the plant cell or tissue transformed in step (i).

The genetic construct may be a vector. The vector in one embodiment is an expression vector.

In another embodiment the genetic construct is a chimeric gene.

The chimeric gene may comprise a GmNARK nucleic acid as described herein, including mutants and variants thereof. The chimeric gene may comprise the GmNARK promoter, or fragments thereof, operatively linked to a transcribable nucleic acid. Such GmNARK promoter may comprise a nucleotide sequence as set forth in SEQ ID NO: 11 or 12, inclusive of fragments thereof. The transcribable nucleic acid may encode GmNARK or GmNARK mutant.

Preferably the GmNARK mutant comprises an amino acid sequence as set for the in SEQ ID NOS : 6-10.

It will also be appreciated that the genetic construct may comprise a nucleic acid having a similar nucleotide sequence as GmNARK, GmNARK mutants or derivative thereof and/or the GmNARK promoter and fragments thereof comprising selected regulatory elements. The similar nucleotide sequence may be that of a GmNARK variant or a different gene, such as GmCLV1A, HAR1 or CLV1. GmCLV1A, HAR1 or CLV1 may comprise one or more analogous mutation as described for GmNARK.

Suitably, the plant cell or tissue used at step (i) may be a leaf disk, callus, meristem, hypocotyls, root, leaf spindle or whorl, leaf blade, stem, shoot, petiole, axillary bud, shoot apex, internode, cotyledonary-node, flower stalk or inflorescence tissue.

Preferably, the plant tissue is a leaf or part thereof, including a leaf disk, hypocotyls or cotyledonary-node.

The plant cell or tissue may be obtained from any plant species including monocotyledon, dicotyledon, ferns and gymnosperms such as conifers, without being limited thereto.

Preferably, the plant is a dicotyledon.

More preferably, the dicotyledon is a legume.

The legume may be a soybean or Lotus aponicus.

Preferably, the soybean is Glycine soja (L. ) or Glycine max (L.) Merr. cv'Bragg'.

The plant may be a monocotyledon such as wheat, rice, barley and maize.

Persons skilled in the art will be aware that a variety of transformation methods are applicable to the method of the invention, such as Agrobacterium tumefaciens-mediated (Gartland & Davey, 1995, Agrobacterium Protocols (Human Press Inc. NJ USA); United States Patent No. 6,037, 522; W099/36637), microprojectile bombardment (Franks & Birch, 1991, Aust. J.

Plant. Physio., 18 471; Bower et al., 1996, Molecular Breeding, 2 239; Nutt et a/., 1999, Proc. Aust. Soc. SugarCane Technol. 21 171), liposome-mediated (Ahokas et al., 1987, Heriditas 106 129), laser-mediated (Guo et al., 1995, Physiologia Plantarum 93 19), silicon carbide or tungsten whiskers (United States Patent No. 5,302, 523; Kaeppler et a/., 1992, Theor. Appl. Genet. 84 560), virus-mediated (Brisson et al., 1987, Nature 310 511), polyethylene- glycol-mediated (Paszkowski et al., 1984, EMBO J. 3 2717) as well as transformation by microinjection (Neuhaus et a/., 1987, Theor. Appl. Genet. 75 30) and electroporation of protoplasts (Fromm et al., 1986, Nature 319 791), all

of which references are incorporated herein.

Microprojection bombardment and Agrobacterium tumefaciens transformation are useful for transforming dicotyledons.

In one form, transformation is by co-precipitating a nucleic acid, such as a plasmid or vector, onto gold particles and using microprojection bombardment to transfer the nucleic acid into a plant tissue.

Preferably, the plant tissue is a leaf or leaf disk.

In another form, transformation is via co-cultivation of a plant part with A. tumefaciens comprising a nucleic acid of the invention.

Preferably, the plant part is a cut hypocotyl or cotyledonary-node.

Preferably, the A. tumefaciens is strain LBA4404.

Preferably, selective propagation at step (ii) is performed in a selection medium comprising genetic as selection agent.

In one embodiment, the expression construct may further comprises a selection marker nucleic acid in the form of an nptll gene.

In another embodiment, a separate selection construct may be included at step (i), which comprises a selection marker nucleic acid in the form of an nptll gene.

The transformed plant material may be cultured in shoot induction medium followed by shoot elongation media as is well known in the art. Shoots may be cut and inserted into root induction media to induce root formation as is known in the art.

It will be appreciated that as discussed hereinbefore, there are a number of different selection agents useful according to the invention, the

choice of selection agent being determined by the selection marker nucleic acid used in the expression construct or provided by a separate selection construct.

Detection of transgene expression The transgenic status of transgenic plants of the invention may be ascertained by measuring transgenic expression of a polypeptide encoded by the transcribable nucleic acid, in the case wherein the transgene is transcribed.

This can be performed using methods applicable to measuring promoter activity.

In one embodiment, transgene expression can be detected by antibodies specific for the encoded polypeptide : (i) in an ELISA such as described in Chapter 11.2 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et a/.

(John Wiley & Sons Inc. NY, 1995) which is herein incorporated by reference; or (ii) by Western blotting and/or immunoprecipitation such as described in Chapter 12 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et a/. (John Wiley & Sons Inc. NY, 1997), which is herein incorporated by reference.

Protein-based techniques such as mentioned above may also be found in Chapter 4.2 of PLANT MOLECULAR BIOLOGY : A Laboratory Manual, supra, which is herein incorporated by reference.

Particularly advantageous protein assays are capable of detecting gus-and nptil-expressing transgenic plants.

The aforementioned protein-based detection methods may take

advantage of"fusion partners"such as glutathione-S-transferase (GST), Fc portion of human IgG, maltose binding protein (MBP) and hexahistidine (HIS6), which are particularly useful for isolation of the fusion polypeptide by affinity chromatography. For the purposes of fusion polypeptide purification by affinity chromatography, relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel-or cobalt-conjugated resins respectively. Many such matrices are available in"kit"form, such as the QIAexpressTM system (Qiagen) useful with (HIS6) fusion partners and the Pharmacia GST purification system.

Fusion partners also include"epitope tags", which are usually short peptide sequences for which a specific antibody is available. Well known examples of epitope tags for which specific monoclonal antibodies are readily available are known in the art.

It will also be appreciated that transgenic plants of the invention may be screened for the presence of mRNA corresponding to a transcribable nucleic acid and/or a selection marker nucleic acid. This may be performed by RT-PCR (including quanitative RT-PCT), Northern hybridization, and/or microarray analysis. Southern hybridization and/or PCR may be employed to detect DNA (the GmNARK promoter, GmNARK mutant, transcribable nucleic acid and/or selection marker) in the transgenic plant genome as described herein.

For examples of RNA isolation and Northern hybridization methods, the skilled person is referred to Chapter 3 of PLANT MOLECULAR BIOLOGY : A Laboratory Manual, supra, which is herein incorporated by reference. Southern hybridization is described, for example, in Chapter 1 of

PLANT MOLECULAR BIOLOGY : A Laboratory Manual, supra, which is herein incorporated by reference.

A selectable marker as described herein is typically used to increase the number of positive transformants before assaying for transgene expression. However, positive transformants identified by PCR and other high throughput type systems (eg microarrays) enable selection of transformants without use of a selectable marker due to a large number of samples that may be easily tested. It may be preferred to avoid use of selectable markers in transgenic plants because of environmental concerns in relation to perceived accidentally release of the selectable marker nucleic acid into the environment.

Herbicide resistant markers, eg. basta, and antibiotic resistant markers, eg ampicillin, are a few selectable markers that may be of concern. PCR may be performed on thousands of samples using primers specific for the transgene or part thereof, the amplified PCR product may be separate by gel electorphoresis, coated onto multi-well plates and/or dot blotting onto a membrane and hybridised with a suitable probe, for example probes described herein including radioactive and fluorescent probes to identify the transformant.

GmNARK Mutants It will be appreciated that with the identification of GmNARK as a systemic regulator, in particular a controller of autoregulation of nodulation, other mutations of this specific nucleic acid are now possible using, for example, molecular biological techniques that are well known in the art and described herein. Such mutations may result in supernodulation as herein described. Mutations may reduce or eliminate kinase activity. Accordingly, the

invention contemplates various forms of mutation including random mutagenesis, include chemical modification of proteins by hydroxylamine, incorporation of dNTP analogs into nucleic acids and PCR-based random mutagenesis such as described herein. Mutagenesis may also be induced by chemical means, such as ethyl methane sulphonate (EMS) and/or irradiation means, such as fast neutron irradiation of seeds as is common in the art and described herein.

GmNARK Knock Out GmNARK may also be knocked out or knocked down. Methods for knocking out a gene are well known and include homologous recombination and gene targeting. General methods for homologous recombination are known, for example as described in chapter 23 of Ausubel et al, supra. In general, a double stranded nucleic acid comprising a nucleic acid sequence homologous to a target nucleic acid, such as GmNARK, is cloned into a genetic construct in the form of a vector. The vector also preferably comprises a selectable marker such as hpt for positive hygromycin B resistance. The vector may further comprise elements for negative selection, eg thymidine kinase (TK), located adjacent to the nucleic acid of interest. TK expression renders a cell sensitive to gancyclovir, and thereby kills the cell. Homologous recombination tends to remove the adjacent TK nucleic acid, whereas random integration tends to preserve the adjacent TK nucleic acid.

Spatial control of a knockout may be provided for example by the Cre/loxP system that is well known in the art. Also, the yeast Flp/FRT system may be used, see for example Fierring et al, 1993, Proc Natl Acad Sci USA 90

8469 and Fierring et al, 1999, Methods of Enzymol 306 42.

In another embodiment, site-selected transposon mutagenesis may also be used to knockout endogenous GmNARK.

Down regulation of GmNARK It will also be appreciated that the transcribable nucleic acid may encode a transcript complementary to an endogenous mRNA transcript, such as used in"antisense"expression. Also contemplated are co-suppression, expression of ribozymes and iRNA. Anti-sense regulation and the use of ribozymes and co-suppression in plants are well known in the art. However, the skilled person is referred to United States Patent 5,759, 829 for an example of antisense technology and to U. S. patent 5, 707,835, U. S. patent 5,747, 335 and U. S. patent 5,840, 874 which each provide examples of ribozyme technology.

With regard to co-suppression, reference is made to U. S. patent 5,283, 184, U. S. patent 5,686, 649 and International Publication W098/53083 for examples of this technology. Each of these patent documents is incorporated herein by reference.

Biological activity of endogenous GmNARK may also be down regulated by expression of one or more GmNARK mutants. A transgenic plant expressing one or more GmNARK mutants may result in a dominant negative effect thereby inducing a phenotype characterised by supernodulation. Not being bound by theory, the GmNARK mutant may compete with a substrate or binding partner of the endogenous wild type GmNARK, thereby reducing or eliminating the biological activity of the endogenous GmNARK.

Detection Methods for Identifying Expression of GmNARK and Mutant

Forms Thereof Identification of wild type and mutant GmNARK has many potential uses, including use for research and screening of plants characterised by modified autoregulation of nodulation, eg supernodulation. It will be appreciated that methods for inducing mutations in GmNARK and related nucleic acids may be combined with detection methods described herein to assist with selection of mutants. GmNARK may be mutated by any suitable means, including those methods described herein. However, it will also be appreciated that wild type GmNARK and/or GmNARK mutants may be identified in one or more plants and/or screened from a population of plants that have not previously been intentionally mutated.

Specific mutants may be selected based on nucleotide sequence, amino acid sequence and/or kinase activity. A high level of specificity for selecting a particular mutant or class of mutant may be possible using the methods described herein. In particular, optimising hybridisation conditions may select for specific nucleotide mutations. Assays measuring kinase activity are useful as they measure GmNARK based on phenotype or function. In some cases, measuring kinase function may be the critical determinant for selecting a mutant or class of mutants.

Nucleic acid based detection of GmNARK and GmNARK mutants With the identification of GmNARK as a regulator of systemic regulation, for example supernodulation, specific nucleotide sequences may be selected for primers, probes and targets. As described above, nucleic acids of the invention may be detected by several different means that are well known in

the art, including for example, Northern blotting, Southern blotting, PCR, RT- PCR, quantiative RT-PCT, real time quantiative RT-PCT and nucleic acid arrays. Such detection methods typically rely on hybridisation properties of a target nucleic acid and a complementary capture nucleic acid. Accordingly, hybridisation and/or wash conditions may be selected to optimise a desired sensitivity of the detection method, for example by selecting a particular temperature and/or salt content of the hybridisation and/or wash solution.

Amplification product As used herein, an"amplification product"refers to a nucleic acid product generated by nucleic acid amplification techniques.

Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include PCR as for example described in Chapter 15 of Ausubel et al. supra, which is incorporated herein by reference; strand displacement amplification (SDA) as for example described in U. S. Patent No 5,422, 252 which is incorporated herein by reference; rolling circle replication (RCR) as for example described in Liu et al., 1996, J. Am. Chem. Soc. 118 1587 and International application WO 92/01813; and Lizardi and Caplan, International Application WO 97/19193, which are incorporated herein by reference; nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et al., 1994, Biotechniques 17 1077, which is incorporated herein by reference ; ligase chain reaction (LCR) as for example described in International Application W089/09385 which is incorporated herein by reference; and Q-p replicase amplification as for example described by Tyagi et al., 1996, Proc. Natl. Acad. Sci. USA 93 5395 which is incorporated

herein by reference. Preferably, amplification is by PCR using primers disclosed herein.

Nucleic Acid Arrav As described above, microarrays may be used to detect expression of GmNARK, including mutated forms thereof. Microarrays are particularly useful for screening a large number of samples. Specific GmNARK, and mutant GmNARK probes may be immobilized on an appropriate substrate as is well understood in the art. This may allow for screening of a plant, plant part (including plant extract) or plant cell for, mutated GmNARK, which may correlate with supernodulation. In this regard, the skilled person is referred to Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds.

Ausubel et al. John Wiley & Sons NY, 2000), International Publication WO00/58516, United States Patent 5,677, 195 and United States Patent 5,445, 934 which provide exemplary methods relating to nucleic acid array construction and use in detection of nucleic acids of interest.

Protein based detection of GmNARK and GmNARK mutants The invention also contemplates antibodies against the isolated GmNARK protein, fragments, variants and derivatives thereof, in particular GmNARK mutants including those describe herein comprising an amino acid sequence as set forth in SEQ ID NOS: 6-10. A peptide fragment of GmNARK may comprise a domain of GmNARK, such as the leucine rich region or the kinase domain, including mutants thereof as herein described. Respective antibodies capable of binding to and thereby detecting wild type and GmNARK mutants may be useful for identifying a plant expressing either wild type or

mutant form of GmNARK. A particular mutation in the GmNARK protein may be detected, for example by an ability to distinguish between an amino acid substitution, namely, V837A; or one or more amino acid deletions. Deletion of one or few amino acids may create a new epitope that a specific antibody may bind to thereby identifying such a mutant. Alternatively, or in addition, antibodies may be used to detect deletion mutants of GmNARK. For example, positive binding of a first antibody to the N-terminal region, such as the Leucine Rich Region, but negative binding (or lack of binding) of a second antibody to the C-terminal region, such as the kinase domain, may indicate a deletion of the kinase domain. Such mutant forms of GmNARK may be correlated to supernodulation of a plant and accordingly may be used to select a plant that is characterised by supernodulation. Analogous mutations in proteins similar to GmNARK may also be detecting using these same or methods modified for a particular protein as would be well known in the art.

A sample to be tested may be obtained from any plant, in particular a plant suspected of comprising GmNARK, homologue or variant thereof. The sample may be obtained from a whole plant, leaf, seed, stem, root or any other suitable plant part and/or extract thereof. The sample may be assayed using any well known technique in the art, including for example Western blot, ELISA (Enzyme Link Immuno-Sorbant Assay) and protein arrays.

Antibodies of the invention may be polyclonal or monoclonal.

Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and

Harlow, E. & Lane, D. Antibodies : A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, which are both herein incorporated by reference.

Generally, antibodies of the invention bind to or conjugate with a polypeptide, fragment, variant or derivative of the invention. For example, the antibodies may comprise polyclonal antibodies. Such antibodies may be prepared for example by injecting a polypeptide, fragment, variant or derivative of the invention into a production species, which may include mice, rabbits or goats, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols that may be used are described for example in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra.

In lieu of the polyclonal antisera obtained in the production species, monoclonal antibodies may be produced using the standard method as for example, described in an article by Köhler & Milstein, 1975, Nature 256, 495, which is herein incorporated by reference, or by more recent modifications thereof as for example, described in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the polypeptides, fragments, variants or derivatives of the invention.

The invention also includes within its scope antibodies that comprise Fc or Fab fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the peptides of the invention. Such scFvs may be

prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091, 513, European Patent No 239,400 or the article by Winter & Milstein, 1991, Nature 349 293, which are incorporated herein by reference.

The antibodies of the invention may be used for affinity chromatography in isolating natural or recombinant polypeptides of the invention. For example, reference may be made to immunoaffinity chromatographic procedures described in Chapter 9.5 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra.

Antibodies may be purified from a suitable biological fluid of the animal by ammonium sulfate fractionation, affinity purification or by other methods well known in the art. Exemplary protocols for antibody purification are given in Sections 10.11 and 11.13 of Ausubel et al., supra, which are herein incorporated by reference.

Immunoreactivity of the antibody against the native or parent polypeptide may be determined by any suitable procedure such as, for example, Western blot, ELISA and protein arrays as are well known in the art.

Kinase activity based detection of GmNARK and GmNARK mutants Assaying for GmNARK kinase activity, or lack or reduction thereof, measures GmNARK kinase is function. Methods for assaying phosphorylation of proteins is well known and methods are described in Unit 18 of Ausubel et al, 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Inc, incorporated herein by reference. Kinase activity may be reduced or eliminated by a mutation in the nucleic acid that results in a modified form of the

GmNARK protein as described above. Such a mutant may, for example, include those mutants described herein, in particular non-sense mutations and amino acid substitutions. A number of different types of mutations may result in a reduction or loss of kinase activity, including mutations in the signal peptide, extracellular domain, transmembrane domain and kinase domain. A mutation in the signal peptide may prevent proper targeting of GmNARK to the cell membrane, a mutation in the extracellular domain (eg in the LRR), for example GmNARK mutants Q106*, K115* and K606*, may prevent proper ligand binding and/or dimerisation when relevant, a mutation in the transmembrane domain may result in improper association with the membrane and a mutation in the kinase domain, for example GmNARK mutants V837A and Q920*, may prevent proper phosphorylation. It will be appreciated that one or more of the abovementioned mutation, in addition to other possible mutations, may result in a change in kinase activity when compared with the wild type protein kinase.

Analogous mutations as describe above may affect a protein having a similar amino acid sequence in a similar manner.

The abovementioned mutations typically result in reducing kinase activity; however, a mutant that increases kinase activity may also be identified.

Ultimately, functionality of the GmNARK protein kinase may have a significant affect on the biological activity of GmNARK and an ability to regulate cell proliferation, namely nodulation, in particular supernodulation. As there are potentially numerous mutations that could result in a reduction or loss of kinase activity, assaying for kinase activity as a result of a mutation has the potential of identifying a mutant that is not anticipated but nevertheless affects kinase

activity. As such, a plant found to have a decreased GmNARK kinase activity correlates with a plant characterised as a GmNARK mutant. Such a mutant may have a phenotype of supernodulation and increased nitrogen fixation.

The kinase domain is an intracellular component of GmNARK.

The overall amino acid sequence of the GmNARK indicates that GmNARK has a substrate specificity consistent with a serine/threonine kinase. Computational analysis for preferred substrate amino acid sequence was performed using the PREDIKIN program developed by B. Kobe (UQ) and indicates the following preferred substrates: (-3) Q/N/R/K/H/A/S/T ; (-2) Q/UR/K/S/A/T/N ; (0) S/T; (+1) T; (+2) Q/N/E/S ; (+3) ANILITIRIK.

In one embodiment, a purified peptide comprising an enzymatically functional part of, or the entire, GmNARK kinase domain is treated by lambda phosphatase dephosphorylation, followed by addition of ATP. The purified peptide may be prepared by recombinant methods as described in the Examples, using fusions such as 6Xhis tag for easy purification via an Ni-NTA column. Plus and minus ATP samples will be analyzed by mass spectrometry after tryptic digestion. Peptide masses will be compared to allow the detection of a phosphorylated residue. The residue amino acid sequence will allow synthesis of a synthetic peptide that may be used as an artificial substrate. Either antibody detection or fluorochrome/biotin labels substrate will permit high through put assay of kinase activity. Parallel studies with the kinase domain will detect transphosphorylation sites on candidate interactors such as ROP (a Rho-like GTPase). Optimization of reaction conditions distinguishing auto-from transphosphorylation sites will permit the assay of in vivo GmNARK

activity using isolated cells, protoplasts, vesicles or other plant parts, preferably comprising at least part of a membrane.

Anti-phosphopeptide antibodies that specifically bind to phosphorylated peptides may also be used to detect a phosphorylation state of GmNARK. Such methods are well known and methods therefor are provided for example in Unit 18.6 of Ausubel et a/, 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Inc, incorporated herein by reference.

Another embodiment of the invention includes labeling cells of a biological sample by culturing the cells with a suitable label, for example 32P ;.

The cells are preferably in a growth stage and accordingly are preferably subconfluent. Following the labeling step, the cells are washed to remove unbound label. The washed cells may then be lysed using a method suitable for the cell being assayed, typically the cell is lysed using a detergent, such as SDS. The labeled proteins may be immunoprecipitated using a GmNARK specific antibody, or antibody to another protein of interest, such a protein sharing amino acid sequence similarity with GmNARK. The immunoprecipitated protein (s) may be assayed for incorporation of label to provide an indication of kinase activity.

Alternatively, or in addition, the labeled proteins may be separated by gel electrophoresis, for example 1D and 2D gel electrophoresis. Proteins from a duplicate gel, or same gel, may be transferred to a membrane and immunoblotted using antibodies specific for the protein of interest, such as GmNARK. Alternatively, or in addition, non-labeled proteins may be detected using [1251] protein A, for example. Also, a shift in protein mobility or migration

pattern during SDS-PAGE may also be an indicator of phosphorylation.

GmNARK Detection Kit for Identifying Expression of GmNARK and Mutants Thereof The present invention also provides a kit for detection of GmNARK in a biological sample. The biological sample may be a plant or plant part, including, nodule, young nodule, root, root tip, stem, leaf, unifoliate leaf, first foliate leaf, second foliate leaf, third foliate leaf, shoot apical meristem, root apical meristem, fruit, seed, flower, embryonic tissue, protoplasts and other plant parts. A kit will comprise one or more agents described above depending upon the nature of the test method employed.

Nucleic Acid Based Kit The present invention also provides a nucleic acid based kit for detection of mutant forms of GmNARK nucleic acid. A kit may comprise one or more particular agents, for example nucleic acids and proteins as described above, depending upon the nature of the test method employed. In this regard, the kits may include one or more nucleic acid for GmNARK and mutant forms of GmNARK (for example deletion mutants, base pair substitution mutants), or fragment thereof, inclusive of primers and probes according to the invention.

Accordingly, the kits may include reagents for detection of labels, positive and negative controls, washing solutions, dilution buffers and the like. For example, a nucleic acid amplification based kit may comprise primers capable of hybridising with target nucleic acids of mutant GmNARK isolated from an individual. Useful primers include those as described herein. The target nucleic acid may comprise a region of known polymorphism, for example at

least one nucleotide deletion, addition or substitution as described herein. The target nucleic acid may be located on a membrane or chip in a pre-processed format. A selection of one or more nucleic acids encoding a particular mutant protein may be located on a same membrane or matrix of a chip. The nucleic acid may be amplified according to methods known in the art, for example PCR.

The amplified nucleic acid can be compared with reference nucleic acids.

Comparison may be by electric charge, molecular weight and/or nucleotide sequence, for example, using gel electrophoresis.

Protein Based Kit The present invention also provides a protein based kit for detection of GmNARK and GmNARK mutants, in a biological sample. The kit may comprise one or more agents described above, including one or more of a protein (eg GmNARK), fragment, variant, derivative, antibody and/or antibody fragment according to the invention. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, dilution buffers and the like. For example, an antibody- based detection kit may include (i) a polypeptide, or fragment or variant thereof according to the invention (which may be used as a positive control), (ii) an antibody according to the invention (preferably a monoclonal antibody) which binds to GmNARK, or mutant thereof, or respective fragment thereof in (i), and (iii) a suitable means for detecting a complex formed between a target (eg.

GmNARK, or mutant, in the sample) and the antibody in (ii), the detection means may include, for example colloidal gold. Antibodies may be polyclonal or monoclonal which bind to different forms of GmNARK. For example, one

antibody may bind to wild type GmNARK and another antibody may bind to a GmNARK mutant as described above. Preferably, the antibodies are monoclonal.

Kinase Based Kit The present invention also provides a kinase based kit for detection of kinase activity, or lack thereof, of GmNARK and GmNARK mutants. The kit may comprise one or more reagents as described above for detecting kinase activity, for example : peptide substrate, ATP, gamma-ATP, antibodies that bind GmNARK, phosphorylated GmNARK, lysis buffers and other buffers, wash solutions, labels, positive and negative controls, dilution buffers and the like. The peptide substrate and antibodies may be prepared as described above.

GmNARK Promoter The present invention provides isolated nucleic acids comprising a nucleotide sequence as set forth in SEQ ID NOS. 11 and 12 and shown in FIGS. 10 and 11, which comprises regulatory elements for controlling expression of endogenous GmNARK.

The term"promoteK'used herein is understood to refer to a regulatory nucleic acid, inclusive of fragments thereof. A"GmNARK promoter" comprises a nucleotide sequence as set forth in SEQ ID NOS: 11 and 12, inclusive of fragments thereof. The GmNARK promoter and fragments comprise regulatory elements, for example those elements shown in tables 4 and 5. The regulatory elements may be recombinantly arranged in any suitable order using well known molecular methods, for example PCR and/or restriction

endonuclease digestion followed by ligation in a desired order. Other regulatory elements known in the art, for example those described herein, may be introduced or combined with the regulatory elements of the GmNARK promoter. Further, by scanning the GmNARK promoter sequence other regulatory elements may be identified using same or similar methods for identifying the present regulatory elements.

The GmNARK promoter is active in response to inoculation with a symbiotic organism or microsymbiont, for example, Bradyrhizobium japonicum or rhizobium. The microsymbiont activation is about 5 fold in the leaf. The induction appears to be leaf specific, which may provide uses wherein herein a transgene is to be expressed in the leaf and activatable via microsymbiont inoculation.

GmNARK transcript level is substantially lower in shoot apical meristem (SAM) as shown in FIG. 2D and also lower in nodules and young leaves (16 day old) second trifoliate leaf compared with 28 day second trifoliate leaf, see FIG. 15. Interestingly, GmNARK is expressed in root tip meristem, but not in nodes or shoot apical meristem. Also, leaves that are spatially located closer to the shoot, eg 16 day second trifoliate leaves have a lower GmNARK expression level than 16 day first trifoliate leaves, which have a lower expression level than 16 day unifoliate leaves, see FIG. 15. This lower GmNARK transcript level is indicative of a decreased GmNARK promoter activity and is consistent with the identification of a negative regulatory element within the GmNARK promoter (identified by comparative genomics with Lotus japonicus).

This decease in GmNARK promoter activity has many potential uses, including uses relating to specific down regulation of a linked nucleic acid transgene expression in shoot apical meristem, nodules and young leaves.

The GmNARK promoter may be operably linked to a nucleic acid to initiate, regulate or otherwise control transcription of the linked nucleic acid or transgene. As such, the GmNARK promoter may be used, for example, if the transgene is to be constitutively expressed in a plant, but expression would have lethal effects in the shoot apical meristem or young leaf tissue. The nucleic acid linked with the GmNARK promoter may be any suitable nucleic acid, including GmNARK and mutants thereof. The linked nucleic acid may express an inhibitory nucleic acid such as RNA for down regulation of an endogenous nucleic acid, wherein inhibition is not desired in the shoot apical meristem. The GmNARK promoter may also regulate expression of nucleic acids similar to GmNARK, such as GmCLV1A, HAR1 and CLV1 and mutants thereof.

Preferably, the GmNARK promoter is capable of initiating, regulating or otherwise controlling transcription of a transcribable nucleic acid operably linked thereto, preferably in a plant or plant part. Accordingly, the GmNARK promoter may form part of a genetic construct, thereby forming a chimeric gene. an expression construct or expression vector, for expression of one or more transgenes appropriately located or inserted into the vector.

Usually, when transgenic expression of a polypeptide is required, a correct orientation of the encoding nucleic acid transgene is in the sense or 5'to 3' direction relative to the GmNARK promoter. However, where antisense

expression is required, the transcribable nucleic acid is oriented 3'to 5'. Both possibilities are contemplated by the expression vector of the present invention, and directional cloning for these purposes may be assisted by the presence of a polylinker.

Transcribable nucleic acids or transgenes, preferably in the form of double-stranded DNA, encompass both non-transcribed and transcribed nucleic acids that encode proteins, and hence comprise a coding region or open reading frame (ORF) as are well understood in the art. In transgenic plants comprising one or more transgenes, encoded protein (s) are usually expressed for the purpose of conferring a desired trait or phenotype such as salt tolerance, drought resistance, pest and disease resistance, altered fruit and flower development and other traits as are well known in the art.

The expression vector may comprise one or more additional elements such as a polylinker, selection marker gene, bacterial origin of replication, antibiotic resistance gene, additional regulatory elements and the like as will be discussed in more detail hereinafter.

Preferably, the expression construct includes a selection marker nucleic acid to allow selective propagation of plant cells and tissues transformed with an expression construct of the invention. Alternatively, the selection marker is included in a separate selection construct. In either case, one or more regulatory elements, as herein described, may be provided to direct expression of the selection marker nucleic acid.

Suitable selection markers include, but are not limited to, neomycin phosphotransferase 11, which confers kanamycin and geneticin/G418

resistance (nptll ; Raynaerts et al., In : Plant Molecular Biology Manual A9: 1-16.

Gelvin & Schilperoort Eds (Kluwer, Dordrecht, 1988), bialophos/phosphinothricin resistance (bar, Thompson et al., 1987, EMBO J. 6 1589), streptomycin resistance (aadA ; Jones et a/., 1987, Mol. Gen. Genet. 210 86) paromomycin resistance (Mauro et a/., 1995, Plant Sci. 112 97), p- glucuronidase (uidA or gus ; Vancanneyt et al., 1990, Mol. Gen. Genet. 220 245) and hygromycin resistance (hmr or hpt, Waldron et al., 1985, Plant Mol.

Biol. 5 103; Perl et al., 1996, Nature Biotechnol. 14 624), green fluorescent protein (gfp ; Haseloff & Amos, 1995, supra) all of which references are incorporated herein.

Selection markers such as described above may facilitate selection of transformants by addition of an appropriate selection agent post- transformation, or by allowing screening of plant tissue that expresses the selection marker by an appropriate assay. This latter approach is particularly applicable to gfp, npUI, luc and gus, for example, which essentially function as screenable"reporter genes", as will be described in more detail hereinafter.

The chimeric gene or expression vector of the present invention may also comprise other gene regulatory elements, such as a 3'non-translated sequence. A 3'non-translated sequence refers to that portion of a gene that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is characterized by effecting the addition of polyadenylic acid tracts to the 3'end of the mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5'AATAAA-3'although

variations are not uncommon.

The 3'nontranslated regulatory DNA sequence preferably includes from about 300 to 1,000 nucleotide base pairs and contains plant transcriptional and translational termination sequences. Examples of suitable 3' non-translated sequences are the 3'transcribed non-translated regions containing a polyadenylation signal from the opaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan et a/., 1983, Nucl. Acid Res. , 11 369) and the terminator for the T7 transcript from the octopine synthase (ocs) gene of Agrobacterium tumefaciens.

Regulator elements include promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator elements. Tanscriptional enhancer elements include elements from the CaMV 35S promoter and octopine synthase (ocs) genes, as for example described in U. S. Patent No. 5,290, 924, which is incorporated herein by reference. It is proposed that the use of an enhancer element such as the ocs element, and particularly multiple copies of the element, may act to increase the level of transcription from adjacent promoters when applied in the context of plant transformation.

Additionally, targeting sequences may be employed to target a protein product of the transcribable nucleic acid to an intracellular compartment within plant cells or to the extracellular environment. For example, a DNA sequence encoding a transit or signal peptide sequence may be operably linked to a sequence encoding a desired protein such that, when translated, the transit

or signal peptide can transport the protein to a particular intracellular or extracellular destination, respectively, and can then be post-translationally removed. Transit or signal peptides act by facilitating the transport of proteins through intracellular membranes, e. g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane. For example, the transit or signal peptide can direct a desired protein to a particular organelle such as a plastid (e. g. , a chloroplast), rather than to the cytoplasm. Thus, the expression construct can further comprise a plastid transit peptide encoding DNA sequence operably linked between a promoter region or promoter variant according to the invention and transcribable nucleic acid. For example, reference may be made to Heijne et a/., 1989, Eur. J. Biochem. 180 535 and Keegstra et a/., 1989, Ann. Rev. Plant Physio. Plant Mol. Biol. 40 471, which are incorporated herein by reference.

Typically, the expression vector of the invention is a plasmid and includes additional elements commonly present in plasmids for easy selection, amplification, and transformation of the transcribable nucleic acid in prokaryotic and eukaryotic cells, e. g. , pUC-derived vectors, pBluescript-derived vectors, pGEM-derived vectors. Additional elements include those which provide for autonomous replication of the vector in bacterial hosts (examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19 and the ColE1 replicon which function in many E. coli. strains), bacterial selection marker genes (ampr, tetr and kanr, for example), unique multiple cloning sites and sequences that enhance transformation of prokaryotic and eukaryotic cells.

The vector may also include an element (s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell. The vector may be integrated into the host cell genome when introduced into a host cell. For integration, the vector may rely on the foreign or endogenous DNA sequence or any other element of the vector for stable integration of the vector into the genome by homologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences.

Preferably, the expression vector of the invention includes a polylinker downstream and adjacent to the GmNARK promoter that facilitates directional cloning of the transcribable nucleic acid into the vector so that the promoter and transcribable nucleic acid are operably linked The plant may be of any taxonomic group, including dicotyledon,

monocotyledon, ferns, lichens, gymnosperms and angiosperms.

In one embodiment, the plant is a dicotyledon.

In one embodiment, the plant is a legume.

Promoter activity assays The activity of GmNARK promoter of the invention and the aforementioned variants, homologs and promoter-active fragments may be measured by any of a number of assays, typically referred to as"reporter assays". Reporter assays involve preparation of an expression construct where a promoter of interest is operably linked to a transcribable nucleic acid in the form of a"reporter gene". The expression construct is then used to transform plant cells or tissues. Promoter activity may be measured in transient assays (such as a few days after transformation) or in plants selectively propagated or regenerated from transformed cells or tissue. Reporter genes are well known in the art and include selectable markers described above as described above, chloramphenicol acetyl transferase (cat; Lindsey & Jones, 1987, Plant Mol. Biol.

10 43), green fluorescent protein and various derivatives thereof (gfp; Haseloff & Amos, 1995, Trends Genet. 11 328), neomycin phosphotransferase (nptil ; Reiss et al., 1984, Gene 30 211), ß-galactosidase (lacZ ; Helmer et al., 1984, BioTechnology 2 520), ß-glucuronidase (gus; Jefferson et al., 1987, EMBO J. 6 3301) and luciferase (luc ; Ow et al., 1986, Science 234 856), each of which is incorporated herein by reference. GUS expression in relation to assessment of GmNARK promoter activity is particularly useful and enzymatic conversion of 4- methylumbelliferyl ß-D-glucuronide to 4-methylumbelliferone is a preferred assay. The skilled person is also referred to Chapter 9.4 of PLANT

MOLECULAR BIOLOGY A Laboratory Manual, Ed. M. S. Clark (Springer- Verlag, Heidelberg, 1997), which is incorporated herein by reference, for examples of specific methods and a general overview of the procedures involved.

In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

GmNARK Isolation and Analysis EXAMPLE 1 Materials and Methods NODULATION TESTS. Nodulation phenotypes were determined as described in Carroll et al, 1985, Proc. Natl. Acad. Sci. U. S. A 82 4164. Plants were inoculated after planting with Bradyrhizobium japonicum strains CB1809 or USDA110, or with a moist peat inoculant Nodulaid 100 (Bio-Care Technology Party Ltd. , Somersby, NSW Australia). It will be appreciated that plants may be inoculated with other suitable symbiotic organisms known to be associated with plants, including plant pathogens, that preferably result in nodule formation.

GENOMIC PCR CONDITIONS. Soybean genomic DNA was extracted from young leaves under established protocol (Carroll et al, 1995, Genetics 139: 407). Genomic amplification of GmNARK and GmCLV1A was done with the same forward primer 5'-GGG TAC TAC TGC AAA GCA AAA TCA GAG-3' (SEQ ID NO: 15) and specific 3'-UTR reverse primers designed to distinguish between two genes. GmNARK reverse primer was 5'-GGG GAA AAT CCT TCG AAT TAC TT-3' (SEQ IS NO: 16) and GmCLV1A reverse primer

was 5'-ACA CAC AAT CGG GAA AAT CCT-3' (SEQ ID NO: 17). PCR conditions were as follows : denaturation at 94° C for 2 min, then ten cycles of 94° C for 15 sec, 60° C for 30 sec and 72° C for 2 min followed by twenty-five cycles of 94° C for 15 sec, 58° C for 30 sec and 72° C for 2 min. Final extension was at 72° C for 10 min.

SOUTHERN BLOT ANALYSIS. Soybean DNA was digested with Ndel and Sacl and blotted on a Nylon membrane (Sambrook et al, 1989, Molecular cloning : a Laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The GmNARK probe was amplified from the wild- type DNA (cv. Bragg) as described above. The 3.6 kb PCR product was gel- purified with QlAquick Gel Extraction Kit (Qiagen, Clifton Hill, Vic, Australia) and quantified visually on a gel with molecular standards. Approximately 50 ng of the PCR product was randomly labelled with 50 uCi (32P) dATP (Amersham Biosciences, Castle Hill, NSW, Australia) and used to probe the blot.

Hybridisation and washings were done at 65° C and the blot was exposed to X- ray film (Kodak, Coburg, VIC, Australia) for 72 hrs.

RNA EXTRACTION. RNA for RT-PCR was extracted from the whole root and shoot tissue of two week old nodulating plants (wild-type G. soja (P1468. 397) and its fast neutron mutant FN37as describe herein using Qiagen RNeasy MIDI kit.

For the Quantitative Real Time PCR seeds of Bragg, nts1007 and nts382 were germinated on filter paper and transferred into vermiculate pots watered with nutrient nitrogen-free solution after two days. Plants were inoculated with Nodulaid 100 at 4 days after planting and allowed to grow for 14

days in a 16/8 h, 24/21° C day/night regime. At the time of harvesting roots had mature nodules. RNA from leaves (includes the unifoliate and first trifoliate leaves) and shoot apical meristem (includes young leaflets of up to 3 mm in length) was extracted with Qiagen RNeasy MIDI kit.

RT-PCR. For each sample about 700 ng of DNAse I-treated RNA was reverse-transcribed and amplified using One Step Access RT-PCR System (Promega, Annandale, NSW, Australia). Conditions were as follows : 48° C for 45 mins, 95° C for 2 mins, then 10 cycles of 95° C for 30 secs, 60° C for 30 secs and 68° C for 30 secs, then 30 cycles of 95° C for 30 secs, 58° C for 30 secs and 68° C for 30 secs. Final extension was at 68° C for 7 min. Primers for both GmNARK and actin were the same as for the quantitative real time PCR (see below).

QUANTITATIVE REAL TIME PCR (S4). cDNA synthesis was done using 5 ug RNA for each sample. RNA was treated in 1X buffer with 2 U of DNAse I (Life Technologies, Mount Waverley, VIC, Australia) in a total volume of 10 pi for 15 min at room temperature. The reaction was stopped by adding 1 ul of 25 mM EDTA, followed by 15 min incubation at 65° C. One microliter of 0. 5 uM oligo (dT) mix (5'-GAC CAC GCG TAT CGA TGT CGA C (T16) V-3') (SEQ ID NO: 4) was added to the reaction and incubated for 10 min at 70° C, then chilled on ice. First strand mix containing 1X buffer, 10 mM DTT, 1.25 mM of each dATP, dCTP, dTTP, dGTP, was added to a total volume of 20 pi and incubated for 5 minutes at 42° C. Then 200 U of SuperScriptTMll reverse transcriptase (Gibco BRL Life Technologies) or water (for the non-reverse transcriptase control) were added and incubated for a further 55 min at 42° C.

The reaction was stopped by incubating at 70° C for 15 min. To remove RNA, 2 U of RNAse H (Gibco BRL Life Technologies) was added and incubated at 37° C for 20 minutes. The final reaction mix was diluted in water to a final volume of 250 ul. Then 5 pi was used as a template in each PCR. The real time PCR primers were: 5'-TGC TCA TCG ATC CAG AAT CA-3' (SEQ ID NO: 18) and 5'-GGG GAA AAT CCT TCG AAT TAC TT-3' (SEQ ID NO: 16) for GmNARK, and 5'-TTA CAA TGA GCT TCG TGT TGA C-3' (SEQ ID NO: 19) and 5'-AAC ATA CAT GGC AGG CAC ATG-3' (SEQ ID NO: 20) for the actin 2/5 gene (control). PCR was carried out in a total volume of 25 ul containing 0.2 uM of each primer, 1X SYBR green PCR master mix (PE Applied Biosystems).

Reactions were amplified in an ABI PRISM 7700 thermocycler as follows : 95° C for 10 min, then 45 cycles of 95° C for 15 sec, 57° C for 30 sec, 60° C for 1 min and a final incubation at 25° C for 2 min.

EXAMPLE 2 Isolation of the NTS-1 locus To elucidate the mechanisms of long distance signal exchange between leaf and nodule primordia, map-based cloning was used to isolate the NTS-1 locus. Mutant alleles were mapped to soybean linkage group H close to restriction fragment length polymorphism (RFLP) marker pA132. A subclone of pA132, pUTG132a, was placed 0.7 cM from NTS-1 in a F2 population of nts382 (G. max Bragg) _ G. soja (P1468. 397) (Landau-Ellis et al, 1991, Mol. Gen.

Genet. 226 221; Kolchinsky et al, 1997, Mol. Gen. Genet. 254 29) and 1.3 cM from NTS-1 in a G. max nts246_ G. soja CPI 100070 population (Searle, 2002, PhD Dissertation, Univ. of Queensland). nts382 and nts246 were identified in

our original mutant screen (Carroll et al, 1985, Natl. Acad. Sci. USA 82 4162).

Amplified fragment length polymorphism (AFLP) marker UQC-IS1 also flanked NTS-1 1.9 cM away (Fig. 1 B) (Searle, 2002, PhD Dissertation, Univ. of Queensland). UQC-IS1 was the closest of 11 AFLP markers shown by bulk segregant analysis and genetic mapping to be linked to NTS-1 (Searle, 2002, PhD Dissertation, Univ. of Queensland). Bacterial artificial chromosome (BAC) clones derived from a soybean P1437. 654 library (Tompkins et al, 1999, Plant Mol. Biol. 41 25) were isolated by filter-hybridization to pUTG132a and UQC- IS1, and were verified to contain either pUTG132a or UQCIS1 by sequencing each marker from the respective BAC clone. Both the pUTG132a and UQC-IS1 BAC contigs were oriented relative to NTS-1 by mapping polymorphic BAC ends on F2 recombinants (Fig. 1 B).

Confirmation of mapping was aided by the fast neutron mutant FN37 (Men et al., 2002, Genome Letters 3 147). Physical mapping of markers and complete BAC sequencing of BAC17107 (135 kb) (Fig. 1B) showed that this mutant contains a chromosomal deletion in the NTS-1 region. The southern and northern deletion breakpoints were localized within the BAC17107 sequence and close to marker UQC-IS4, respectively (Fig. 1 B). Arrangement of putative open reading frames from BAC17107, sequenced BAC ends, and markers in the NTS-1 region showed contiguous microsynteny to Arabidopsis chromosomes 2 and 4 (Fig. 1B). Seven genes (three from the northern contig and four from the southern contig) were syntenic between the NTS-1 region and Arabidopsis. BAC92D22 contained three expressed sequence tags, highly

syntenic with Arabidopsis chromosome 2, but was not demonstrated to overlap with either the northern or southern contig.

For the UQC-IS1 contig, UQC-IS2 (southern end of BAC 95P14) mapped 1.2 cM away from the locus (Fig. 1 B). UQC-IS2 was used to identify additional BAC clones, of which BAC3K21 was demonstrated to extend towards NTS-1 by AFLP fingerprinting. BAC end UQC-IS3 was cloned and used to identify additional BAC clones of which BAC129E8 was shown by AFLP fingerprinting to extend further towards NTS-1. BAC end UQC-IS4 from BAC129E8 was cloned and used to select BAC75M10, which was AFLP fingerprinted in comparison to BAC129E8. One AFLP marker called UQC-IS5, 650 bp in length and located within BAC 75M10, was cloned and sequenced, revealing complete identity to a known gene called GmCLV1 B (27). This gene encodes a predicted protein showing 75% amino acid similarity to the shoot meristem-expressed CLAVATA1 of Arabidopsis (4). clv1 mutants of Arabidopsis have increased numbers of undifferentiated stem cells in shoot and floral meristems, and extra organs within flowers (4).

GmCLV1 B was a good candidate for NTS-1 because it mapped to the correct region, was absent in deletion mutant FN37 (FIG. 2), and the homologous CLV1 is involved in control of cell proliferation in shoot meristems.

The full-length genomic sequence of the candidate NTS-1 gene including about 600 bp upstream of the translation start, was obtained (Acc no. AY16665). In view of the difficulty of transforming soybean, confirmation of the gene was obtained by sequencing an allelic series rather than by complementing the mutant phenotype. Sequencing of GmClv1B from several wild-type and mutant

lines (Table 1) identified changes in the coding sequence that strongly indicated it was the gene responsible for control of nodule meristem proliferation (FIG. 1).

This gene was renamed GmNARK (Glycine max Nodule Autoregulation Receptor Kinase) to reflect its putative biochemical and developmental function in root nodulation.

EXAMPLE 3 GmNARK Protein and Mutation Analysis GmNARK encodes a predicted receptor-like protein kinase (RLK) composed of a 24 amino acid-long N-terminal signal peptide (MRSCVCYTLLLFIFFIWLRVATCS ; SEQ ID NO: 13), an extracellular domain composed of 19 tandem copies of a 24 amino acid leucine-rich repeat (LRR), a transmembrane domain (TRVIVIVIALGTAALLVAVTVYM ; SEQ ID NO: 14) and finally, a C-terminal cytoplasmic kinase domain. A 49 amino acid island interrupts the LRR domain between repeats 11 and 12. Overall, the protein contains 15 potential N-glycosylation sites.

The type of mutational change was correlated with the severity of the nodulation phenotype (Table 1; FIG. 1C). Allele nts1007 (causing a greater than 10-fold increase in nodule number) is a nonsense mutation that truncates the protein at glutamin residue 106 (Q106*), eliminating most of the LRRs and the entire protein kinase domain. The Q106* mutation was confirmed in the Australian variety PS55, carrying the nts1007 allele. Alleles nts246, en6500 and nts382, conferring extreme phenotypes almost identical to nts1007, also truncate the protein by nonsense mutations; nts246 (K115*) is located immediately downstream of nts1007 also in the LLR domain, en6500 (K606*) is

immediately upstream of the transmembrane domain and nts382 is in the kinase domain (Q920*). The deletion mutant FN37 also has an extreme supernodulation phenotype. As all five mutations (FN37 deletion, three receptor nonsense and a kinase nonsense) showed indistinguishable extreme supernodulation, the loss of the kinase activity is sufficient to confer the extreme phenotype. In contrast is the weak allele nts1116, conferring 2-3 fold increased nodulation. It is caused by a transition of valine to alanine at position 837, also in the kinase domain (FIG. 1C). Thus gene identification was confirmed by the characterisation of six independent mutant alleles, in which the predicted impact of the molecular alteration of the protein is precisely reflected in the severity of the phenotype. We confirmed that gene structure and amino acid sequence are conserved in wild-type soybean cultivars Clark, Williams, Bragg, G. soja P1468. 397 and G. soja CPI100070. Some silent mutations exist in the wild G. soja lines.

In contrast to CLV1 that is present as a single copy in Arabidopsis, in soybean GmNARK (same as GmCLV18) is strongly homologous to a duplicated gene called GmCLV1A (Yamamoto et al, 2000, Biochim. Biophys.

Acta 1491 333). GmNARK and GmCLV1A were previously investigated in a study of stem fasciation (Yamamoto et al, 2000, Biochim. Biophys. Acta 1491 333). Both genes were reported to be wild-type in fasciated soybean mutants but linked to pA381-1 (Yamamoto et al, 2000, Biochim. Biophys. Acta 1491 333), a RFLP marker in the vicinity of NTS-1 (24). The GmCLV1A gene differs from GmNARK by only about 10% at the nucleotide level, but a greater sequence divergence in the 3'UTRs allowed gene-specific amplification

(Yamamoto et al, 2000, Biochim. Biophys. Acta 1491 333). Interpretation of PCR and Southern analyses was aided by the availability of deletion mutant FN37, lacking GmNARK and several NTS-1-linked markers (FIGS. 1B, 2A, B).

GmNARK, but not GmCLV1A, is located on overlapping BAC clones BAC75M10 and BAC112J23 (FIG. 2B). GmCLV1A is only weakly detected in Southern analysis of FN37, presumably by virtue of a duplicated copy of GmCL\/M elsewhere the genome.

RT-PCR analysis with GmNARK 3'UTR-specific oligonucleotide primers was also used to determine tissue-specific transcript levels. While Arabidopsis CLV1 expression is restricted to the shoot apical meristem (SAM) (Clark et al, 1997, Cell 89 575), GmNARK is expressed in nodulated roots and shoots of wild type (FIGS. 2C, 2D). No GmNARK transcript was detected in FN37 (FIG. 2C). Quantitative RT-PCR also revealed that leaf GmNARK transcript levels are substantially higher than those in the SAM tissue (FIG. 2D) for wild type, nts1007 and nts382. Such expression of GmNARK is consistent with a primary role of the leaf in AON (Delves et a/, 1992, Plant, Cell and Environm. 15 249; Francisco and Harper, 1995, Plant Sci 107 167; Delves et a/., 1986, Plant Physio. 82 588).

GmNARK contains a single intron (465 bp) in the kinase domain, in precisely the same position as the intron (79 bp) in CLV1 (Clark et a/, 1997, Cell 89 575). The similarity of gene structure and protein sequence suggests that GmNARK shares functional and evolutionary similarities with CLV1 (FIG.

1D). CLV1 controls stem cell proliferation in shoot meristems and mutations lead to apical and floral meristem changes, whereas GmNARK functions in the

leaf and exerts long distance control of nodulation with no detectable SAM (FIG.

2E), floral or leaf phenotypes. Also, GmNARK displays differential tissue- specific expression to CLV1. It is therefore likely that (a) GmCLV1A is the immediate functional CLV1 orthologue, and (b) GmNARK is a duplicated version of CLV1, but with a different expression pattern and divergent function involved in long distance control of nodulation.

It is intriguing that GmNARK is most similar to CLV1, while two receptor-like kinase genes in the regions on chromosomes 2 and 4 of Arabidopsis syntenic with the soybean NTS-1 region are much more distantly related (FIG. 1D). There is no synteny between the NTS-1 region of soybean and the vicinity of CLV1 (data not shown). One possible explanation for this finding is that a localized gene recombination or conversion-like event may have occurred in evolution involving the CLV1 orthologue and another receptor-like kinase gene, such as to change the chromosomal location of CLV1 in either Arabidopsis or soybean.

Our findings suggest evolutionary mechanisms for the development of the root nodule symbiosis. Duplication of genes followed by divergence in function is a common theme in evolution (Venter et al., Science 291 1304). Ancestral duplication of a gene controlling stem cell proliferation in the SAM may have led to a variant mechanism in which shoot control of cell proliferation is extended to root tissue. Research in legumes into CLAVATA- related signalling may facilitate the understanding of key developmental processes such as nodulation that are absent in the model plant Arabidopsis.

Fast neutron mutant FN37 Analysis EXAMPLE 4 Materials and Methods Fast Neutron (FN) Mutagenesis and Isolation of Supernodulating Soybean Mutant FN37 About 15,000 soybean G. soja seeds (P1468. 397) were irradiated with a dosage of 8 Gy. M1 plants were self-pollinated and M2 seeds were harvested and analysed in bulk. Briefly, M2 plants were inoculated three days after sowing with microsymbiont Bradyrhizobium japonicum strain USDA 110 (approximately 1010 bacteria per plant) and were cultured in sand/vermiculite (1: 2 ratio). The plants received 1.2 L of nitrogen-free Herridge nutrient solution (Herridge, 1997, Carbon and nitrogen nutrition of two annual legumes (dissertation). Perth (WA): University of Western Australia) three times a week.

The nodulation phenotype of plants was scored 4 weeks after germination.

Putative mutants were transferred into new pots and supplied with full-nutrient solution (Herridge, 1997, Carbon and nitrogen nutrition of two annual legumes (dissertation). Perth (WA): University of Western Australia). After screening about 5000 M2 plants, one mutant with highly increased nodule number was found. This mutant was advanced through five generations of self-pollination and visual selection of progeny and showed a stable supernodulating phenotype. The mutant (designated FN37) was then subjected to genetic and molecular analysis (FN37 seeds are available from the corresponding author upon request).

Grafting Experiments

All four shoot/root, wild-type/mutant combinations were tested with at least four plants per graft. A protocol described by Delves et al, 1986, Plant Physiol 82 588 and Delves et al, 1992, Plant Cell Environ 15 249, was used with some modifications. Plants were grafted 2 weeks after sowing by wedge-shaped grafts in the hypocotyls with the cotyledons left on the scion.

Graft junctions were externally supported by short well-fitting Tygon tubes.

Plants were then covered with plastic bags or cut 2 L soft drink bottles and inoculated four days after grafting with either B. japonicum USDA110 strain or Nodulaid 100 (Bio-Care Technology Pty Ltd. , Somersby, NSW, Australia). The bottle lids were slowly opened and then discarded to permit venting and hardening off. Plastic covers were removed 1 week after and plants were harvested 9-10 weeks after germination and scored for nodulation phenotype.

BAC DNA Isolation, Fingerprinting, and End Sequencing Soybean BAC libraries (Meksem et a/, 2000, Theor. Appl. Genet.

101 747; Tomkins et al, 1999Plant Mol. Biol. 41 25; Danesh et al, 1998, Theor.

Appl. Genet. 96 196) arrayed as individual duplicated clones on 2205£2205 cM Nylon filters (4x4 gridding pattern) were screened by standard Southern hybridization procedures (Sambrook et al, 1989, Molecular cloning : a laboratory manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY).

Hybridization with radiolabeled probes was carried out for 20 hours at 65°C.

Filters were washed at 65. C twice with 2X SSC/0.1% SDS and twice with 2X SSC/0. 1X SSC (each wash was 20 min) and signals were detected by autoradiography. Supercoiled BAC DNA was isolated from 6-10 ml LB cultures as described by plasmid alkali lysis protocol (Sambrook et al, 1989, Molecular

cloning : a laboratory manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY). For insert size determination, 1 ! 11 of each BAC was digested with Notl followed by fractionation using PFGE. For fingerprinting, 2 zizi of BAC DNA was digested with either EcoRl or Hindlil and electrophoresed in a 1% agarose overnight at 2 V/cm. DNA bands were visualized by staining the gel with SYBRa Gold dye (Molecular Probes Inc., OR, USA). For each clone we normally observed 20-25 DNA bands in the range of 0.4-10 kb. BACs were considered as overlapping if they shared >70% of the bands. Based on the presence of either common or unique DNA bands, overlapping BACs were assembled into contigs. After fingerprinting, all bands were manually called and the sum of the band sizes from each gel lane was divided by the BAC size (estimated by the Notl digestion/PFGE) in order to find the proportion of the BAC DNA represented by fingerprinting (proportion was usually about 65- 70%). Based on this coefficient, the approximate lengths of overlaps between BACs as well as unique, nonoverlapping portions of the BACs were determined. BAC ends were sequenced using standard ABI chemistry using 1 Ll of BAC DNA, 16 pI reaction mixture (ABI/PE #402122, ABI, USA), and 50 pM of either T7 or M13 reverse (or Sp6) sequencing primer. The final reaction volume for cycle sequencing was 40 pI, and sequencing PCR was performed for 99 cycles as recommended by the BACPAC sequencing protocol (Children's Hospital Oakland Research Institute, CA, USA, http : //www. chori. org/bacpac/home. htm). Sequencing reactions were separated on an ABI 377 sequencer at the Australian Genome Research Facility, Brisbane, Australia.

PCR and Southern Analysis of Wild-Tvpe and FN37 Soybean genomic DNA was isolated as described in (Carroll et al, 1995, Genetics 139 407). The sense and antisense oligos were designed based on the end sequences of BACs mapped close to NTS-1 gene. PCR thermal cycling consisted of 5 min at 95°C, then 35 cycles of 15 s at 94°C, 30 s at a specific annealing temperature, and 2 min at 72°C. The final extension was 7 min at 72°C. Oligos for pUTG132a (GeneBank accession number Z26335) were 5'-GCA GCA GTG TTG GGC ATG TCT CT (SEQ ID NO: 21) and 5'-CTG CAG AAT TGG ATT CCC AAA AGC (SEQ ID NO: 22) (annealing temperature 60°. C); for UQCIS1 (accession number AY043286) : 5'-GTC CCA AAC ATA GCC AAT GGA ATC ACG AC (SEQ ID NO: 23) and 5'-GGT AGA GGT GAC TTA GAC AGA GGT G (SEQ ID NO: 24) (annealing temperature 55°. C). All BACs described below come from the soybean (G. max cv. P1437654) GM_PBa BAC library (Tomkins et al, 1999Plant Mol. Biol. 41 25). Primers for the T7 ("southern") end of BAC17107 (accession number BH919817): 50-GCT TCA AAT CGC AGC ACA ATT (SEQ ID NO: 25) and 5-TTT AGA TTT ACA TCA AGA ACT A (SEQ ID NO: 26) (annealing temperature 55°C) ; for UQG- AS1 (Sp6 end of BAC17107, accession number BH919818): 5'-ATT TGA ATA TGC ATT GTT TTA AGT G (SEQ ID NO: 27) and 5'-CGT CTT CTT'AAA TCC TCA AAT TAC TA (SEQ ID NO: 28) (annealing temperature 56°C) ; for T7 ("southern") end of BAC164M23 (accession number BH919816): 5'-TGA ATA CAT AAA CCC CCA TAG ATG C (SEQ ID NO: 29) and 5'-GGT CAC TAT CAG AAA GAA CGA AGC (SEQ ID NO: 30) (annealing temperature 60°. C); for UQG-AS2 [Sp6 ends of either BAC164M23 or BAC156F11 (accession number

BH919814) ]: 5'-ATTA GTG TCC GAG TTT'AAT CTA C (SEQ ID NO: 31) and 5'-TCA GAA TTG GTA ATT AAA GCA ATT C (SEQ ID NO: 32) (annealing temperature 55°C) ; for UQC-IS2 (T7 end of BAC95P14) : 5'-AGC TTG TTA GAC CTG AAG GAT GTT C (SEQ ID NO: 33) and 5'-TAT TAC TTA GTC AAC GTG AAA TCT CC (SEQ ID NO: 34) (annealing temperature 60°C) ; for UQC- IS4 (T7 end of BAC129E8): 5'-AAA CAC ACA CAC TTT CTA CTA AGG TGG (SEQ ID NO: 35) and 5'-TTT GAA TCT CTG TGT TTC GAT GTG (SEQ ID NO: 36) (annealing temperature 60°C).

For Southern hybridization, soybean genomic DNA was digested with restriction endonucleases (15 pl of DNA per digestion) followed by 18 h electrophoresis in 1% agarose. Blotting and hybridization were performed in conditions identical to the BAC library screening (Sambrook et al, 1989, Molecular cloning : a laboratory manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY), and the hybridization signals were detected either by a Phospholmager (Molecular Dynamics, Castle Hill, NSW, Australia) or by autoradiography.

EXAMPLE 5 Results and Discussion Mutant Isolation Eight Gy FN mutagenesis of soybean G. soja (P1468. 397) seeds gave nearly 60% lethality in seedling growth. Of the surviving M1 plants, most had normal morphology. For example, chlorophyll deficient leaf sectors were rarely observed. About 5000 M2 plants were analyzed for nodulation abnormalities. FN37 was the only supernodulation isolate. Several putative

non-nodulating mutants were also detected and are presently being tested for stability.

EXAMPLE 6 GmNARK Mutant Characterization Mutant FN37 showed increased nodule number compared to the wild-type parent (FIG. 3). To identify whether the phenotype of the FN37 mutant was conferred by aboveground parts (as previously shown for nts246, nts382, nts1007, and other allelic mutants (Delves et al, 1986, Plant Physiol. 82 588; Francisco and Harper, 1995, Plant Sci. 107 167) the present investigators performed reciprocal grafting experiments. Supernodulation was consistently conferred to root stocks by mutant scions, whereas wild-type scions grafted on any root stocks resulted in wildtyp nodulation (FIG. 3). Therefore, the supernodulation phenotype of FN37 was controlled by the plant scion suggesting that this mutant was part of the existing class of autoregulationmutants previously described in soybean and L. japonicus (Delves et al, 1986, Plant Physiol. 82 588 ; Francisco and Harper, 1995, Plant Sci. 107 167; Wopereis et al, 2000, Plant J. 23,97).

EXAMPLE 7 Genetic Mapping Since the FN37 mutant phenotypically resembled allelic EMS supernodulating mutants nts246, nts382, and nts1007 (Carroll ef a/, 1985, Proc.

Natl. Acad. Sci. USA 82 4162; Delves et al, 1986, Plant Physiol. 82 588), the possible relationship between FN37 and EMS mutant genomes was investigated. The phenotype of the EMS mutants is conferred by single

recessive mutations in the NTS-1 gene. We previously showed that in the C16 cross G. max (nts382) £ G. soja (P1468. 397), NTS-1 maps between two RFLP markers, pUTG132a located 0.7 cM"south"of NTS-1 and marker pA381-1 located on the"north"side (Kolchinsky et al, 1997, Molec. Gen. Genetics 254 29; Men and Gresshoff, 2001, Plant J. Physio. 158,999). A new cross between G. max (nts246) and G. soja (Pi 100. 070) was generated. This cross was named UQnts. We then implemented an AFLP approach (Vos et al, 1995, Nucleic Acids Res. 23 4407) on the UQnts F2 segregating population to identify additional markers distal to pUTG132a. Eleven linked AFLP markers were found [34], of which marker UQC-IS1 was shown to be the closest and mapped 1.5 cM"north"from NTS-1 (genetic map on the left part of FIG. 4). In the UQnts segregating population, the pUTG132a marker was mapped further (1.2 versus 0.7 cM) from NTS-1, when compared to the C16 population (genetic map in FIG. 4).

EXAMPLE 8 BAC Isolation Four publicly available soybean BAC libraries (Meksem et al, 2000, Theor. Appl. Genet. 101 747 ; Tomkins et al, 1999, Plant Mol. Biol. 41 25; Danesh et al, 1998, Theor. Appl. Genet. 96 196; Salimath and Bhattacharyya, 1999, Theor. Appl. Genet. 98 712) were screened with pUTG132a and UQC- IS1 probes. Eighteen and fifteen positive BACs were identified, respectively.

Most of the BACs (11 for pUTG132a and 8 for UQC-IS1) were derived from the G. max cv. P) 437654 GMPBa BAC library (Tomkins et al, 1999, Plant Mol.

Biol. 41 25). The positive BACs were characterized for insert size and Hindlll

fingerprinting (FIG. 5). By comparing BAC fingerprints (lanes 1-8 in the agarose gel stained with SYBRe Gold), the most informative BACs (lanes 3 and 5) were easily found, because they contained all bands represented by the other six clones and had extra DNA fragments of about 1.2 kb (lane 3) and 1.3 kb (lane 5) (FIG. 5). The informative BACs selected from the fingerprinting experiments were then subjected to end sequencing. Sequenced BAC ends were converted into sequence tagged sites and used in the next round of BAC library screenings. The whole screening procedure was repeated three times for each contig. No additional BAC clones extended further north from BAC156F11 were found in any of the four BAC libraries (125£ soybean genome equivalents) (Meksem et al, 2000, Theor. Appl. Genet. 101 747; Tomkins et al, 1999, Plant Mol. Biol. 41 25; Danesh et al, 1998, Theor. Appl. Genet. 96 196; Salimath and Bhattacharyya, 1999, Theor. Appl. Genet. 98 712). One BAC was, however, found that extended further"south"from BAC129E8 and contained the NTS-1 locus, but this BAC did not overlap with the"south"contig (Searle et al, 2002, Science 299 109). The central part of FIG. 4 shows the"south"and"north" contigs built by aligning three of the most informative BACs containing pUTG132a and UQCIS1 markers, respectively.

EXAMPLE 9 Delineation of the Deletion To define the extent of the FN37 deletion, we used PCR probes developed from markers and BAC ends mapped close to NTS-1. First, the PCR primers for the pUTG132a marker, as well as both ends of the pUTG132a- positive BAC17107 (135 kb long), were used to amplify both wild-type and

FN37 DNA. Data showed that the"south" (ST7) end of BAC17107 was present in both genomes, whereas both pUTG132a and"north"end of BAC17107 were deleted in FN37 (FIGS. 6A and 6B). The investigators then performed the same analysis with loci derived from both ends of BAC156F11 and BAC164M23.

PCR results indicated that all end clone sequences of BAC156F11 (FIG. 6B) and 164M23 (data not shown) were deleted in FN37. The PCR data were con. rmed by simultaneous Southern hybridization with two labeled probes derived from BAC ends (FIG. 7A). One of the fragments, namely UQG-AS2, was derived from BAC156F11 and deleted from FN37 DNA based on the PCR test.

The second probe derived from the ST7 end of BAC17107 and gave a positive PCR result on FN37 DNA. The results of the Southern hybridization revealed signals present in or deleted from FN37 DNA (FIG. 7A). Based on the size of BAC156F11 (140 kb), and assuming from the fingerprinting that BAC164M23 extended 10-20 kb south from BAC156F11, preliminaryestimation of DNA loss from the"south"contig in FN37 was >150 kb.

In order to obtain more information on the genomic environment of NTS-1 and to determine physical/genetic distance relationships for this area of the soybean genome, we have sequenced and annotated the entire 135 kb long BAC17107. From the sequencing data, the physical distance between pUTG132a and UQGAS1 is 83.7 kb. On the other hand, mapping of F2 soybean recombinants in the interval between pUTG132a and UQG-AS1 showed that the genetic distance between these two markers is 0.3 cM (Searle, 2002, dissertation, Brisbane (QLD): University of Queensland, Australia), giving a physical/genetic distance ratio in this region of soybean genome as 279

kb/cM. Since the ST7 end of BAC17107 was still present in the FN37 DNA, we recently have derived PCR probes for every 10 kb region of BAC17107 starting from the ST7 end. This analysis showed that the deletion start was located <40 kb from the"south"end of BAC17107, based on the 10 kb resolution (data not shown). Similarly, BAC ends derived from the"north"contig were PCR-tested on wild-type and FN37 DNA. Previous BAC fingerprinting as well as PCR analysis, hybridization, and genetic mapping of BAC ends showed that the orientation of the"north"contig relative to NTS-1 was as illustrated in FIG. 4 (Men and Gresshoff, 2001, Plant J. Physio. 158, 99; Searle, 2002, dissertation, Brisbane (QLD): University of Queensland, Australia). PCR and hybridization experiments demonstrated the presence of all"north"contig markers located between UQC-IS1 and UQC-IS3 the in FN37 DNA (data not shown). The north end of the deletion was determined with the T7 end of BAC129E8 (UQC-IS4 in FIG. 4). UQC-IS4 was partially deleted from the FN37 genome as determined by PCR (data not shown) and rearranged as shown by Southern hybridization (FIG. 7B).

UQC-IS4 has high homology to several plant vacuolar proton pump ATPase genes and most probably represents a member of a gene family in the soybean genome as illustrated by the presence of multiple bands on the Southern blot (FIG. 7B). Additionally, the presence of multiple UQCIS4 paralogs detected in this experiment is consistent with the amphidiploid nature of soybean and the presence of multigene families (Shoemaker et al, 1996, Genetics 144 329). Since other markers north of UQCIS4, such as ESTs and genes located on BAC95P14 and BAC3K21, also showed duplications but

neither loss nor rearrangement in FN37 (data not shown), it was evident that the region of UQC-IS4 defined the"north"end of the chromosomal deletion.

Complex rearrangements detected by this Southern hybridization indicate that the deletion break point is located within UQC-IS4, because both gain of new bands (enzymes Hind))) and EcoRI) and loss of a fragment (enzyme Ncol) were observed between wild-type and FN37 DNA.

Because of the presence of the ST7 end of BAC17107 and markers UQC-IS1, UQC-IS2, and UQC-IS3 in the FN37 genome, PCR haplotyping of FN37 DNA with BAC ends enabled us to determine the"north" and"south"sides of the deletion and also to confirm the orientation of both contigs relative to NTS-1. As estimated by analysis of the"south"contig, the genetic/physical distance ratio in this region is 279 kb/cM. Genetic distance between pUTG132a and UQC-IS3 is 1. 8 cM (Men and Gresshoff, 2001, Plant J.

Physio. 158,999 ; Searle, 2002, dissertation, Brisbane (QLD): University of Queensland, Australia), and physical distance between UQC-IS3 and UQC-IS4 is about 40 kb based on BAC129E8 analysis. Therefore the estimated size of the deletion in FN37 mutant is 460 kb, assuming physical collinearity between G. max and G. soja genomes.

EXAMPLE 10 Kinase Assay The kinase domain, defined by the SMART database (Schultz et al, 1998) was cloned into the MCS of 6X His tag vector pGEM-T Easy (Promega) forming a His tag construct that was selected using blue-white detection. The His tag construct was transformed into pET15b E coli cells for

expression after IPTG induction from the T71acZ promoter. Expressed kinase domain was purified via Ni-NTA agarose columns. Purification was carried out under native conditions. Purified enzyme and crude cell lysate were analyzed by gel electrophoresis and strained for total protein using Coomassie Blue stain. The same cell was blotted and probed by antibody against the His tag domain. Purified protein and crude extract were assayed for in vitro kinase activity using gamma labeled ATP, followed by fluorimetry of separated proteins.

The results shown in FIG. 8 indicated that the E. coli expressed His tagged kinase domain retains autophosphorylation activity.

Promoter Nucleotide Sequence Analysis EXAMPLE 11 PLACE (Higo et al, 1999, Nucleic Acids Research 27 297) is a WV\/\N-accessible database of nucleotide sequence motifs found in plant cis-acting regulatory elements. Regulatory elements are show in FIGS. 10 and 11 and in Tables 4 and 5. The search for the potential cis-element motifs on GmNARK promoter was performed using the homology search tool, SIGNAL SCAN program, available at the PLACE Web site (http ://www. dna. affrc. go. jp/htdocs/PLACE). Many motifs having sequences homology to plant TATA boxes were found; however, the mapping of the transcription start site by S1 nuclease assay (Ruiz de Almodóvar et al., 2002) will be performed according to the TATA boxes found not further than 200 bp upstream of the translation start site. The confirmation of the transcription start site will be performed using primer extension analysis

(Sambrook et al, 1989, Molecular cloning : a Laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). In addition to TATA boxes, an 8-bp potential negative regulatory region (NRR) homology to NRR in promoter region of Brassica napus extA extension gene (Elliott and Shirsat, 1998) was found between-301 to-308 bp upstream of the translation start site of GmNARK. The experiment done by Elliott and Shirsat (1998) revealed that removal of this region led to expression in all tissues within the stem internode, petiole and root in tobacco. Interestingly, the exactly matched sequence was also found at approximately the same location on the promoter of HAR1.

Whether or not this element is responsible for the negative regulation of GmNARK will be experimentally tested in this study. The further in silico finding of motifs on GmNARK promoter was performed by a comparison of GmNARK promoter and HAR1 promoter using Multiple EM for Motif Elicitation (MEME) (Bailey and Elkan, 1994). The results from MEME will assist in the determination of deletion sites in the promoter truncation experiments.

Promoter truncation or promoter deletion analysis will assess functional regions of regulatory control element. Deletion of nucleotides located 5'of the GmNARK gene may be fused to a reporter gene, such as GUS or green fluorescent protein (GFP) and expression patterns of the reporter are examined. Internal deletions and point mutations within the functional promoter regions can help localize specific cis-acting regulatory elements.

Any suitable plant or plant material may be transformed and selection of an appropriate material for transformation may be selected by a person skilled in the art. Such plant material may include for example, transient

or stable transformation of soybean protoplasts (as described by Kim et al., 1995) or immature seeds (lida et al., 1995) or introduced the promoter/reporter gene constructs into species that had rapid and efficient transformation systems such as tobacco (Schoeffl et al., 1989; Susuki et al., 1993; Sadka et al., 1994; Buzeli et al., 2002), cowpea (Susuki et al., 1993), and Lotus comiculatus if the organs of interest are nodules (Jorgensen et al., 1988, 1991; Stougaard et a/. 1990).

Lotus japonicus is a model legume used to study legume- Rhizobium symbiosis (Handberg and Stougaard, 1992; Jiang and Gresshoff, 1997; Schauser et al., 1999). Transformation of L. japonicus hypocotyls using Agrobacterium tumefaciens is relatively efficient via shoot organogenesis (Handberg and Stougaard, 1992). This method has been further optimized.

The frequency of the gene transfer is reported to be as high as 70% and the time used to produce fertile transgenic plants was as short as 4 months (Stiller et al., 1997).

Preliminary QRT-PCR data (not shown) revealed a similarity between transcript levels of GmNARK and that of HAR1 previously reported by Nishimura et a/. (2002) and Krusell et a/. (2002). Therefore, L. japonicus transformation system may be useful for GmNARK promoter deletion analysis since soybean itself has been regarded as recalcitrant to transformation.

Soybean transformation is commonly via A. tumefaciens- mediated T-DNA delivery into regenerable cells in the axillary meristems of the cotyledonary node. The method was initiated by Hinchee et a/. (1988) and recently improved by Olhoft and Flagel (2003). Combining strategies to

enhance A. tumefaciens-mediated T-DNA delivery into cotyledonary-node cells with the development of selection system based on hygromycin B was reported to significantly increase the efficiency of soybean transformation from an average of 0.7% to 16.4% (Olhoft and Flagel, 2003).

The GmNARK promoter/reporter gene constructs will be first introduced into both L. japonicus and soybean. If there is no major difference between the expression patterns of a reporter gene driven by GmNARK promoter constructs in L. japonicus and soybean, internal deletion and/or point mutation of GmNARK promoter will be tested mainly in L. japonicus.

Identification of specific protein-DNA binding sites will be performed using electrophoretic mobility shift and deoxyribonuclease-I footprinting assays.

EXAMPLE 12 Quantitative real-time RT-PCR RNA will be extracted from roots, nodules, mature leaves, young leaves and SAMs of inoculated soybean plants using SDS/Phenol extraction method. DNA contamination will be removed by the use of DNase I treatment following by chloroform extraction. The quantification of DNase-treated RNA will be determined by measuring the absorption at 260 nm. The first-strand cDNA synthesis will be performed using the combination of random primers and oligo (dT) mix as the primers. The quantitative real-time PCR reaction will be carried out in an ABI PRISME 7700 Sequence Detection System according to the manufacturer's instructions. Primers will be designed using Primer Express 1.0 software (PE Applied Biosystem). Ribosomal RNA will be used as internal control for the normalization of the data.

EXAMPLE 13 Bioinformatics The 4289-bp sequence of Glycine max nodule autoregulation receptor kinase gene (GmNARK) including 616 bp upstream of the translation start site was sequenced and deposited in the GenBank with accession number AY166655 (Searle et al., 2003). The 616 bp of GmNARK promoter and 5' untranslated region from GenBank and another 1.4 kb of GmNARK promoter were analysed for the potential cis-acting regulatory elements using the PLACE database (Higo et a/., 1999). A sequence comparison of the 2-kb of the GmNARK promoter and the 2-kb sequence upstream of the translation start site of L. japonicus HAR1 (LjHAR1) (GenBank accession number AP005667) was performed using MEME (Bailey and Elkan, 1994). The results are summarized in FIGS. 10 and 11 and tables 4 and 5.

EXAMPLE 14 Mapping transcription start site S1 nuclease analysis S1 nuclease analysis will be performed as described by Ruiz de Almodóvar et a/. (2002). A 60-bp antisense oligonucleotide spanning the potential transcription start site will be designed based on the potential TATA boxes found on the promoter region. This oligonucleotide will be labeled using the polynucleotide kinase and [y-32P] ATP and then hybridized to soybean total RNA. S1 nuclease will be used to digest the non-hybridized single-stranded RNA. The digested product and non-digested (control) labeled oligonucleotide will be analyzed on an 8% denaturing polyacrylamide gel at the same time.

Primer extension The transcriptional start site of GmNARK will be confirmed by using primer extension analysis as described by Sambrook et a/. (1989). The primer designed according to sequence approximately 100 bases downstream from the 5'-end of the GmNARK will be radioactively labeled and used to anneal soybean total RNA. The extension will be performed using reverse transcriptase. The reaction products will be separated on an 8% denaturing polyacrylamide gel and visualized by autoradiography. Sequencing reactions using the same primer will be run at the same time as size standards.

Promoter-reporter constructs The 2.0-kb promoter of GmNARK will be amplified by PCR using plasmid DNA derived from BAC 75M10 as the template. The forward primer was designed to anneal at the-1965 bp uptream of the translation start site.

The reverse primer was designed to flank the translation start site and carry Ncol restriction site at the first codon. The PCR product will be directly inserted into pCR2.1 Vector using a TA Cloning Kit (Invitrogen). The presence and orientation of the PCR product will be analyzed by restriction enzyme digestion.

The plasmid DNA from the clone that contains the correct orientation of PCR product will be partially digested with Ncol. The 1965-bp promoter of GmNARK will be translationally fused to the p-giucuronidase (GUS) gene containing catalase intron in plasmid pUbiGUSP/us (Vickers et a/., 2003). Full-length and different truncated promoter constructs of GmNARK promoter will be generated based on the results of bioinformatics analysis (FGI. 17) using restriction enzyme digestion and PCR. The constructs will be introduced into polylinker

site of plasmid pGreenO179 (Hellens et a/., 2000). The transformation of Agrobacterium tumefaciens strain LBA4404 will be performed according to Hellens et al. (2000).

EXAMPLE 15 Plant transformation Transient transformation Leaves of soybeans, Glycine max (L.) Merr. cv'Bragg', will be collected and surface-sterilized. The plasmids containing promoter-reporter constructs will be co-precipitated onto gold particles and used to bombard the leaves as described by Vickers et al. (2003).

Hvpocotvl transformation of Lotus iaponicus L. japonicus ecotype Gifu B-129 will be used in this study.

Hypocotyl transformation will be performed as described by Stiller et a/. (1997).

Scarified and sterilized seeds were germinated in darkness. Hypocotyls from these seeds will be cut and co-cultivated with A. tumefaciens strain LBA4404 harboring plasmids with GmNARK promoter-GUS reporter constructs. The explants will be then transferred to regeneration medium and incubated for 5 d and subsequently transferred to selection medium for 4 weeks. Shoot induction and shoot elongation will be achieved on shoot induction medium (SIM) and shoot elongation medium (SEM) respectively. Well-grown shoots will be cut off and inserted into root induction medium (RIM) and incubated under the appropriate conditions for a week.

Cotyledonary-node transformation of soybean The cotyledonary-node method will be performed as described by

Olhoft and Flagel (2003). Soybean seeds from the cultivar Bragg will be gas sterilized and then grown 5-6 days on germination medium. Two explants will be obtained by removing the roots and the majority of the hypocotyls below the cotyledonary node, separating the cotyledons, and finally cutting vertically through the remaining hypocotyls. The epicotyl will be subsequently removed and both the axillary bud and the cotyledonary node will be wounded by cutting ten times perpendicular to the hypocotyls. The explants will be then inoculated in the suspension of A. tumefaciens strain LBA4404 carrying plasmids with GmNARK promoter-GUS reporter constructs, placed on solid co-cultivation medium containing sterilized 1 mM sodium thiosulfate, 8.8 mM L-cysteine and 1 mM DTT and incubated at 25°C in the dark. After 5 days of incubation, the explants will be transferred into solidified SIM for the first 14 days. The newly developed shoots will be sub-cultured to fresh SIM containing selective agent.

Cotyledons will be excised from the callus/shoot pad after 28 days on SIM and the callus will be transferred to SEM containing selective agent. Elongated shoots will be placed into RIM. Rooted plants will be transferred into soil and grown in the greenhouse.

EXAMPLE 16 Plant culture and nodulation induction Plants cultured in vermiculite pots will be watered with B&D nutrient solution (Broughton and Dilworth, 1971) three times a week. Plants will be supplied with appropriate concentration of KN03 whenever it is necessary.

Induction of nodulation in L. japonicus and soybean will be performed using diluted 3-d-old culture of Mesorhizobium loti strain NZP2235 and

Bradyrhizobium japonicum strain CB1809 in YMB medium respectively.

EXAMPLE 17 GUS assays Fluorimetric analysis of GUS activity will be performed according to Jefferson et al. (1987). Plant extracted will be prepared from different tissues of transgenic plants and assayed by enzymatic conversion of 4- methylumbelliferyl ß-D-glucuronide to 4-methylumbelliferone. Protein quantification in the extracts will be done as described by Bradford (1976). GUS activity will be expressed as umol 4-MU per mg protein per hour. The average GUS activity and standard error will be calculated using at least five biological replicates per construct. Histochemical localization of GUS activity will be performed as described by Jefferson (1987) using 5-bromo-4-chloro-3-indolyl-D- D-glucuronic acid (X-gluc) as chromogenic substrate. The samples will be photographed on Nikon SMZ800 Stereomicroscope or sectioned and photographed on Nikon E600 Compound Microscope using SPOT RT Colour Digital Cooled camera.

EXAMPLE 18 Mapping specific protein-DNA binding sites Nuclear extracts will be prepared from soybean leaves as described by Jacobsen et a/. (1990). Electrophoretic mobility shift and DNase-I footprinting assay will be performed according Tang et al. (2001).

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by

those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.

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Table 1 Genetic allelel supernodulation mutation reference2 background phenotype G. max Bragg nts382 extreme Q920* 11,12 G. max Bragg nts1007 extreme Q106* 11 G. max Bragg nts1116 intermediate V837A 11 G. max PS55 nts1007 extreme Q106* 11 G. max nts246 x nts246 extreme K115* 11 G. soja CPI 1000703 G. max Enrei en6500 extreme K606* 15 G. soja PI468. 397 FN37 extreme deletion 16 Table 2 RT+ Actin GmNARK #Ct Ratio Sample R1 Ct R2 Ct Ave R1 Ct R2 Ct Ave Bragg leaf 25.22 25.58 25.4 26.16 26.11 26.13 0.73 20.9 nts10071eaf 24. 66 25 24. 83 25.51 26.12 25.81 1.9 9.3 5 nts382 leaf 24.33 24.27 24.3 26.02 26.39 26.2 0.98 17.5 Bragg SAM 21.48 21.26 21.37 26.45 26. 53 26.49 5. 12 1 nts1007 21.96 21.73 21. 84 28.74 27.62 28.18 5.34 0. 85 SAM 5 nts382 SAM 23. 08 22.22 22.65 27.88 28.1 27.99 6.33 0. 43

Table 3 RT-Actin GmNARK Samples R1 R2 R1 R2 Bragg leaf 32.48 32.52 36.43 36. 27 nts10071eaf 34. 56 35. 08 45 45 nts382 leaf 31.01 32.6 39.48 34.66 Bragg SAM 30.04 32.58 36.85 34.27 nts 1007 SAM 35. 28 34. 94 45 45 nts382 SAM 31. 46 31.08 34. 42 36.01 Table 4 Identifier Accession Description (1) ROOTMOTIFTAPOX1 S000098 rolD promoter of "ATATT"Agrobacterium rhizogenes ; Expression of the rolD-gus (+) 53, 78, 89, 255, 340 gene in mature transformed (-) 77, 98, 228, 339, 474 tobacco was strong in roots (strongest in the root elongation (LjHAR1 : many) zone and in vascular tissue, and much less in the root apex), and much lower in stems and leaves. (2) NRRBNEXTA S000242"NRR (negative regulatory "TAGTGGAT"region)"in promoter region of Brassica napus (B. n.) extA +) 309 extensin gene ; Removal of this region leads to expression in all (LjHAR1 : (+) 331) tissues within the stem internode, petiole and root (in tobacco) ; Extensin is one of cell wall proteins that play an important role in the plant defense response. Plt Mol Bio 37 : 675-687 (3) GT1 CONSENSUS S000198 Consensus GT-1 binding site in many light-regulated genes, e. g., RBCS from many species, (-) 267, 653, 356, 357, 384 bean CHS15 ; GT-1 can stabilize the TFIIA-TBP-DNA (LjHAR1 : (+) 2, (-) 374) (TATA box) complex ; Binding of GT-1-like factors to the PR-1 a promoter influences the level of SA-inducible gene expression (4) CAATBOX1 S000028"CAAT promoter consensus "CMT"sequence"found in legA gene of pea ; Sequences responsible (+) 227, 265 for the tissue specific promoter (-) 193, 257, 342, 417, 646 activity of a pea legumin gene in tobacco (LjHAR1 : many) (5) SEF4MOTIFGM7S S000103"SEF4 binding site" ; Soybean consensus sequence found in "RTTTTTR"5'upstream region (-199) of beta-conglycinin (7S globulin) (-) 65, 94, 171, 527 gene (Gmg17. 1) ;"Binding with SEF4 (soybean embryo factor (LjHAR1 : (+) 73) 4)" (6) TATABOX4 S000111"TATA box" ; TATA box found in the 5'upstream region of "TATATAA" (+) 568 sweet potato sporamin A gene (LjHAR1 : NO) (7) ARFAT S000270 ARF (auxin response factor) binding site found in the "TGTCTC" (-) 612 promoters of primary/early auxin response genes of A. t. ; AuxRE ; Sequence found in (LjHAR1 : NO) NDE element in Soybean SAUR (Small Auxin-Up RNA) 15A gene promoter ; Involved in auxin responsiveness ; Found in D1 or D4 element in Soybean GH3 promoter (8) PROLAMINBOXOSGLUB1 S000354"Prolamine box"found in the rice GluB-1 gene promoter ; "TGCAAAG" (+) 595 Involved in quantitative regulation of the GluB-1 gene (LjHAR1 : NO) (9) TATABOX5 S000203"TATA box" ; TATA box found in the 5'upstream region of pea "TTATTT" (-) 530 (Pisum sativum) glutamin synthetase gene ; a functional (LjHAR1 : (-) 257) TATA element by in vivo analysis (10) RYREPEATGMGY2 S000105 RY repeat motif ; Present in the "CATGCAT"S000100 5'region of the soybean (G. m.) RYREPEATLEGUMINBOX glycinin gene (Gy2) "CATGCAY" (+) 448, (-) 26 RY repeat (CATGCAY)"or legumin box found in seed- (LjHAR1 : (-) 703) storage protein genes in legume such as soybean (G. m.) ; (11) CATATGGMSAUR S000370 Sequence found in NDE "CATATG"element in soybean (G. m.) (+) 429, 661 SAUR (Small Auxin-Up RNA) (-) 429, 661 15A gene promoter ; Involved in auxin responsiveness (LjHAR1 : NO) (12) CACGCAATGMGH3 S000368 Sequence found in D4 element "CACGCAAT"in Soybean (G. m.) GH3 gene (-) 417 promoter ; Showed constitutive activity with TGTCTC element (LjHAR1 : NO) (ARFAT) ; Confers auxin inducibility ; Binding site of nuclear protein TBOXATGAPB S000383"Tbox"found in the Arabidopsis (13) thaliana (A. T.) GAPB gene "ACTTTG"promoter ; Mutations in the "Tbox"resulted in reductions of (+) 372 light-activated gene transcription ; GAPB encodes (LjHAR1 : (-) 480) the B subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GADPH) of A. T. (14) PYRIMIDINEBOXHVEPB1 S000298"Pyrimidine box"found in the "TTTTTTCC"barley EPB-1 (cysteine (+) 355 proteinase) gene promoter ; Required for GA induction (LjHAR1 : NO) (15) ASF1MOTIFCAMV S000024"ASF-1 binding site"in CaMV "TGACG"35S promoter ; ASF-1 binds to (+) 334 two TGACG motifs ; Found in HBP-1 binding site of wheat histone H3 gene ; TGACG motifs are found in many promoters and are involved in transcriptional activation of (LjHAR1 : NO) several genes by auxin and/or salicylic acid ; May be relevant to light regulation ; Binding site of tobacco TGA1 a ; TGA1 a and b show homology to CREB ; TGA6 is a new member of the TGA family ; Abiotic and biotic stress differentially stimulate "as-1 element"activity (16) POLLEN 1LELAT52 S000245 One of two co-dependent "AGAAA"regulatory elements responsible (+) 247 for for (-) 41, 177, 269, 655 pollen specific activation of tomato (L. e.) lat52 gene ; (LjHAR1 : many) AGAAA and TCCACCATA (S000246) are required for pollen specific expression (17) BOXCPSAS1 S000226 Box C in pea asparagine "CTCCCAC"synthetase (AS1) gene ; AS1 is negatively regulated by light ; (+) 209 Box C binds with nuclear proteins, which was competed (LjHAR1 : (-) 343) by a putative repressor element RE1 (18) SEBFCONSSTPR10A S000391 Binding site of the potato "YTGTCWC"silencing element binding factor (-) 152 (SEBF) gene found in promoter of pathogenesis-related gene (LjHAR1 : NO) (PR-1 Oa) ; Similar to the auxin response element (19) GTGANTG10 S000378"GTGA motif'found in the "GTGA"promoter of the tobacco (N. t.) (+) 152 (-) 298 late pollen gene g10 which shows (LjHAR1 : many) homology to pectate lyase and is the putative homologue of the tomato gene lat56 (20) POLARSIG1 S000080"PolyA (polyadnylation) signal" ; "AATAAA"poly A signal found in legA (+) 140 gene of pea, rice alpha- (-) 56, 60, 650 amylase (LjHAR1 : (+) 253) (21) GATABOX S000039"GATA box" ; GATA motif in CaMV 35S promoter ; Binding "GATA"with ASF-2 ; Three GATA box repeats were found in the (+) 48 promoter of Petunia (P. h.) (-) 106 chlorophyll a/b binding protein, Cab22 gene ; Required for high (LjHAR1 : many) level, light regulated, and tissue specific expression ; Conserved in the promoter of all LHCII type I Cab genes SEF1 MOTIF S000006"SEF1 (soybean embryo factor (22)"ATATTTAWW"1)"binding motif ; sequence (+) 53, 89 found in 5'-upstream region (- 640 ;-765) of soybean beta- conglicinin (7S globulin) gene (23) NTBBF1ARROLB S000273 NtBBF1 (Dof protein from "ACTTTA"tobacco) binding site in (-) 22 Agrobacterium rhizogenes (A. r.) rolB gene ; Found in regulatory domain B (-341 to- 306) ; Required for tissue- specific expression and auxin induction (24) TATABOX2 S000109"TATA box" ; TATA box found in "TATAAAT"the 5'upstream region of pea (+) 496 legA ; sporamin A of sweet potato

Table 5 AUXIN response ARFAT (7) CACGCAATGMGH3 (showed constitutive activity with ARFAT) (12) CATATGGMSAUR (11) ASF1 MOTIFCAMV (15) SEBFCONSSTPR10A (18)-similar to auxin response element NTBBF1ARROLB (23) Light-regulated gene GT1 CONSENSUS (3) TBOXATGAPB (13) ASF1 MOTIFCAMV (15) BOXCPSAS1 (17)-negatively GATABOX (21) Negatively regulatory region NRRBNEXTA (2) Root ROOTMOTIFTAPOX1 (1) Legume GT1CONSENSUS (3) see light CAATBOX1 (4) SEF4MOTIFGM7S (5)

ARFAT (7) see auxin TATABOX5 (9) RYREPEATGMGY2 and RYREPEATLEGUMINBOX (10) CATATGGMSAUR (11) see auxin CACGCAATGMGH3 (12) see auxin BOXCPSAS1 (17) see light POLARSIG1 (20) SEF1MOTIF (22) TATABOX2 (24) Pollen POLLEN1LELAT52 (16) GTGANTG10 (19) Pathogenesis SEBFCONSSTPR1 OA (18) GA PYRIMIDINEBOXHVEPB1 (14) Quantitative PROLAMINBOXOSGLUB1 (8) TATA TATABOX4 (6) TATABOX5 (9) see legumessss TATABOX2 (24) see legume Table 6 Expression Expression Expression rRNA level Actin level NARK level FTF16 17. 53 FTF16 21. 51 FTF16 25. 98 FTF28 17. 21 FTF28 20. 54 FTF28 25. 59 NOD16 16. 17 NOD16 22. 89 NOD16 27. 28 NOD28 17. 12 NOD28 25. 16 NOD28 28. 02 04 RT16 23. 54 RT16 25. 85 RT28 17. 37 RT28 21. 26 RT28 24. 79 SAM16 19. 10 SAM16 25. 71 SAM16 33. 03 SAM28 16. 87 SAM28 21. 12 SAM28 28. 15 STF16 17. 46 STF16 22. 14 STF16 29. 22 STF28 17. 16 STF28 20. 39 STF28 25. 13 TTF28 17. 68 TTF28 21. 22 TTF28 25. 87 UF16 17. 23 UF16 23. 26 UF16 24. 72 UF28 17. 29 UF28 19. 40 UF28 24. 24 YNOD28 17. 12 NOD28 22. 62 NOD28 27. 11 Table 7 Avr-Avr- Template Replicate1 Replicate2 Avr Rep1 Rep2 NOD16 0. 133 0.147 0.140 0.007 0.007 YNOD28 0. 300 0. 261 0. 280-0. 020 -0.019 NOD28 0. 151 0. 154 0. 153 0. 001 0. 001 RT16 0.817 1.220 1.000 0.183 0.220 RT28 1. 311 1. 772 1. 527 0. 216 0. 246 UF16 1.195 1.798 1.468 0.273 0. 329 FTF16 0. 706 0.825 0.764 0.058 0.061 STF16 0. 092 0. 078 0. 085-0. 007-0. 007 UF28 1.973 2.216 2.094 0.121 0.122 FTF28 0. 708 0. 927 0. 811 0. 103 0. 115 STF28 0. 993 1. 145 1. 068 0. 075 0. 077 TTF28 0. 947 0. 843 0. 895-0. 052-0.052 SAM16 0.023 0.015 0. 018-0.005-0. 004 SAM28 0. 132 0. 110 0. 121-0.011-0. 010 Table 8 Template Replicate1 Replicate2 Avr Avr-Rep1 Avr-Rep2 NOD16 0. 244 0. 269 0. 256 0.012 0. 013 NOD28 0.259 0. 225 0.241-0. 017-0. 016 NOD28 0. 665 0. 673 0. 669 0. 004 0. 004 RT16 0. 819 1. 220 1. 000 0. 181 0. 220 RT28 0. 405 0. 546 0. 470 0.065 0. 076 UF16 1. 45C 2. 173 1. 775 0. 325 0. 398 FTF16 0. 232 0. 270 0. 251 0. 018 0. 020 STF 16 0. 047 0. 040 0. 044-0. 004-0. 003 UF28 0. 194 0. 217 0. 205 0. 011 0. 012 FTF28 0. 152 0. 198 0. 174 0. 022 0. 025 STF28 0.200 0. 230 0. 214 0.014 0. 015 TTF280. 2360. 210 0.222-0. 014-0.013 SAM16 0.043 0. 027 0.034-0. 009-0.007 SAM28 0. 051 0. 042 0. 046-0. 004-0. 004