PSEUDOMONAS IN PLANTS

CROSS-REFERENCING

This application claims the benefit of U.S. provisional application serial no.

62/548,913, filed on August 22, 2017, which application is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. 2016-67012- 25106 awarded by the United States Department of Agriculture. The government has certain rights in the invention.

BACKGROUND

Plant pathogens are a significant problem for agriculture, resulting in an estimated 10% decrease in crop yields despite control measures (Oerke, 2005). Utilizing genetic resistance to protect plants is generally preferable over chemical methods, which can be more expensive and pose risks to human or environmental health (Jones et al., 2014;

Vincelli, 2016); however in many cases genetic resistance is not available. The identification of the genetic basis of disease resistance pathways can allow for the creation of resistant crop varieties either through breeding or transgenic approaches (Dangl et al., 2013; Rodriguez- Moreno et al., 2017). As pathogens may evolve to overcome resistance mediated by a single resistance gene, it's desirable to utilize several independent resistance pathways against the same pathogen to confer durable resistance. The identification of resistance genes, particularly those that have broad specificity across many pathogen species, therefore remains a significant focus of plant pathology research.

Plant bacterial pathogens in the genus Xanthomonas contain a Type III Secretion System, which is used to deliver effector proteins into the plant cell (Rossier et al., 1999). These effector proteins can function to suppress the immune system of the plant or to manipulate the metabolism of the host to promote pathogenesis (Giirlebeck et al., 2006; Block et al., 2008; Toruno et al., 2016). While effector proteins are typically beneficial to the pathogen, if the plant contains a perception pathway capable of detecting a particular effector protein a strong immune response can be triggered, known as Effector Triggered Immunity (ETI) (Alfano and Collmer, 2004; Chisholm et al., 2006; Jones and Dangl, 2006). An ETI response is often associated with a visible cell death response known as the hypersensitive response. Identification of the genes responsible for specific ETI pathways can allow for engineering disease resistance into susceptible crop varieties.

Many ETI responses in plants are mediated by a member of the nucleotide-binding leucine -rich repeat (NLR) protein family, with the typical plant genome encoding between 100 and 600 of these proteins (Jones et al., 2016). The recognition of an effector protein by an NLR may be through a direct physical interaction or may involve additional protein components. NLR proteins can be divided into groups based on their domain architecture, as well as genetic dependencies required for their function. The two N-terminal domains common on plant NLR proteins are the Toll-like interleukin- 1 receptor (TIR) domain on TIR-NLRs (TLRs) and the coiled coil domain on CC-NLRs. All NLR proteins appear to depend on the protein SGT1 for function, whereas the TLRs require a functional EDS1 protein and a subset of the CC-NLRs require the NRC proteins (Wiermer et al., 2005;

Shirasu, 2009; Wu et al., 2016). These genetic dependencies can be used to help determine which family or clade of genes may be responsible for mediating the perception of a particular effector protein.

The effector protein XopQ from Xanthomonas, and the close-homolog HopQl from Pseudomonas, are widely distributed and highly conserved among various species in these genera. XopQ and HopQl have been shown to suppress the immune system of the plant and promote pathogen virulence (Li et al., 2013a; Sinha et al., 2013; Hann et al., 2014; Teper et al., 2014; Gupta et al., 2015). The mechanism by which XopQ/HopQl suppress immunity is not fully understood and is complicated by the observation that XopQ recognition in N. benthamiana can suppress visible cell death responses independently from the virulence activity of XopQ (Adlung and Bonas, 2017). The protein has homology to nucleoside hydrolases and a structural study suggested that XopQ can hydrolyze a molecule with a ribosyl group (Yu et al., 2014), as demonstrate by in vitro hydrolase activity on the substrate 4-nitrophyenyl β-D-ribofuranoside (Gupta et al., 2015). A recent study demonstrated that HopQl can hydrolyze the cytokinin precursor iP-riboside 5 '-monophosphate (iPRMP) in vitro and activate cytokinin signaling in vivo, suggesting this as the mechanism for immune suppression (Hann et al., 2014). An alternative hypothesis is that XopQ/HopQl virulence function occurs through direct targeting of 14-3-3 proteins. Both HopQl and XopQ have been shown to interact with 14-3-3 proteins in vivo following phosphorylation of a 14-3-3 binding site and that this interaction is important for the virulence function (Li et al., 2013a; Teper et al., 2014). As 14-3-3 proteins have a role in the plant immune system (Oh et al., 2010), it has been proposed that one or more of the 14-3-3's are targets of XopQ/HopQl and that the virulence function is achieved through modification, degradation or sequestration of these proteins (Teper et al., 2014).

Both XopQ and HopQl are recognized in the plants N. benthamiana and N. tabacum and trigger an avirulence response (Wei et al., 2007; Schwartz et al., 2015). The recognition of XopQ/HopQl in Nicotiana species is not dependent on interaction with host 14-3-3 proteins, suggesting that 14-3-3 proteins are not involved in the recognition pathway (Li et al., 2013a). Perception of XopQ/HopQl in N. tabacum is independent of the putative active site of the protein (Li et al., 2013b; Adlung and Bonas, 2017).

SUMMARY

The gene Roql from Nicotiana benthamiana has been unexpectedly found to mediate the perception of the plant pathogen effector XopQ/HopQl and thereby enhance resistance to pathogens containing this or a similar gene, including species of Xanthomonas,

Pseudomonas and Ralstonia, in other plant species. Based on this discovery, a non-Nicotiana transgenic plant comprising an exogenous polynucleotide encoding Nicotiana benthamiana Roql or a variant thereof is provided, as well as seeds from the same.

Also provided is method for enhancing the resistance of a non-Nicotiana plant to a plant pathogen strain, species or pathovar containing an effector recognized by Roql. This method may comprise: (a) introducing an exogenous polynucleotide encoding Nicotiana benthamiana Roql or variant thereof into the plant cell; and (b) regenerating a transgenic plant from the plant cell.

These and other inventions are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. Fig. 1. Effect of XopQ perception on bacterial growth and disease symptoms. X. euvesicatoria and X. gardneri wild type and XopQ knockout strains were infiltrated into N. benthamiana leaves at low inoculum (Οϋδοο = 0.0001). Bacterial growth was assayed at six days post infiltration and disease symptoms were imaged at thirteen and nine days post infiltration for Xe and Xg respectively. Error bars indicate standard deviation from three replicates. CFU - colony forming units.

Fig. 2. GFP-based reporter for immune activation. GFP-TMV was co-expressed with an empty vector or XopQ transiently using Agrobacterium in wild type and edsl-1 mutant N. benthamiana leaves. The leaves were imaged three days post infiltration under white light (left) or long-wave UV (right) using a handheld digital camera.

Fig. 3. GFP-TMV reporter in VIGS leaves of N. benthamiana. Viral-Induced Gene Silencing was used to downregulate either the GUS gene (as a negative control, top) or Roql (bottom). XopQ was transiently expressed along with the TMV-GFP reporter using

Agrobacterium and the plants were imaged at four days post infiltration under white light (left) or long- wave UV (right).

Fig. 4. Transient expression of Roql and XopQ. Roql and XopQ were transiently expressed using Agrobacterium in leaf tissue of N. tabacum, N. sylvestris and B. vulgaris. The Agrobacterium was infiltrated at a total Οϋδοο of 0.5 and the plants were imaged at six days post infiltration for N. tabacum and N. sylvestris, and nine days post infiltration for B. vulgaris.

Fig. 5: Co-immunoprecipitation of XopQ and Roql. XopQ-3xFlag, ATRl-3xFlag and HopQl-3xFlag were transiently co-expressed with either Rppl-6xHA or Roql-6xHA in the N. benthamiana edsl mutant. Western blots with a-HA primary antibody (top) and a- Flag primary antibody (middle) show proteins pulled down with anti-Flag beads for each combination. A Western blot of the input protein extract (prior to precipitation) is shown on the bottom. The Arabidopsis NLR protein RPP1 and its cognate effector ATR1 were included as controls. Predicted molecular weights are as follows: XopQ-3xFlag 53kD, HopQl-3xFlag 52kD, ATRl-3xFlag 33kD, RPPl-6xHA 145kD, and Roql-6xHA 159kD.

Fig. 6: XopQ phylogenetic tree. XopQ homologs were identified by BLAST search and used to generate a phylogenetic tree. Sequences identified with * were cloned and tested for their ability to be recognized by Roql. All tested homologs of XopQ elicited a Roql- dependent immune response. Genbank accession numbers for the various sequences are in brackets next to the species name.

Fig. 7 (A-B): Roql gene models. (A) The gene models for Roql from Nicotiana benthamiana, the highly similar ortholog from Nicotiana tabacum and the putative pseudogene from Nicotiana sylvestris are shown. The Roql gene contains five exons (depicted as white-filled box) and four introns. The predicted CDS for Roql is 3921 bp and the gene spans a predicted 9.6 kb from the start codon to the stop codon. The N. sylvestris gene contains a predicted stop codon in exon 2 and is missing part of exon 1 and most of exon 2 (B, aberrant sequences boxed). An aberrant, 8.7 kb insert is present between exonl and exon2 in the N. sylvestris gene. The genomic sequence from N. benthamiana was obtained from Benth Genome website, the N. tabacum sequence from Sol Genomics and the N. sylvestris sequence was obtained from NCBI. Accession numbers for the genome scaffolds are shown in parentheses for each. SEQ ID NOS. 2-7.

Fig. 8: Roql perception of XopQ/HopQl alleles. The indicated XopQ/HopQl alleles were cloned and transiently expressed along with the GFP-TMV reporter and either Roql or an empty vector in Nicotiana s_y/vestris. The leaves were imaged at six days post infiltration under white light and long- wave UV to view the visible HR response and viral GFP expression. Agrobacterium harboring the expression constructs was infiltrated at an Οϋδοο of 0.05 for each of the three TMV-GFP constructs and 0.3 for the other elicitor / receptor constructs. Xe - X. euvsicatoria, Xg - X. gardneri, Xam - X. axonipodis pv. manihotis, Xcc - X. campestris pv. campestris, Xoo - X. oryzae pv. oryzae, EV - empty vector, Xaj - X. axonopodis pv. juglandis, Xcitrii - X. citrii, Psp - P. syringae pv. phaseolicola, Pst - P. syringae pv. tomato, Xfa - X. fuscans subsp. aurantifolii.

Fig. 9: Roql mediates recognition of the Ralstonia solanacearum effector RipB. Two alleles of the Ralstonia solanacearum effector RipB (WP_003278485.1 and CAD13773.2) we cloned and transiently expressed in N. benthamiana using Agrobacteirum. Expression of both RipB alleles resulted in a visible immune response in wild type plants but was not observed plants homozygous for loss of function mutations in the Roql gene. This indicates that Roql mediates recognition of the Ralstonia solanacearum effector RipB. : Fig. 10: Roql confers resistance to several bacterial pathogens in tomato.

Xanthomonas euvesiactoria (Xe), Xanthomonas perforans (Xp), Xanthomonas gardneri (Xg) and Pseudomonas syringae pv tomato (Ps) were infiltrated into leaf tissue of wild type and Roql tomato plants. Bacterial growth was quantified after four days (Ps) or six days (Xe, Xp, Xg). The tomato plants expressing Roql were found to have approximately tenfold fewer bacteria, indicating that Roql confers resistance to these bacteria.

Fig. 11: Visible disease symptoms were imaged at six days post infiltration of Pseudomonas syringae pv tomato at an Οϋδοο of 0.0001. Wild type tomato leaves (left) have visible necrotic disease lesions in the infiltrated region, marked with an arrow and outline in black marker. The Roql tomato plants (right) do not develop disease lesions, indicating that they are resistant to this pathogen.

Fig. 12: The Roql signaling pathway depends on EDS1 and NRG1. Viral Induced Gene Silencing (VIGS) was used to silence the EDS1 and NRG1 genes in N. benthamiana. Relative to the negative control, these plants were unable to perceive transiently expressed XopQ as observed by the lack of a visible immune response. This indicates that both EDS1 and NRG1 are required for Roql -dependent perception of XopQ. DEFINITIONS

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

All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.

It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

As used herein, "resistance" is a relative term in that the presence of a polypeptide of the invention (i) reduces the disease symptoms of a plant comprising the gene (R (resistance) gene) that confers resistance, relative to a plant lacking the R gene, and/or (ii) reduces pathogen reproduction or spread on a plant or within a population of plants comprising the R gene and/or (iii) would act to confer resistance to a particular pathogen in the absence of other resistance mechanism(s). Resistance as used herein is relative to the "susceptible" response of a plant to the same pathogen. Typically, the presence of the R gene improves at least one production trait of a plant comprising the R gene when infected with the pathogen, such as grain yield, when compared to an isogenic plant infected with the pathogen but lacking the R gene. The isogenic plant may have some level of resistance to the pathogen, or may be classified as susceptible. Thus, the terms "resistance" and "enhanced resistance" are generally used herein interchangeably. Furthermore, a polypeptide of the invention does not necessarily confer complete pathogen resistance, for example when some symptoms still occur or there is some pathogen reproduction on infection but at a reduced amount within a plant or a population of plants. Resistance may occur at only some stages of growth of the plant, for example in adult plants (fully grown in size) and less so, or not at all, in seedlings, or at all stages of plant growth. By using a transgenic strategy to express an polypeptide in a plant, the plant of the invention can be provided with resistance throughout its growth and development. Enhanced resistance can be determined by a number of methods known in the art such as analysing the plants for the amount of pathogen and/or analysing plant growth or the amount of damage or disease symptoms to a plant in the presence of the pathogen, and comparing one or more of these parameters to an isogenic plant lacking an exogenous gene encoding a polypeptide of the invention. The terms "non-Nicotiana plant" and "not a Nicotiana plant" refer to a plant that is not a member of the Nicotiana genus of herbaceous plants and shrubs also referred to as "tobacco" plants.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a

contradiction.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes a plurality of such nucleic acids and reference to "the compound" includes reference to one or more compounds and equivalents thereof known to those skilled in the art, and so forth.

The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used.

Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, A., Principles of Biochemistry 3 ^rd Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5 ^th Ed., W. H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Provided herein is a transgenic plant comprising an exogenous polynucleotide encoding a polypeptide that is at least 80% identical to (e.g., at least 90% identical to, at least 94% identical to, at least 95% identical to, at least 98% identical to or the same as) the Nicotiana benthamiana Roql sequence of SEQ ID NO: 1, wherein the plant is not a

Nicotiana plant.

SEQ ID NO: l provides the amino acid sequence of the Roql protein from Nicotiana benthamiana and is shown below. Roql orthologs may be found in other species and utilized herein. MLTSSSHHGRSYDVFLSFRGEDTRKTFVGHLFNALIEKGIHTFMDDKELKRGKSISSELM KA IGESRFAWVFSKNYASSTWCLEELVKILEIHEKFELIWPVFYDVDPSTVRKQNGEYAVCF TKFEANLVDDRDKVLRWREALTKVANISGHDLRNTYNGDESKCIQQILKDIFDKFCFSIS IT NRDLVGIESQIKKLSSLLRMDLKGVRLVGIWGMGGVGKTTAARALFNRYYQNFESACFLE DV KEYLQHHTLLYLQKTLLSKLLKVEFVDCTDTEEMCVILKRRLCSKKVLWLDDVNHNDQLD K LVGAEDWFGSGSRIVITTRDMKLLKNHDVHETYEIKVLEKDEAIELFNLHAFKRSSPEKE FK ELLNLWDYTGGLPLALKVLGSLLYKEDLDVWISTIDRLKDNPEGEIMATLKISFDGLRDY E KSIFLDIACFFRGYNQRDMTALFHASGFHPVLGVKTLVEKSLIFILEDKIQMHDLMQEMG RQ IAVQESPMRRIYRPEDVKDACIGDMRKEAIEGLLLTEPEQFEEGELEYMYSAEALKKTRR LR ILVKEYYNRGFDEPVAYLPNSLLWLEWRNYSSNSFPSNFEPSKLVYLTMKGSSI IELWNGAK

RLAFLTTLDLSYCHKLIQTPDFRMITNLERLILSSCDALVEVHPSVGFLKNLILLNM DHCIS LERLPAI IQSECLEVLDLNYCFNLKMFPEVERNMTHLKKLDLTSTGIRELPASIEHLSSLEN LQMHSCNQLVSLPSSIWRFRNLKISECEKLGSLPEIHGNSNCTRELILKLVSIKELPTSI GN LTSLNFLEICNCKTISSLSSSIWGLTSLTTLKLLDCRKLKNLPGIPNAINHLSGHGLQLL LT LEQPTIYERLDLLRI IDMSWCSCISSLPHNIWMLKFLRILCISYCSRLEYLPENLGHLEHLE

ELLADGTGILRLPSSVARLNKLEVLSFRKKFAIGPKVQYSSSMLNLPDDVFGSLGSL GSWK LNLSGNGFCNLPETMNQLFCLEYLDITFCQRLEALPELPPSIKELYVDEHLALRIMEDLV IK CKELNLIAVTKIEYQNFYRWLDSIWSDVSELLENSQKQQLDDMLQLIPFSYLSTAKREEV LK IVIHGTRIPEWFRWQDRSATTMSVNLPEYWYTENFLGFAICCSCCFYHSARSYDVEFEGS MH HYNYDSSYWKEYEEPSYDFYERDSIEITAKLTPRHKGMRTEELKKVCSFSMNVLRRATAV PN MCFAFFPFNSLCHISNLQANNPNDYGIFETCLSPGDIRHRGKQWGFNLVYKDETGGSVTH EM LINR*

The transgenic plant may be monocotyledonous or dicotyledonous. Target plants include, but are not limited to, the following: cereals (for example, wheat, barley, rye, oats, rice, maize, sorghum and related crops); grapes; beet (sugar beet and fodder beet); pomes, stone fruit and soft fruit (mango, kiwi, apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and black-berries); leguminous plants (beans, lentils, peas, soybeans); oil plants (rape or other Brassicas, mustard, poppy, olives, sunflowers, safflower, flax, coconut, castor oil plants, cocoa beans, groundnuts); cucumber plants (marrows, cucumbers, melons); fibre plants (cotton, flax, hemp, jute); citrus fruit (oranges, lemons, grapefruit, mandarins); vegetables (spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, peppers, potatoes, paprika); lauraceae (avocados, cinnamon, camphor); or plants such as maize, cassava, nuts (walnut), coffee, sugar cane, tea, vines, hops, turf, bananas and natural rubber plants, as well as ornamentals (flowers, shrubs, broad-leaved trees and evergreens, such as conifers). In many embodiments, the plant may be susceptible to infection by one or more species of Xanthomonas, Pseudomonas, Ralstonia, or another pathogen containing an effector with homology to XopQ recognized by Roql (e.g., one or more species of Xanthomonas and one or more species of Pseudomonas) without the exogenous polynucleotide. In some cases the plant may be a hybrid.

As would be apparent, the transgenic plant has enhanced resistance to at least one species of Xanthomonas, Pseudomonas, Ralstonia, or another pathogen containing an effector with homology to XopQ which is recognized by Roql (e.g., one or more species of Xanthomonas and one or more species of Pseudomonas), relative to a control plant that is otherwise identical to the transgenic plant but does not contain the exogenous

polynucleotide. Xanthomonas/ Pseudomonas species to which the Roql provide resistance to include those that encode a XopQ/HopQl polypeptide. These polypeptides appear to be present in many pathogenic strains of Xanthomonas I Pseudomonas and, as such, Roql is believed to provide resistance to one or more of the bacteria listed in Fig. 6, including, but not limited to, Xanthomonas gardneri, Xanthomonas perjorans, Xanthomonas euvesicatoria, Xanthomonas oryzae pv oryzae, Xanthomonas oryzae pv. oryzicola, Xanthomonas hortorum, Xanthomonas campestris, Xanthomonas axonopodis, Xanthomonas citri, Xanthomonas arboricola, Xanthomonas asicola, Xanthomonas fragariae, Xanthomonas sacchari and Pseudomonas syringae, as well as Acidovorax citrulii, Xanthomonas translucens and Ralstonia solanaceamm. A list of Xanthomonas and Pseudomonas to which the plants should have enhanced resistance is set forth in Bull et al. J. Plant Pathology (2010 92: 551- 591, which is incorporated by reference for that list.

In some embodiments, the amino acid sequence of the polypeptide may be at least 80% identical to (e.g., 90% identical to, 94% identical to, at least 95% identical to, at least 96% identical to, at least 97% identical to, at least 98% identical to, at least 99% identical to, or 100% identical to) the amino acid sequence of a wild type Roql polypeptide from tobacco, e.g., a Roql polypeptide from N. benthamiana, N. tabacum, N. attenuata, or N. tomentosiformis (the sequences for which are available in Genbank as accession numbers ATD14363.1, XP_019226668.1, XP 009615050.1, and XP_016467297.1).

Methods for making transgenic plants are very well known in the art, as are the choices for promoters and other regulatory regions (see, e.g., US20160076050,

US20170218386 and US20160208279). As such, the present plants may be readily implemented by adapting any suitable method. In some embodiments, the exogenous polynucleotide is operably linked to a promoter. The promoter can be exogenous to the plant or endogenous to the plant. In some embodiments, the plant may be made by replacing a coding sequence in the genome of the plant with the exogenous polynucleotide.

Also provided is a seed of a transgenic plant described above. These seeds may be made by selfing the plant or crossing the plant with another plant of the same species to produce, e.g., hybrid seed.

Also provided is a population of at least 100 of the transgenic plants, e.g., at least 1,000, or at least 10,000 of the transgenic plants, growing in a field.

Also provided is a method for enhancing the resistance of a non-Nicotiana plant to at least one species of Xanthomonas or Pseudomonas (e.g., one or more species of

Xanthomonas and one or more species of Pseudomonas). In some embodiments, this method may comprise: (a) introducing an exogenous polynucleotide encoding a polypeptide that is at least 90% identical (e.g., at least 95%, at least 98%, or 100% identical) to the Roql sequence of SEQ ID NO: 1 into a plant cell that is from a plant that is susceptible to infection by Xanthomonas and/or Pseudomonas; and (b) regenerating a transgenic plant from the plant cell.

In some embodiments, the amino acid sequence of the polypeptide may be at least 80% identical to (e.g., at least 90% identical to, at least 95% identical to, at least 95% identical to, at least 96% identical to, at least 97% identical to, at least 98% identical to, at least 99% identical to, or 100% identical to) the amino acid sequence of a tobacco Roql polypeptide, e.g., a Roql polypeptide from N. benthamiana, N. tabacum, N. attenuata, or N. tomentosiformis. (the sequences for which are available in Genbank as accession numbers ATD14363.1, XP_019226668.1, XP 009615050.1, and XP_016467297.1).

Roql activity appears to require a functional and compatible NRG1 and/or EDS1 gene. As such, a functional NRG1 and/or EDS1 gene can be added to plants that do not already have a functional NRG1 and/or EDS1 gene or that contain a version of these genes which is not compatible with Roql, in order for Roql to be active in immune activation.

As noted above, methods for making transgenic plants are very well known in the art, as are the choices for promoters and other regulatory regions (see, e.g., US20160076050, US20170218386 and US20160208279). As such, the present plants may be readily implemented by adapting any suitable method.

EMBODIMENTS

A transgenic plant comprising an exogenous polynucleotide encoding a polypeptide that is at least 80% identical to the Nicotiana benthamiana Roql sequence of SEQ ID NO: 1, wherein the plant is not a Nicotiana plant.

The transgenic plant of any prior embodiment, wherein the plant has enhanced resistance to at least one species of Xanthomonas, Pseudomonas, Ralstonia, or another pathogen containing an effector with homology to XopQ which is recognized by Roql relative to a control plant that is otherwise identical to the transgenic plant but does not contain the exogenous polynucleotide.

The transgenic plant of any prior embodiment, wherein the plant has enhanced resistance to at least one species of Xanthomonas and at least one species of Pseudomonas relative to a control plant that is otherwise identical to the transgenic plant but does not contain the exogenous polynucleotide.

The transgenic plant of any prior embodiment, wherein the plant is a monocot.

The transgenic plant of any prior embodiment, wherein the plant is a dicot.

The transgenic plant of any prior embodiment, wherein the amino acid sequence of the polypeptide is at least 90% identical to the amino acid sequence of the Nicotiana benthamiana Roql sequence of SEQ ID NO: l

The transgenic plant of any prior embodiment, wherein the amino acid sequence of the polypeptide is at least 94% identical to the amino acid sequence of the Nicotiana benthamiana Roql sequence of SEQ ID NO: l

The transgenic plant of any prior embodiment, wherein the amino acid sequence of the polypeptide is at least 95% identical to the amino acid sequence of the Nicotiana benthamiana Roql sequence of SEQ ID NO: l

The transgenic plant of any prior embodiment, wherein the amino acid sequence of the polypeptide is identical to the amino acid sequence of the Nicotiana benthamiana Roql sequence of SEQ ID NO: 1.

The transgenic plant of any prior embodiment, wherein the exogenous polynucleotide is operably linked to a promoter.

The transgenic plant of any prior embodiment, wherein the promoter is exogenous to the plant or wherein the promoter is endogenous to the plant.

A seed of a transgenic plant of any prior embodiment.

A population of at least 100 plants of any prior embodiment, growing in a field.

A method for enhancing the resistance of a non-Nicotiana tobacco plant to at least one species of Xanthomonas, Pseudomonas, Ralstonia, or another pathogen containing an effector with homology to XopQ which is recognized by Roql (e.g., enhancing the resistance to at least one species of Xanthomonas and at least one species of Pseudomonas), comprising: (a) introducing an exogenous polynucleotide encoding a polypeptide that is at least 80% identical to the Nicotiana benthamiana Roql sequence of SEQ ID NO: 1 into the plant cell, wherein the plant cell is from a plant that is susceptible to infection by

Xanthomonas, Pseudomonas, Ralstonia, or another pathogen containing an effector with homology to XopQ which is recognized by Roql; and (b) regenerating a transgenic plant from the plant cell.

The method of any prior method embodiment, wherein the plant has enhanced resistance to at least one species of Xanthomonas or Pseudomonas relative to a control plant that is otherwise identical to the transgenic plant but does not contain the exogenous polynucleotide.

The method of any prior method embodiment, wherein the plant has enhanced resistance to at least one species of Xanthomonas and at least one species of Pseudomonas) relative to a control plant that is otherwise identical to the transgenic plant but does not contain the exogenous polynucleotide.

The method of any prior method embodiment, wherein the plant is a monocot.

The method of any prior method embodiment, wherein the plant is a dicot.

The method of any prior method embodiment, wherein the amino acid sequence of the polypeptide is at least 90% identical to the amino acid sequence of the Nicotiana benthamiana Roql sequence of SEQ ID NO: l.

The method of any prior method embodiment, wherein the amino acid sequence of the polypeptide is at least 94% identical to the amino acid sequence of the Nicotiana benthamiana Roql sequence of SEQ ID NO: l.

The method of any prior method embodiment, wherein the amino acid sequence of the polypeptide is at least 95% identical to the amino acid sequence of the Nicotiana benthamiana Roql sequence of SEQ ID NO: l

The method of any prior method embodiment, wherein the amino acid sequence of the polypeptide is identical to the amino acid sequence of the Nicotiana benthamiana Roql sequence of SEQ ID NO: 1.

The method of any prior method embodiment, wherein the exogenous polynucleotide is operably linked to a promoter.

The method of any prior method embodiment, wherein the promoter is exogenous to the plant or wherein the promoter is endogenous to the plant.

The transgenic plant, seed, or method of any prior embodiment, wherein the plant is a cereal (for example, wheat, barley, rye, oats, rice, maize, sorghum and related crops); a grape; a beet (sugar beet and fodder beet); a pome, a stone fruit and a soft fruit (mango, kiwi, apple, pear, plum, peach, almond, cherry, strawberry, raspberry and black-berry);

leguminous plant (bean, lentil, pea, soybean); oil plant (rape or other Brassicas, mustard, poppy, olive, sunflower, safflower, flax, coconut, castor oil plant, cocoa bean, groundnut); cucumber plant (marrow, cucumber, melon); fibre plant (cotton, flax, hemp, jute); citrus fruit (orange, lemon, grapefruit, mandarin); a vegetable (spinach, lettuce, asparagus, cabbage, carrot, onion, tomato, pepper, potato, paprika); a lauraceae (avocado, cinnamon, camphor); or a plant such as maize, cassava, nuts (walnut), coffee, sugar cane, tea, vines, hops, turf, banana or a natural rubber plant, or an ornamental (flower, shrub, broad- leaved tree or evergreen).

In any embodiment, the exogenous polynucleotide may additionally provide resistance to other pathogen species containing an effector with homology to XopQ which is recognized by Roql, such as species of the genus Ralstonia, e.g, Ralstonia solanacearum. As such, in transgenic plant embodiments, the plant may have enhanced resistance to at least one species of Xanthomonas, at least one species of Pseudomonas and at least one species of Ralstonia, relative to a control plant that is otherwise identical to the transgenic plant but does not contain the exogenous polynucleotide

EXAMPLES

Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.

In this study the perception pathway of XopQ in N. benthamiana was investigated. Using the CRISPR/CAS9 system (Jinek et al., 2012), a stable edsl mutants of N.

benthamiana was created which were unable to perceive XopQ/HopQl, thereby implicating a TLR in the perception of these effectors as was recently reported (Adlung and Bonas, 2016). A reverse genetic screen was conducted using Viral Induced Gene Silencing (VIGS) of the TLRs in N. benthamiana. A single TLR was identified which is required for the perception of XopQ. Biochemical experiments suggest that this protein, named Recognition of XopQ (Roql), directly interacts with XopQ and HopQl. Expression of Roql in other plant species is sufficient to enable the perception of diverse XopQ/HopQl/RipB alleles, suggesting this gene may be useful for engineering resistance against Xanthomonas,

Pseudomonas, Ralstonia and other pathogens containing a Roql -recognized homolog of XopQ in many different crop species.

Results

Perception of XopQ depends on EDSl. The Xanthomonas effector protein XopQ and the close homolog HopQl from Pseudomonas have previously been shown to trigger an avirulence response in N. benthamiana, indicating the presence of an immune perception pathway capable of detecting XopQ. To test if this pathway is dependent on edsl , a growth assay using wild type Xanthomonas euvesicatoria and the XopQ knockout was performed on both N. benthamiana wild type and edsl mutant plants. Wild type X. euvesicatoria grew approximately 100-fold more on the edsl mutant than the wild type plants, whereas the XopQ knockout grew to similarly high levels on both the wild type and edsl plant (Figure 1). Similar but less pronounced results were observed for X. gardneri, which grew 10-fold more in the absence of either EDS1 or XopQ (Figure 1).

Development of a fluorescence-based assay for XopQ perception. Unlike some recognized effector proteins that give a strong visible cell death response when transiently expressed, expression of XopQ in wild type N. benthamiana typically gives a mild chlorotic phenotype not well suited for a screen (Figure 2). The disease symptoms observed to be associated with a loss of XopQ recognition do give a strong visible response (Figure 1), but this assay was found to be inconsistent on Viral-Induced Gene Silencing (VIGS) plants, possibly due to only a partial knockdown of the target gene. An alternative assay was designed in which the XopQ protein was transiently expressed along with a Tobacco Mosaic Virus replicon containing a GFP gene (Marillonnet et al., 2005). In wild type plants XopQ perception resulted in immune activation and a lack of visible GFP from the viral replicon (Figure 2). Lack of XopQ perception in the edsl mutant allowed for strong GFP

fluorescence to be observed using a handheld long-wave UV light. This phenotype was very robust in the edsl mutants and consistent, though weaker, in EDS1 VIGS plants. This assay was employed to a reverse genetic screen of the TLR genes in N. benthamiana to identify the immune recognition receptor for XopQ.

Identification of a TLR required for XopQ perception. VIGS constructs were designed to target all TLR genes in N. benthamiana. Nine VIGS constructs were cloned with fragments of up to four TLRs each. In this initial screen one of the nine constructs, targeting three distinct TLRs, was found to consistently prevent perception of XopQ and allow for expression of the GFP reporter. Individual VIGS constructs were made for these three candidate genes and one was found to prevent XopQ perception (Figure 3). The gene targeted by this construct was named Recognition ofXopQl (Roql). Putative orthologs of Roql were identified in several other Nicotiana species but were absent in Solanum lycopersicum, Solanum tuberosum, Capsicum annuum and all other non-Nicotiana species examined.

Roql is sufficient to allow perception of XopQ in Nicotiana sylvestris and Beta vulgaris. While highly conserved orthologs of Roql were identified in several other

Nicotiana species including N. tabacum, N. attenuata, and N. tomentosiformis, the copy of Roql from N. sylvestris was found to contain an aberrant sequence disrupting the first and second exons, as well as a stop codon in a conserved part of the second exon (Figure 7). Disruption of the endogenous Roql gene in N. sylvestris is consistent with a previous report that this species is unable to perceive XopQ (Adlung and Bonas, 2016). Transient expression of the N. benthamiana Roql gene along with XopQ in N. sylvestris resulted in a visible hypersensitive response that was not present when either Roql or XopQ were expressed alone (Figure 4). To determine if Roql can be used to enable perception of XopQ in plants outside of the Nicotiana genus, Roql was co-expressed with XopQ in Beta vulgaris. This resulted in a chlorotic effect similar to that observed when XopQ is expressed in wild type N. benthamiana leaves (Figure 4).

Roql co-immunoprecipitates with XopQ. The recognition of HopQl in N.

tabacum has previously been shown to be independent of HopQl activity (Li et al., 2013b). It was hypothesized that Roql may directly interact with XopQ instead of "Guarding" a protein or molecule that is modified by this effector. To test this, a series of co- immunoprecipitation experiments in edsl-1 N. benthamiana were performed. Roql-6xHA was found to be pulled down by both XopQ-3xFlag and HopQl-3xFlag, but not by ATR1- 3xFlag, an effector known to interact with the TLR RPP1 (Figure 5) (Krasileva et al., 2010). ATRl-3xFlag was found to be able to pull down RPPl-6xHA, consistent with previous results, but not Roql-6xHA.

Roql can mediate the perception of diverse proteins with homology to XopQ. Homologous alleles of XopQ, HopQl and RipB are widely distributed among

Xanthomonas, Pseudomonas and Ralstonia species that are pathogenic on various crops. This suggests that Roql may be useful to engineer resistance into these crops species, but depends on Roql being able to recognize these diverse alleles. To sample the diversity of XopQ proteins a phylogenetic tree was generated and select XopQ and HopQl alleles for transient expression. Out of thirteen XopQ / HopQl alleles cloned, all elicited a with a Roql -dependent immune response in N. benthamiana or N. sylvestris (Figure 6, Figure 8).

Expression of Roql confers disease resistance against Pseudomonas syringae.

With reference to Fig. 11, wild type tomato leaves (left) and Roql -expressing tomato leaves (right) were infiltrated with Pseudomonas syringae pv. tomato. Six days after inoculation, disease symptoms are clearly visible in the infiltrated area in the wild type leaves (left), whereas no symptoms are visible on the Roql -transgenic plants (right). The infiltrated parts of the leaves were delimitated with black marker and marked with an arrow. Circular wounds visible in the leaves are from the inoculation procedure and not a result of disease symptoms.

Roql confers resistance to pathogens containing a recognized homolog of XopQ.

With reference to Fig. 10, bacterial pathogens Xanthomonas euvesicatoria (Xe), Xanthomonas perforans (Xp), Xanthomonas gardneri (Xg) and Pseudomonas syringae pv. tomato (Ps) were infiltrated into the leaves of wild type tomato plants and tomato plants expressing Roql. After six days, the leaf tissue was homogenized in buffer and plated to determine bacterial abundance. Approximately ten- fold fewer bacteria were detected in the Roql tomato leaves compared to the wild type, indicating that Roql confers disease resistance to these pathogens. All of these bacterial have a homolog of XopQ that is recognized by Roql. Error bars indicated standard deviation.

Roql activity depends on EDSl and NRGl. With reference to Fig. 11, XopQ was transiently expressed in N. benthamiana leaf tissue using Agrobacterium. Following infiltration, leaves were covered in foil for three days before imaging. Activation of Roql leads to a strong immune activation, visible as a cell death response (top) in VIGS negative control plants. Silencing either EDSl or NRGl blocks Roql activation, indicating that both may be required Roql activity and that functional and compatible versions of these genes may need to be added along with Roql into a plant species to enable Roql function.

Methods

CAS9 mediated knockout of EDSl and Roql. Guides to target the N. benthamiana EDSl and Roql genes were designed and cloned into entry plasmids containing the CAS9 gene and the Arabidopsis thaliana U6-26 promoter to drive guide expression. LR reactions were performed to move the guide and CAS9 cassette into a binary vector which was used for stable transformation into N. benthamiana by Agrobacterium co-cultivation.

Transformed plants were genotyped by PCR and Sanger sequencing, and homozygous knockout lines were obtained from the Tl generation

Viral Induced Gene Silencing. Target TLR genes from N. benthamaiana were identified using a BLAST search of a transcript database (Nakasugi et al., 2013). The predicted protein sequences of identified genes were aligned and manually curated to remove gene fragments and pseudogenes. Candidate TLRs were targeted for silencing by cloning approximately 300 bp into the TRV2 VIGS vector (Liu et al., 2002) using restriction ligation cloning with the enzyme Bsal. The plasmids were transformed into Agrobacterium tumefaciens strain GV3101 and infiltrated into N. benthamiana plants along with TRV1 at an OD600 of 0.2 each. The plants were infiltrated at 4-6 weeks of age and phenotyped 2-4 weeks after infiltration.

GFP-reporter assay for XopQ perception. Agrobacterium harboring the vectors pICH2011, pICH14011, and pICH7410 (Marillonnet et al., 2005) was mixed to a final OD ₆ oo of 0.05 each along with an Agrobacterium strain for transient expression of XopQ at an OD600 of 0.3. The mixture was infiltrated into leaf tissue and imaged under long-wave UV light to visualize GFP expression.

Growth assay. To assay bacterial growth, Xanthomonas was grown overnight in NYG (0.5% peptone, 0.3% yeast extract, 2% glycerol) with 100 μg / mL rifampcin on a shaker at 30 °C. The cultures were spun down at 10,000 g, washed once with 10 mM MgCh, and infiltrated into leaf tissue at Οϋβοο = 0.0001. Leaf punches were collected at 6 days post infiltration, homogenized, and serially diluted before plating on NYG agar plates with 100 μg / mL rifampcin and 50 μg / mL cycloheximide. Colonies were counted 3-4 days after plating.

Transient expression using Agrobacterium. Agrobacterium cultures were grown overnight in LB with selection at 30 °C on a shaker. Cells were pelleted by centrifugation at 10,000 g and resuspended in infiltration buffer (10 mM MgCh, 10 mM MES pH 5.6). The cells were diluted to the appropriate Οϋδοο and infiltrated into leaf tissue using a needleless syringe.

Plant material and bacteria strains. Nicotiana sylvestris was obtained from Select Seeds Co. in Union, Connecticut USA ("Woodland Tobacco 346"). Nicotiana tabacum and Beta vulgaris "Detroit Dark Red, Morse's Strain" (Plantation Products, Norton

Massachusetts, USA) were used for transient expression. X. euvesicatoria 85-10 and X. gardneri 153 were used for pathogen assays. The X. euvesicatoria 85-10 AXopQ knockout was constructed previously (Schwartz et al., 2015). Agrobacterium tumefaciens strains GV3101 and C58C1 were used for transient expression and VIGS.

XopQ knockout in X. gardneri. 1000 bp regions upstream and downstream of the XopQ coding sequence were cloned into the pLVC18 plasmid (Lindgren et al., 1986) containing a SacB counter-selectable marker. This plasmid was conjugated into X. gardneri and single crossover events were selected for by tetracycline resistance (10 μg / mL) and PCR. Colonies were plated onto NYG media containing 5% sucrose to select for a second crossover event and screened by PCR to identify XopQ deletion strains.

Co-immunoprecipitation assay. Each tissue sample (0.5 g) was ground with mortar and pestle to a homogeneous powder in liquid nitrogen. The samples were suspended in 1 mL of the protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 0.2% Nonidet P-40, 6 mM β-mercaptoethanol, IX protease inhibitor cocktail).

Samples were centrifuged twice (10 min, 14,000 rpm, 4 °C). The supernatant was then transferred to a new tube with 10 μΐ, Flag beads (A2220, Sigma) and incubated at 4 °C for 3 hours. The samples were centrifuged (1 min, 1,000 g) then washed three times with 1 mL of the protein extraction buffer. The protein was eluted by boiling for 5 min in 50 μΐ of 3X Laemmli buffer, centrifuged and loaded for Western blot. For the anti-Flag Western, the primary antibody was F7425 (Sigma) and the secondary antibody was A0545 (Sigma).

Discussion

Mechanism of Roql recognition of XopQ. Co-immunoprecipitation experiments showed that Roql can interact with either XopQ/HopQl as determined by

immunoprecipitation after transient co-expression in N. benthamiana (Figure 5). Roql did not interact with ATR1, indicating that there is specificity and that Roql is not interacting with the beads during the procedure. Furthermore, XopQ and HopQl did not interact with the TLR protein RPP1, indicating these proteins have some specificity to Roql and are not able to pull down all TLR proteins. This data is consistent with the model that Roql directly interacts with XopQ during activation. While the various XopQ / HopQl alleles have significant amino acid variation, especially at the Ν terminus, there are highly conserved regions which could mediate Roql recognition (data not shown). X. euvesicatoria XopQ and P. syringae pv. tomato HopQl share 65% amino acid identity with the variable Ν terminal region removed. While the EDS 1 protein is required for perception of XopQ, it is not required for interaction between Roql and XopQ or RPP1 and ATR1. This data supports a model in which EDS 1 is involved in the signaling process downstream of the TLR proteins and is not required for expression or proper folding of TLRs. NRG1 has been demonstrated to be required for the function of other TLRs and is believed to be a downstream signaling component(Peart et al., 2005; Qi et al., 2018).

Distribution and specificity of Roql.The Roql gene is highly conserved in several Nicotiana species but was not detected in any species outside this genus. This suggests that Roql evolved within the Nicotiana lineage and is consistent with the previous observation that XopQ failed to elicit an immune response when expressed in non-Nicotiana species (Adlung and Bonas, 2016). The wide distribution and high conservation of XopQ homologs in Xanthomonas,Pseudomonas and other pathogens may be a consequence of the narrow distribution of the Roql gene. The observation that co-expression of Roql with various

XopQ homologs can elicit a Roql -dependent immune response indicates that Roql is able to recognize the different homologs despite the various amino acid differences (Figure 6, Figure 8). Use of Roql to confer resistance against pathogens with homologs of XopQ.

Because Roql is able to recognize diverse XopQ homologs (Figure 6, Figure 8), this gene likely can be used in many crop species to confer resistance against pathogens containing these or similar effector genes. Because Roql likely directly interacts with XopQ, Roql may be able to function in a distantly related plant species without the requirement for the transfer of additional signaling components (Wulff et al., 2011). This is supported by the observation that co-expression of Roql and XopQ triggers a visible reaction, presumably an immune response, in Beta vulgaris (Figure 6). Roql activity does depend on the downstream signaling components such as EDS1 (Figure 1, Figure 2, Figure 12) and SGT1, and NRG1 (Figure 12). These genes are found in many plant species and therefore in some cases, including tomato, the endogenous EDS1 and NRG1 genes are sufficient for Roql function. In other species, particularly in plants more distantly related from the Solanaceous plants, exogenous copies of EDS1 and/or NRG1 may be required to enable Roql function. References

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Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.