WO2013055867A1

WO2013055867A1 - Genes involved in stress response in plants

Info

Publication number: WO2013055867A1
Application number: PCT/US2012/059673
Authority: WO
Inventors: Insuk LEE; Young-Su Seo; Edward Marcotte; Pamela Ronald
Original assignee: The Regents Of The University Of California; Board Of Regents, The University Of Texas System
Priority date: 2011-10-14
Filing date: 2012-10-11
Publication date: 2013-04-18

Abstract

Provided herein are genes involved in stress resistance (e.g., resistance to a pathogen), genetically modified plants with elevated or reduced expression of these genes, and methods of making genetically modified plants with enhanced stress resistance.

Description

GENES INVOLVED IN STRESS RESPONSE IN PLANTS

BACKGROUND OF THE INVENTION

[00011 Rice XA2I is a pathogen recognition protein (PRR) encoding a typical receptor kinase that confers resistance to strains of Gram-negative bacterium {e.g., Xanthomonas oryzae pv. oryzae (Xoo)), that contain the microbe associated molecular pattern AvrXA21 (Song et al, Science 270, 1804-6 (1995)). The intracellular non-RD kinase domain of XA21 possesses intrinsic kinase activity (Liu et al, J Biol Chern 277, 20264-9 (2002)). XA21 and like proteins are also referred to as pathogen associated molecular pattern (PAMP) receptors, which typically recognize a broad range of pathogens, so long as the pathogen presents the recognized molecular pattern.

[0002] Provided herein are ROX (Regulator of XA21 mediated immunity) proteins that are involved in XA21 -mediated disease and stress resistance. The ROX proteins were discovered using a genome-scale gene interaction model that allows for identification of functionally interacting genes that would not necessarily be determined based on sequence and expression data alone.

BRIEF SUMMARY OF THE INVENTION

[0003] Provided herein are genes involved in XA21 mediated immunity to pathogens and resistance to stress. For example, provided here in are transgenic plants that express

heterologous ROX1 or ROX2, or that express reduced levels of ROX3. Further provided are expression vectors for generating these plants, and methods of conferring pathogen and stress resistance in plants of various species using these vectors.

[0004] In some embodiments, provided herein is a transgenic plant comprising a heterologo expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a Regulator of XA21 mediated immunity 1 (R.OX1) polypeptide, wherein the plant has enhanced stress or disease resistance compared to the plant lacking the expression cassette. In some embodiments, the plant expresses the ROX1 polypeptide at a higher level than a plant lacking the expression cassette, e.g., at least 50%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or higher level than a plant lacking the expression cassette. In some embodiments, the expression is at least 1.5-fold, 2-fold, 3-fold, 4- fold, 5-fold higher than in a plant lacking the expression cassette. In some embodiments, the polypeptide has a sequence that is at least 90% or 95% identical to SEQ ID NO:3. In some embodiments, the polypeptide comprises a functional fragment or variant of the sequence of

SEQ ID NO:3. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:3. In some embodiments, the promoter is tissue-specific, cell type-specific, or inducible. In some embodiments, the promoter is a wild type ROX1 promoter. In some embodiments, the transgenic plant has higher thiamine synthesis than a plant lacking the expression cassette. [0005] In some embodiments, the plant is a monocot. In some embodiments, the plant is a dicot. In some embodiments, the plant is selected from the group consisting of rice, maize, wheat, soybeans, cotton, canola, turfgrass, and alfalfa. Further provided is an isolated plant cell from a plant as described above.

[0006] In addition, provided herein is an expression cassette comprising a promoter operabiy linked to a polynucleotide encoding a ROX1 polypeptide, wherein expression of the expression cassette in a plant increases the level of ROX1 polypeptide expression of the plant and enhances disease resistance of the plant compared to a plant in which the expression cassette is not expressed. In some embodiments, the polypeptide has a sequence that is at least 90% or 95% identical to SEQ ID NG:3, In some embodiments, the polypeptide comprises a functional fragment or variant of the sequence of SEQ ID NO:3. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:3. In some embodiments, the promoter is tissue-specific, cell type-specific, or inducible. In some embodiments, the promoter is a wild type ROX1 promoter.

[0007] Further provided are methods of enhancing the stress resistance of a plant comprising introducing an expression cassette as described above into one or more plants, wherein the expression cassette comprises a promoter operabiy linked to a polynucleotide encoding a ROX1 polypeptide. In some embodiments, the stress resistance is resistance to a pathogen. In some embodiments, the method further comprises selecting a plant having increased resistance to a pathogen or pathogens from the one or more plants into which the nucleic acid comprising the expression cassette has been introduced, as compared to the resistance of a plant lacking the expression cassette. In some embodiments, the polypeptide has a sequence that is at least 90% or 95% identical to SEQ ID NO:3. In some embodiments, the polypeptide comprises a functional fragment or variant of the sequence of SEQ ID NO:3. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:3. In some embodiments, the promoter is tissue-specific, cell type-specific, or inducible. In some embodiments, the promoter is a wild type ROXl promoter. In some embodiments, the plant expresses the ROXl polypeptide at a higher level than a plant lacking the expression cassette, e.g. , at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or higher level than a plant lacking the expression cassette. In some embodiments, the expression is at least 1 .5-fold, 2-fold, 3-fold, 4-fold, 5-fold higher than in a plant lacking the expression cassette. [0008] In some embodiments, provided herein is a transgenic plant comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a Regulator of XA21 mediated immunity 2 (ROX2) polypeptide, wherein the plant has enhanced stress or disease resistance compared to the plant lacking the expression cassette. In some embodiments, the plant expresses the ROX2 polypeptide at a higher level than a plant lacking the expression cassette, e.g. , at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or higher level than a plant lacking the expression cassette. In some embodiments, the expression is at least 1.5-fold, 2-foid, 3-fold, 4- fold, 5-fold higher than in a plant lacking the expression cassette. In some embodiments, the polypeptide has a sequence that is at least 90% or 95% identical to SEQ ID NO:6. In some embodiments, the polypeptide comprises a functional fragment or variant of the sequence of

SEQ ID NO:6. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:6. In some embodiments, the promoter is tissue-specific, cell type-specific, or inducible. In some embodiments, the promoter is a wild type ROX2 promoter.

[0009] In some embodiments, the plant is a monocot. In some embodiments, the plant is a dicot. In some embodiments, the plant is selected from the group consisting of rice, maize, wheat, soybeans, cotton, canola, turfgrass, and alfalfa. Further provided is an isolated plant cell from a plant as described above.

[0010] In addition, provided herein is an expression cassette comprising a promoter operably linked to a polynucleotide encoding a ROX2 polypeptide, wherein expression of the expression cassette in a plant increases the level of ROX2 polypeptide expression of the plant and enhances disease resistance of the plant, compared to a plant in which the expression cassette is not expressed. In some embodiments, the polypeptide has a sequence that is at least 90% or 95% identical to SEQ ID NO:6. In some embodiments, the polypeptide comprises a functional fragment or variant of the sequence of SEQ ID NO:6. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:6. In some embodiments, the promoter is tissue-specific, cell type-specific, or inducible. In some embodiments, the promoter is a wild type ROX2 promoter.

[0011] Further provided are methods of enhancing the stress resistance of a plant comprising introducing an expression cassette as described above into one or more plants, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding a ROX2 polypeptide. In some embodiments, the stress resistance is resistance to a pathogen. In some embodiments, the method further comprises selecting a plant having increased resistance to a pathogen or pathogens from the one or more plants into which the nucleic acid comprising the expression cassette has been introduced, as compared to the resistance of a plant lacking the expression cassette. In some embodiments, the polypeptide has a sequence that is at least 90% or 95% identical to SEQ ID NO:6. In some embodiments, the polypeptide comprises a functional fragment or variant of the sequence of SEQ ID NO:6. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:6. In some embodiments, the promoter is tissue-specific, cell type-specific, or inducible. In some embodiments, the plant expresses the ROX2

polypeptide at a higher level than a plant lacking the expression cassette, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%), 70%, 80%, 90%, 100%, 150%, 200% or higher level than a plant lacking the expression cassette. In some embodiments, the expression is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold higher than in a plant lacking the expression cassette.

[0012] In some embodiments, provided herein is a genetically modified plant that expresses a reduced amount or reduced activity Regulator of XA21 mediated immunity 3 (ROX3) compared to a wild type plant, wherein the genetically modified plant has enhanced stress or disease resistance compared to the wild type plant. In some embodiments, the plant is a ROX3 knockdown plant. In some embodiments, the reduced amount or reduced activity ROX3 is tissue or cell type-specific. Further provided is an isolated cell from, the genetically modified plant.

[0013] Also provided is a transgenic plant, comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to an RNAi polynucleotide specific for Regulator of XA21 mediated immunity 3 (ROX3) polynucleotide, wherein the plant. expresses ROX3 polypeptide at a reduced level compared to a plant lacking the expression cassette and wherein the plant has enhanced disease resistance compared to the plant lacking the expression cassette. In some embodiments, the plant expresses the ROX3 polypeptide at a level less than 0.1%, 0.5%, 1%, 5%, 10%, 20%, 25%, 50% or lower level than a plant lackmg the expression cassette. In some embodiments, the expression is at least 1 .5-fold, 2-fold, 3-fold, 4- fold, 5-fold, 10-fold, 20-fold, or 100-fold lower than in a plant lacking the expression cassette. In some embodiments, the polypeptide has a sequence that is at least 90% or 95% identical to SEQ ID NO:9. in some embodiments, the polypeptide comprises a functional variant of the sequence of SEQ ID NO:9. In some embodiments, the polypeptide comprises the sequence of SEQ) ID NO:9. In some embodiments, the promoter is tissue-specific, cell type-specific, or inducible.

[0014] In some embodiments, the plant is a monocot. In some embodiments, the plant is a dicot. In some embodiments, the plant is selected from the group consisting of fice, maize, wheat, soybeans, cotton, canola, turfgrass, and alfalfa. Further provided is an isolated plant cell from a plant as described above.

[001S] Further provided is an expression cassette comprising a promoter operably linked to an RNAi polynucleotide specific for ROX3 polynucleotide, wherein expression of the expression cassette in a plant reduces the level of ROX3 polypeptide expression of the plant and enhances disease resistance of the plant compared to a plant in which the expression cassette is not expressed. In some embodiments, the polypeptide has a sequence that is at least 90% or 95% identical to SEQ ID NO: 9. In some embodiments, the polypeptide comprises a functional variant of the sequence of SEQ ID NO:9. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:9. In some embodiments, the promoter is tissue-specific, cell type- specific, or inducible. [0016] Further provided are methods of enhancing the stress resistance of a plant comprising introducing an expression cassette as described above into one or more plants, wherein the expression cassette comprises a promoter operably linked to RNAi polynucleotide specific for ROX3 polynucleotide, wherein expression of the expression cassette in a plant reduces the level of ROX3 polypeptide expression of the plant. In some embodiments, the stress resistance is resistance to a pathogen. In some embodiments, the method further comprises selecting a plant having increased resistance to a pathogen or pathogens from the one or more plants into which the nucleic acid comprising the expression cassette has been introduced, as compared to the resistance of a plant lacking the expression cassette. In some embodiments, the polypeptide has a sequence that is at least 90% or 95% identical to SEQ ID NQ:9. In some embodiments, the polypeptide comprises a functional variant of the sequence of SEQ ID NO: 9. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:9. In some embodiments, the plant expresses the ROX3 polypeptide at a level less than 0, 1 %, 0.5%, 5 %, 5%, 10%, 20%, 25%, 50% or lower level than a plant lacking the expression cassette. In some embodiments, the expression is at least 1 .5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 100-fold lower than in a plant lacking the expression cassette.

BRIEF DESCRIPTION OF THE DRAW I NGS

[0017| Figsire 1: RNA quantification and phenotypes of transgenic lines altered for expression of LOC_Os01g70580 (Roxl), LOC_Os02g21510 (Rox2), and LOC_Os06gl2530

(Rox3) Quantification of mRNA for (a) LOC Os()lg70580 (Roxl) mRNA in Tl progeny of XA21 -LOC OsO 1 g70580 (Roxl) RNA12- 10 and 2- 1 1 independently transformed lines, (c)

LOC _Os02g21510 (Rox2) mRNA in TO progeny of XA21-LOC _Os02g21510 (i?ox?)RNAi 1 , 3, and 4 independently transformed lines, (e) LOC Os06g 12530 (Rox3 jmRNA in TO progeny of XA21 -LOC _Os06gl2530 (Rox3) RNAil and 3 independently transformed lines, along with Kit- XA21 controls. Leaf lesion lengths measured after Xoo challenge for (b) Tl progeny of XA21 - LOC_. Os01g70580 (Roxl) RNAi 2-2, 2-7, 2- 10, 2-1 1 and 2- 12 lines, (d) TO progeny of XA21 - LOC _Os02g21510 (Rox2) RNAi 1 , 3 and 4 lines, (f) TO progeny of XA21 -LOG _. OsOiSg 12530 (Rox3) RNAi 1 , 3 and 4 lines. RT-PCR was performed using specific primers for each tested candidate gene. Ubiquitin mR A was an internal control. The presence or absence of the transgene is indicated by (+) or (-). Leaves were inoculated when the plants were 5 weeks old and lesion lengths were measured at 14 day after inoculation. Each bar represents the average and standard deviation from three tested leaves.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

[0018] Pathogen recognition receptors (PRRs) are key components of the innate immune responses in animals and plants (Vivanco et al. (2002) Nat Rev Cancer 2, 489-501 ; Ausubel (2005) Nat Immunol 6, 973; Robatzek et al (2006) Genes Dev 20, 537-42; Jones et al (2006) Nature 444, 323-9; Asai et al (2002) Nature 415, 977-83; Chen et al. (2006), Plant J 46, 794- 804; Sun et al. (2004) Plant J 37, 517-27). The rice PRR, XA21 , confers resistance to

Xanihomonas oryzae pv. oryzae (Xoo) and other pathogens that carry the pathogen associated molecular pattern of AvrXA21 (Song et al (1995) Science 270, 1804).

[0019] Provided herein are genes that participate in XA 1 mediated pathogen resistance.

These include ROXl and ROX2, which are positive regulators of X.A21 mediated immunity (i.e., enhancers of pathogen and stress resistance), and ROX3, which is a negative regulator of X.A21 mediated immunity (i.e., an inhibitor of pathogen and stress resistance). II. Definitions

[0020] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4^th ed. 2007); Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Raven et al PLANT BIOLOGY (7^th ed. 2004). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0021] The term ROXl (Regulator of XA21 mediated immunity 1 ; LOC_Os01g70580) refers to the polypeptide of SEQ ID NO:3 and functional fragments thereof, and homologs (interspecies orthoiogs or intraspecies paralogs), functional variants, and allelic variants therof. In some embodiments, ROXl is substantially identical to SEQ ID NO:3. Polynucleotides encoding these polypeptides are also referred to as ROXl (or Roxl ). SEQ⁾ ID NO:2 and substantially identical sequences are examples of a polynucleotide sequence that encodes for ROXl protein. ROXl functions to enhance stress and disease resistance, e.g., pathogen resistance mediated by XA21 , and interacts with XA21 in a yeast two-hybrid assay. ROXl also has thiamine

pyrophosphokinase activity and acts to increase thiamine (vitamin Bl) synthesis in a plant.

[0022| The term ROX2 (Regulator of XA21 mediated immunity 2 ; LOG Os02g21510) refers to the polypeptide of SEQ ID NO:6 and functional fragments thereof, and homologs (interspecies orthoiogs or intraspecies paralogs), functional variants, and allelic variants therof. In some embodiments, ROX2 is substantially identical to SEQ ID NO: 6. Polynucleotides encoding these polypeptides are also referred to as ROX2 (or Rox2). SEQ ID NO:5 and substantially identical sequences are examples of a polynucleotide sequence that encodes for ROX2 protein. ROX2 functions to enhance stress and disease resistance, e.g., pathogen resistance mediated by XA21 , and interacts with XA21 in a yeast two-hybrid assay.

[0023] The term ROX3 (Regulator of XA21 mediated immunity 3; LOC_Os06g .12530) refers to the polypeptide of SEQ ID NO:9 and functional fragments thereof, and homologs (interspecies orthologs or intraspecies paralogs), functional variants, and allelic variants therof. in some embodiments, ROX3 is substantially identical to SEQ ID NO:9. Polynucleotides encoding these polypeptides are also referred to as ROX3 (or Rox3). SEQ ID NO: 8 and substantially identical sequences are examples of a polynucleotide sequence that encodes for ROX3 protein. ROX3 functions to inhibit stress and disease resistance, e.g., pathogen resistance mediated by XA21, and interacts with XB12 and XB12IP-1 in a yeast two-hybrid assay.

[0024] The term "transgenic," e.g., a transgenic plant or plant tissue, refers to a recombinantiy modified organism with at least one introduced genetic element. The term is typically used in a positive sense, so that the specified gene is expressed in the transgenic organism. However, a transgenic organism can be transgenic for an inhibitor}' nucleic acid, i.e., a sequence encoding an inhibitory nucleic acid is introduced. The introduced polynucleotide can be from the same species or a different species, can be endogenous or exogenous to the organism, can include a non-native or mutant sequence, or can include a non-coding sequence.

[0025] In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or sense suppression) one of skill will recognize that a polynucleotide sequence need not be identical and can be "substantially identical" to a sequence of the gene from which it was derived. [0026] The term "knockdown," with reference to a particular gene, describes an organism that is genetically modified to delete the gene, reduce expression of the gene (e.g., to less than 1, 5, 10, or 20% of wild type expression), or to express a non-functional gene product. The term gene knockdown is used synonymously with gene knockout or gene deficient.

[0027] "Enhanced (or improved or increased) stress resistance" refers to an increase in the ability of a plant to survive or thrive in stress (either biotic or abiotic) conditions. Enhanced resistance can be specific for a particular stressor, e.g., drought, limited nutrient supply, disease, or pathogen, or can be increased resistance for multiple stressors. Typically, enhanced resistance is determined relative to a control, e.g., a wild type plant, or otherwise non-resistant plant.

[0028] "Enhanced (or improved or increased) disease resistance" refers to an increase in the ability of a plant to prevent or reduce pathogen infection or to survive or thri ve once infected. Enhanced resistance can be specific for a particular pathogen species or genus, or can be increased resistance to all pathogens (e.g., systemic acquired resistance). Typically, enhanced resistance is determined relative to a control, e.g., a wild type plant, or otherwise non-resistant plant.

[0029] The term "plant" includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. Plants that can be used in the presently described methods includes those amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. Plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, hapioid and hemizygous can also be used.

{00301 A polynucleotide or polypeptide sequence is "heterologous to" an organism or a second sequence if it originates from a different species, or, if from the same species, it is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety). Similarly, a heterologous expression cassette includes sequenee(s) that are from a different species than the ceil into which the expression cassette is introduced, or if from the same species, is genetically modified.

[0031] "Recombinant" refers to a genetically modified polynucleotide, polypeptide, ceil, tissue, or organism. For example, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is one that has been manipulated using well known methods. A recombinant expression cassette comprising a promoter operably linked to a second

polynucleotide (e.g. , a coding sequence) can include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et at, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (5989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc. (1994-5998)). A recombinant expression cassette (or expression vector) typically comprises polynucleotides combinations that are not found in nature. For instance, human manipulated restriction sites or plasmid vector sequences can fiank or separate the promoter from other sequences. A recombinant protein is one that is expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide). [00321 The term "exogenous," in reference to a polypeptide or polynucleotide, refers to polypeptide or polynucleotide which is introduced into a cell or organism (e.g., plant) by any means other than by a sexual cross. A plant that expresses an exogenous polypeptide or polynucleotide, I.e., one that is not native (i.e., not endogenous) to the plant is referred to as a transgenic or genetically modified plant. Examples of introducing exogenous sequences are described herein, e.g. , Agrobacterium- edisted transformation, biolistic methods,

electroporation, etc.

[0033] "Pathogens" include, but are not limited to, virases, bacteria, nematodes, fungi and insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, CA (1988)).

[0034] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. The monomer is typically referred to as a nucleotide. Nucleic acids can include modified nucleotides that permit correct read through by a polymerase and do not significantly alter expression of a polypeptide encoded by that nucleic acid.

{0035] The terms "antisense," "inhibitory nucleic acid," "inhibitory polynucleotide,"

"interfering polynucleotide," and "interfering nucleic acid" are used generally herein to refer to R A targeting strategies for reducing gene expression. These strategies include R Ai, siRNA, shRNA, dsRNA, etc. Typically, the antisense sequence is identical to the targeted sequence (or a fragment thereof), but this is not necessary for effective reduction of expression. For example, the antisense sequence can have 85, 90, 95, 98, or 99% identity to the complement of a target RN A or fragment thereof. The targeted fragment can be about 10, 20, 30, 40, 50, 10-50, 20-40, 20-100, 40-200 or more nucleotides in length. The terms "RNAi polynucleotide specific for," "si NA polynucleotide specific for," "shRNA specific for" a given polynucleotide, and like terms, refer to an antisense or other interfering polynucleotide that inhibits expression of the given polynucleotide in a sequence specific manner, as described in more detail herein.

[0036] The term "RNAi" refers to RNA interference strategies of reducing expression of a targeted gene, RNAi technique employs genetic constructs within which sense and anti-sense sequences are placed in regions flanking an intron sequence in proper splicing orientation with donor and acceptor splicing sites. Alternatively, spacer sequences of various lengths can be employed to separate self-complementary regions of sequence in the construct. During processing of the gene construct transcript, intron sequences are sp iced-out, allowing sense and anti-sense sequences, as well as splice junction sequences, to bind forming double-stranded

RNA. Select ribonucleases then bind to and cleave the double-stranded RNA, thereby initiating the cascade of events leading to degradation of specific mRNA gene sequences, and silencing specific genes. The phenomenon of RNA interference is described and discussed in Bass, Nature 41 1 : 428-29 (2001); Elbahir et al. Nature 411 : 494-98 (2001); and Fire et al., Nature 391 : 806-11 (1998); and WO 01/75164, where methods of making interfering RNA also are discussed.

[0037] The term "siRNA" refers to small interfering RNAs, that are capable of causing interference with gene expression and can cause post-transcriptional silencing of specific genes in cells, e.g., in plant cells. The siRNAs based upon the sequences and nucleic acids encoding the gene products disclosed herein typically have fewer than 100 base pairs and can be, e.g., about 30 bps or shorter, and can be made by approaches known in the art, including the use of complementary DNA strands or synthetic approaches. Typical siRNAs have up to 40bps, 35bps, 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integer thereabout or therebetween. Tools for designing optimal inhibitory siRNAs include that available from DNAengine Inc. (Seattle, WA) and Ambion, Inc. (Austin, TX).

[0038] A "short hairpin RNA" or "small hairpin RNA" is a ribonucleotide sequence forming a hairpin turn which can be used to silence gene expression. After processing by cellular factors the short hairpin RNA interacts with a complementary RNA thereby interfering with the expression of the complementary RNA. [0039] The phrase "nucleic acid sequence encoding" refers to a nucleic acid which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA, and the RNA sequence that is translated into protein. The nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences. A coding sequence can include degenerate codons (relative to the native sequence) or sequences that provide codon preference in a specific host cell,

{0040] The term "promoter" refers to regions or sequence located upstream and/or downstream from the start of transcription and which are in volved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A plant promoter can be a nucleic acid sequence originally isolated from a plant, but promoters not initially isolated from a plant can be used to drive expression as well,

{00411 An "expression cassette" refers to a nucleic acid construct, which when introduced into a host ceil (e.g., a plant ceil), results in transcription and/or translation of a RNA or polypeptide, respectively. An expression cassette typically includes a sequence to be expressed, and sequences necessary for expression of the sequence to be expressed. The sequence to be expressed can be a coding sequence or a non-coding sequence (e.g., an inhibitory sequence). Generally, an expression cassette is inserted into an expression vector to be introduced into a host cell. The expression vector can be viral or non-viral.

[0042] The terms "transfection" and "transformation" refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0043] The term "gene" refers to a segment of D^'NA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene (e.g., promoters, enhancers, etc.). A "gene product" can refer to either the mRNA or protein expressed from a particular gene.

[0044] The words "complementary" or "complementarity" refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity can be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.

[0045] The terms "protein", "peptide", and "polypeptide" are used interchangeably to denote an amino acid polymer or a set of two or more interacting or hound amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0046] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, γ-carhoxygiutamaie, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups

(e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a. manner similar to a naturally occurring amino acid. The terms "non-naturally occurring amino acid" and "unnatural amino acid" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0047] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the iUPAC-IUB Biochemical

Nomenclature Commission, Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes,

[0048] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservati vely modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0049] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution fables providing functionally similar amino acids are well known in the art. Conservatively modified variants can include polymorphic variants, interspecies homologs (orthologs), intraspecies homologs

(paralogs), and allelic variants.

[0050] The terms "identical" or percent "identity," in the context of two or more nucleic acids or proteins, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%), 85%, 90%, 91%, 92%, 93%, 94%», 95%, 96%, 97%, 98%, 99%, or higher identity over a. specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov/BLAST/. Such sequences are then said to be "substantially identical." This definition also refers to, and can be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Optimal alignment, of such sequences can be carried out by any of the publicaliy available algorithms or programs for determining sequence identity and alignment, e.g., BLAST. ΪΪΙ, Recombinant techniques ssid expression vectors

[0051] Sequences for a ROX gene can be used to prepare an expression cassette for expressing a ROX polypeptide in a transgenic plant, directed by a native or heterologous promoter. In some embodiments, the ROX gene is ROX1 or ROX2, e.g., to enhance pathogen resistance of the plant. In some embodiments, the ROX gene is ROX3, e.g., to study resistance mechanisms in the plant.

[0052] Any of a number of means kno wn in the art can be used to dri ve ROX expression in plants. Any tissue or cell type can be targeted, such as shoot vegetative organs/structures {e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit. Alternatively, ROX expression can be conditioned to only occur under certain conditions (e.g., using an inducible promoter).

[0053] To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of pl ant cel ls are prepared . Techniques for transforming a wide variety of higher plant species are known see, e.g., Weising et at Ann. Rev. Genet. 22:421 -477 (5988). A DNA sequence coding for the desired polypeptide (e.g., ROX 1 or ROX2), can be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.

[0054] For example, a plant promoter can direct expression of the ROX polypeptide in all tissues a transgenic plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the - or 2'- promoter derived from T-DNA of Agrobacterium t mqfacien , and other transcription initiation regions from various plant genes known to those of skill.

{00551 T e plant promoter can alternatively direct expression of the ROX polypeptide in a specific tissue (tissue-specific promoters) or under certain environmental conditions (inducible promoters). Examples of tissue-specific promoters are those specific to leaf or guard cells (including but not limited to those described in WO/2005/085449; U.S. Patent No. 6,653,535; Li et al. , Sci China C Life Sci. 2005 Apr;48(2): 181-6; Husebye, et al. , Plant Physiol, April 2002, Vol. 128, pp. 1 180- 1 188; and Plesch, el al , Gene, Volume 249, Number 5 , 56 May 2000 , pp. 83-89(7)). Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light.

[0056| A polyadenylation region at the 3'-end of the coding region can be included. The polyadenylation region can be derived from a ROX gene, from a variety of other plant genes, or from T-DNA.

[0057] The polypeptide can also comprise a tag that facilitates detection or purification of the polypeptide. The tag may be added to the N-terminal or C-terminal region of the polypeptide or internally within the polypeptide. Examples of suitable tags include, but are not limited to, Myc, FLAG, HA, His, giutathione-S-transferase (GST), tandem affinity purification (TAP), and fluorescent protein (e.g., GFP, YFP, EGFP, RFP, DsRed) tags.

[0058| The vector comprising the control and coding sequences typically comprise a marker gene that confers a selectable phenotype on plant cells. For example, the marker can encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.

[0059| 1° some embodiments, the promoter that is operably linked to a polynucleotide encoding a ROX polypeptide increases the level of expression of ROX in a transgenic plant relative to that of a wild type (non-transgenic) plant.

Native promoters

[0060| 1° some embodiments, expression of a ROX nucleic acid is directed by its own native promoter or a portion thereof. In some embodiments, a "portion" of a promoter comprises a continuous length of a promoter sequence that is from about 100 nucleotides in length to about 10,000 nucleotides in length. In some embodiments, a portion of a promoter comprises a continuous length of a promoter sequence that is from about 500 nucleotides in length to about 5,000 nucleotides in length. The term "native" means the naturally occurring promoter sequence that directs expression of the endogenous gene in a plant. A native promoter that is operably linked to a polynucleotide of interest, e.g. a ROX polynucleotide, directs expression of the polynucleotide of interest in those cell and tissue types, or during those environmental conditions and states of development or ceil differentiation, in which the native promoter would drive expression under physiological conditions. [0061] A native promoter can be operably linked to an identical or substantially similar polynucleotide as is normally expressed by the native promoter. For example, the native ROX 1 or RQX2 promoter in rice can be operably linked to a polynucleotide encoding the rice ROX 1 or R.OX2 polypeptide; the native ROX! or ROX2 promoter in maize may be operably linked to a polynucleotide encoding the maize ROX1 or ROX2 polypeptide orthoiog; etc. Alternatively, the native promoter can be operably linked to a polynucleotide that is not identical, but is substantially similar, to the polynucleotide that is normally expressed by the native promoter. For example, the native RQXl or ROX2 promoter in rice can be operably linked to a polynucleotide encoding the maize ROXl or ROX2 polypeptide orthoiog, soybean ROX! or ROX2 polypeptide orthoiog, wheat ROX ! or ROX2 polypeptide orthoiog, etc. inducible promoters

[0062| Alternatively, a plant promoter can direct expression of the ROX polynucleotide under the influence of environmental or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light. Such promoters are referred to herein as "inducible" promoters.

[0063] Exemplary inducible promoters include those promoters that are specifically induced upon infection by a virulent pathogen. Selected promoters useful in the invention are discussed in PCX application WO 99/43824, and include promoters from: a. lipoxygenases (e.g., Peng et al, J. Biol. Chem. 269:3755-3761 (1994)), b. peroxidases (e.g., Chittoor et al. Molec. Plant-Microbe Interact. 10:861-

871 (1997)),

c. hydroxymethylgiutaryl-CoA reductase,

d. phenylalanine ammonia lyase,

e. giutathione-S-transferase,

f. chitinases (e.g., Zhu et al. Mol. Gen. Genet. 226:289-296 (1991)), g. genes involved in the plant respiratory burst (e.g., Groom et al. Plant J. !0(3):5!5-522 (1996)); and

h. pathogenesis-related (PR) protein promoters. [0064] Alternatively, plant promoters which are induced upon exposure to plant hormones, such as auxins, are used to express the ROX polynucleotide. For example, the invention can use the auxin-response elements El promoter fragment (AuxREs) in the soybean (Glycine max L) (Liu (1997) Plant Physiol. 1 15:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) Mol. Plant Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen ( 1996) Science 274: 1900-1902),

[0065] Plant promoters inducible upon exposure to chemical reagents (e.g., herbicides or antibiotics) can be used in a plant to express a ROX polynucleotide. For example, the maize In2- 2 promoter, activated by benzenes ulfonamide herbicide safeners, can be used (De Veylder ( 1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. A ROX coding sequence can also be under the control of, e.g., a tetracycline- inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 1 1 :465-473); or, a salicylic acid-responsive element (Stange ( 1997) Plant J. 1 1 : 1315- 1324; IJknes et al.. Plant Cell 5: 159- 169 (1993); Bi et al. Plant J. 8:235-245 (1995)).

[0066] Examples of useful inducible regulatory elements that can be used with the expression cassettes described herein include copper-inducible regulatory elements (Mett et al., Proc. Natl. Acad. Sci. USA 90:4567-4571 (1993); Furst et al.. Cell 55:705-717 (1988)); tetracycline and chlor-tetracycline-inducible regulatory elements (Gatz et ah, Plant J. 2:397-404 (1992); Roder et ah, Mol. Gen. Genet. 243:32-38 (1994); Gstz, Meth. Cell Biol. 50:41 1 -424 (1995)); ecdysone inducible regulatory elements (Cbxistopherson et ah, Proc. Natl. Acad. Sci. USA 89:6314-6318 (1992); Kreutzweiser et al., Ecotoxicol. Environ. Safety 28: 14-24 (1994)); heat shock inducible regulatory elements (Takahashi et ah. Plant Physiol. 99:383-390 (1992); Yabe et ah, Plant Cell Physiol. 35: 1207- 1219 (1994); Ueda et ah, Mol. Gen. Genet. 250:533-539 (1996)); and lac operon elements, which are used in combination with a constitutively expressed lac repressor to confer, for example, IPTG-inducible expression (Wilde et al, EMBO J. 1 1 : 1251 -1259 (1992)). An inducible regulatory element, useful in the transgenic plants of the invention also can be, for example, a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et ah. Plant Mol. Biol. 17:9 (1 991)) or a light-inducible promoter, such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et ah, Mol. Gen. Genet. 226:449 (1991 ); Lam and Chua, Science 248:471 (1990)). Tissue-specific promoters

[0067] The plant promoter used in the ROX protein expression cassette can also be tissue- specific. Tissue specific promoters are transcriptional control elements that are only active in particular cell types or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues.

[0068| Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue, or epidermis or mesophyil. Reproductive tissue-specific promoters may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or some combination thereof. In some embodiments, the promoter is cell-type specific, e.g., guard cell-specific.

[0069] Other tissue-specific promoters include seed promoters. Suitable seed-specific promoters are derived from the following genes: MAC! from maize (Sheridan (1996) Genetics 142 : 1009- 1020); Cat3 from maize (GenBank No . L05934, Abler ( 1993) Plant Mol. Biol.

22: 10131 -1038); vivparous-1 from Arabidops is (Genbank No. U93215); atmycl from

Arabidopsis (Urao ( 1996) Plant Mol. Biol. 32:571-57; Conceicao (1994) Plant 5:493-505); napA from Brassica napus (GenBank No. J02798, Josefsson (1987) JBL 26: 12196-1301); and the napin gene family from Brassica napus (Sjodahl ( 1995) Planta 197:264-271 ). [0070] A variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers, can also be used to drive ROX polypeptide expression. For example, promoters controlling patatin, the major storage protein of the potato tuber, can be used, see, e.g., Kim (1994) Plant Mol Biol. 26:603-615; Martin (1997) Plant J. 1 1 :53-62. The ORF13 promoter from Agrobacterium rhizogenes that exhibits high activity in roots can also be used (Hansen (1997) Mol. Gen. Genet. 254:337-343. Other useful vegetative tissue-specific promoters include: the tarin promoter of the gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (Bezerra (1995) Plant Mol. Biol. 28: 137-144); the curculin promoter active during taro corm development (de Castro (1992) Plant Cell

4: 1549-1559) and the promoter for the tobacco root-specific gene TobRB7, whose expression is localized to root meristem and immature central cylinder regions (Yamamoto (1991) Plant Cell 3:371 -382). [0071] Leaf-specific promoters, such as the ribulose triphosphate carboxylase (RBCS) promoters can be used. For example, the tomato RBCS 1 , RBCS2 and RBCS3A genes are expressed in leaves and light-grown seedlings, only RBCS1 and RBCS2 are expressed in developing tomato fruits (Meier (1997) FEBS Lett, 415:91). A ribulose bisphosphate

carboxylase promoters expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels, described by Matsuoka (1994) Plant J. 6:31 1 , can be used. Another leaf- specific promoter is the light harvesting chlorophyll a/b binding protein gene promoter, see, e.g., Shiina ( 1997) Plant Physiol. 115:477; Casal (1998) Plant Physiol. 1 16: 1533. The Arahidopsis thaliana myb-related gene promoter (AtmybS) described by Li (1996) FEBS Lett. 379: 1 17, is leaf-specific. The AtmybS promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds.

AtmybS niRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage. A leaf promoter identified in maize by Busk (1997) Plant J. 11 : 1285, can also be used. [0072] Another cl ass of useful vegetative tissue-specific promoters are meristematic (root tip and shoot apex) promoters. For example, the " SHOOTMERISTEMLESS" and "SCARECROW" promoters, which are active in the developing shoot or root apical meristems can be used (Di Laurenzio (1996) Cell 86:423-433: Long (1996) Nature 379:66-69) Another useful promoter is that which controls the expression of 3-hydroxy-3- methylglutaryl coenzyme A reductase HMG2 gene, whose expression is restricted to meristematic and floral (secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues (see, e.g., Enjuto (1995) Plant Cell 7:517). Also useful are knl -related promoters from maize and other species which show meristem-specific expression, see, e.g., Granger (1996) Plant MoL Biol. 31 :373-378; Kerstetter (1994) Plant Cell 6: 1877; Hake (1995) Philos. Trans. R. Soc. Land. B. Biol. Sci.

350:45. For example, the Arahidopsis thaliana KNAT1 promoter can be used (see, e.g., Lincoln (1994) Plant Cell 6: 1859).

[0073] One of skill will recognize that a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some (generally very low) expression in other tissues as well. [0074] In some embodiments, ROX3 activity or expression is reduced or eliminated, e.g., to improve disease resistance in a plant. In some embodiments, the activity or expression of ROX1 or ROX2 is reduced or eliminated, e.g., to study disease resistance or the XA21 pathway. A number of methods can be used to inhibit, mutate, or inactivate expression of a ROX protein in plants. For instance, antisense technology can be conveniently used to inactivate gene expression. To accomplish this, a nucleic acid segment from the desired gene is cloned and operahly linked to a promoter such that the antisense strand of RNA will be transcribed. The expression cassette is then transformed into plants and the antisense strand of RNA is produced. In plant cells, antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the polypeptide of interest (Sheehy et al., Proc. Nat. Acad. Sci. USA,

85:8805-8809 (1988); Pnueli et al, The Plant Cell 6: 175- 186 (1994); and Hiatt et al., U.S. Patent No. 4,801 ,340).

[0075] The antisense nucleic acid sequence transformed into plants will be substantially identical to at least a portion of the endogenous gene or genes to be repressed. The sequence, however, does not have to be perfectly identical to inhibit expression. Thus, an antisense or sense nucleic acid molecule encoding only a portion of a ROX protein, or a portion of the ROX mRNA (including but not limited to untranslated portions of the mRNA) can be useful for producing a plant in which the ROX protein expression is suppressed. The vectors are optionally designed such that the inhibitory effect applies specifically to the targeted polynucleotide, and does not affect expression of other genes. In situations where endogenous ROX3 is to be inactivated, one can target the antisense sequence to ROX3 sequences (e.g., tail or untranslated mRNA sequences) not found in other proteins within the family of genes exhibiting homology to ROX3 (e.g., nud genes).

[0076] For antisense suppression, the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the mtroduced sequence need not have the same intron or exon pattern, and homology of non-coding segments can be equally effective. For example, a sequence of between about 30 or 40 nucleotides can be used, and in some embodiments, a sequence of at least about 20, 50, 100, 200, or 500 nucleotides can be used.

[0077] Catalytic RNA molecules or ribozymes can also be used to inhibit, expression of a ROX gene. Ribozymes that specifically pair with a target RNA and cleave the phosphodiester backbone at a specific location can be designed to functionally inactivating the target RNA . In carrying out this cleavage, the ribozyme is not itself altered, and can cleave other molecules, making it a true enzyme. The inclusion of a rihozyme sequence in an antisense RNA confers RNA-cleaving acti vity, thereby increasing the activity of a given antisense construct,

[0078] A number of classes of ribozymes have been identified. One class of ribozym.es is derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RN As from avocado simblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodifiorum mottle virus and subterranean clover mottle virus. The design and use of target RNA-specific ribozymes is described in Haseloff et al. Nature, 334:585-591 (1988).

[0079| Another method of suppression is sense suppression (also known as co-suppression). Introduction of expression cassettes in which a nucleic acid is configured in the sense orientation with respect to the promoter has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes (Napoli et ., The Plant Cell 2:279-289 (1990); Flaveli, Proc. Natl. Acad. Set, USA 91 :3490-3496 (1994); Kooter and Mol, Current Opin. Biol. 4: 166-171 (1993); and U.S. Patents Nos. 5,034,323, 5,231 ,020, and 5,283,184).

[0080] Generally, the inhibitory polynucleotide will be substantially identical to the targeted endogenous sequence. This minimal identity will typically be greater than about 65% to the target ROX polynucleotide sequence, but a higher identity can more effectively reduce expression of the endogenous sequences. In some embodiments, sequences with greater identity are used, e.g., at least about 80, at least about 95%, or 100% identity are used. The effect of the inhibitor}' polynucleotide can be designed and tested so as to not significantly affect expression of other proteins.

[0081] Endogenous gene expression can also be suppressed by means of RN A interference (RNAi) (and indeed co-suppression can be considered a type of RNAi). RNAi uses a double- stranded RN A having a sequence identical or substantially identical to the sequence of the target gene. When a double-stranded RNA having a. sequence identical or similar to that of the target gene or portion thereof (i.e., an RNAi construct) is introduced into a cell, the expressions of both the introduced inhibitory polynucleotide and target endogenous gene are suppressed. The double-stranded RNA can be formed from two separate complementary RNAs or a single RNA with internally complementary sequences that form a double-stranded hairpin RNA. RNAi is effective in plants (Chuang et al. (2000) Proc. Natl. Acad. Sci. USA 97: 4985; Waterhouse et al, Proc. Natl. Acad. Sci. USA 95: 13959-13964 (1998); Tabara el al. Science 282:430-431 (1998); Matthew, Camp Fund. Genom. 5: 240-244 (2004); I .u. et al, Nucleic Acids Research

32(21):el 71 (2004)). For example, to reduce expression of a ROX protein using RNAi, a double-stranded RNA having the sequence of an mRNA encoding a RO protein, or a substantially identical sequence (including those engineered not to translate the protein), or fragment thereof, is introduced into a plant. The resulting plant can then be screened for a phenotype associated with the target protein and/or by monitoring steady-state RN A levels for transcripts encoding the protein. Although the sequence used for RNAi need not be completely identical to the target gene, they are typically substantially identical, e.g., at least 70%, 80%, 90%, 95%, 98%), or more identical to the target sequence.

[00821 The RNAi polynucleotide can encompass the full-length target RNA or to a fragment of the target RNA. In some cases, the fragment will have fewer than 20, 50, 100, 200, 300, 400, or 500 nucleotides corresponding to the target sequence, in addition, in some embodiments, the fragment is at least, e.g., 10, 15, 20, 50, 100, 150, 200, or more nucleotides in length.

[00831 Further provided are expression vectors that express short interfering RN A (siRNA) or mall hairpin RNAs (shR A) to carry out sequence-specific silencing (Brummelkamp el al., Science 296:550-553 (2002), and Paddison, et al.. Genes & Dev. 16:948-958 (2002)). Post- transcriptional gene silencing by double-stranded RNA is discussed in further detail by

Hammond et al Nature Rev Gen 2: 110-119 (2001), Fire et al. Nature 391: 806-811 (1998) and Timmons and Fire Nature 395: 854 (1998).

[0084] Expression of an endogenous plant gene can also be suppressed by recombinant expression of a micro RNA that suppresses a target ROX gene. Artificial micro RNAs are single- stranded RNAs (e.g., between 18-25 mers, generally about 21 mers), that are not normally found in plants and that are processed from endogenous miRNA precursors. MicroRNA sequences are designed according to the determinants of plant mRNA target, such that the artificial micro RNA specifically silences its intended target gene(s) (Schwab et al, The Plant Cell 18: 1121-1133 (2006), as well as the internet-based methods described therein; US Patent Publication No.

2008/0313773).

[0085] Methods for introducing genetic mutations into plant genes and selecting plants with desired traits (e.g., pathogen resistance) are well known and can be used to introduce mutations or to knock out ROX gene (e.g., a ROX3 gene). For instance, seeds or other plant material can he treated with a mutagenic insertional polynucleotide (e.g., transposon, T-DNA, etc.) or chemical substance, according to standard techniques. Such chemical substances include, but are not limited to, the following: diethyl sulfate, ethylene imine, ethyl methanesulfonate and N- nitroso-N-ethyliirea. Alternatively, ionizing radiation from sources such as X-rays or gamma rays can be used. Plants having a mutated RO gene can then be identified , for example, by phenotype or by molecular techniques.

[0086] Modified protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art and described for instance, in Sambrook et al, supra. Hydro xylamine can also be used to introduce single base mutations into the coding region of the gene (Sikorski et al., Meth. EnzymoL, 194:302-318 (1991)). For example, the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain. [0087] Alternatively, homologous recombination can be used to modify (e.g., to render nonfunctional) or knockout the targeted gene (e.g. , ROX3) in vivo (see, generally, Grewal and lar, Genetics, 146: 1221-1238 (1997); Xu et al., Genes Dev., 10:241 1 -2422 (1996)). Homologous recombination is routinely used in plants (Puchta et al. , Experientia, 50:277-284 (1994);

Swoboda et al, EMBO 1, 13:484-489 (1994); Offringa et al, Proc. Natl. Acad. Sci. USA, 90:7346-7350 (1993); and enipin et al . Nature, 389:802-803 (1997)).

[0088] In applying homologous recombination technology to a ROX gene, mutations in selected portions the gene (including 5 ' upstream, 3' downstream, and intragenic regions) are generated in vitro, and then introduced into the desired plant using standard techniques.

Dicistronic gene targeting vectors (Mountford et al., Proc. Natl. Acad. Sci. USA, 91 :4303-4307 (1994); and Vaulont et al., Transgenic Res., 4:247-255 (1995)) can be used to increase the efficiency of selecting for altered ROX gene expression in transgenic plants. The mutated gene will interact with the target wild-type gene in such a way that homologous recombination and targeted replacement, of the wild-type gene will occur in transgenic plant cells, resulting in suppression of ROX protein expression and activity.

TV, Genetically modified and transgenic plants

[0089] Provided herein are transgenic plants comprising recombinant expression cassettes (e.g., for expressing a ROX! or ROX2 polypeptide, or an ROX3 inhibiting polynucleotide). In some embodiments, a transgenic plant is generated that contains a complete or partial sequence of a polynucleotide that is derived from a species other than the species of the transgenic plant. Transgenic plants include the plant or plant cell in which the expression cassette is introduced, as well as progeny of such plants or plant cells that contain the expression cassette, including the progeny that have the expression cassette stably integrated in a chromosome.

[0090] in some embodiments, a transgenic plant comprising a recombinant expression cassette for expressing a ROX1 or ROX2 polypeptide express ROX1 or ROX2 at higher levels than a plant lacking the recombinant expression cassette. In some embodiments, the transgenic plant comprising a recombinant expression cassette for expressing a ROXl or ROX2 polypeptide express ROXl or ROX2 at about 1.5, 2, 3, 4, 5, 10, or higher- fold levels than a plant lacking the recombinant expression cassette. In some embodiments, the transgenic plant comprising a recombinant expression cassette for expressing a ROXl or ROX2 polypeptide express ROXl or ROX2 in a range of 1.5-2.5, 1.5-5, 2-5, 5-10, or higher-fold levels than a plant lacking the recombinant expression cassette. [0091 j In some embodiments, a transgenic plant comprising a recombinant expression cassette for expressing a ROX3 inhibiting polynucleotide expresses ROX3 at a lower level than a plant lacking the recombinant expression cassette. In some embodiments, the transgenic plant comprising a recombinant expression cassette for expressing a ROX3 inhibiting polynucleotide express ROX3 at less than 20% of the level of a plant lacking the recombinant expression cassette, e.g., less than 10, 5, 2.5, or 1% of the level or lower than a plant lacking the expression cassette. In some embodiments, the ROX3 inhibiting polynucleotide is an siRNA, shR A, snR A or other antisense sequence specific for inhibiting the ROX3 sequence endogenous to the transgenic plant.

[0092] In some embodiments, the transgenic plants described above in increased disease resistance compared to a plant lacking the recombinant expression cassette, wherein the transgenic plants have about the same growth as a plant lacking the recombinant expression cassette. In some embodiments, the transgenic plant has no more than about a. 10% reduction in growth compared to a plant lacking the recombinant expression cassette.

[0093] A recombinant expression vector comprising a ROXl or ROX2 coding sequence, or a ROX3 inhibitory sequence, driven by promoter can be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as

electroporation and microinjection of plant cell protoplasts, or the DNA construct can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. Alternatively, the DNA construct may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrohacterium tumefaciens host vector. The virulence functions of the Agrohacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. While transient expression is encompassed by the invention, expression is typically effected by inserting an appropriate expression cassettes into the plant genome, e.g., such that at least some plant offspring also contain the integrated expression cassette.

[0094] Microinjection techniques are also useful for this purpose. These techniques are well known in the art. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski el al. EMBO J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al. Pro Natl. Acad. ScL USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al Nature 327:70-73 (1987).

(0095| Agrohacterium tumefaciens-mQdi&ted transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example, Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. ScL USA 80:4803 ( 1983).

[0096] Transformed plant ceils derived by any of the above transformation techniques can be cultured to regenerate a whole plant that possesses the transformed genotype and thus the desired phenotype such as enhanced disease resistance. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture grow h medium, typically relying on a biocide a.nd/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al, Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, expiants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987). (0097] As explained above, further provided are genetically modified plants with reduced or non-detectable ROX3 activity or expression, and methods for generating a mutation in an endogenous R.OX3 gene that reduces or eliminates R.OX3 activity or expression, or a ROX3 gene knockout (ROX3 knockdown, ROX3 deficient plant or cell, etc.). Accordingly, provided are plants and plant cells in which one or both ROX3 alleles are knocked out or mutated to significantly reduce or lack detectable R.OX3 activity. In plants having more than a diploid set of chromosomes (e.g. tetraploids), all alleles can be inacti vated, mutated, or knocked out.

[0098] One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. [0099| The expression cassettes described herein can be used to confer disease resistance on essentially any plant. Thus, the invention has use over a broad range of plants, including species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurhita, Daucus, Fragaria, Glycine, Gossypium, Helianlkus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Mains, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Primus, Raphanus, Secale, Senecio, Sinapis, Solatium, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea. In some embodiments, the plant is selected from the group consisting of rice, maize, wheat, soybeans, cotton, canola, turfgrass, and alfalfa. In some embodiments, the plant is an ornamental plant. In some embodiments, the plant is a vegetable- or fruit-producing plant. In some embodiments, the plant is a monocot. In some embodiments, the plant is a dicot.

V. Selection of lants with enhanced stress or disease resistance

[0100| Plants with enhanced resistance can be selected in a number of ways. In some embodiments, stress resistance is determined by observing and comparing plants in drought, high density, or limited light or nutrient conditions (e.g., abiotic stress conditions). For example, a plant that is genetically modified as described herein can be compared for enhanced resistance

(e.g., survival, growth, oxygen production, etc) to a. wild type plant in these conditions. Another method of selecting plants with enhanced resistance is to determine resistance of a. plant to a specific plant pathogen, e.g., by comparing the effect of a pathogen on a transgenic or genetically modified plant as described herein to that in a wild type plant. Possible pathogens include, but are not limited to, viruses, bacteria, nematodes, fungi and insects (see, e.g., Agrios, Plant

Pathology (Academic Press, San Diego, CA) (1988)). Typically, determining resistance to a pathogen comprises visual observation, e.g., comparison of lesion formation, growth, or survival in a transgenic or genetically modified plant to that of a wild type plant (e.g., wild type for a ROX gene).

[0101] Generally, for pathogens such as X. oryzae pv oryzae (Xoo), the effect of exposure to the pathogen can be compared to determine which plants are more resistance. For example, Xoo is known to cause lesions on the plant surface. Plants that develop fewer or smaller lesions upon exposure to Xoo would be considered more resistant than plants that develop a comparatively larger number of lesions or lesions of a larger size. One of skil l wil l understand ho w to compare resistance in plants using known effects of plant pathogens (e.g., reduced growth, leaf appearance, reduced productivity).

[0102] Another method of selecting plants with enhanced resistance is to determine resistance of a plant to a specific compound that induces Systemic Acquired Resistance. Such compounds include, but are not limited to, salicylic acid, 2,6-dichloroisonicotinic acid (INA),

henzofhiadiazoie (BTH), and probenazole (see, e.g., Ward et al., 1991; Gorlach et al., 1996; Schweizer et al., 1999; and Morris et al., 1998)). The resistance responses of a plant can vary depending on many factors, including the pathogen, compound, or plant that is used. Generally, enhanced resistance is measured by the reduction or elimination of disease symptoms when compared to a control plant. In some cases, however, enhanced resistance can also be measured by the production of the hypersensitive response (HR) of the plant (see, e.g., Staskawicz et al. (1995) Science 268:661). Plants with enhanced resistance can produce an enhanced

hypersensitive response relative to control plants.

[0103] Enhanced resistance can also be determined by measuring the increased expression of a gene operably linked a defense related promoter. Measurement of such expression can be measured by quantifying the accumulation of RNA or subsequent protein product (e.g., using northern or western blot techniques, respectively (see, e.g., Sambrook et al. and Ausubel et al.). An alternate strategy for measuring defense gene promoter expression involves operably linking a reporter gene to the promoter. Reporter gene constructs allow for ease of measurement of expression from the promoter of interest. Examples of reporter genes include: β-gal, GUS (Jefferson et al, (1987) EMBO J 6:3901 -3907), green fluorescent protein, luciferase, and others. [0104] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence database entries, internet sites, patents, and patent applications cited herein are incorporated by reference in their entireties for all purposes.

A. Example 1: Materials and methods

[0105] Plasmid construction for yeast two-hybrid (Y2H) experiments and generation of transgenic plants. Full-length cDNAs for 16 RiceNet candidates from a rice cDNA or KOME (Knowledge-based Oryza Molecular biological Encyclopedia; Kikuchi el al. (2003) Science 301 :376) cDNA library were amplified using specific primers (see Table 3). The cDNAs were cloned into pENTR/D TOPO (Invitrogen) according to the manufacturer instructions, and the sequences confirmed by sequencing. Positive clones were then moved by Gateway LR Clonase (Invitrogen) into the yeast two hybrid vector pNlexA carrying the BD domain or pB42AD containing the AD domain (Ciontech) for yeast two hybrid assays. Five positive clones were moved into the Ubi-Cl 300 binary vector in order to generate high expressing transgenic lines. The amplified partial fragment (350-450bp) for four RiceNet candidates from a rice cDNA or KOME cDNA using primers was cloned and recombined into the pANDA binary vector for generation of RNAi silencing transgenic lines using the same procedure.

[0106] Yeast two-hybrid (Y2H)anaIysis. The yeast two-hybrid was performed as described in Seo el al (201 1) PLoS Genet 7:el 002020. Briefly, purified plasmid DNAs from BD vector-bait genes and AD vector-prey genes were transformed into the yeast, pEGY48/p8op-LacZ

(Ciontech), using the kit Frozen-EZ yeast transformation II (Zymo Research). The strength of each protein-protein interaction was determined by color development of a yeast colony two days after streaking on media containing X-gal. An empty vector was used as a negative interaction control. XA21 -XB3, a pair of known strong interactors was used as a positive control (Seo et al.). An interaction was considered significant and reproducible if similar results were observed in 2 or 3 independent assays. The results of yeast two hybrid analysis are summarized in Table 2.

[0107] Generation of transgenic plants. A transgenic rice line carrying the Xa21 gene under its native promoter in the Kitaake genetic background was used to validate the RiceNet-guided predictions. Rice transformation was carried out as described in Chern el at (2001 ) Plant J. 27: 101. All generated transgenic lines were tested and confirmed by PCR with gene-specific and/or construct-specific primers. At least 3-7 independent transformed plants were generated for each line except for the Xa21 -LOC_Os02g21510 RNAi line. [0108] Expressless quantification and Xoo inoculation methods. Altered expression of the transgenes in the transgenic lines was validated by semi-quantitative reverse transcription PCR (Figure 1). For X, oryzae pv oryzae (Xoo) inoculation, a bacterial suspension (OD₆₀o of 0.5) of Xoo strain PX099, which carries Ax21 activity was used. Five week-old transgenic plants were inoculated with Xoo via the scissors-dip method (Song et at (1995) Science 270: 1804). B. Example 2: Genetic and functional analysis of ROX (Regulator of XA21- niediated immunity) genes

[0109] To identify candidate genes governing the rice defense response, RiceNet was queried with 1 5 member proteins of the XA21 interactome with clear phenotypes. The RiceNet predicted proteins were tested for interaction with 24 members of the XA25 interactome, including XA21 .

[0110] Fi ve of the genes in the interactome (indicated in Table 1 ) were further prioritized based on their direct interactions with XA21 (LOC_Os01g70580, LOC_Os01 g70790,

LOC_Os02g21510, and LOC_Os03g20460) or the presence of a motif present in animal genes that govern the inflammatory response (LOC_Os06gl2530). [0111] Transgenic analysis using altered expression mutant plant lines was used to detect an altered immune response. Over-expression (ox) and RNAi constructs were generated for each of the five selected genes, except for LOC__Os06gl2530, for which only an over-expression construct was generated. The constructs were introduced these into a homozygous Kitaake- XA21 rice line using a hygromycin selectable marker. Resistance to Xanthomonas oryzae pv, oryzae (Xoo) was determined by measuring lengths of water-soaked lesions 14-21 days after inoculation.

[0112] Three out of five genes (indicated in bold italic) displayed altered immune responses. No differences were observed between the control Kitaake-XA21 line and the ox or RNAi transgenic lines for LOC_Os01 g70790 or LOC Os03g2046(). Three of the lines (XA21- LOC Os01g70580 RNAi, XA21-LOC _Os02g21510 RNAi, and XA21 -LOC _Os06gl2530 ox) showed resistance to Xoo in transgenic plants (Figure 1 ). Transgenic lines silenced for

LOC_Os01 g70580 and LOC_Os02g21510 showed enhanced susceptibility to Xoo, as did the ox line for LOC_Os06gl 2530. These genes are designated Roxl (Regulator of XA21 -mediated immunity), Rox2, and Rox3, respectively. The phenotypes are heritable for two generations, the RNA accumulation correlates with the overexpression or knockdown, and the transgene cosegregates with the altered phenotypes in progeny analyses.

[0113] Roxl: Tl progeny containing the LOC_Os01 g70580 RNAi construct displayed enhanced susceptibility to Xoo (lesion lengths of 3-7 cm +/- i .3 cm) as compared with the Kitaake-XA2i control (2 cm +/- 0.8 cm) (Figure l b). Leaf lesion lengths correspond to bacterial growth in planta (Seo et al. (201 1) PLoS Genet. 7:el002020; Chern et al. (2001 ) Plant J.

27: 101). The Tl lines (2-2, 2-7, 2-10, and 2-11) that displayed highly enhanced susceptibility to Xoo as compared with the Kitaake-XA21 control were self-pollinated. The resulting T2 progeny lines segregated for the enhanced susceptibility phenotypes.

[0114| T2 transgenic plants carrying the LOC OsO 1 g70580 RNAi construct (2-2, 2-7, 2-10, and 2-11) displayed enhanced susceptibility to Xoo (lesion lengths of 7-11 cm +/- 1.5 cm), whereas the T2 progeny 2-7-4 and 2-7-10 that do not contain the LOC Os01 g7058G RNAi construct displayed no phenotypic alteration (lesion lengths of 2-3 cm +/- 0.5 cm) (Figure la). The knock-down of gene expression was correlated with the enhanced susceptibility phenotype in LOC_Os01g70580 RNAi 2-10 and 2-11 lines (Figure la-b). The results show that

LOC Os01g70580 (now designated Roxl, Regulator of XA21 -mediated immunity) is a positive regulator of XA21 -mediated immunity.

[0115] The genomic, coding, and protein sequences for ROX1 are shown as SEQ ID NQsi l-3, respectively. ROX1 is annotated as a thiamine pyrophosphokinase (TP ). TP catalyzes the transfer of a pyrophosphate group from ATP to vitamin B 1 (thiamine) to form the coenzyme thiamine pyrophosphate (TPP). Treatment with thiamine and TPP induces resistance to rice pathogens including Xoo (Ann et al. (2005) Plant Physiol. 138: 1505).

[0116] Rox2: From the three independent XA21 -LOC Os02g21510 RN Ai lines generated, T 1 progeny from two of the lines (TO lines 3 and 4) displayed decreased accumulation of

LOC Os02g21510 niRN A. The reduced mRNA levels correlated with susceptibility to Xoo (lesion lengths of 4-5 cm +/- 0.5 cm) (Figure lc-d) in these lines. Control Kitaake-XA21 displayed shorter lesions (lesion lengths of 2 cm +/- 0.6 cm) (Figure Id). Tl progeny from two lines (3 and 4) as well as T2 progeny from line 3-1 expressing the LOC_Os02g21510 RNAi construct displayed enhanced susceptibility to Xoo (lesion lengths of4-7 cm +/- 1.1 cm) (Figure 1 d). The results show that LOC_Os02g2151 0, designated ROX2, is also a positive regulator of XA21 -mediated immunity. [0117] The genomic, coding, and protein sequences for ROX2 are shown as SEQ ID NOs:4-6, respectively. ROX2 is annotated as a member of the NOLl/NOL2/sun gene family. Human homologs have been implicated in Williams-Beuren syndrome, a developmental disorder associated with haploinsufficiency of multiple genes at 7ql 5.23 (Merla et al. (2002) Hum. Genet. 1 10:429). This family was not known to function in innate immunity in plants. [0118| Rox3 The resistance response of three independent XA21-LOC_Os06g 12530 overexpression lines was tested. Two Tl progeny (from TO lines 1 and 3) were selected based on increased accumulation of LOC Os06gl2530 mR^'NA. These lines displayed enhanced susceptibility to Xoo (lesion lengths of 4-5 cm +/- 0.5 cm) (Figure 1 e-f). Tl progeny from two TO lines (1 and 3) and T2 progeny from one Tl line (1-15) carrying the LOC Os06g 12530 overexpression construct exhibited enhanced susceptibility (lesion lengths of 4-8 cm +/- 1.3 cm) compared with XA21 plants (lesion lengths of 1.8 cm +/- 0.7 cm) (Figure If). The results indicate that LOC Os06gl253(), designated ROX3, serves as a negative regulator of XA21- mediated immunity.

[0119] The genomic, coding, and protein sequences for ROX3 are shown as SEQ ID NOs:7-9, respectively. ROX3 is annotated as a nuclear migration protein, nudC. nudC plays a role in cell division through the regulation of cytoplasmic dynein (Riera & Lazo (2009) Cell Mot Life Sci 66:2383). The presently disclosed ROX genes were not known to play a role in innate immunity to plant pathogens, and sequence homology alone would not have suggested these functions Table 1 : Genes predicted to be involved in Xa21 -mediated immunity

Table 2: Results of yeast two hybrid tests between XA21 mteractome proteins and candidates XA21 regulators predicted by RiceNet.

* Strength of protein-protein interaction is indicates by intensity of colony color development; +. weak; ++, medium; +++, strong.

Table 3: Sequences of forward (F) and reverse (R) primers used for cloning cDNAs

Gene name TIGR Locus ID Primer sequence

N3^0X LOC OsO 1 §70790 F 5 ATGGCGCAGAGGACGCTGGA 3

R 5 CTAGAAATCGAAGCCACCGCC 3

N6^0X ! x H ^' _Os02g2 i 510 F 5 ATGGCCGACGCTAGGTCCGC

R 5 CTAAATCTGTAATTTAGTGAACTTGGC 3

N9^0X LOC_ _Os03g20460 F 5 AI^'GCTTCTCACGCGAAGGTTC 3

R 5 CTAGATATTAACCGTACGTTTGTG 3

N17K ^0X LOG _Os06g 12530 F 5 ATGGCGATCATCTCCGACTTC 3

R 5 CTAAGCTATTTTTGGATTGGAGAAGTC 3

2p LOG _Os()lg70580 F 5 ATGC^'CGCTGCC^'GACXjArGACC 3

R 5 TTC:AGAGC:CAGGI GATTTC:T 3

N3p LOC_. _Os01g70790 F 5 CCA GCC GCC GCG GGG C 3

R 5 CTAGAAATCGAAGCCACCGCC 3

N6p LOC_. Os02g2 i 5 i 0 F 5 ATGGCCGACGCTAGGTCCGC 3

R 5 AAC ATC AAC GCC AGT GAG AGA 3

N9p LOG _Os03g20460 F 5 ATGCTTCTCACGCGAAGGTTC 3

R 5 GAA ATG CAT CCC CTC CCT CTC 3

p primers used to amplify 350-450 nt cDNA fragment to generate silencing (RNAi) construct ^ox primers used to amplify construct for overexpression Informal Sequence Listing

Genomic sequence for OX1 (LOC_Os01g70580; SEQ ID NO: i)

GTTCTACATCTTGCCTCGCCGTGCACCGCAGTCCGCCGCACTACCAGCGCCGGCGCT AC CGTCGTTACCGAC GCAGCCTCGC:CTACACTACACCC CGZ TCX:TTC C C CAAC:CACCACCG CAGCAAATACACCGCCGCGGCGCCGCGCCTCTTGGAGGCTTCGACGCGCGCCCAAAAAGC

GACCGCGGCTTCCCGTTCCCGftA⁽^^

CCAATCGAATCGCCTCTCCG CCTCCGCGGCGGCGGGCCGrCCATGCCGCTGCCGACGATG ACCCACTCCTCCTCCTTCCTCCGCCTCCCCGCC^CCTCCTCGCCGCACCCGCCGCCCGCG GATGATGCTTCCGCGG CCTACGCCGTCGTCGTCCTCAACCAG CGCCTGCCCCGCTTCGCC CCCCTCCTCTGGGACCGCGGTATCCTTCTTCTTCTTTGCCTCGATGATGCCCTTCGTTCG CTTTTTTAGGTTTGGTGTGGGCTGGCTGACTGATGATGACGTGGTTTGTGGTGTGTCGTG GTGGGTGGCGAGAGCAGCGCGGCTGCGGGTGTGCGCCGACGGGGGTGCCAACCGGGTGTT CGATGGAATGCCGGAGCTGCTGCCCGCCGAGGACCCGGACCAGGTCCGCATGAGGTGACC GATAGTTTCCAGCTTTATTTATCCCCCCCCCCCCCCCCCCCCCCCTACTGTTCTTTGTTT GCATGTGTCCTCTCCTCGCGCGTTTTGAGTCAGGCTTGGCGGTCATGATGGAA GCGTGG GAGGCGAGTGAATCTTGTTTTCGTTAGTATTTCATTATGCAGAGGAGTGAAACTGTAAAT GTTGTCATGGAAGCGAGTACTGTCTAGCTGAACCAGATACCA CTAGTTAAGTGTTATTTC TCGATATGTTTATCTCCCATGCTTTACATGTTTCCGTTATTATATGTTTAAGCAAATGCT GAAGTTCCCCTGATATGTTCAAGTTGAGTAATTTAGTATGCAACACCTTATAGA GTTAA TTTCCTGTTGGGTTATGGTTTAGTGCTTTAGCTAACGAAGTGCTCTTTCATTTGATGAAC TCTGTTAAAAGTTTTGTTCTTATCTGCZ TGATTGCTAGTTTTGTTTTCGGTGZ GCTTAGA ATAACATTCTTAAATTAGCCGAAGTTGCCCTCACAGACCTGAATGTACGCGATATTGCGC CAAAGGCTATCTGAAGCCAC7iCAAAATTACTCAAAATCTGGGATGA7»CTACAGGTCTAAA GCAGCTTCAACTTGTGTCTTCTTCACTATGAACTTTTTTTTCCTATCTTTGGTTGCCAAA GGTTTGGGAGACATTTATGCATCCAACAGAGGAGCATGGCTAGTTGATTTGCCATACCTG GTAGTTGGCAAGAGAC'rGT' i'i'i'CITIAAATTGTTGGTTGGGTT'TAAAGACAGCTTCTTTG TTGGGTGT CAT TATTTATTAATTTGTGCTCATCTTCCTGCAGGTAC7iAGCCAGATGTG ATCAAAGGGGATATGGACTCAATAAGGTCCAGAAG GAAGG'AATATTATTCCAATCTGG A ACTATTTTAAAAATCACTTCAAGTTGAAACTAGCTGCTAAGGCTGGATGTTAAACCAGCJi ATACTGTATGCAGCAAC'rGi'CCAGGCAATTTTCACTGTAATGTGCAGAAAIAAATTGATG TTGCTGGATGCAGTATTTTTGTATGTAATATATATTTGGAATTTTGGATGTCACAGGCTC ACAGTTTAAGTCTTCTAATTTAGATGACTTTACTAATTACAGTAATATGTGAAGAATTAT TTTCAATTTCCTTTGTGAAATCAGCATACTGAAATGATTTACAGTGACTAGGTTTTAAAC A ATCAATGTCCTTAAAAATCAGGAAGGTATAAACAGCATGCTTTATTTATATTTTAGCA TTGCTGTTTAGGTAATCTGTATCTAAGAAAACAAAATGTGTATTGCATGGCCTGATACTG TATTATCTTTTTTTTTATTGAAATTGCATGCTCAGAGTACTGCTATATGTACGTCTCTAT TTGTCTTTCTGGATATGTTGTTGATTGTCCACAAGAAGCTCATCTGCTTAACTAAAATAT GTGCATTTTGTTTTGACCTGTTGACTGCTGTATATGTTATAACCACTTTCGAGAGGAACG ATAGTAAGTTCAATAATGGGAGATCCGTATTTAGTTCAAGTTTTTTTTTTCTTTGTTTGC TATACACAGCTGA GCAATCTGCTTCAAGAATTAATATGTAA CCTCCTAATTCTGATGAG CTTGACATTGTTCTTTGCCATCTAATAACACGCTGTTCTTCTGTTGCAGGGTGCCGAAAT AGTTGATGAATCACATGATCAGGACACCACTGACCTGCATZ\Z ATGTGTATCATTTATCAC

CAGAAATCCACCTGGCTCTGAAGAGTCTAATGTACTGAGCTCAATTGTTAATCATAACAA GZ\ATTTCAGAAC:CACCGACCACACAACATTAGC CTTACA,AATTTTACTGTATTCTTTATC TGCAGCTGTATATTCTTGT C TGGTGCACTTGGAGGAAGRGRTTCGA CATGAGATGGGGA ACATCAACGTGTTATATCGCTTCTCAAACATCAGGATTGTCCTCCTGTCTGATGACTGCT CAATCTTTCTGCTCCCAAAGACGCACAGCCACGAGATCCATATCGAGCGATCAATCGAGG GTCX:TCATTGTGGATTGATTCCAATGGGTTCAC CATCGGCTAGCACTACCACCACAGGGC TCCGGTGGAATTTAG!G!TATAAGCATCAAACTTTACAGTTCTTTGT G ATCATGTATGGC AACAGCATCTGAACTGATAGTCATTCTGCACTTTGTCTTCAGATAATACTAGCATGAGCT CGGTGGACTGATAAGCACATCTAACATCGTGGAAGAGGAAA CAGTTAGGATCACTTCAG ATTCTGATCTGATTTGGACAATATCACTCCGGAACTGAGCTTTAAATTACCCATAAATGT A CTTGATCCAAATTTTGGCTCCTTAGTATAGCATGGATGGGCTACATTTTTGTAACTTA GATTTTTTTTTCACTGATCTACTGTTAGTAAACAGGAAAGGTATCCATATGTGTAAGCCT CTTGGACATGCATCCATTATAGAAAAAAATATTATTAACTGTGTTGTTCAATTCTCA Coding sequence for ROX1 (LOC_Os01 g70580; SEQ ID NO:2)

ATGCCGCTGCCGACGA GACCCACTCCTCCTCC CCTCCGCCTCCCCGCCACCTCCTCG CCGCACCCGCCGCCCGCGGA GATGCTTCCGCGGCCTACGCCGTCGTCGTCCTCAACCAG CGCCTGCCCCGCTTCGCCCCCCTCCTCTGGGACCGCGCGCGGCTGCGGGTGTGCGCCGAC GGGGGTGCCAACCGGGTGTTCGATGGAATGCCGGAGCTGCTGCCCGCCGAGGACCCGGAC CAGG'RCCGCAI'GAGGTACAAGCCAGATGTGATCAAAGGGGATATGGACTCAATAAGGCCA GZ\AGTGAAGGAATATTATTCCAATC GGGTGCXGAAATAGTTGATGAATCACATGATCAG GACACCACTGACCTGCATAAATGTGTATCATTTATCACCAGAAATCCACCTGGCTCTGAA GAGTCTAATCTGTATATTCTTGTTCTTGGTGCACTTGGAGGAAGGTTCGATCATGAGA G GGGAACATCAACGTGTTATATCGCTTCTCAAACATCAGGA TGTCCTCCTGTCTGATGAC TGCTCAATCTTTCTGCTCCCAAAGACGCACAGCA¾.CGAGATCCATATCGAGCGATCAATC GAGGGTCCTCATTGTGGATTGATTCCAA GGGTTCACCATCGGCTAGCACTACCACCACA GGGCTCCGGTGGAATTTAGATAATACTAGCATGAGCTACGGTGGACTGATAAGCACATCT AACATCGTGGAAGAGGAAACAGTTAGGATCACTTCAGATTCTGATCTGATTTGGACAATA TCACTCCGGAACTGA

Protein sequence for ROX1 (LOC_Os01 g70580; SEQ ID NO:3)

PLPT THSSSFLRLPATSSPI-IPPPADDASAAYAVWLNQRLFRFAPLLVJDRARLRVCAD GGANR.VFDGMPELLPAEDPDQVRMRYKPDVIKGDMDSIRPEVKE¥YSNLGAEIVDE8HDQ DTTDLHKCVSFITRNPPGSEESNLYILVLGALGGRFDKE GNINVLYRFSNI .IVLLSDD CSIFLLPKTHSHEIHIERSIEGPKQJLIPMGSPSASTTTTGLRW LDNTSMSYGGLISTS NIVEEETVRITSDSDLIWTISLRN

Genomic sequence for ROX2 (LOC _Os02g21510; SEQ ID NO:4)

AAAACCG7IGATATCCAATCCGGCCCTTCTCATG7IGTTGTCAGTCACGCATCGGCGCCGCC CTCCGGCCAGCAGCGACTCGCCGACACATCTTCAAAGAGTCCCGCCGTCGTATGCGTGGC TCCCCTCCCCGGCCTCTCGTCTCGTGGGAGGTCTGGATCGACCGCTTCCTCCGCTCGTCG CGGCGAATCCAACCAACGCCGGCGTCCCTCCGCGCCGCAGCTTCCTGTGCTGAATCTCGC CGCTCCCCGAGTCCGGGAACGCACGGAGCGGGCGCGGCGAGCGGCTGGCACCTTGGTTCA AA CTCGCCGCGAAACTTCTAGTAGCAGGCAGCGGTTTAAATCTCGAATTTATCCATTTT GGTGTTCGGCTGAGCCCCCATGGCCGACGCTAGGTCCGCCCCTGAATCGCTGCCGCCCGC GTTTCTCGAG CCTCCAGGAGAATGGCTTGGACCCAA GATGTATTCCATGGTCGACAC CATCCCACGATACATAAGGTGAGGAGGCAGCCTGAGATTGATTCAGAGCTACTTATGCTC TGATGAATCACCGGAAGGCTTTCATAA TGTTATTGTCAGTTTACTTGCCTTGCATTGCG TTGAGTTTGCTTTGTGATTCCAAGGCTAAAACCAGGCA_^TGGAGCCCCATATTCCAGAGAT TCAGAGCGAGCTGACGTGCCATCTCAACAAGGTTTCATGGCTGCCAGATTTCTACGCAAT TCCGCCTCAAGTTCAGATTGCTAGCTCCATGGCCTATCAGCAAGGAAAGGTGACAAGACA TTAAAATTTACTTTTGATTCTGTTCTTTTTTTACAGGCCAGCGCA/ACGCATTGGATTGC ATAATCGGTTAATGGTATCCTAGATTAAC GAATGTTATCTGG G ATGCAATTTTAT TTTTATTCTATG,AAGTGTTTGCCAGATZ\ATTTATCX:TAGGTAGAACTCTTAAZ\ACATCTT ACTTTTATGTGCAAGAGAGAAATTGTCTCAAGTACTCCTTAGTCCTTATAACACTACTTG TACTGTTACAGGZTZ\ZAGCTGCTGTGCAATCATGTCTTATCZTGTGTTCTTTTATTTGCT

CCAT'rGCi'i'CCI'GCTAAGACATTTTAAATACAi'i'i'GGCI'AGATCTATGGAATTGATGCAG

CTTCTGGAGCTGCTGTCTTAGCAC TGZTGTTCAAC:CAGGAGAACATGTTCTGGACCTCT GTGCTGCCCCAGGTAATTAAI'I'CAAI'ATTGTTATATATTACATCACACCAGI'ATTTTATC TGTGGCAGATGTCTACX:GTGTATAACAATGGCCCTTTGCTZGACTTGTTTTTTTGCGCCA CATGTATTCAGCAGTAATGGAAATACCTAGTTTTCTCCATAAAACACAAGAGTAATTATC TTTCCTGATTGGTTGATCTTTGGGGA-AGTAACTGCTCTCTCTGCATCGTTGGATTGAACC AAATCTGTTCAGCCCCAAACCAGATTTA GGATTGACACAGTCTTTAAGTTTGAAATATA TGTTGAACTTTATTCTCAAATAGGTTTGATATAGCATTGTTATATTACATATACATTGCA AAATTGGTGATGGTCAAACTCGAACGCCATTTTTGCATGA TGTTTTGTCATAAGTCTGT AGTTTCTGTTAGATTATTTGTTA.GTTGTGGAATTATTCTGAATGATGTGCTCATATCATG TGCAAGGTGCAAAGCTTTGCATGCTTGCAGATATGCTTGGAGGCAGAGGTTCTCTGACTG GCGTTGATGTTGCAAAGCACCGTCTTGCGGCCTGTCGTACGATGTTGCAGAAGTATTCTC TCGGAGATCGTTGTCGTCTCTTTGTTGCCGATGGGACTTCA TTTCTATATTACCTGTGA ACTCTAGCTTGGGTAATGGAGAAGGTAATAGTTTGCCTCTTTAAACAGAGTAAAATCCTT

C :TTTCTGTC TZ TTGCTCATGGAGCACTTGAGTAGATGCZ TCTGGTTGCTTATCTATCA

CAGTGATCAACTTGTCATAAGGACAATGTGTAAGTACATTGTCGG'AATGGACTTCAAAGAGA

CGTGGAAAGACCGACAAJ AGTCTAAAAAGGCAAGAATGGCGGGTTC^

CAACTTCAGAACCTGAAC'RGAIAIACTATGGCAAACATTCAGGAT IAG I'I'GGATTACGCA

AATGTGATGCTATTCGTCCATCAGCTGATGATGAAGCCCAGACATCTGGCTATGACAAAG

TAT^IΓCCACAG AACCTTA ACAA ACTGTCC^IΓΊ^,Ί^,CΊ'Ί'Ί'CTTCAGTACAΆ GCTGT^IΓ^IΓ^IΓΊ^,Ί^,

TAGTGTTACTGAAAACAAACTAATGATATTTTTAGTTTTTTATCTGTGCACTTTGATCAT

TTTAGGAAGGAAATGTCTTGTGATTTAGTGTGCTATCTTGAAAAGTTTGAAGACCTAGCC

ATTCCAAATGTTGCACGCTTAAATCATGACATAAATCTAGTGTCTCGTTTAGTCATCAAG

CACTCAATACA CTTATTGAGAACTACC TTCCC GTCCAATAAAGTATTCATGTTT

ATAACTTTGTGTATGTAACATTTTGACTCTTGATAGTTTCCTTTCTATAATTAAGCAGTG

CTTCTGTGTCCACCAGAAAATTAACTTGCATGCTCACCAGAGAATCAGCTTGTGCAAATA

TTTAAAGTAGTACAATTAGTAGTGGAGTTTCATGTAGATGCAAATCAGGTCTTTGAACTT

CTCTTAATATTGCGTTGTTACTTTGTGAGAGAGAAATGAAGCACTCTTCTTATGCAACAT

ACTTTTCCAGCAGGAATATCCTTTGGCAGTTTATTTCACAGCTTGGATAATA.TTTTTAGT

TTGTTATGTTTC^V\Z TGCACACAGGTCCTAGTTGATGCAGZ\ATGTACTCATGATGGATCC

ATAAAGCACATCCAAAAATTTGAGTTCTGGGGATGGAAAACCTTAGATCGTCGTGTACTT

GZ TGCAGAAAGAACTGATGACCTGC TCATCTTCAGGTACTACTTTCAGTCTTGATGCTT GCTTGAAGGGTTGACAGTTGGCACCCAATCAAGCTATGTACATTT iAGAIAIAATTACTT ATCTCGTTAGCATGTGAGTGCACAATTTCATGAGTTTCATCCGTTGTGCAGTTACGCCTT CTCACTAJ^ GGTTTTAAATTATTGAAAACTGG GGATCACTAGTATATAGTACTTGCAGG TGAGTA7»TCTCTTCACATGCAATTCCAATATTTGATGCCTCCTCAATCGCTTCCCAGCTG TGTAGTCACCTTTTCCTATTGCATTTCGTGTCTTACTTAACTACTGATGGCTTGCGTTGA

,AATAGCTTGACAGTTGCACAAAATGAG,AATGTGGTTCAACAGTTTCTTTGCA,AACACTCT

TCAGCAGGTACAGAA AGCCGTGTTCCTCATTTCAGTATTA GTATATCCCTTATATTTT TACTTTGAAGATGAATATTGTTATTTTTCTCAAATGGACTGATTAAATTGATTGTGGTAC TTCTAGTCAGGAAATCTTTAGTAGTGTACTTGTTCTCACTTCTTGGTGTGGTTAAGTGAA CCTTACTTATA.ACAAGATTATTTAGTTTGTCTTGGTTCA.TTGAAGTTGTCAAAGTTCAAC AGATCTGCCCTGA GA CCTTCTGGGATTTCTTTTGCAAACA GCAGAGCTACAAAAGATT GATTCAGCTGACAGTTGGCCTTGCAGAAGTGGAAGCATCTTCAAAACATTGCGATTTGAT CCTGCAACTTCACAAACGAGTGGCCTTTTTGTTGCCAAGTTCACTAAATTACAGATTTAG AGCATATCTGATCAGTCTCCATGTAGTTCTAAAGATGTGCATTTAACTAGTATATGATTC CATGTGCIICGTTAGATGTTCACAAGTAACTGTATGCTTCAAATTCCCGGAGCCATCACTG TTATGGTTTGCGAAGCAAAGTGGTGCAAACTGTCAGCTATGCGAATCTAAACTTGCAATT TGGAAGGTAAAAGCTTGGTTTCAGTTAACA

Coding sequence for ROX2 (LOC_Os02g21510; SEQ ID NO: 5)

ATGGCCGACGC Z GGTCCGC C CX:TGAATCGCTGCCGCCCGCGTTTCTCGAGTTCCTCCAG GAGAATGGCTTGGACCCAATGATGTATTCCATGGTCGACACCATCCCACGATACATAAGG CTAAAACC7IGGCATGGAGCCCCATATTCCAGAGATTCAGAGCGAGCTGACGTGCCATCTC AACAAGGTTTCATGGCTGCCAGATTTCTACGCAATTCCGCCTCAAGTTCAGATTGCTAGC TCCATGGCCTATCAGCAAGGA?^VAGATCTATGGAATTGATGCAGCTTCTGGAGCTGCTGTC TTAGCACTTGATGTTCAACCAGGAGAACATGTTCTGGACCTCTGTGCTGCCCCAGGTGCA

Z\Z GCTTTGCATGCTTC AGATATGC TGGAGGCAGAGGTTCTCTGACTGGCGTTGATGTT GCAAAGCACCGTCTTGCGGCCTGTCGTACGATGTTGCAGAAGTATTCTCTCGGAGATCGT TGTCGTCTCTTTGTTGCCGATGGGACTTCATTTTCTA.TA.TTACCTGTGAACTCTAGCTTG GGTAATGGAGAAGGA CAACTTGTCATAAGGACAATGGAAGTACATTGTCGGAATGGACT TCAA-AGAGATCGTGGAAAGACCGACA-AAAGTCTAAAAAGGCAAGAATGGCGGGTTCACCT CA CTAACATCAACTTCAGAACCTGAACTGATATACTATGGCAAACATTCAGGA TAGTT GGATTACGCAAATGTGATGCTATTCGTCCATCAGCTGATGATGAAGCCCAGACATCTGGC TATGACAAAG CCTAGTTGATGCAGAATGTACTCA GA GGATCCATAAAGC C TCCAA AAATTTGAGTTCTGGGGATGGAAAACCTTAGATCGTCGTGTACTTGATGCAGAAAGAACT GA GACCTGCTTCATCTTCAGTTACGCCTTCTCACTAATGGTTTTAAATTATTGAAAACT GTGVRGGA CA.CTAGTATATAGTACTTGCAGTCTTGA.CAGTTGCACAAAATGAGAATGRTGG CAACAGTTTCTTTGCAAJ CACTCTTCAGCAGAGCTAC^

TGGCCTTGCAGAAGTGGCAGCATCTTCAAAACATTGCGATTTGATCCTGCAACTTCACAA CGAGTGGCCTTTTTGTTGCCAAGTTCACTAAATTACAGATTTAG Protein sequence for ROX2 (LOC_Os02g21510; SEQ ID NO:6)

MADARSAPE SLP PAFLEFLQENGLDPMMY SIWD I PRYI RLKPGMEPHI PE I QSELTCHL NKVSWL PDFY I PPQVQIAS SMAYQQGKI YGI DAASGAAVLALDVQPGEKVLDLCAAPGA KLCMLADMLGGRGSLTGVDVAKKRLAACRTMLQKYSLGDRCRLFVADGTSFSILPVNSSL GNGEGS CHJ DNGSTL SEWTS RS ORQKSKKARMAGS PHLTSTSEPEL IYYGKHSGLV GLRKCDAIRPSADDEAQTSGYD VLVDAECTHDGSIKlilQKFEFWGWKTLDRRVLDAERT DDLLHLQLRI JTNGFKLLKTGGSIA'YSTCSLTVAQNE VQQFI ^KHS SAET QKI DSADS WPCRSGSIFKTLRFDPATSQTSGLF'VAKFTKLQI

Genomic sequence for ROX3 (LOC_Os06gl 2530; SEQ ID NO:7)

CCTCGAAAACCCTCGCGAATCTGAGATCCCCAAGAATTCCTTCTCGAAAACCCTAGAGGA GAGCGAGAGAGAAGAGGAGGCGGCGGCGGCGGCGGCGGAAGCACATGGCGA CATCTCCG ACTTCCAGGAGGAAGAGGCTCCGCCGCGGCAGCAGCAGCAACCGGCTTCGGTGGCGGCGG CGGCGGGGAGCGGCGACGAGGTCCTCGCGGCGGAGC GGAGCGGAGGGGCGGCGCGATCC CCTTCCTCCAGGCGGCAATCGACGTGGCGAGGCGGCGGTCCGACCTGTTCCGCGACCCCT CCGCGGTGAGCAGGGTCACATCGATGGCGTCGGCGGCGAGGGCGGTGGTGGAGGCGGAGG

AGAGGAAGGCGAGGGAGGCCAAGAGGAAGGCGGAGGAGG CGGAGAGGAAGGCGGCCGAGG

CCGAGAGGAAGGCGAAGGCGCCCGCGGAGCCGAAGCCGGAGAGCTCGGCGGGGAAGGATA GCATGGAGGTGGACAAGAAGGAGGAGGGCAACGTGAGGAGTAAGTAC I'CI'GI'TCCCATGC CCCCTGCAATTGTTZ\ATACTGCTGAGTTTTAGGGTTTTGZ TGCTAAATGTGCTTTAGATA TTGATGAC I'ITGCTTGTCAGGGATTTAGGGATC I' I'GAITATTTGCGTGATGCTAATACAA ACTTTTTTTTGTTGTTTTAGTACCTTAATTTCTGTCTGCTACTGCGGTAGGTTCTTTTTG TCTCTATAGAACTCTTGAT I'AAGGCIAATATTGGTTTATATGATC IAI' I'CCI'CACAACAT ACTTAGTTTTC Z\Z\Z\Z ATAACATAGGTATACTTCGTAGTTGGAAGATGGATTTGCTGCTT AGCTAT GCTGCTGATTACCTCTGG6TAAAACAATACTGGTGCATACGGAGTA6TAT AGGAATTAGAATAAACTTCATGTA.TTCATTATTTATTATAAGTAAATTCCCA.TTGCAATT GTCAAATTGATGCAGAACAAAGGTATCTGTTTCCATTAGATTTGAAGGCCAATTTCAATC TGGATGAATTTTACTTTGGTTTGCTCAATTGATGGGGCTTGCAGAAAGTTGGTTTTGTGC TATGCTATATCCCGAGAAACAACTGAACTTAATTTGATATAACCATGTAATCTTACCTGT AGTAGTCCTTTCATTTGCCTATTGGCGAAATAATTTGCCCCAATTTTTGGAGATCAATTG ATTGGCGTCTAACATAATCCTCTTCTATGAAACAGAACCAAATGCTGGCAATGGTCTTGA TTTGGAGAAGTA.CTCTTGGATTCAGCAATTGCCAGA.GGTTACCATCACCGTACCTGTTCC TCAAGGAACGAAGTCAAGGTTTGTCGTCTGTGATATTAAGAAGAACCATCTGAAGGTTGG GCTGAAGGGCCAGCCTCCTATCATTGATGTAAGTACCAACTACTATAAACTGATGATTTT TCTTGTGAATGCATTTGTTTTGGGAAAAAAGCACATTTTCTCTGAATCTGATA GTGGTG TTCTAATTGTCTAATTGTGGCTCTTTCTGGACAGGGTGAGCTTTTCAAACCAGTCAAAGT TGATGACTGCTTCTGGAGCATAGGTATATGATACAAGTC:CTTCACTTTTACGZ\AGTGAC C TTTTAAAATACTCTCIACCTTAGTTCLATTAC GACAAAAAAATCAT GATTCTGATAATA TCTTCAGAGGATGGZ\AAATCGCTGTC:CATTTTGCTGACAZ\AGCAAAATCAAATGGAGTGG TGGAAATCCGTAGTGAAGGGTGACCCTGAAGTTGATACACAGAAGGTGGAACCGGAGAAC AGCAAACTCGCTGATTTGGATCCAGAGACTCGACIIAACTGTGGAGAAGATGATGGTAAGT TGAGCAATCCTTGATCCATAAACAAI'CTGAGCTAGAGAACTTCACAAI' I'GIACAAAGCAT GGTGAGCTAAATZ TTTTTCX TCTATTGTC AGTTTGATC V CGCCAGAAGCAGATGGGTC TTCCAACAAGTGATGAAATGCAGAAGCIAAGACATGCTCAAGAAATTCATGGCTCAGGTAA TTCGCAGCAGATGATCAAACTGTGTATGCATTATTTCGTTAACCGCCTATTACGCTTCAG TTA CCTGACTGAACTTTTTTTTTGTTACAGCACCCGGAGATGGACTTCTCCAA GCAAAA

ATAGCTTAGA-ACTTATAAATCGAGAGTGACCAGCAGTA_^TGGAGAAGGTTGGAGATGCTGA TGGAGAACTACA TTCATCTCGTTCTTAGTGTCTGTCGA GGATGGTTTACGGGTAGTG TCTGTCGATGGATGGTTTACGGGTTTAGCCGTGAGGTGATTGGCCTCACGTCATTTTGTG AA TTTGTGTTCTTCAGAAACGTGAATCCGTGCTGTTTCCTGAACCTTTGAATGGATAGT AAAATCAGTTGCACCCCAGTG

Coding sequence for ROX3 (LOC_Os06g 12530; SEQ ID NO: 8)

ATGGCGATCATCTCCGACTTCCAGGAGGAAGAGGCTCCGCCGCGGCAGCAGCAGCAACCG GCTTCGGTGGCGGCGGCGGCGGGGAGCGGCGACGAGGTCCTCGCGGCGGAGCTGGAGCGG AGGGGCGGCGCGATCCCCT CCTCCAGGCGGCAATCGACGRTGRGCGAGGCGGCGGTCCGAC CTGTTCCGCGACCCCTCCGCGGTGAGCAGGGTCACATCGATGGCGTCGGCGGCGAGGGCG GTGGTGGAGGCGGAGGAGAGGAAGGCGAGGGAGGCCAAGAGGAAGGCGGAGGAGGCGGAG AGGAAGGCGGCCGAGGCCGAGAGGAAGGCGAAGGCGCCCGCGGAGCCGAAGCCGGAGAGC TCGGCGGGGAAGGATAGCATGGAGGTGGACAAGAAGGAGGAGGGCAACGTGAGGAAACCA AATGCTGGCAATGGTCTTGATTTGGAGAAGTACTCTTGGATTCAGCJATTGCCAGAGGTT ACCATCACCGTACCTGTTCCTCAAGGAACGAAG CAAGGTTTGTCGTCTGTGATATTAAG AAGAACCJITCTGAAGGTTGGGCTGAAGGGCCAGCCTCCTATCATTGATGGTGAGCTTTTC AAACCAGTCAAAGTTGATGACTGCTTCTGGAGCATAGAGGATGGAAAATCGCTGTCCATT TTGCTGACAAAGCAAAATCAAATGGAGTGGTGGAAATCCGTAGTGAAGGGTGACCCTGAA GTTGATACACAGAAGGTGGAACCGGAGAACAGCAAACTCGCTGATTTGGATCCAGAGACT CGACAAACTGTGGAGAAGATGATGTTTGATCAACGCCAGAAGCAGATGGGTCTTCCAACA AGTGATGAAATGCAGAAGCAAGACATGCTCAAGAAATTCATGGCTCAGCACCCGGAGATG GACTTCTCCAATGCAAAAATAGCTTAG

Protein sequence for ROX3 (LOC_ Os06gl2530; SEQ ID NO:9)

MAI ISDFQEEEAPPRQQQQPASVAAAAGSGDEVLAAELERRGGAI PFLQAAIDVARRRSD LFRDPSAVSRVTSMASAARAVVEAEERKAREAKRKASSAERKAAEAERKA APAE PKPES SZ GKDSMEVDKKEEGNVRKPNAGNGLDLEKYSWIQQLPEVTITVPVPQGTKSRFVVCDIK HLKVGLKGQPPIIDGELFKPV VDDCF SIEDGKSLSILL KQNQMEWWKSVVKGDPE VDTQKVEPENSKLADLDPETRQWEKMMFDQRQKQMGLPTSDEMQ QDMLKKFMAQHPEM DFS AKIA

Claims

WHAT IS CLAIMED IS: 1. A transgenic plant comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a Regulator of XA21 mediated immunity 1 (ROXl ) polypeptide, wherein the transgenic plant expresses the ROXl polypeptide at, a higher level than a plant lacking the expression cassette and wherein the transgenic plant has enhanced disease resistance compared to the plant lacking the expression cassette. 2. The transgenic plant of claim 1, wherein the polypeptide has a. sequence that is at least 90% identical to SEQ ID NO:3 , 3. The transgenic plant of claim 1, wherein the polypeptide has a. sequence comprising SEQ ID NO: 3. 4. The transgenic plant of any one of the preceding claims, wherein the promoter is tissue-specific, cell type-specific, or inducible. 5. The transgenic plant of any one of the preceding claims, wherein the plant expresses the ROXl polypeptide at a level at least 2-fold higher than the plant lacking the expression cassette. 6. A transgenic plant cell from the transgenic plant of any one of the preceding claims. 7. An expression cassette comprising a promoter operably linked to a polynucleotide encoding a Regulator of XA21 mediated immunity 1 (ROXl) polypeptide, wherein expression of the expression cassette in a plant increases the level of ROXl polypeptide expression of the plant and enhances disease resistance of the plant compared to a plant in which the expression cassette is not expressed. 8. The expression cassette of claim 7, wherein the polypeptide has a sequence that is at least 90% identical to SEQ ID NO:3. 9. The expression cassette of claim 7, wherein the polypeptide has a sequence comprising SEQ ID NO:3. 10, The expression cassette of any one of claims 7-9, wherein the promoter is tissue-specific, cell type-specific, or inducible. 11 , A method of enhancing plant stress resistance, the method comprising: introducing a nucleic acid comprising an expression cassette into one or more plants, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding a Regulator of XA21 mediated immunity 1 (ROXl ) polypeptide: and

from the one or more plants into which the nucleic acid comprising the expression cassette has been introduced, selecting a plant having increased stress resistance as compared to the resistance of a plant lacking the expression cassette. 12. The method of claim 1 1, wherein the polypeptide has a sequence that is at least 90% identical to SEQ ID NO:3. 13. ^'The method of claim I I, wherein the polypeptide has a sequence comprising SEQ ID NO: 3. 14. The method of any one of claims 11-13, wherein the promoter is tissue- specific, cell type-specific, or inducible. 15. The method of any one of claims 11-14, wherein the one or more plants into which the nucleic acid comprising the expression cassette has been introduced expresses the ROXl polypeptide at a level at least 2-fold higher than the plant lacking the expression cassette. 16. The method of any one of claims 1 1-15, wherein the stress resistance is resistance is to a pathogen. 17. A transgenic plant comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a Regulator of XA21 mediated immunity 2 (ROX2) polypeptide, wherein the transgenic plant expresses the ROX2 polypeptide at a higher level than a plant lacking the expression cassette and wherein the transgenic plant has enhanced disease resistance compared to the plant lacking the expression cassette. 18, The transgenic plant of claim 17, wherein the polypeptide has a sequence that is at least 90% identical to SEQ ID NO:6. 19, The transgenic plant of claim 17, wherein the polypeptide has a sequence comprising SEQ ID NO:6. 20. The transgenic plant of any one of claims 17-19, wherein the promoter is tissue-specific, cell type-specific, or inducible. 21. The transgenic plant of any one of claims 17-20, wherein the transgenic plant expresses the ROX2 polypeptide at a level at least 2-fold higher than the plant lacking the expression cassette. 22. A plant cell from the transgenic plant of any one of claims 17-21. 23. An expression cassette comprising a promoter operably linked to a polynucleotide encoding a Regulator of XA21 mediated immunity 2 (ROX2) polypeptide, wherein expression of the expression cassette in a plant increases the level of ROX2 polypeptide expression of the plant and enhances disease resistance of the pl ant compared to a plant in which the expression cassette is not expressed. 24, The expression cassette of claim 23, wherein the polypeptide has a sequence that is at least 90% identical to SEQ ID NO:6. 25, The expression cassette of claim 23, wherein the polypeptide has a sequence comprising SEQ ID NO:6. 26, The expression cassette of any one of claims 23-25, wherein the promoter is tissue-specific, cell type-specific, or inducible. 27, A method of enhancing plant stress resistance, the method comprising: introducing a nucleic acid comprising an expression cassette into one or more plants, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding a Regulator of X.A21 mediated immunity 2 (ROX2) polypeptide; and from the one or more plants into which the nucleic acid comprising the expression cassette has been introduced, selecting a plant having increased stress resistance as compared to the resistance of a plant lacking the expression cassette, 28. The method of claim 27, wherein the polypeptide has a sequence that is at least 90% identical to SEQ ID NO : 6. 29. The method of claim 27, wherein the polypeptide has a sequence comprising SEQ ID NO: 6. 30. ^'The method of any one of claims 27-29, wherein the promoter is tissue- specific, cell type-specific, or inducible. 31. The method of any one of claims 27-30, wherein the one or more plants into which the nucleic acid comprising the expression cassette has been introduced expresses the ROX2 polypeptide at a level at least 2-fold higher than the plant lacking the expression cassette. 32. The method of any one of claims 27-31 , wherein the stress resistance is resistance to a pathogen. 33. A genetically modified plant that expresses a reduced amount or reduced activity of Regulator of XA21 mediated immunity 3 (ROX3) compared to a wild type plant, wherein the genetically modified plant has enhanced disease resistance compared to the wild type plant. 34. The genetically modified plant of claim 33, wherein the genetically modified plant is a ROX3 knockdown plant. 35. The genetically modified plant of claim 33 or 34, wherein the reduced amount or reduced activity of ROX3 is tissue or cell type-specific. 36. A plant cell from the genetically modified plant of any one of claims 33- 35. 37. A transgenic plant comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to an RNAi polynucleotide specific for Regulator of XA2.1 mediated immunity 3 (ROX3) polynucleotide, wherein the transgenic plant expresses R0X3 polypeptide at a reduced level compared to a plant lacking the expression cassette and wherein the transgenic plant has enhanced disease resistance compared to the plant lacking the expression cassette. 38. The transgenic plant of claim 37, wherein the ROX3 polypeptide has a sequence that is at least 90% identical to SEQ ID NO:9. 39. The transgenic plant of claim 37, wherein the ROX3 polypeptide has a sequence comprising SEQ ID NO: 9. 40. ^'The transgenic plant of any one of claims 37-39, wherein the promoter is tissue-specific, ceil type-specific, or inducible. 41. The transgenic plant of any one of claims 37-40, wherein the transgenic plant expresses the ROX3 polypeptide at a. level at least 2-fold lower than the plant lacking the expression cassette. 42. A transgenic plant cell from the plant of any one of claims 37-41. 43. An expression cassette comprising a promoter operablv linked to an RNAi polynucleotide specific for Regulator of XA21 mediated immunity 3 (ROX3) polynucleotide, wherein expression of the expression cassette in a plant reduces the level of ROX3 polypeptide expression of the plant and enhances disease resistance of the plant compared to a plant in which the expression cassette is not expressed. 44. The expression cassette of claim 43, wherein the ROX3 polypeptide has a sequence that is at least 90% identical to SEQ ID NO:9. 45. The expression cassette of claim 43, wherein the polypeptide has a sequence comprising SEQ ID NO:9. 46. The expression cassette of any one of claims 43-45, wherein the promoter is tissue-specific, cell type-specific, or inducible. 47. A method of enhancing plant stress resistance, the method comprising: introducing a nucleic acid comprising an expression cassette into one or more plants, wherein the expression cassette comprises a promoter operablv linked to an RNAi polynucleotide specific for Regulator of XA2I mediated immunity 3 (R.OX3) polynucleotide; and

from the one or more plants into which the nucleic acid comprising the expression cassette has been introduced, selecting a plant having increased stress resistance as compared to the resistance of a plant lacking the expression cassette, 48. The method of claim 47, wherein the R.OX3 polypeptide has a sequence that is at least 90% identical to SEQ ID NO:9, 49. The method of claim 47, wherein the ROX3 polypeptide has a. sequence comprising SEQ ID NO: 9. 50. ^'The method of any one of claims 47-49, wherein the promoter is tissue- specific, cell type-specific, or inducible. 51. ^'The method of any one of claims 47-50, wherein the one or more plants into which the nucleic acid comprising the expression cassette has been introduced expresses the ROX3 polypeptide at a level at least 2-fold lower than the plant lacking the expression cassette. 52, The method of any one of claims 47-51, wherein the stress resistance is resistance to a pathogen.