WO2010071897A2 - Engineering heat-stable disease resistance in plants - Google Patents

Abstract

Disclosed are nucleic acid molecules which encode heat-stable plant resistance polypeptides having NB-LRR structural motifs, where the polypeptide confers heat- stability to a plant defense response. The invention further involves transgenic plants and transformed host cells that express these nucleic acid molecules and exhibit enhanced disease resistance over a wide range of temperatures.

Description

ENGINEERING HEAT-STABLE DISEASE RESISTANCE IN PLANTS

Statement as to Federally Funded Research This work was supported by grant number 2005-35100- 16044 from the United

States Department of Agriculture. The Government has certain rights in the invention.

Background of the Invention The invention relates to plant disease resistance.

Plant diseases cause crop losses that are devastating to world food supply and human well being. Plants have evolved defense mechanisms to ward off diverse biotic attacks to ensure their survival and fitness. This plant immunity occurs at multiple levels and can be largely divided into two branches (1, 31). One is a general resistance responding to molecules common to many pathogens and the other is a 'cultivar-specific' resistance responding to pathogen virulent factors. The latter resistance is induced upon a specific recognition of the pathogen race-specific avirulence (Avr) gene by the disease resistance (R) gene of the host plant (32). This 'gene-for-gene' interaction leads to rapid and efficient defense responses including a form of programmed cell death named hypersensitive response (HR) to restrict the growth of pathogens (53). A number of R genes have been molecularly cloned in many species and the largest class of R proteins contains 'nucleotide-binding' (NB) and leucine-rich repeat (LRR) domains (8, 9). A direct or indirect interaction between Avr proteins and the R proteins activates downstream signaling events leading to local HR and systemic acquired resistance throughout plants.

Plant defense responses are modulated by a number of environmental factors including light, humidity, and temperature. Temperature variations influence disease resistance to bacteria, fungi, virus, and insects (34). A high temperature often suppresses disease resistance in plants, and this phenomenon is named 'heat masking'. This heat sensitivity has been reported in a number of disease resistance responses mediated by R genes such as the JV gene in tobacco (35), Mi in tomato (6), RPW8 in Arabidopsis (36), and SNCl in Arabidopsis (7). There is accordingly a need in the art for plants having increased tolerance to temperature thereby avoiding high temperature-suppression of a resistance response. Genes conferring temperature insensitivity are consequently needed for disease resistance management at high temperatures. The present invention provides such genes and includes methods for conferring disease resistance to plants at temperatures at which defense responses are typically diminished or masked due to heat sensitivity.

Summary of the Invention It has been found that temperature modulates plant defense responses through disease resistance proteins. As is described below, through a genetic screen, a causal temperature sensitive component in plant disease resistance has been identified, and this component is a NB-LRR type of i?-like gene SNCl from a crucifer, Arabidopsis thaliana. In particular, specific mutant forms of SNCl have been demonstrated to induce heat-stable disease resistance. Demonstrating the general applicability of this discovery, similar modifications created in another R gene N were also shown to confer heat-stable disease resistance to tobacco mosaic virus. This discovery provides a methodology to engineer or breed plants or both with an ability to mount defense responses over a range of temperatures.

In general, the invention features an isolated nucleic acid molecule which includes a nucleotide sequence encoding a polypeptide including: (a) a nucleotide binding (NB) domain; and (b) a leucine rich repeat (LRR) domain, wherein the polypeptide confers a heat-stable defense response to a plant pathogen. In one example, the polypeptide includes an LRR domain that includes a sub-domain that confers the heat stable defense response. In preferred embodiments, the LRR domain that confers a heat-stable defense response to a plant pathogen includes an E/L(K or R)LD motif or an EZL(K or Y)LV(N or D) motif.

In other preferred embodiments, the nucleotide sequence is operably linked to a promoter that drives expression in a plant cell. Useful promoters include constitutive promoters, inducible promoters, or a tissue-specific promoter. In preferred embodiments, tissue-specific promoters include root-, leaf-, or seed-specific promoters. Exemplary promoters are described herein. In other preferred embodiments, the isolated nucleic acid molecule is composed, either wholly or partially, of synthetic nucleotide building blocks. In still other preferred embodiments, codon usage of the nucleotide sequence is optimized for expression in a plant.

In preferred embodiments, the expressed polypeptide confers disease resistance on a plant expressing the polypeptide at both 22°C and 28⁰C or at both 22°C and 3O⁰C. In other embodiments, the expressed polypeptide confers disease resistance on a plant expressing the polypeptide at 23⁰C, 24⁰C, 25 ⁰C, 26⁰C, 27°C, 28⁰C, 29⁰C, 30⁰C, 31°C, 32⁰C, 33°C, 34°C, 35⁰C, 36°C or a higher temperature.

In other aspects, the invention features a vector, a cell, or a plant or plant component which includes any of the aforementioned isolated nucleic acid molecules. Exemplary vectors, cells, and plants are described herein.

In still other aspects, the invention features a method of conferring resistance to a pathogen in a plant or a plant component, the method includes the steps of: (a) transforming a plant cell with an isolated nucleic acid molecule that encodes a polypeptide which includes: (i) a NB domain; and (ii) a LRR domain, wherein the polypeptide confers a heat-stable defense response to a plant pathogen; and (b) regenerating a plant or plant component from the transformed plant cell, wherein the plant or plant component exhibits resistance to the pathogen. In one example, the polypeptide includes an LRR domain that confers the heat stable defense response. In preferred embodiments, the expressed polypeptide confers disease resistance on a plant expressing said polypeptide at a wide range of temperature (such as 23°C, 24°C, 25 °C, 26⁰C, 27⁰C, 28⁰C, 29⁰C, 30°C, 3 TC, 32°C, 33°C, 34°C, 35⁰C, 36⁰C or a higher temperature).

In other preferred embodiments, the plant expressing the heat-stable NB-LRR polypeptide has an increased level of resistance compared to a corresponding control plant at an elevated temperature of the control plant.

In still other embodiments, the nucleic acid molecule is introduced through breeding or through transformation or both. Preferably, the plant is a monocot or a dicot and the pathogen is a bacterial, fungal, nematode, insect, or a viral pathogen. Exemplary plants useful in the invention include, without limitation, crucifers such as Arabidopsis thaliana, Populus trichocarpa (black cottonwood), Solanum tuberosum (potatoes), Cucumis melo (Oriental melon), Medicago sativa (alfalfa), Populus balsamifera (balsam poplar), Phaseolus vulgaris (French bean), Glycine max (soybeans), Medicago truncatula, Nicotiana tabacum (tobacco), Nicotiana glutinosa, Solanum lycopersicum (tomato), Solarium tuberosum subsp. andigena (potato), Lens culinaris (lentil), Linum usitatissimum (flax), Vitis vinifera (wine grape), Oryza sativa Indica Group (Indian rice), or ϋryza sativa Japonica Group (Japanese rice), Zea mays (corn), (barley), and (wheat). In another aspect, the invention features a substantially-pure polypeptide which includes: (a) a NB domain; and (b) a LRR domain, wherein the polypeptide confers a heat-stable defense response to a plant pathogen. Preferably, the polypeptide confers disease resistance on a plant expressing said polypeptide at both 22°C and 28°C or at both 22⁰C and 3O⁰C. In one example, the substantially-pure polypeptide includes an LRR domain that confers the heat-stable defense response. In other embodiments, the substantially-pure polypeptide confers disease resistance on a plant expressing the polypeptide at 23⁰C, 24⁰C, 25 ⁰C, 26°C, 27⁰C, 28⁰C, 29⁰C, 3O⁰C, 31⁰C, 32⁰C, 33⁰C, 34°C, 35⁰C, 36°Cor a higher temperature.

By "heat-stable defense response" is meant a NB-LRR polypeptide mediated pathogen defense response in a plant which occurs at a temperature at which the plant fails to mount the pathogen defense response in its absence. Such a temperature is higher than the temperature at which the plant mounts the pathogen defense response without the presence of the polypeptide. For example, a heat-stable defense response is preferably 2⁰C, 3⁰C, 4⁰C, or even 5⁰C higher than the temperature at which the plant fails to mount the pathogen defense response in the absence of the polypeptide; more preferably, the heat-stable defense response is 6⁰C, 7⁰C, or even 8°C higher than the temperature at which the plant fails to mount the pathogen defense response in the absence of the polypeptide; and most preferably, the heat-stable defense response is 9⁰C, 1O⁰C, or 11⁰C (or even 12⁰C or more) higher than the temperature at which the plant fails to mount the pathogen defense response in the absence of the polypeptide. By a "substantially pure polypeptide" is meant a polypeptide such as a heat- stable NB-LRR disease resistance polypeptide (for example, a SNC 1-3, SNCl E640R, N Y646K, or N Y638K N648D polypeptide or a polypeptide described in Figure 9 or Figure 10) that has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a heat-stable NB-LRR disease resistance polypeptide. A substantially pure heat-stable NB-LRR disease resistance polypeptide may be obtained, for example, by extraction from a natural source (for example, a plant cell); by expression of a recombinant nucleic acid encoding a heat-stable NB-LRR disease resistance polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By "isolated nucleic acid molecule" is meant a nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule is derived from, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

By "derived from" is meant isolated from or having the sequence of a naturally-occurring sequence (for example, a cDNA, genomic DNA, synthetic DNA, or combination thereof). By "masking temperature" is meant a temperature at which a defense response to a pathogen is ineffective to provide a plant with resistance to such pathogen.

By "polypeptide" or "protein" is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation). By "plant cell" is meant any self-propagating cell bounded by a semipermeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

By "plant component" is meant a part, segment, or organ obtained from an intact plant or plant cell. Exemplary plant components include, without limitation, somatic embryos, leaves, stems, roots, flowers, tendrils, fruits, scions, seeds, and rootstocks.

By "crucifer" is meant any plant that is classified within the Cruciferae family. The Cruciferae include many agricultural crops, including, without limitation, rape (for example, Brassica campestris and Brassica napus), broccoli, cabbage, brussel sprouts, radish, kale, Chinese kale, kohlrabi, cauliflower, turnip, rutabaga, mustard, horseradish, and Arabidopsis.

By "transgene" is meant any piece of a nucleic acid molecule (e.g., DNA) which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene having sequence identity to an endogenous gene of the organism.

By "transgenic" is meant any cell which includes an isolated nucleic acid molecule (e.g., a DNA sequence) which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic plants and the DNA (for example, a transgene) is inserted by artifice into the nuclear or plastidic genome. A transgenic plant according to the invention may contain one or more of the isolated nucleic acid molecules described herein.

By "pathogen" is meant an organism whose infection of viable plant tissue elicits a disease response in the plant tissue. Such pathogens include, without limitation, bacteria, mycoplasmas, fungi, oomycetes, insects, nematodes, viruses, and viroids. Examples of such plant diseases caused by these pathogens are described in Chapters 11-16 of Agrios, Plant Pathology, 3rd ed., Academic Press, Inc., New York, 1988. Examples of bacterial pathogens include, without limitation, Erwinia (for example, E. carotovora), Pseudomonas (for example, P. syringae), and Xanthomonas (for example, X. campepestris and X. oryzae).

Examples of fungal or fungal-like disease-causing pathogens include, without limitation, Alternaria (for example, A. brassicola and A.solanϊ), Ascochyta (for example, A. pisϊ), Botrytis (for example, B. cinerea), Cercospora (for example, C. kikuchii and C. zaea-maydis), Colletotrichum sp. (for example, C. lindemuthianum), Diplodia (for example, D. maydis), Erysiphe (for example, E. graminisf.sp. graminis and E. graminis f.sp. hordeϊ), Fusarium (for example, F. nivale and F. oxysporum, F. gramϊnearum, F. solani, F. monilforme, and F. rosewή), Gaeumanomyces (for example, G. graminis f.sp. triticϊ), Helminthosporium (for example, H. turcicum, H. carbonum, and H. maydis), Macrophomina (for example, M. phaseolina and Maganaporthe grisea), Nectria (for example, N. heamatocacca), Peronospora (for example, P. manshurica, P. tabacina), Phoma (for example, P. betae),

Phymatotrichum (for example, P. omnivorum), Phytophthora (for example, P. cinnamomi, P. cactorum, P. phaseoli, P. parasitica, P. citrophthora, P. megasperma f.sp. sojae, and P. infestans), Plasmopara (for example, P. viticola), Podosphaera (for example, P. leucotrichά), Puccinia (for example, P. sorghi, P. striiformis, P. graminis f.sp. tritici, P. asparagi, P. recondita, and P. arachidis), Puthium (for example, P. aphanidermatum), Pyrenophora (for example, P. tritici-repentens), Pyricularia (for example, P. oryzeά), Pythium (for example, P. ultimum), Rhizoctonia (for example, R. solani and R. cerealis), Scerotium (for example, S. rolfsiϊ), Sclerotinia (for example, S. sclerotiorum), Septoria (for example, S. lycopersici, S. glycines, S. nodorum and S. tritici), Thielaviopsis (for example, T. basicola), Uncinula (for example, U. necator), Venturia (for example, V. inaequalis), and Verticillium (for example, V. dahliae and V. albo-atrum).

Examples of pathogenic nematodes include, without limitation, root-knot nematodes (for example, Meloidogyne sp. such as M. incognita, M. arenaria, M. chitwoodi, M. hapla, M. javanica, M. graminocola, M. microtyla, M. graminis, and M naasϊ), cyst nematodes (for example, Heterodera sp. such as H. schachtii, H. glycines, H. sacchari, H. oryzae, H. avenae, H. cajani, H. elachista, H. goettingiana, H. graminis, H. mediterranea, H. mothi, H. sorghi, and H. zeae, or, for example, Globodera sp. such as G. rostochiensis and G. pallida), root-attacking nematodes (for example, Rotylenchulus reniformis, Tylenchuylus semipenetrans, Pratylenchus brachyurus, Radopholus citrophilus, Radopholus similis, Xiphinema americanum, Xiphinema rivesi, Paratrichodorus minor, Heterorhabditis heliothidis, and Bursaphelenchus xylophilus), and above-ground nematodes (for example, Anguina funesta, Anguina tritici, Ditylenchus dipsaci, Ditylenchus myceliphagus, and Aphenlenchoides besseyϊ).

Examples of viral pathogens include, without limitation, tobacco mosaic virus (TMV), tobacco necrosis virus (TNV), potato leaf roll virus, potato virus X, potato virus Y, tomato spotted wilt virus, and tomato ring spot virus. As discussed herein, several genes encoding heat-stable NB-LRR disease resistance polypeptides has been identified, isolated or engineered, and characterized. Accordingly, the invention provides a number of important advances and advantages for the protection of plants against pathogens. For example, the invention facilitates an effective and economical means for in-plant protection against plant pathogens at temperatures at which heat masking or sensitivity to heat occurs. Such protection against pathogens reduces or minimizes the need for traditional chemical practices (for example, application of fungicides, bactericides, nematicides, insecticides, or viricides) that are typically used by farmers for controlling the spread of plant pathogens and providing protection against disease-causing pathogens. In addition, because plants which include heat-stable NB-LRR disease resistance polypeptides are less vulnerable to pathogens and their diseases, the invention further provides for increased production efficiency, as well as for improvements in quality and yield of crop plants and ornamentals. Thus, the invention contributes to the production of high quality and high yield agricultural products, for example, fruits, ornamentals, vegetables, cereals and field crops having reduced spots, blemishes, and blotches that are caused by pathogens; agricultural products with increased shelf-life and reduced handling costs; and high quality and yield crops for agricultural (for example, cereal and field crops), industrial (for example, oilseeds), and commercial (for example, fiber crops) purposes. Furthermore, because the invention reduces the necessity for chemical protection against plant pathogens, the invention benefits the environment where the crops are grown. Genetically-improved seeds and other plant products that are produced using plants expressing the genes described herein also render farming possible in areas previously unsuitable for agricultural production due to, for example, temperature extremes and global climate change.

Constructs

As is discussed above, the invention relates to vectors, especially plasmids, and other vectors common in genetic engineering, that contain the above-described isolated nucleic acid molecules encoding a heat-stable NB-LRR polypeptide.

Preferably, such nucleic acid molecules contained in the vectors are operably linked to regulatory elements that provide for the transcription and expression of a polypeptide or protein in eukaryotic cells. Typically, the recombinant DNA molecule containing the isolated nucleic acid molecule will include in an operably linked manner, in the 5' to 3' direction of transcription, a transcription initiation control region capable of promoting, initiating and/or modulating transcription and translation in a plant cell (a promoter region), the nucleic acid molecule encoding a heat-stable NB-LRR polypeptide, and a transcription and translation termination region

(otherwise referred to as a 3' untranslated region). As used herein, a first nucleic acid sequence is "operably linked" with a second nucleic acid sequence when the sequences are so arranged that the first nucleic acid sequence affects the function of the second nucleic-acid sequence. Preferably, the two sequences are part of a single contiguous nucleic acid molecule and more preferably are adjacent. A "recombinant" nucleic acid or "recombinant DNA molecule" is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. The terms "recombinant DNA construct," "recombinant vector," "expression vector" or "expression cassette" refer to any agent such as a plasmid, cosmid, virus, BAC

(bacterial artificial chromosome), autonomously replicating sequence, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleotide sequence, derived from any source, capable of genomic integration or autonomous replication, comprising a DNA molecule in which one or more DNA sequences have been linked in a functionally operative manner.

As used herein, a "promoter" refers to a DNA sequence that binds an RNA polymerase (and often other transcription factors as well) and promotes transcription of a downstream DNA sequence. The resulting transcribed sequence can be an RNA that has function, such as rRNA (ribosomal RNA) or tRNA (transfer RNA). Often, the RNA produced is a heteronuclear (hn) RNA that has introns that are spliced out to produce an mRNA (messenger RNA). A "plant promoter" is a native or non-native promoter that is functional in plant cells. Constitutive promoters are functional in most or all tissues of a plant throughout plant development. Tissue-, organ- or cell- specific promoters are expressed only or predominantly in a particular tissue, organ, or cell type, respectively. Specific expression is used herein to refer to any promoter that provides an increased expression in a single tissue or developmental stage, or under a particular environmental condition, but causes expression, even significant expression, in other tissue(s), or developmental stage(s), or environmental condition(s).

Temporally regulated promoters are functional only or predominantly during certain periods of plant development or at certain times of day. Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example, by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, cold, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners. For the most part, any plant promoter can be used as a 5' regulatory sequence to modulate expression of a particular gene or genes, such as a plant RNA polymerase II promoter. When fused to heterologous DNA sequences, such promoters typically cause the fused sequence to be transcribed in a manner that is similar to that of the gene sequence with which the promoter is normally associated. Promoter fragments that include regulatory sequences can be added (for example, fused to the 5' end of, or inserted within, an active promoter having its own partial or complete regulatory sequences). Alternatively, heterologous regulatory sequences can be added to the 5' upstream region of an inactive, truncated promoter, e.g., a promoter including only the core TATA and, sometimes, the CCAAT sequences. Promoters include distinct cis-acting transcriptional regulatory elements, which can confer a different aspect of the overall control of gene expression. The promoter sequences of the present invention may contain cis elements that modulate gene expression. Cis elements can be part of the promoter, or can be upstream or downstream of said promoter. Cis elements (or groups thereof), acting at a distance from a promoter are often referred to as repressors or enhancers. Enhancers act to upregulate the transcriptional initiation rate of RNA polymerase at a promoter, while repressors act to decrease said rate. In some cases, the same elements can be found in a promoter and an enhancer or repressor. Cis elements are generally sites where transcription factors bind to the DNA and modulate the rate at which RNA polymerase binds to the promoter.

Examples of constitutive promoters that are active in plant cells include but are not limited to the nopaline synthase (NOS) promoters; the cauliflower mosaic virus (CaMV) 19S and 35S (sometimes called 35S herein); the tobacco mosaic virus promoter; the figwort mosaic virus promoters; and actin promoters, such as the Arabidopsis actin gene promoter.

The term "tissue-specific promoter" means a regulatory sequence that causes an enhancement of transcription from a downstream gene in specific cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues. 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, leaves or stems, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue. Reproductive tissue specific promoters may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed coat-specific, pollen-specific, petal-specific, sepal-specific, style-specific, or some combination thereof. One skilled in the art will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to expression in other tissues as well.

In a preferred embodiment of the invention, the nucleic acid molecule encoding a heat-stable NB-LRR polypeptide is operably linked to a tissue-specific promoter, or promoter that directs expression in the leaf tissue or a region therein. Other examples of possible suitable regulatory sequences include introns, 3' non-coding regions such as poly A sequences, insulator regions and the like. Molecular biological techniques for identifying, obtaining and using such regulatory elements in combination with the nucleic acid molecules of the present invention are known in the art.

Transformation Methods and Transgenic Plants or Plant Components

The present invention further relates to transgenic plant cells and transgenic plants having been transformed to contain and express a heat-stable NB-LRR polypeptide. "Transformed," "transfected," or "transgenic" refers to a cell, tissue, organ, or organism into which has been introduced a foreign nucleic acid, such as a recombinant vector. A transgenic plant according to the invention may contain one or more of the nucleic acid molecules described herein. Preferably, the introduced nucleic acid is integrated into the genomic DNA of the recipient cell, tissue, organ or organism such that the introduced nucleic acid is inherited by subsequent progeny. A "transgenic" or "transformed" cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing such a "transgenic" plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a recombinant construct or vector. The method of transformation is not critical to the current invention and various methods of plant transformation are currently known and available. For example, the introduction of DNA sequences into plants and/or plant cells can be accomplished by Agrobacterium mediated transformation, viral vector mediated transformation, electroporation, and microprojectile bombardment mediated transformation (particle gun or biolistics methods). The DNA sequence may also be transformed directly into the plastid genome by plastid transformation.

Floral dip transformation is also useful for introducing a construct into a plant. Here Agrobacterium carrying a genetic construct is applied to a flowering plant such as Arabidopsis that subsequently set seeds, and transgenic plants are then selected among the progeny seedlings according to standard methods.

Whole plants can be regenerated, for example, from single cells, callus tissue, or leaf discs transformed with a plant expression vector according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant; according to standard techniques.

This invention is applicable to dicotyledonous and monocotyledonous species and is readily applicable to new and/or improved transformation techniques. For efficient production of transgenic plants from plant cells or plant tissue, it is desirable that the plant tissue used for transformation possess a high capacity for regeneration. Techniques are known in the art to regenerate plants from transformed plant tissue cultures of plant cells.

The transgenic plants produced in accordance with this invention may, as previously stated, be any dicotyledonous or monocotyledonous species and are preferably from useful species such as maize (corn), peas, soybean, alfalfa, cassava, potato, cotton, and cereals (e.g., barley, oats, rye, triticale and wheat). Com refers to Zea Mays and all species and varieties that can be bred with it. Wheat refers to all of Triticum aestivum varieties including but not limited to spring, winter, and all facultative wheat varieties. Wheat includes any other wheat species, including but not limited to durum wheat (Triticum durum), spelt (Triticum spelta), emmer (Triticum dicoccum). and wild wheat (Triticum monococcum). Wheat also includes any species that can be bred with any of the aforementioned wheat species. Soybeans refers to Glycine max and any species or variety that can be bred with it. Rice refers to Oryza sativa and any species or variety that can be bred with it. Barley refers to Hordeum vulgare and any species or variety that can be bred with it. Oats refers to Avena sativa and any species or variety that can be bred with it. Canola is a coined name recently given to seed, oil, and meal produced by genetically modified rapeseed plants, oilseed rape (Brassica napus L.) and turnip rape (B. campestris L), herein canola includes all rapeseed plants and organisms that can be bred with them. Agrobacterium tumefaciens as used herein includes all strains and types of this species. Cotton refers to all plants in the genus Gossypium and all plants that can be bred with them. A method according to the invention comprises introducing a DNA molecule containing a promoter functional in plant cells operably linked to a nucleic acid sequence encoding a heat-stable NB-LRR polypeptide (exemplary isolated DNA molecules are described in Figures 8, 9, 10, and 11), and producing a plant (as well as fertile progeny plant of such a plant) from the transformed plant cell. Progeny includes fertile descendants of a particular plant or plant line.

The invention further relates to the production of transgenic seed. "Transgenic seed" means a plant seed whose nucleus has been altered by the incorporation substantially pure nucleic acid molecule encoding a polypeptide comprising a NB- LRR that confers a heat-stable defense response; for example a NB-LRR polypeptide that includes a domain which includes a heat- stable subdomain of the LRR, the polypeptide modulating a defense response in a plant expressing such polypeptide, e.g., by transformation as described herein. The term "transgenic plant" is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains a nucleus with the recombinant DNA in its genome.

The invention further relates to a transgenic plant component. By "plant component" is meant a part, segment, or organ obtained from an intact plant or plant cell. Exemplary plant components include, without limitation, somatic embryos, leaves, stems, roots, flowers, tendrils, fruits, scions, seeds, tubers, and rootstocks.

Other features and advantages of the invention will be apparent from the following Drawings, Detailed Description, and the Claims.

Brief Description of the Drawings

Figures 1 A-IE show that the intlO2 mutant has a temperature-insensitive defense response.

Figure IA shows the dwarf phenotype of sncl-1 at 22⁰C is suppressed by a higher temperature of 28⁰C. Shown arc 4-week-old plants.

Figure IB shows the intlO2 mutation enables sncl-1 to retain resistance to a virulent pathogen at 28⁰C. Shown is the growth of P seudomonas syringae pv tomato (Pst) DC3000 in the wild type, sncl-1, and int 102-1 sncl-1 at 22⁰C and 28⁰C.

Figure 1C shows the int 102 mutation confers a dwarf phenotype to sncl-1 at 28°C. Shown are 3-week-old plants.

Figure ID shows that the expression of defense genes is upregulated in int 102-1 sncl-1 at both 22⁰C and 28⁰C and this upregulation in intlO2-l sncl-1 is dependent on PAD4. Expression of PRl and SNCl is analyzed by RNA blot.

Figure IE shows the dwarf phenotype of int 102-1 sncl-1 at 28⁰C is suppressed by pad4 and nahG. Shown are wild type, intl 02-1 sncl-lpad4, and int 102-1 sncl-1 nahG grown at 28⁰C after bolting.

Figures 2A-2C show that the sncl-3 mutation confers temperature-insensitive activity to SNCl.

Figure 2A shows that the hypersensitive response (HR) is induced by SNCl with the sncl-3 mutation at 28°C . Four p35S: :SNC1 : GFP constructs : SNCl wild- type (WT), SNCl-I, SNCl-3 and SNCl -4 (containing both sncl-1 and sncl-3 mutations), were infiltrated via Agrobaterium (agro-infϊltrated) into Nicotiana benthamiana (Nb) leaves. Shown are leaves at 3 days after infiltration. HR is indicated by arrows. Figure 2B shows the subcellular localization of the SNCl :GFP proteins in Nb at 22°C and 28⁰C. Infiltrated leaves as described in Fig 2A were analyzed by fluorescent microscopy one day before the onset of HR. SNC 1-3 and SNC 1-4 but not SNCl-I were retained in the nucleus at 28⁰C. SNClWT had a weaker signal and a longer exposure time was used to take the image.

Figure 2C shows the subcellular localization of the SNCl :GFP proteins in Arabidopsis thaliana protoplasts. The same set of four constructs as in Fig 2A were transformed into Arabidopsis protoplasts and the GFP signals were observed at 12 hours after transformation by fluorescent microscopy. SNC IWT was exposed for a longer time than the other three samples.

Figure 2D shows a nucleus export signal (NES) abolishes the activities of SNCl-3 and SNCl-4. SNC1-3.GFP, SNC1-3:GFP:NES, SNC1-4:GFP, and SNCl- 4: GFP: NES were agro-infiltrated in Nb and their HR inducing activities were assayed at 22°C and 28°C. The left half of each leaf was infiltrated with the SNCl :GFP fusion, and the right half of the leaf was infiltrated with the corresponding SNCl :GFP fusion with NES. Shown are leaves at 3 days after infiltration. HR indicated by arrows was only observed with fusion proteins without NES. Abbreviations: WT, SNC1WT:GFP; -1, SNCl-I :GFP; -3, SNC1-3:GFP; -4, SNCl- 4:GFP; -3:NES, SNC1-3:GFP:NES; -4:NES, SNC1-4:GFP:NES.

Figure 3 A-3C show that the M0S3 and M0S6 genes are required for the SNC 1-3 protein activity.

Figure 3Λ shows that the dwarf phenotype of sncl-4 is suppressed by mos3 or mosό. Shown are 3 -week-old seedlings of the wild type, sncl-4 mos3, sncl-4 mosό, and sncl-4 at 22⁰C and 28°C.

Figures 3 B and 3 C show the subcellular localization of the SNCl-I :GFP protein at 22°C (Fig. 3A) and SNC1-3:GFP at 28°C (Fig. 3C) in wild-type (wt), mos3, mosό, andpad4 plants. SNC1WT:GFP, SNCl-I :GFP, and SNC1-3:GFP were expressed at 22°C or 28⁰C in protoplasts isolated from the mos3, mosό, and pad4 mutant plants and the GFP signals were observed at 12 hours after transformation by fluorescent microscopy. The SNCl -I and SNC 1-3 proteins were present in the nucleus in thepad4 mutant as in the wild type, but were mostly in the cytosol and plasma membrane in mos3 and mosό. Abbreviations: wt, wild type; others are the same as in Fig 2.

Figures 4A-4B show that the mutations in the N gene confer temperature- insensitive defense responses. Figure 4A shows HR induced by the WT and three mutant N genes at 22⁰C and 3O⁰C in Nt. The WT and mutant N genes were agro-infiltrated in Nt together with its elicitor p50. The WT N gene induced HR at 22⁰C but not 3O⁰C, while the Y646K, and Y646KN648D mutants induced HR at both temperatures. Figure 4B shows the localization of N-citrine in Nb at 22⁰C and 3O⁰C when co-expressed with p50. The N-citrine chimeric gene was agro-infiltrated in Nb with p50, and the citrine signal was monitored up to three days. The nuclear localization of the N-citrine protein was observed at 22⁰C but not at 3O⁰C.

Figure 5 shows that the nucleus export signal (NES) suppresses the nuclear localization of the SNC 1-3 and SNC 1-4 proteins. Shown are the localization of SNC1-3:GFP, SNC1-4:GFP, SNC1-3:GFP:NES, and SNC 1-4: GFPrNES proteins expressed in Nb at 28⁰C. While SNC1-3:GFP and SNC1-4:GFP were mostly localized to the nucleus, SNC1-3:GFP:NES and SNC1-4:GFP:NES had reduced expression in the nucleus and are also found in the cytosol and the plasma membrane. Abbreviations are the same as in Fig 2.

Figure 6 shows that the MOS3 and M0S6 genes are required for the SNC 1-3 and SNC 1-4 activities. Subcellular localization of SNC1-3:GFP at 22⁰C (A) and SNC1-4:GFP proteins at 22°C (B) and 28⁰C (C) in wild type (wt), mos3, mosό, and pad4. SNC1-3:GFP, and SNC1-4:GFP were expressed at 22⁰C and 28⁰C in protoplasts isolated from the mos3, mosό, and pad4 mutant plants, and the GFP signals were observed at 12 hours after transformation by fluorescent microscopy. The SNC 1-3 and SNC 1-4 proteins were present in the nucleus in the pad4 mutant as in the wild type, but were mostly in the cytosol and plasma membrane in mos3 and mosό at both temperatures. Abbreviations: wt, wild type; others are the same as in Fig 2.

Figure 7 shows the amino acid sequences of SNCl proteins. The TIR, NB- ARC, and LRR domains are spectively presented in italics, Bold, and double- underline. Mutations of sncl-1, sncl-3, and sncl-5 are highlighted.

Figure 8 shows a list of 500 homologous sequences of SNCl found in Arabidopsis thaliana. Genbank accession numbers are shown.

Figure 9 shows a list of SNCl homologs in species other than Arabidopsis. A list of 500 homologous sequences of SNCl found in non- Arabidopsis thaliana species. Genbank accession numbers are shown. Figure 10 shows R mutations useful for conferring heat-stable disease resistance. A list of mutations in select R proteins useful for conferring heat-stable disease resistance is shown.

Figure 11 shows an alignment of R genes and modifications to render R genes useful for conferring a heat-stable defense response in select plants.

Detailed Description Overview The plant immune system consists of multiple layers of responses to various biotic attacks, enabling a success in plant survival, fitness and reproduction (1, 2). These defense responses are modulated by abiotic environmental factors such as light, humidity, and temperature (3-7). As is mentioned above, a high temperature suppresses a number of plant defense responses rendering a normally resistant plant susceptible to pathogen attacks. This temperature sensitivity poses a challenge to agriculture and yet the molecular mechanisms underlying this temperature modulation are unknown. Below it is demonstrated that the disease resistance (R) gene known to recognize pathogen effectors (8, 9) is a temperature sensor conferring temperature sensitivity in disease resistance. Further, specific residue alterations in the SNCl and the N proteins counteract this temperature effect and sustain defense responses at high temperatures. These findings are useful to generate effective plant immune responses within a broader temperature range.

As is discussed in greater detail below, an Arabidopsis thaliana mutant sncl-1 was used as a starting strain to isolate mutants that retain enhanced disease resistance at higher temperatures, sncl-1 has a missense mutation in the NB-LRR type of R protein SNCl, as well as a constitutive defense response and a dwarf phenotype at normal growth temperature 22°C but is disease susceptible with a normal growth phenotype at a higher temperature such as 28°C. This mutant screen yielded mutants with a sncl-1 dependent growth and defense mutant phenotype at both 22⁰C and 28⁰C. One mutant identified from this screen was cloned as a new allele (sncl-4) of SNCl with an E640 mutated to K (referred as the snc 1-5 mutation) in the SNCl protein (Table 1). This sncl-3 mutation alone is sufficient to induce enhanced disease resistance at both low and high temperatures without the original sncl-1 mutation. This finding revealed for the first time that an alteration in the R protein rather than other players in plant innate immunity is sufficient to confer temperature insensitivity. Thus, the R gene is a temperature sensor in defense responses. In addition, specific mutations in the R proteins SNCl alter temperature sensitivity, making them active at both low and high temperatures. The molecular basis for this temperature sensitivity in SNCl was also examined. Temperature was found to influence the subcellular localization of the SNCl protein: the nuclear localization of SNCl correlated with its biological activity. The SNCl-I protein that is active only at 22⁰C is concentrated in the nucleus at 22⁰C but is out of the nucleus at 28°C while the SNC 1-3 protein that is active at both 22⁰C and 28⁰C stays in the nucleus at both temperatures. The addition of a nucleus export signal to the SNC 1-3 protein resulted in exclusion of the SNC 1-3 protein from the nucleus, leading to a loss of SNCl activity at 28⁰C. Thus, temperature modulation of R protein localization is the basis for temperature sensitivity of R proteins and consequently temperature sensitivity of disease resistance. These findings in the Arabidopsis SNCl have a general application for disease resistance mediated by other NB-LRR R proteins. For example, the tobacco N gene (77) mediates resistance to cauliflower mosaic virus, and this resistance is suppressed by a higher temperature of 3O⁰C. Cognate mutations of sncl-3 when introduced into the N gene enable a heat-stable resistance (Table 1), indicating a universal mechanism in temperature sensitivity of disease resistance. Although the molecular consequences induced by these mutations are not well understood, it is believed that a change of the charges from negative to positive around the E640 residue in the LRR region alters the conformation of the R protein or its interaction with other proteins. By mutating E640 to R in SNCl a heat-stable HR (Table 1) was obtained, supporting this hypothesis.

The details of the aforementioned experiments now follow.

Experimental

A genetic screen in sncl-1 (10) for mutants defective in high temperature suppression of defense responses was carried out. The wild-type Arabidopsis plants do not have constitutive defense responses as these responses usually compromise plant growth and sometimes cause cell death. The Arabidopsis sncl-1 mutant has a constitutive defense response and a dwarf phenotype under normal growth temperature 22°C due to a missense mutation in the linker region of SNCl (10), and both phenotypes are suppressed by a higher growth temperature such as 28°C (7) (Fig IA, B). One such mutant isolated, int (insensitive to temperature) 102-1, has a sncl-1 dependent dwarf phenotype at both 22⁰C and 28⁰C (Fig 1C). The int 102-1 sncl-1 mutant is as resistant to Pseudomonas Syringae pv. tomato (Pst) DC3000 as sncl-1 at 22⁰C, but retains resistance at 28°C in contrast to sncl-1 (Fig IB). Therefore, the intlO2-lsncl-l mutant indeed has a temperature-insensitive (heat-stable) constitutive defense response. Consistently, the expressions of PRl and SNCl, two genes responsive to an increase of salicylic acid (SA) associated with enhanced disease resistance (7, 12), were higher in intlO2-lsncl-l than in the wild type or sncl-1 at

28°C (Fig ID). Both the defense and the growth phenotypes of intl 02-1 mcl-1 can be suppressed by nahG (13) coding a SA degradation enzyme and by pad4 (14) defective in defense responses (Fig ID, E, and data not shown), further demonstrating an upregulation of defense responses in the mtl 02-1 sncl-1 mutant at 28°C. We cloned the INTl 02 gene based on its tight linkage to SNCl in a mapping population (online methods). Sequencing the SNCl gene in intlO2-lsncl-l revealed a G to A point mutation causing a change of glutamic acid to lysine (named as sncl- 3) at amino acid residue 640 in the LRR domain of SNCl (Table 1).

Table 1. Mutations isolated or generated in the SNC1 and the N proteins.

Genes starting residue # Sequences HR at high temperatures

SNC1 WT 639 EELD no

SNC1-3 EKLD yes

SNC1 E640R ERLD yes

N WT 637 EYVN no

N Y646K EKVN yes, weak

N N648D EYVD yes

N Y646K N648D EKVD yes

Shown are the aligned regions of SNC1 and N proteins with mutated residues underlines The wild-type and the mutant genes were tested for HR-inducing activities at 28⁰C (for SNC1) and 3O⁰C (for N)

The int!02-lsncl-l mutant is therefore named sncl-4, and it contains both the sncl-1 and sncl-3 mutations. To confirm that the sncl-3 mutation is responsible for the temperature-insensitive disease resistance phenotype, we generated transgenic plants carrying the genomic fragments of either the wild-type or the mutant SNC 1-4 genes in the wild-type CoI-O plants. However, transgenic plants carrying the wild- type SNCl gene exhibited a dwarf phenotype similar to those carrying the SNCl -4 mutant gene (data not shown), and this auto-activation of the SNCl as a transgene is likely due to a loss of transcriptional repression imposed at its endogenous chromosomal location (15). We subsequently developed a transient assay for the SNCl gene activity in Nicotiana benthamiana (Nb) by the hypersensitive response (HR), a form of programmed cell death induced by the activation of R proteins. The SNCl protein was tagged by the green fluorescent protein (GFP) at the carboxyl- terminus and expressed under the strong CaMV 35 S promoter. This p35S::SNCl:GFP fusion gene was expressed in Nb leaves by Agrobacterium- mediated infiltration (agro-infiltration), and HR symptoms such as collapsed leaves was monitored at 22⁰C and 28⁰C respectively over a period of 3 days after infiltration. In contrast to the wild-type SNCl (SNCl WT) gene that did not cause any HR, the SNCl-I mutant gene induced HR at 22⁰C but not at 28⁰C while the SNCl-3 and SNCl-4 genes each induced HR at both temperatures (Fig 2A). Thus, sncl-3 is indeed the mutation responsible for the intlO2-lsncl-l phenotype. Furthermore, the sncl-3 mutation alone (without sncl-1) is sufficient to induce temperature-insensitive defense responses. This result indicates that temperature sensitivity of disease resistance is controlled by the R gene rather than other regulatory components and that a mutation in an NB-LRR type of R gene is sufficient to confer temperature- insensitive disease resistance. To further investigate the mechanism underlying temperature sensitivity of defense responses, we carried out a suppressor screen in the heat-stable sncl-4 mutant background. Mutants that regained high-temperature inhibition of defense responses were isolated and named rit (revertant ofint). One such mutant ritl sncl-4 had a sncl- 1 like phenotype: dwarf at 22⁰C but wild-type like at 28⁰C suggesting a regaining of temperature sensitivity. Correlated with the growth defect, the ritlsncl-4 has enhanced disease resistance to virulent pathogen Pst DC3000 at 22⁰C and this elevated defense is suppressed at 28⁰C. At 22⁰C, Pst DC3000 had a similar growth reduction in ritlsncl-4 as in sncl-1 and sncl-4 compared to the wild-type Col. At 28⁰C, sncl-4 exhibited an inhibition of bacterial growth to a similar extent as at 22°C. In contrast, ritl sncl-4 lost the inhibition of bacterial growth at 28⁰C and supported bacterial growth to a similar extent as sncl-1 and the wild type. Therefore ritl indeed reverses the heat-stable resistance phenotype to the heat-sensitive phenotype. We found that ritl is an intragenic suppressor of sncl-4. There is no phenotypic segregation in the F2 progenies of a cross of ritl sncl-4 with wild-type Col or Ler grown at 28⁰C indicating that the ritl is very closely linked to the SNCl gene. Sequencing the entire SNCl genomic fragment in the ritl sncl-4 mutant identified a G to A point mutation resulting in a serine substitution of glycine at amino acid residue 380 (Fig. 7). We named this G380S mutation sncl-5 and this new allele with sncl-1, sncl-3, and sncl-5 mutations as sncl-6 (Fig. 3C). This glycine residue resides immediately after the putative GxP or GLPL motif in the NB-ARC domain. This motif was previously identified as important for nucleotide binding and mutations in residues close to the motif might compromise activation of NB-LRR proteins (30).

A second intragenic suppressor named rit4 was identified from the same rit screen. This mutant was independent of ritl as it was isolated from a different mutagenesis pool and had an additional phenotype unrelated to defense. Interestingly, we found the same G to A alteration resulting in a G380S mutation as in rit4. That two independent but identical mutations result in the same rit phenotype confirms that sncl-5 is indeed the mutation responsible for reverting the temperature insensitivity of sncl-4.

This conclusion is further supported by the SNCl -6 activity in the Nb transient expression system. The sncl-5 mutation was introduced into the p35S::SNCl-4:GFP construct to create p35S::SNCl-6:GFP. While SNCl-4 induced HR in Nb at both 22°C and 28°C, SNCl -6 induced HR only at 22°C but not at 28°C. Thus sncl-5 mutation appears to be a suppressor of the heat-stable SNC 1-4 activity specifically at 28°C and it does not significantly suppress SNCl-4 activity at 22⁰C. This notion is further supported by the failure of inhibiting the SNCl-I 22C activity with the sncl-5 mutation at 22°C (data not shown).

With the identification of different forms of SNCl conferring defense responses of different temperature sensitivity, we conclude that the NB-LRR gene SNCl is the temperature sensitive component causing temperature sensitivity of the whole defense responses it induces. An elevated temperature inhibits plant immunity probably through the very early component of the signaling pathways that is the NB-LRR genes.

We further investigated the molecular basis underlying the temperature sensitivity of the NB-LRR type of R gene SNCl. A correlation was observed between the nuclear localization of the SNCl protein and its HR-inducing activity in Nb at both temperatures. A very weak GFP signal was detected in the cytosol and the plasma membrane for the SNCl WT:GFP fusion protein (Fig 2B). The SNC1-1:GFP mutant fusion protein was localized to the nucleus at 22⁰C (Fig 2B), confirmed by nucleus staining with 4',6-diamidino-2-phenylindole (DAPI) (data not shown), but it was found in the cytosol and the plasma membrane at 28⁰C (Fig 2B). In contrast, both SNC1-3:GFP and SNC1-4:GFP were localized to the nucleus at both 22⁰C and 28⁰C (Fig 2B). The localization of these SNCl proteins was confirmed in a transient expression system in Arabidopsis thaliana. When expressed at 22⁰C in Arabidopsis protoplasts, SNCl-I :GFP, SNC1-3:GFP, and SNC1-4:GFP, but not SNC1WT:GFP, were localized predominantly in the nucleus (Fig 2C). At 28⁰C, only SNC1-3:GFP and SNC1-4:GFP, but not SNCl WT:GFP or SNCl-IrGFP, were found in the nucleus (Fig 3B, 3C, Fig 6). It appears that temperature influences the localization of the SNCl protein and the sncl-3 mutation causes a nuclear localization of SNCl even at a higher temperature. To determine whether the nuclear localization of SNCl is the cause or the consequence of the enhanced defense responses at elevated temperatures, we added a nucleus export signal (NES) (16) to the SNCl : GFP fusions and expressed these constructs in Nb leaves. The addition of NES largely reduced the nuclear localization of SNC1-3:GFP and SNC1-4:GFP at 28⁰C, resulting in their expression in cytosol and plamsma membrane as well (Fig 5). Neither SNC1-3:GFP:NES nor SNCl-

4:GFP:NES induced HR in Nb leaves, in contrast to SNC1-3:GFP and SNC1-4:GFP (Fig 2D). Thus, nuclear localization is critical for the mutant SNCl proteins to induce defense responses at a higher temperature. This finding is consistent with the emerging finding that nuclear localization of some R proteins is essential for them to confer disease resistance (17, 18).

Nuclear-cytosyl partition of proteins has critical roles for diverse plant developmental signaling and environmental responses, and a few genes involved in the nuclear-cytosyl shuttling processes are shown to be required for defense responses (19, 20). For instance, mutations in M0S3 (encoding a putative nucleoporin Nup96) or M0S6 (encoding a putative importin α3) could suppress enhanced disease resistance in sncl-1 (21, 22), although the exact mechanisms are not well understood. We tested whether genes involved in nuclear-cytosol transport are essential for defense responses induced by the SNCl mutant protein at high temperatures. The mosS and mosό mutations were each introduced into the sncl-4 mutant, and both the sncl-4 mos3 and the sncl-4 mosό double mutants exhibited a wild-type growth phenotype at 22°C and 28⁰C (Fig 3A), indicating a requirement of functional MOS3 and MOS6 for the SNCl-4 activities at both temperatures. To determine whether M0S3 and MOS6 regulate SNC 1 -mediated defense response through SNC 1 localization, we expressed different forms of p35S::SNCl:GFP in protoplasts isolated from the wild-type, mos3, and mosό plants. The nuclear localization of SNCl-I at 22⁰C in the wild type was largely suppressed by the mos3 and mosό mutations (Fig 3B), indicating that M0S3 and M0S6 likely regulate sncl-1 mediated defense through the localization of the SNCl-I protein. Similarly, nuclear localization of the SNC 1-3 and SNC 1-4 proteins at 22⁰C and 28°C in the wild type were greatly reduced in the mos3 and mosό mutants, and the SNC 1-3 and SNC 1-4 proteins had enhanced cytosol and plasma membrane localization in the two mutants (Fig 3C, and Fig 6). Thus, the activities of SNC 1-3 and SNC 1-4 at a high temperature requires the two genes involved in nuclear-cytosyl transport. In contrast to the mos3 and mosό mutations, the loss-of-function mutation of PAD4 that functions downstream of R protein activation did not alter the nuclear localization of the SNCl mutant proteins although it suppresses the sncl mutant phenotype similarly to mos3 and mosό (Fig IE, 3B, 3C, and Fig 6). These data indicate that M0S3 and MOS6 mediate SNCl- induced defense responses via regulating the R protein SNCl . It further demonstrates that nuclear localization of the SNCl protein at high temperature is the cause rather than the consequence of heat-stable defense responses.

To determine whether the finding on temperature sensitivity in defense responses induced by the Arabidopsis SNCl is a general phenomenon for disease resistance, we introduced similar mutations of sncl -3 to a tobacco R gene N that confers resistance to tobacco mosaic virus (TMV). It is well known but not understood how the N-containing plants lose resistance at a higher temperature of 3O⁰C (11). We altered two residues in the Ν protein by site-directed mutagenesis: one is the Y646K mutation corresponding to the sncl-3 mutation (E640K) of SΝC1 and the other is the Ν640D mutation corresponding to D642 of SNC 1 (Table 1). When co-expressed with its elicitor p50 in Nicotiana tabacum (Nb) by agro-infiltration, the wild-type N gene triggered HR at 22°C but not at 3O⁰C (Fig 4A, Table 1). In contrast, the three mutant N genes with Y646K, N648D, or Y646K N648D mutations induced HR in Nb at both 22⁰C and 30⁰C together with the N elicitor p50 (Fig 4A, Table 1). These mutations do not appear to confer constitutive auto-activities because they did not cause HR in the absence of p50 (data not shown). Thus, the N gene is responsible for the temperature sensitivity of TMV resistance, indicating that other NB-LRR type of R genes function as temperature sensors in disease resistance and that temperature sensitivity can be altered by specific mutations in the R proteins to make heat-stable disease resistance.

We determined whether temperature sensitivity of the N gene is correlated with the N protein localization similar to that of the 57VCi gene. When expressed together with p50 in N. benthamiana, the N-citrine fusion protein was localized to the nucleus at 22⁰C (Fig 4B), consistent with previous findings (18). However, no signal could be detected in the nucleus when plants were incubated at 3O⁰C (Fig 4B). This indicates that nuclear localization of activated wild-type R protein(s) is subject to temperature modulation, similarly to that of the active form of the mutant R protein such as SNCl-I.

The molecular basis of temperature-insensitive mutants of R proteins is not totally understood. This is not merely due to an over-activation of R proteins because the SNCl-I mutant protein apparently has the same activity as SNC 1-4 at 22⁰C and yet has no activity at 28⁰C. The E640K mutation in SNCl can potentially induce local post-translational protein modification such as ubiquitination, sumoylation, acetylation. and biotylation. Especially the 'EKID' sequence around sncl-3 and intriguingly the 'LKIG' sequences around sncl-1 are predicted to be sumoylation sites (http://www.abgent.com/tools/sumoplot_login). However, the N648D alone in N without the Y646K mutation is sufficient to induce HR at high temperatures, indicating that sumoylation is not the basis or the only basis for the activity. We therefore tested whether a charge change rather than a local protein modification is responsible for temperature insensitivity by introducing an E640R mutation to SNCl (Table 1). Similar to SNC1-3:GFP, SNC1E64OR:GFP induced HR at 28°C and was localized to the nucleus when expressed in Nb (data not shown). Both E to K and E to R mutations add positive charges to the region and could potentially generate a local nuclear localization signal (23). To test whether these mutations generate such a signal or are sufficient to induce nuclear localization, we created shorter versions of the SNCl :GFP proteins consisting of the linker region and the LRR domain of the SNCl protein. Neither the sncl-3 nor the sncl-1 mutations conferred a nuclear localization to the shorter SNChGFP fusion proteins (data not shown), indicating that these mutations do not function autonomously to induce SNCl nuclear localization. The R protein activation involves alteration of intra-molecular interactions, with opening of the NB domain and possibly allowing interaction of the amino- terminal domain with downstream signaling molecules (24, 25). It is possible that a high temperature can influence the intra-molecular interactions and keep the R protein in an inactive closed form. A protein 3-D structure modeling suggested that the SNCl E640 and a few nearby residues including D642 could form a more negatively charged pocket and this pocket might have a direct contact with positively charged residue(s) in the ARC domain. The E640K/R mutations could disrupt the interaction between the LRR domain and the ARC domain, leading to an active conformation of the SNCl protein at a high temperature. Alternatively, these mutations could alter the interaction of SNCl with downstream signaling molecules, leading to an active signaling event at high temperatures.

The above-described experimental results were obtained using the following materials and according to the following methods.

Material and Methods The Arabidopsis thaliana plants were grown on soil at 22°C or 28°C under constant light for morphological phenotypic analysis, and under a photoperiod of 12 hour (hr) light/12 hr dark for disease resistance test. Arabidopsis seedlings used for protoplast transformation were grown on solid medium under a photoperiod of 8 hr light/ 16 hr dark. The Nicotiana tabacum (Nt) and Nicotiana bethamiana (Nb) plants were grown in the greenhouse. For chemical mutagenesis, the sncl-1 seeds were treated with 0.25% EMS (ethane methyl sulfonate) for 12 hours. For cloning the INT102 gene, the intlO2-lsncl-l mutant in CoI-O was crossed to the Ws-2 wild type and standard mapping method (26) was used to identify the intervals containing the INT102 gene. The p35S::SNCl :GFP construct consists of the strong 35S CaMV promoter, the genomic fragment of the SNCl coding region (from the translation start site to the last amino acid), and the green fluorescent protein (GFP) fused in frame to the carboxyl -terminus of SNCl . Site-directed mutagenesis was carried out with 'QuikChange' kit according to manufacture's instruction (Stratagene). The Arabidopsis protoplasts were generated from plate-grown seedlings according to methods by Zhai and Vatamaniuk (in press). RNA blot analysis, pathogen resistance test, and transient expression in Nb and Nt was performed as described (7, 27, 28).

Plant material and growth condition. The Arabidopsis thaliana plants were grown on soil at 22⁰C or 28°C under constant light with a relative humidity between 40% and 60% for morphological phenotypic and gene expression analysis. Arabidopsis seedlings used for protoplast transformation were grown on solid medium with 1/2 MS salts, 2% sucrose, and 0.8% agar and under a photoperiod of 8 hr light/ 16 hr dark. Nicotiana tabacum (Nt) and Nicotiana bethamiana (Nb) plants were grown in the greenhouse.

Mutant screen. The sncl-1 seeds were treated with 0.25% EMS (ethane methyl sulfonate) for 12 hours. Approximately 40,000 M2 plants (derived from 4,000 Ml) were screened at 28⁰C for the sncl-1 like dwarf phenotype. Putative mutants were then crossed to the wild-type Col-0, and the F₂ population was analyzed to determine whether they arc sncl-1 dependent or not.

Map-based cloning. The mapping population was created by crossing the int 102-1 mutant in the CoI-O accession to the wild type of the Ws-2 accession. Dwarf plants among the F₂ progenies grown at 28⁰C were chosen for mapping. Bulked segregation analysis was preformed on pools of 40 plants with SSLP, CAPS, and dCAPS markers between CoI-O and Ws-2 (26). The INT 102 gene was found to be linked to marker VRN2 that is 1 cM from the SNCl gene. Further analysis of 240 dwarf plants revealed no recombination between INT 102 and SNCl, indicating a tight linkage of the two genes.

Generation of constructs. A 5.5 kb fragment containing the SNCl coding region and the 3'UTR was isolated from the BAC clone F5D3 from ABRC. A

BamHI restriction site was added to this genomic fragment before the stop codon of SNCl via polymerase chain reaction (PCR). The NcoI-BamHI fragment containing the SNCl coding region without the stop codon was inserted into pSAT-Nl vector (29) to generate the p35S: .SNCl :GFP construct. The SNCl-I, SNC 1-3, and SNCl-4 mutations were introduced into p35S::SNCl :GFP through site-directed mutagenesis with the 'QuikChange' kit according to manufacture's instruction (Stratagene). The cassettes of various p35S:. SNCLGFP were excised at the PI-PspI sites and cloned into the pHPT binary vector (29) to generate pHPT-SNCl, pHPT-SNCl-l,pHPT- SNCl -3, andpHPT-SNCl-4 constructs.

Transient expression in Nb and Nt. Each of these above binary vectors was then transformed into Agrobαcterium tumefαciens stain C58C1 (30). Cultures of the transformed A. tumefαciens were grown overnight to OD₆₀O of 1.0 in liquid LB. Cells were then collected by centrifugation and resuspended in the induction medium (10 mM MES, pH5.7, 10 mJM MgCl₂, 200 μM acetosyringone) to OD₆₀₀ of 0.5. After sitting at room temperature for 3 hrs, the Agrobacterial cells were infiltrated into the abaxial surface of Nb or Nt leaves using 1 ml needleless syringes. Infiltrated plants were subsequently incubated at 22⁰C or 28⁰C before infiltrated leaves were examined for GFP signals under a microscope (model BX61 , Olympus) within a 48 hr period after inoculation.

RNA analysis. Total RNAs were extracted using Tri Reagent (Molecular Research, Cincinnati, OH) from leaves of 3-week-old plants. Twenty micrograms of total RNAs per sample were used for RNA gel blot analysis. Pathogen resistance assay. P.syringαe pv. tomato DC3000 was grown overnight in the KB medium and resuspended at 10⁸ ml in a solution of 1 OmM MgCl₂ and 0.02% Silwet L-77. Two- week-old seedlings were dip inoculated with bacteria and kept covered for Ih. The amount of bacteria in plants was analyzed at Ih after dipping (day 0) and 3 days after dipping (day 3). The aerial parts of three inoculated seedlings were pooled for each sample and three samples were collected for each genotype at one time point. Seedlings were ground in 1 ml of 10 mM MgCl₂ and serial dilutions of ground tissue were used to determine the number of cfu per miligram of leaf tissues.

Engineering Heat-Stable Disease Resistance

As is discussed above, the invention involves engineering heat-stable disease resistance in plants where a heat-labile race-specific disease resistance is normally found. In general, mutant forms of an NB-LRR type of R protein that are responsible for this race-specific resistance are generated. Alternatively, mutant forms in natural populations are identified and isolated according to standard methods, followed by introducing such isolated R variants into plants, rendering such plants disease resistant against one or more pathogens at a temperature which would typically mask such a defense repsonse. Such R variants are useful to modify temperature sensitivity in disease resistance. Furthermore, R proteins may be altered at specific sites to generate an R protein which not only mediates disease resistance but one that is also heat-stable rendering a plant resistant to disease above its heat masking temperature. As is also discussed above, the invention features methods for conferring disease resistance to a plant at a temperature at which such a disease response is suppressed, thereby attenuating heat masking. Such methods include, without limitation, overexxpression or ectopic expression a heat-stable NB-LRR disease resistance polypeptide (such as SNC 1-3, SNCl E640R, N Y646K, N648D or N Y738K N648D or any polypeptide described in Figure 9 and Figure 10).

To test the ability of a construct described herein to confer disease resistance to a pathogen, transgenic plants expressing such construct are planted and disease resistance is compared to non-transformed plants using techniques familiar to those of skill in the art at masking temperatures. Plants expressing a heat-stable NB-LRR polypeptide have an "increased level of resistance" as compared to a control plant at the "masking temperature" of the control plant, and are taken as being useful in the invention. Plants expressing a heat-stable NB-LRR polypeptide confer a heat-stable defense response to a plant pathogen, which is monitored and assessed according to standard methods known in the art and described herein. By "increased level of resistance" is meant a level of resistance to a disease-causing pathogen in a non- naturally occurring plant (or cell or seed thereof) which is greater than the level of resistance in a control plant (for example, a non-transgenic plant or wild-type or a transgenic plant or a wild-type plant with a heat-labile NB-LRR polypeptide). In preferred embodiments, the level of resistance in a non-naturally occurring transgenic plant of the invention is at least 5% to 20% (and preferably 30% or 40%) greater than the resistance exhibited by a control plant. In other preferred embodiments, the level of resistance to a disease-causing pathogen is 50% greater, 60% greater, and more preferably even 75% or 90% greater than a control plant; with up to 100% or greater above the level of resistance as compared to a control plant being most preferred. For example, a disease resistance response may be measured at 23⁰C, 24⁰C, 25 ⁰C, 26⁰C, 27⁰C, 28⁰C, 29⁰C, 3O⁰C, 31⁰C, 32⁰C, 33⁰C, 34⁰C, 35⁰C, or greater relative to the masking temperature of a control plant which does not express a heat-stable NB-LRR disease resistance polypeptide. The level of resistance is measured using conventional methods such as those described herein. For example, the level of resistance to a pathogen may be determined by comparing physical features and characteristics (for example, plant height and weight) or by comparing disease symptoms (for example, delayed lesion development, reduced lesion size, leaf wilting and curling, water-soaked spots, amount of pathogen growth, and discoloration of cells) of the non-naturally occurring plant (e.g., a transgenic plant).

In one example, transgenic tobacco plants were engineered to express a heat- stable defense response by modifying N to confer a heat-stable disease resistance. Tobacco plants transformed with N genes with Y646K or Y646KN648D modifications were generated. Plants were infected with tobacco mosaic virus at 3O⁰C for seven (7) days. After 7 days, plants were shifted to 22°C and analyzed for the spread of TMV occurred at 30⁰C. Transgenic tobacco expressing N genes with Y646K or Y646KN648D were found to be resistant to TMV as compared to control tobacco plants expressing the wild-type N gene at 3O⁰C. The invention also involves rendering heat-labile NB-LRR resistance genes heat-stable. This generally involves (1) identification of a heat-labile NB-LRR type of R gene, (2) Alignment of the protein sequence of Rs to those of SNCl and N to identify LRR domain with identity to the heat-stability domain of SNCl ; (3) Introduction of cognate mutations of snc 1-3 into the Rs gene by site-directed mutagenesis; and (4) Assaying for temperature sensitivity.

1) Identification of the NB-LRR type of R gene responsible for resistance to a particular pathogen.

For heat-labile disease resistance, an R gene (referred to here as Rs) responsible for this resistance is identified. This is accomplished in a number of ways known in the art, for example, by map-based cloning exploring natural variations among different accessions of a plant species. Additional Rs genes are available in the art (See Figures 8 and 9). Rs genes may also be identified in databases (See Figures 8 and 9). Cloning and isolation of Rs is accomplished using standard methods known in the art. 2) Align the protein sequence of Rs to those of SNCl and N and identify the corresponding residue of E640 of SNCl.

Most sequence analysis programs have a function for protein sequence alignment and can all be used for this purpose. For instance, NCBI has 'Blast 2' for aligning two sequences and Vector NTI has an alignment function for two or more sequences. NB-LRR proteins have CC or TIR domains at the N-terminus, ARC- linker domains in the middle, and LRR domains at the C-terminus. The ARC-linker and LRR domains usually have high sequence similarities among different R proteins and are aligned easily.

3) Introduction of cognate mutations oisncl-3 into the Rs gene by site- directed mutagenesis.

The corresponding region of SNCl 640ELD in the Rs protein is altered into KLD. This is achieved by commercially available site-directed mutagenesis kits available from a number of companies. For instance, 'Quick-change' from Invitrogen uses a complementary pair of primers with desired mutations a polymerase-chair reaction (PCR) to replace the wild-type sequences. The mutated Rs genes are sequenced to confirm that the desired mutations but not others are indeed introduced into the Rs protein. 4) Test the temperature sensitivity of the mutant Rs gene.

For those Rs genes where a transient assay for their activities are available (as for N and SNCl), the assay can be used to determine whether the mutant Rs gene confers disease resistance at a high temperature. For those Rs genes without a transient assay, procedures in step 6) are used. Such an assay involves co-infiltrating the pathogen elicitor gene and the plant Rs gene into Nicotiana benthamiana through agrobacterium and monitoring hypersensitive responses (HR) at different temperatures. If the mutant Rs gene induces HR at high temperature while the wild- type Rs does not, the mutant gene indeed harbors the heat-stable property. If no such activity is achieved, a screen for such a mutation is carried out as described below. 5) Isolate heat-stable mutations in the Rs gene

Random-mutagenesis, for example, is also used to create a mutant library of the Rs gene. This is achieved according to standard techniques, for example, through error-prone PCR or error-prone replication using XIl -red cells. The DNA fragment coding for the linker and the LRR domains is subject to such mutagenesis so that every fragment will have 0.1-0.5% of the nucleotides altered. These mutant fragments are ligated with the rest of the Rs gene to reconstitute the full-length Rs genes. Other regions of an Rs gene may be mutagenized these include the CC or TIR domains at the N-terminusand ARC-linker domain. Any region of an Rs gene, in general, may be mutagenized to generate a library of mutants useful for analysis. A library consisting of these mutated Rs genes is then screened to identify mutants that have HR causing activity at high temperatures described in step 4 above according to standard methods. 6) If a transient assay for Rs gene activity is not available, the mutant Rs gene or the library of mutant Rs gene is transformed into disease susceptible plants and transgenic plants are assayed for disease resistance at elevated temperature. Those with heat-stable resistance are identified and selected according to the methods described herein. The invention also features the following:

1) Breed plants with heat-stable resistance

Variations in a plant species (either wild varieties or cultivars) provide resources for R gene variations. NB-LRR genes are the most rapidly evolved compared to other genes in Arabidopsis (37) and this is likely true in other plant species. It is possible that one particular variety has already evolved to harbor heat- stable disease resistance by evolving a mutation in the Rs gene. The invention accordingly provides a quick screen for such an R variant to assist plant breeding.

PCR is then used to amplify the linker and LRR regions of the Rs gene from different plant varieties. Rs genes with positively charged residues (K, R, or H) at the position corresponding to 640E in SNCl are good candidate for variant conferring heat-stable resistance. These varieties can be tested directly for disease resistance at high temperatures.

Once the heat-stable resistance conferred by an Rs variant is found, this Rs variant are introduced into crops by crossing the two varieties and isolating those with most of the elite traits together with the Rs variant in the F2 population with marker assisted selection.

Other examples of engineering a heat-stable defense response in crop plants include the following. To provide a heat-stable defense response in barley to combat powdery mildew caused by Blumeria graminis, R genes such as Mlal and Mla6 are modified to express polypeptides (as compared to wild type R polypeptides) having Y613K G615D and V636K, respectively. In maize, R genes such as RpI-D and Rp3 are respectively modified compared to wild type R polypeptides as follows: A676K E678D and A613K H615D to engineer a resistance polypeptide that combats leaf rust caused by Puccinia sorghi. Rice plants expressing a modified NB-LRR polypeptide (XaI : E1086K El 088D) are useful for providing resistance to bacterial blight caused by Xanthamonas oryzae in a heat-stable defense response. Modification of the rice resistance gene Pi-b to express a NB-LRR polypeptide having Yl 130K Sl 132D compared to wild type provides resistance to rice blast caused by Magnaporthe grisea. Leaf rust in wheat caused by Puccinia triticina may be combated in a heat- stable defense response by modification of the Lr21 protein as follows: M967K N969D. Powdery mildew in wheat caused by caused by Blumeria graminis is addressed by expression of a Pm3 polypeptide mutated as follows: V628K. Phytophthora infestans is a fungal pathogen that causes late blight in potato.

Modification of a potato R gene in a transgenic potato, such as Rl (Q 1203K) is useful for providing resistance to late blight in potato. In another example, transgenic tomato plants expressing Cf-2 (F243K F245D) are useful for providing resistance to leaf mould caused by Cladosporium fulvum. Figure 11 summarizes the aforementioned modifications to wild type polypeptides to confer resistance in a variety of plants to various pathogens. Figure 11 also shows alignment of R genes with SNCl.

Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the claims.

Other Embodiments

The invention provides methods of identifying, isolating, and using a nucleic acid molecule which encodes a NB-LRR disease resistant polypeptide which includes a heat- stable subdomain of the LRR.

A polypeptide that includes (a) a nucleotide binding (NB) domain and (b) a leucine rich repeat (LRR) domain, wherein the LRR domain includes a sub-domain that confers a heat-stable defense response to a plant pathogen includes variant polypeptides derived from a particular NB-LRR polypeptide by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C- terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the NB-LRR polypeptide. Such variants may result from, for example, from human manipulation. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the art. Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred.

Thus, the NB-LRR polypeptide genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the NB-LRR polypeptides encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired activity. The deletions, insertions, and substitutions of the NB-LRR polypeptide sequence encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays such as those described herein.

The nucleic acid molecules are optimized for enhanced expression in plants. In this manner, the genes or gene fragments can be synthesized utilizing plant- preferred codons according to methods well known in the art. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used. Variant nucleotide sequences and proteins also encompass sequences and protein derived from a mutagenic and recombinant procedure such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create a new polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Strategies for such DNA shuffling are known in the art.

By "variants" is intended substantially similar sequences. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the reference protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis which encode the reference protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention will have at least 40%, 50%, 60%, preferably 70%, more preferably 80%, even more preferably 90%, most preferably 99%, and single unit percentage identity to the native nucleotide sequence based on these classes. For example, 40%, 41%, 42% and the like, up to at least the 90% class. Variants may also include a full length gene corresponding to an identified gene fragment.

Nucleic acid molecules which are substantially identical to any of the nucleic acid molecules described herein which encode NB-LRR polypeptides having a heat- stable LRR subdomain are included within the scope of the invention (for example, a SNCl-3, SNCl E640R, N Y646K, , or N Y646K N648D polypeptide or any of the polypeptides described in Figure 10).

By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 40-50%, preferably 85%, more preferably 90%, and most preferably 95% homology to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides. Generally, nucleotide sequences will again have at least 40%, 50%, 60%, preferably 70%, more preferably 80%, even more preferably 90%, most preferably 99%, and single unit percentage identity to the NB-LRR nucleotide sequence (or polypeptide) based on these classes. For example, 20%, 25%, 30%, 35%, 40%, 41%, 42% and the like, up to at least the 90% class. Sequence identity is typically measured using sequence analysis software

(e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Plant cells useful in the invention as sources of nucleic acid molecules encoding heat-stable NB-LRR polypetides as well as recipients for transformation using isolated nucleic acids molecules include preferably those with an agronomic, horticultural, ornamental, economic, or commercial value, and more preferably is include, without limitation, Acacia, alfalfa, apple, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, balsam poplar, banana, barley, beans, beet, black cottonwood, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassaya, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, Clementines, clover, coconut, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, flax, French bean, garlic, gourd, grape, grapefruit, honey dew, Indian rice, Japanese rice, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, maize, melon, mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion, orange, oriental melon, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat, yams, and zucchini cell. References

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What is claimed is:

Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising: (a) a nucleotide binding (NB) domain; and

(b) a leucine rich repeat (LRR) domain, wherein said polypeptide confers a heat-stable defense response to a plant pathogen.

2. The isolated nucleic acid molecule of claim 1, wherein said LRR domain includes a sub-domain that confers said heat stable defense response.

3. The nucleic acid molecule as in any one of the preceding claims, wherein said nucleotide sequence is operably linked to a promoter that drives expression in a plant cell.

4. The nucleic acid molecule as in any one of the preceding claims, wherein said promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

5. The nucleic acid molecule as in any one of the preceding claims, wherein said tissue-specific promoter is a root-specific promoter, a leaf-specific promoter, or a seed-specific promoter.

6. The nucleic acid molecule as in any one of the preceding claims, wherein said nucleic acid molecule is synthetic.

7. The nucleic acid molecule as in any one of the preceding claims, wherein the codon usage of the nucleotide sequence is optimized for expression in a plant or plant component.

8. The nucleic acid molecule as in any one of the preceding claims, wherein the LRR sub-domain that confers a heat-stable defense response to a plant pathogen includes an E/L(K or R)LD motif or an E/L(K or Y)LV(N or D) motif.

9. The nucleic acid molecule as in any one of the preceding claims, wherein the polypeptide is SNC 1-3, SNCl E640R, N Y646K, or N Y646K N648D polypeptide.

10. The nucleic acid molecule as in any one of the preceding claims, wherein the polypeptide is a homolog of SNCl , wherein said homolog includes said LRR sub- domain that confers a heat-stable defense response to a plant pathogen.

11. The nucleic acid molecule as in any one of the preceding claims, wherein said polypeptide is described in Figure 9, Figure 10, or Figure 11.

12. The nucleic acid molecule as in any one of the preceding claims, wherein the polypeptide is a homolog of N, wherein said homolog includes said LRR sub- domain that confers a heat-stable defense response to a plant pathogen.

13. A vector comprising any one of the nucleic acid molecules as in any one of the preceding claims.

14. A cell comprising any one of the nucleic acid molecules as in any one of the preceding claims.

15. A plant or plant component comprising any one of the nucleic acid molecules as in any one of the preceding claims.

16. A method of conferring resistance to a pathogen in a plant or plant component, the method comprising the steps of:

(a) transforming a plant cell with a nucleic acid molecule of any one of claims 1-12 that encodes the polypeptide comprising: (i) a nucleotide binding (NB) domain; and

(ii) a leucine rich repeat (LRR) domain, wherein said polypeptide confers a heat-stable defense response to a plant pathogen; and

(b) regenerating a plant or plant component from said transformed plant cell, wherein said plant exhibits resistance to said pathogen.

17. The method of claim 1, wherein said LRR domain includes a sub-domain that confers said heat stable defense response.

18. The method of as in any one of the preceding claims, wherein the plant or plant component expressing a heat-stable NB-LRR polypeptide has an increased level of resistance compared to a corresponding control plant at the masking temperature of the control plant or control plant component.

19. The method as in any one of the preceding claims, wherein said nucleotide sequence is operably linked to a promoter that drives expression in a plant cell.

20. The method as in any one of the preceding claims, wherein said promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

21. The method as in any one of the preceding claims, wherein said tissue- specific promoter is a root-specific promoter, a leaf-specific promoter, or a seed- specific promoter.

22. The method as in any one of the preceding claims, wherein said nucleic acid molecule is synthetic.

23. The method as in any one of the preceding claims, wherein the codon usage of the nucleotide sequence is optimized for expression in a plant or plant component.

24. The method as in any one of the preceding claims, wherein the LRR sub- domain that confers a heat-stable defense response to a plant pathogen includes an EL(K or R)LD motif or an EL(K or Y)LV(N or D) motif.

25. The method as in any one of the preceding claims, wherein the nucleic acid molecule is introduced through breeding.

26. The method as in any one of the preceding claims, wherein the nucleic acid molecule is introduced through transformation.

27. The method as in any one of the preceding claims, wherein said plant or plant component is a monocot or a dicot.

28. The method as in any one of the preceding claims, wherein said pathogen is a bacterial, a insect, a nematode, a fungal, or a viral pathogen.

29. A substantially-pure polypeptide comprising:

(a) a nucleotide binding (NB) domain; and

(b) a leucine rich repeat (LRR) domain, wherein polypeptide confers a heat-stable defense response to a plant pathogen.

30. The polypeptide of claim 29, wherein said LRR domain includes a sub- domain that confers said heat stable defense response.

31. The polypeptide as in any one of the preceding claims, wherein the polypeptide, when expressed in a plant, is localized in the plant cell nucleus.

32. The polypeptide as in any one of the preceding claims, wherein the LRR sub-domain that confers a heat-stable defense response to a plant pathogen includes an EL(K or R)LD motif or an EL(K or Y)LV(N or D) motif.

33. The polypeptide as in any one of the preceding claims, wherein the polypeptide is SNC 1-3, SNCl E640R, N Y646K, or N Y646K N648D polypeptide.

34. The polypeptide as in any one of the preceding claims, wherein the polypeptide is a homolog of SNCl, wherein said homolog includes said LRR sub- domain thai confers a heat-stable defense response to a plant pathogen.

35. The polypeptide as in any one of the preceding claims, wherein said polypeptide is described in Figure 9, Figure 10, or Figure 11.

36. The polypeptide as in any one of the preceding claims, wherein the polypeptide is a homolog of N, wherein said homolog includes said LRR sub-domain that confers a heat-stable defense response to a plant pathogen.

37. A plant of plant component comprising a substantially-pure polypeptide comprising:

(a) a nucleotide binding (NB) domain; and (b) a leucine rich repeat (LRR) domain, wherein said polypeptide confers a heat-stable defense response to a plant pathogen.

38. The plant of plant component of claim 37, wherein said LRR domain includes a sub-domain that confers said heat stable defense response.

39. Use of a plant of plant component comprising a substantially-pure polypeptide comprising:

(a) a nucleotide binding (NB) domain; and (b) a leucine rich repeat (LRR) domain, wherein said polypeptide confers a heat-stable defense response to a plant pathogen.

40. Use of the plant or plant component of claim 39, wherein said LRR domain includes a sub-domain that confers said heat stable defense response.