WO2000078944A1

WO2000078944A1 - Methods to design and identify new plant resistance genes

Info

Publication number: WO2000078944A1
Application number: PCT/US2000/016461
Authority: WO
Inventors: Steven R. Scofield
Original assignee: Dna Plant Technology Corporation
Priority date: 1999-06-17
Filing date: 2000-06-15
Publication date: 2000-12-28
Also published as: AU5614100A

Abstract

The present invention discloses methods for the identification of plant resistance genes that are capable of rendering a plant of interest more resistant to a pathogen of interest. Using the methods of the invention, one may design resistance genes that encode polypeptides capable of recognizing a pathogen elicitor molecule and that are capable of inducing a protective reaction in the plant of interest, for example, a hypersensitive response. Pathogen elicitor molecules for which a corresponding resistance gene can be isolated using the disclosed methods include molecules expressed by a pathogen of interest for which no corresponding resistance gene exists. Thus, the methods of the present invention facilitate the generation of plants with enhanced resistance to a pathogen of interest.

Description

METHODS TO DESIGN AND IDENTIFY NEW PLANT RESISTANCE GENES

1. FIELD OF THE INVENTION

The present invention relates to methods for the design and identification of plant resistance genes. The resistance genes obtained with the disclosed methods are capable of enhancing the resistance of a plant to a pathogen of interest. The resistance genes obtained with the disclosed methods can be used to generate new pathogen-resistant plants.

2. BACKGROUND OF THE INVENTION

Plants play a critical role as nutrients for animals, including humans, and for the production of substances useful as pharmaceuticals, cosmetics and the like. The steady growth in the world's population results in increasing needs for plant crops. This increased need must be satisfied with reduced soil resources available to agriculture. Increased crop yield can be provided with existing soil resources by engineering plant species that grow better and that are more resistant to plant pathogens.

Plants are subject to threats by numerous pathogens, e.g., fungi, bacteria, viruses, insects and nematodes. A small fraction of pathogens succeed in invading plant tissue and thereby cause disease. In many cases, the plant initiates a rapid response to the pathogen, i.e., hypersensitive response, resulting in localized cell and tissue death (i.e., necrosis) at the site of invasion of the pathogen. This localized reaction prevents the spreading of the invasion by the specific pathogen and is followed by the plant being resistant to pathogens in general and throughout the plant over a period of days, i.e., systemic acquired resistance. Genetic analysis of plant disease resistance mediated through the hypersensitive response has demonstrated the requirement of a dominant, or semidominant, plant resistance gene ("R gene") and a corresponding dominant pathogen avirulence gene or elicitor gene. This mode of resistance is referred to as gene-for-gene resistance. An elicitor from a pathogen ("pathogen elicitor") is typically a peptide or polypeptide, e.g., a phytopathogen avirulence ("Avr") gene product. Recognition of the pathogen elicitor by the R gene product is believed to be a vital first step in provoking a hypersensitive response to protect the plant from a large scale invasion of the pathogen. For background on plant pathogen resistance and plant R genes, see, e.g., Baker et αl, 1997, Science 276:726-733; Bent, 1996, The Plant Cell 8:1757-1771; Innes, 1998, Curr. Opinion in Plant Biology 1:229-304; Staskawicz et αl., 1995, Science 268:661-667; Ellis et αl., 1998, Curr. Opinion in Plant Biology 1 :288-293. A number of R genes have been isolated and characterized. However, many crops lack useful R genes for some important pathogens. Also, new pathogens are created in different ways, for example, through mutations in existing pathogens. Thus, R genes that provide effective protection to a plant from a particular pathogen may no longer be effective once the pathogen has mutated to an extent that prevents R gene products in the plant from recognizing the newly created pathogen.

Thus, methods to engineer and identify new R genes that are capable of rendering a plant resistant to a pathogen of interest would be highly desirable. Such an engineered R gene may be expressed in a plant to render it resistant to a pathogen comprising the pathogen elicitor which the engineered R gene was designed to recognize. Such methods would allow one to protect plants from the challenge by any new or known pathogen.

Previous attempts to increase pathogen resistance in plants include the expression of the tomato Mi- 1.2 nematode resistance gene in a nematode-susceptible tomato line. The resulting transgenic tomato plants showed resistance to the root knot nematodes M. javanica strain VW4 and M. incognita strain VW6 in most of the transgenic plants but not against M. javanica strain VW5, thus resembling the specificity of the Mi gene in wild-type plants (Milligan et α/., 1998, The Plant Cell 10:1307-1319).

In another study, plant resistance was induced by activating an inactive transgene encoding the Cf-9 R gene product through excision of a transposable element from that gene in a plant that expressed Avr9, a Cf-9 elicitor (WO 95/31564). Also, the plant Prf R gene was overexpressed in tomato plants, leading to enhanced resistance to P. s. pathovar tomato strain DC3000, X. c. pv. vesicatoria strain 56, R. solanacearum strain 82 bacterial pathogens and TMV viral pathogen (Oldroyd et al, 1998, Proc. Natl. Acad. Sci. USA 95:10300-10305). In another attempt to increase plant resistance, glucose oxidase was expressed in potato plants to generate H₂O₂ a reagent produced during plant defense responses, through glucose oxidation. H₂O₂ elevation in transgenic potato plants was shown to increase resistance to E. carotovora subspecies carotovora and P. infestans (Wu et al, 1995, The Plant Cell 7:1357-1368). Transgenic rice plants were generated expressing the potato proteinase inhibitor II gene, rendering the plants more resistant to pink stem borer larvae of Sesamia inferens (Duan et al, 1996, Nature Biotechnology 14:494-498). Also, resistance to G. pallida was enhanced in transgenic potato plants expressing cowpea trypsin inhibitor (U.S. Patent No. 5,494,813). The expression of a protein that disrupts the feeding structure of plant nematode pathogens is suggested in U.S. Patent No. 5,866,777 and the expression of lytic proteins in apple tree plants is discussed in U.S. Patent No. 5,824,861. U.S. Patent No. 5,856,154 addresses the overexpression of pathogenesis related proteins with a chemically regulatable expression system to protect a plant from an oomycete pathogen. The expression of proteins to repel fungus plant pathogens was discussed in U.S. Patent No. 5,750,874.

In another attempt to increase plant resistance, the cryptogein elicitor from P. cryptogea pathogen was expressed in tobacco plants as a transgene with a pathogen inducible promoter. The transgenic plants exhibited increased resistance upon infection with the fungi P. p. v. nicotianae, T. basicola, E. cichoracearum or B. cinerea (Keller et al. , 1999, The Plant Cell 11 :223-235). The inducible expression of an R gene specific pathogen elicitor in a transgenic plant was also suggested in U.S. Patent No. 5,866,776. In another study, transgenic A. thaliana plants were generated that overexpressed A. thaliana NPR1, a regulatory protein for systemic acquired resistance. The resulting transgenic plants showed enhanced resistance to the bacterial pathogen P. s. pv. maculicola ES4326 and the oomycete pathogen P. parasitica strain Noco (Cao et al, 1998, Proc. Natl. Acad. Sci. USA 95:6531-6536; see, also, WO 98/06748 and WO 97/49822). Also, the expression of the H. halobium bacterio-opsin proton pump in potato plants induced an increased resistance similar to systemic acquired resistance (Abad et al, 1997, Molec. Plant Microbe Interact. 10:635-645).

However, there is a need for an effective method of engineering and identifying R genes that specifically recognize a chosen pathogen elicitor and that provide enhanced resistance to a pathogen comprising the chosen pathogen elicitor. Methods that effectively employ polynucleotide mutagenesis techniques, including DNA shuffling (see, e.g., Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91:10747-10751; Crameri et al, 1998, Nature 391. '288-291), to engineer new R genes capable of enhancing the resistance of a plant to a pathogen of interest would be highly desirable. Citation of a reference in this or in any section of the specification shall not be construed as an admission that such reference is prior art to the invention.

3. SUMMARY OF THE INVENTION

The present invention describes methods for the engineering and identification of plant resistance genes capable of enhancing the resistance of a plant to a pathogen of interest. The described methods preferably employ DNA shuffling for the engineering of new R gene polynucleotides and a high throughput screening approach in transgenic plants for the identification of engineered R gene polypeptides with recognitional specificity for an elicitor from a pathogen of interest. The transgenic plants used in the preferred screening approach express a pathogen elicitor that is introduced into the plant by the pathogen of interest during infection of the plant and multiple R gene polynucleotides are expressed, e.g., transiently, in those transgenic plants. Using this screening approach in transgenic plants, desired R gene products are preferably identified by visually screening the plants for necrosis.

In one embodiment, the methods of the invention facilitate the engineering and identification of a plant R gene capable of enhancing the resistance of a plant to a pathogen of interest, i.e., a pathogen-specific R gene. An R gene engineered and identified using a method of the invention, when expressed in a plant, is capable of initiating a defensive response, e.g., a hypersensitive response, in the plant upon exposure of the plant to a pathogen of interest. Using the methods of the invention, one may engineer and identify an R gene effective to enhance pathogen resistance in a plant to a pathogen. For example, one may identify a new or mutated pathogen (or, a known and unmutated pathogen) against which a plant species of interest shows little or no resistance, and one may engineer and identify an R gene capable of providing resistance to that pathogen in the plant species of interest. In a preferred embodiment, a pathogen-specific R gene engineered and identified using the methods of the present invention is used to make a plant that is resistant to that pathogen.

In one embodiment, the method of the invention comprises the generation of a collection (i.e., library) of R genes through mutagenesis, i.e., a collection of mutagenized R genes or R gene variants (e.g., natural or synthetic), and the selection of an R gene from said collection that is capable of enhancing a plant's resistance to a pathogen of interest. In one aspect, polynucleotide sequences corresponding to one or more R genes are subjected to mutagenesis to generate a collection of mutated R genes. Such mutagenesis of one or more R genes is preferably random or substantially random. In a preferred aspect, mutagenesis of R genes in the methods of the invention is carried out by DNA shuffling. In another aspect, mutagenesis of R genes is concentrated on a domain, e.g., the leucine-rich repeat domain, of R gene products. R gene polynucleotides that can be used in the methods of the invention to generate a pathogen-specific R gene include, but are not limited to, R genes encoding cytoplasmic proteins, membrane-bound proteins, transmembrane proteins, kinase proteins, receptor proteins, leucine-rich region proteins and leucine zipper proteins. The collection of mutagenized R genes is screened for their ability to enhance the resistance of a plant to a pathogen of interest, which may be any chosen or designated pathogen (i.e., pathogen-specific R gene). The pathogen or an elicitor from the pathogen may be used to screen for a desired R gene in a functional assay. Pathogens to which a pathogen-specific R gene may be designed include, but are not limited to, bacteria, viruses, fungi, insects and nematodes. A pathogen-specific R gene is generated with the methods of the invention by screening a collection of mutagenized R genes with the pathogen of interest or with a pathogen elicitor derived from said pathogen as a screening agent. In a preferred embodiment, the collection of mutagenized R genes is screened by expressing the pathogen elicitor in a transgenic plant, for example, of the plant species of interest. In a preferred aspect, the transgenic plant expresses the pathogen elicitor so that the elicitor concentrates in the apoplastic space (i.e., the space between cells or intercellular space) of the plant. In another preferred aspect, one or more mutagenized R genes are expressed in the transgenic plant that expresses the pathogen elicitor. Such screening provides a means for selecting new recognitional specificity between an R gene and a corresponding elicitor. A preferred mode of screening is detection by visual observation of necrosis. The order of the steps of the invention may be varied as appreciated by the skilled artisan. For example, one may screen a collection of mutagenized R genes by an in vitro screen or by using a cell culture based screening method. Such an in vitro or cell culture based screen may be used to identify R gene products capable of recognizing a pathogen elicitor expressed by a pathogen of interest following infection of a plant. Or, for example, one may screen a collection of mutagenized R genes in a plant for their ability to enhance resistance of the plant to the pathogen of interest. Screening R genes in plants may be carried out in addition to in vitro and/or cell culture screening.

When screening in plants by expressing a polynucleotide for an elicitor from a pathogen of interest and polynucleotides specific for one or more R genes in a plant, such elicitor and R gene polynucleotides may be introduced into the plant serially, in either order, or simultaneously. Also, the expression of such elicitor and R gene polynucleotides may be, for example, constitutive, inducible or transient, just as long as there is some period of time in which the expression products of the polynucleotides encoding the pathogen elicitor and the R gene or genes are present in the plant or plants, such that a resistance response can be detected. Screening in plants may be carried out, for example, by expressing the mutagenized R genes in the plant and then supplying the pathogen or the pathogen elicitor to that plant. Or, for example, one may supply a pathogen of interest or a pathogen elicitor, expressed by the pathogen of interest, to a plant and then express one or more mutagenized R genes in that plant. Or, for example, one may express an elicitor from a pathogen of interest and one or more mutagenized R gene polynucleotides in a plant simultaneously.

The screening of R gene polynucleotides in accordance with the preferred embodiments of the invention is carried out using high throughput screening techniques. Such high throughput screening preferably is capable of processing at least about 50,000 R gene polynucleotides, more preferably at least about 100,000 R gene polynucleotides, even more preferably at least about 200,000 R gene polynucleotides and most preferably at least about 500,000 R gene polynucleotides, in order to identify one or more desired R gene polynucleotides.

In another embodiment of the invention, transgenic plant material expressing a pathogen elicitor may be used to identify DNA sequences that encode a known R gene from a library of genomic or cDNA clones prepared from a plant that contains the desired R gene. In a preferred embodiment, a method is described for screening for a plant resistance gene capable of enhancing the resistance of a plant of interest when exposed to a pathogen elicitor of interest comprising (a) introducing into the plant of interest the pathogen elicitor of interest; (b) expressing in the plant of interest one or more mutagenized plant resistance gene polynucleotides; and (c) identifying one or more of said mutagenized plant resistance gene polynucleotides capable of enhancing the resistance of said plant of interest to said pathogen of interest; wherein steps (a), (b) and (c) are carried out using the same plant; wherein steps (a) and (b) can be carried out in either order; and wherein step (c) is carried out following steps (a) and (b). In another preferred embodiment, a method is described for generating a transgenic plant comprising introducing an expressible plant resistance gene polynucleotide into cells of a plant, wherein said plant resistance gene polynucleotide is screened for using a method comprising (a) introducing into the plant of interest the pathogen elicitor of interest; (b) expressing in the plant of interest one or more mutagenized plant resistance gene polynucleotides; and (c) identifying one or more of said mutagenized plant resistance gene polynucleotides capable of enhancing the resistance of said plant of interest to said pathogen of interest; wherein steps (a), (b) and (c) are carried out using the same plant; wherein steps (a) and (b) can be carried out in either order; and wherein step (c) is carried out following steps (a) and (b). In a preferred embodiment, a method is described for generating a plant resistance gene capable of enhancing the resistance of a plant of interest to a pathogen of interest comprising (a) constructing a library of two or more mutagenized plant resistance gene polynucleotides; and (b) screening said plant resistance gene polynucleotides for their ability to enhance the resistance of a plant of interest to said pathogen of interest when at least one of said mutagenized plant resistance gene polynucleotides is expressed in said plant of interest.

In another preferred embodiment, a method is described for generating a transgenic plant comprising introducing an expressible plant resistance gene polynucleotide into cells of a plant, wherein said plant resistance gene polynucleotide is generated using a method for generating a plant resistance gene capable of enhancing the resistance of a plant of interest to a pathogen of interest comprising (a) constructing a library of two or more mutagenized plant resistance gene polynucleotides; and (b) screening said plant resistance gene polynucleotides for their ability to enhance the resistance of a plant of interest to said pathogen of interest when at least one of said mutagenized plant resistance gene polynucleotides is expressed in said plant of interest. In a preferred embodiment, a method is described for generating a plant resistance gene capable of enhancing the resistance of a plant of interest to a pathogen of interest comprising (a) identifying one or more plant resistance gene polynucleotides; and (b) screening said plant resistance gene polynucleotides for their ability to enhance the resistance of said plant of interest to said pathogen of interest when at least one of said plant resistance gene polynucleotides is expressed in said plant.

In another preferred embodiment, a method is described for generating a transgenic plant comprising introducing an expressible plant resistance gene polynucleotide into cells of a plant, wherein said plant resistance gene polynucleotide is generated using a method for generating a plant resistance gene capable of enhancing the resistance of a plant of interest to a pathogen of interest comprising (a) identifying one or more plant resistance gene polynucleotides; and (b) screening said plant resistance gene polynucleotides for their ability to enhance the resistance of said plant of interest to said pathogen of interest when at least one of said plant resistance gene polynucleotides is expressed in said plant.

4. DETAILED DESCRIPTION OF THE INVENTION

Pathogen resistance is an important property in plants and a useful tool for the protection of plants, especially crop plants. Plant R genes are key in mediating and effecting pathogen resistance in plants. R gene products are believed to be receptors for a plant to detect a pathogen elicitor and, thus, initiate a protective response to the corresponding pathogen. The term "plant", as used herein, includes whole plants, plant parts, individual plant cells, groups of plants cells (e.g., cultured plant cells) and progeny thereof. The term "enhance" when used to describe an increase of resistance of a plant to a pathogen or a pathogen elicitor, as used herein, includes the increase of the resistance of a plant that may have no resistance, or some resistance or substantial resistance to the pathogen or a pathogen elicitor prior to effecting the increase in resistance using the methods of the present invention. The term "pathogen elicitor", as used herein, means any molecule expressed in a pathogen, e.g., a polypeptide, that can elicit a resistance response, e.g., a hypersensitive response, in a plant if that plant expresses an R gene product that corresponds to that pathogen elicitor molecule, i.e., the R gene product can recognize (e.g., bind to, interact with, detect or has recognitional specificity for) that elicitor molecule expressed in a pathogen. Thus, the term "pathogen elicitor", as used herein, includes a molecule expressed by a pathogen for which no R gene product capable of recognizing that molecule exists, either in a plant of interest or in any plant, but for which a corresponding R gene can be engineered and identified using the methods described herein. The terms "pathogen of interest" and "pathogen elicitor of interest", as used herein, mean a pathogen or a pathogen elicitor against which a plant is to be made more resistant and for which a corresponding R gene is to be engineered and/or identified using the methods of the invention. Thus, as used herein, a "pathogen of interest" and a "pathogen elicitor of interest" refer to a pathogen and a pathogen elicitor which were chosen as a target for the methods of the present invention, i.e., they refer to a pathogenic agent to which an R gene o product with recognitional specificity for the chosen pathogen and/or pathogen elicitor is engineered and/or identified using the methods of the invention. The term "hypersensitive response", as used herein, means the rapid collapse of a limited area of plant tissue following infection by a pathogen, e.g., an avirulent pathogen, usually accompanied by, for example, a transient burst of hydrogen peroxide production, cell wall reinforcement through 5 callose deposition and lignifϊcation, accumulation of phytoalexins, and or the activation of defense-related genes (see, e.g., Hammond-Kosack et al, 1996, The Plant Cell 8:1773- 1791). The term "necrosis", as used herein, refers to a condition that is characterized by dead or discolored plant tissue. The interaction of R gene products with a pathogen elicitor has been shown to involve interaction of the R gene product and the pathogen elicitor. See, 0 e.g., Scofield et al, 1996, Science 274:2063-2065; Tang et al, 1996, Science 274:2060- 2063. Thus, structural compatibility of the R gene product (i.e., "corresponding R gene") with the pathogen elicitor (i.e., "corresponding elicitor") is believed to be important in effecting plant protection against a pathogen. Also, one or more domains of an R gene product may be responsible for the recognition of a pathogen elicitor. See, e.g., Anderson et al, 1997, The Plant Cell 9:641-651; Ellis et al, 1999, The Plant Cell 11:495-506; Erickson et al, 1999, Phil. Trans. R. Soc. Lond. 354:653-658.

The present invention presents methods that facilitate the engineering and identifying of a pathogen-specific R gene. A pathogen-specific R gene engineered and identified using the methods of the invention recognizes a pathogen elicitor and, when expressed in a plant, enhances resistance of that plant to a pathogen comprising the pathogen elicitor. Thus, using the methods of the invention, a pathogen elicitor may be used to engineer and identify an R gene that renders a plant more resistant upon exposure to the elicitor. R genes made using the methods of the invention may be expressed in a plant to generate a new transgenic plant line. As the methods of the invention facilitate the design of R genes capable of recognizing a pathogen elicitor, one may use the methods of the invention to design new plant lines with increased resistance to a pathogen of interest as compared to a wild-type plant.

Plant pathogen elicitors include any molecule found in a pathogen, for example, a protein. Plant pathogen elicitors are molecules from a pathogen that trigger a defensive response in a plant, for example, a hypersensitive response. Typically, a pathogen elicitor is a molecule that is secreted by the pathogen or is otherwise, in whole or in part, on the outside of a pathogen, so that it can be detected by a receptor, e.g., an R gene product, from the plant.

The methods of the invention facilitate the identification of a pathogen elicitor and the design and identification of an R gene that is specific to that elicitor. For example, where new pathogens are discovered, or where known pathogens are found to have changed in some way (e.g., through one or more mutations), one may identify a suitable pathogen elicitor and use that elicitor molecule to screen for a corresponding R gene. In a preferred aspect, a corresponding R gene is capable of enhancing resistance in a plant to a pathogen comprising the pathogen elicitor of interest. Thus, any new or altered or known pathogen can be rendered less destructive to a plant by engineering one or more R genes capable of providing enhanced resistance in the plant to the pathogen. Therefore, new plant lines can be generated by expressing one or more R genes made using the methods of the invention in a plant. The new plant lines made using the methods of the invention can therefore be designed to be resistant to a pathogen of interest.

4.1. PLANT PATHOGENS AND PATHOGEN ELICITORS

Plant pathogens include, but are not limited to, bacteria, viruses, fungi, nematodes and insects. A pathogen may infect a plant and cause severe damage to that plant, including death. Upon infection, a plant may initiate a protective reaction to the pathogen, e.g., a hypersensitive response, depending on whether the plant can recognize the pathogen. Molecules that originate from the pathogen and that are recognized by a plant resistant to that pathogen are called pathogen elicitors. Pathogen elicitors are molecules of any type, for example, proteins, and they may be secreted or only partially exposed to the environment of the pathogen. Also, pathogen elicitors may be inserted into plant cells by the pathogen, for example, as observed in bacteria.

Pathogens of the various classes may change, for example, through mutagenesis. Also, new pathogens may arise that were not previously encountered by a plant species. For example, when a plant (e.g., a crop, a fruit, a vegetable, etc.) is introduced into a continent (for example, through importation), a plant species is likely exposed to pathogens it has not encountered before. In order to render a plant species resistant to a pathogen it has not encountered before, one may design an R gene capable of enhancing resistance of the plant to the new pathogen, i.e., a pathogen-specific R gene. A pathogen-specific R gene, in a preferred embodiment, is designed to recognize a pathogen elicitor from the pathogen of interest. A pathogen elicitor may be provided to screen for an R gene product that recognizes the elicitor and, thus, is specific to a pathogen comprising the elicitor, for example the pathogen of interest.

Pathogen elicitors may be selected based on their location, size, molecular nature, accessability, stability, half-life, etc., as appreciated by the skilled artisan. Any molecule useful as a pathogen elicitor may be used for the design and identification of a pathogen- specific R gene. In a preferred aspect, a protein is isolated and characterized from a pathogen and used in the methods of the invention to engineer and identify a pathogen- specific R gene. Such a protein is preferably a secreted protein, a transmembrane protein, an extracellular membrane-bound protein or any other protein that is accessible to a plant molecule, for example, a protein that is not located solely inside the pathogen. A pathogen elicitor protein may be an unmodified or a modified (i.e., processed) protein, e.g., a glycoprotein, a phosphorylated protein, an acylated protein.

The terms "protein", "polypeptide" and "peptide" are used interchangeably throughout the specification and claims. These terms also encompass glycosylated proteins, e-, glycoproteins.

The identification and characterization of a pathogen elicitor is carried out using techniques known in the art of biotechnology as described below.

4.1.1. ISOLATION OF A PATHOGEN ELICITOR A pathogen elicitor may be isolated from the pathogen of interest either in association with or outside (i.e., separate from) tissue from an infected plant. Many pathogen elicitors are only expressed by a pathogen when that pathogen infects a plant (i.e., infection-dependent elicitor expression), thus those pathogen elicitors are preferably isolated from an infected plant. The choice of the source from which to isolate the pathogen elicitor depends on a variety of factors known to the skilled artisan, for example, the complexity of the pathogen, the mode of infecting a plant by the particular pathogen (e.g., does the pathogen deposit the pathogen elicitor in an environment of the plant from which it can easily be isolated), etc.

All protein purification methods known to the skilled artisan may be used for the purification of a pathogen elicitor. Such techniques have been extensively described in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Volume 152, Academic Press, San Diego, CA (1987); Molecular Cloning: A Laboratory Manual, 2d ed., Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989); Current Protocols in Molecular Biology, John Wiley & Sons, all Vols., 1989, and periodic updates thereof); New Protein Techniques: Methods in Molecular Biology, Walker, J.M., ed., Humana Press, Clifton, N.J., 1988; and Protein Purification: Principles and Practice, 3rd. Ed., Scopes, R.K., Springer-Verlag, New York, N.Y., 1987; Parvez et al, 1985, Progress in HPLC, Vol. 1, Science Press, Utrecht, The Netherlands. In general, techniques including, but not limited to, ammonium sulfate precipitation, centrifugation, ion exchange, gel filtration, gel filtration molecular exclusion chromatography, reverse-phase chromatography (and the HPLC or FPLC forms thereof), differential solubility, and hydrophobic interaction chromatography may be used to purify the pathogen elicitor.

4.1.1.1. ISOLATION OF A PATHOGEN ELICITOR FROM BACTERIA A variety of bacteria are capable of infecting plants. Typically, these bacteria use macromolecule delivery systems to introduce their pathogen elicitors into plant cells. Bacterial pathogen elicitors may be isolated from bacteria or from plant cells following infection.

A variety of bacteria are capable of causing diseases in plants including, but not limited to, Xanthomonas, Pseudomonas, Erwinia, Clavibacter and Streptomyces (see, e.g., Agrios, 1988, "Plant Pathology", Academic Press). Typically, bacteria use macromolecule delivery systems to introduce elicitors into plant cells so that these molecules are not found in the media in which cells are grown or in the apoplastic fluid of infected plants. Thus, these molecules have to be isolated from the cytoplasm of plant cells. The quantities of these elicitor molecules that bacteria transfer into plant cells is small. Therefore, a preferred way of providing a bacterial pathogen elicitor to design an R gene that enhances plant resistance to the bacterial pathogen of interest, is to deliver the entire bacterial pathogen into a plant (e.g., through infection) and to screen for an R gene capable of providing resistance to any of the pathogen elicitors provided in this way. See, e.g., Mindrinos et al, 1994, Cell 78:1089-1099, which describes a similar approach for providing a bacterial pathogen elicitor. 4.1.1.2. ISOLATION OF A PATHOGEN ELICITOR FROM VIRUSES

Viruses are typically small organisms that consist of a limited number of molecular species. Viral plant pathogen elicitors include, but are not limited to, any viral proteins, for example, replicase proteins, proteases, movement proteins or coat proteins.

The complete nucleotide sequence for many plant pathogenic viruses has been determined and is known to the skilled artisan. All proteins expressed by a plant virus can serve as a pathogen elicitor for the design of a corresponding R gene provided those proteins are accessible to the R gene product in the plant infected by the virus. The accessibility of a viral protein can be determined using routine methods known in the art as described herein.

In a preferred embodiment, when engineering and identifying an R gene that corresponds to a protein from a virus of interest, the viral pathogen elicitor is provided by expressing the viral genome in a transgenic plant in each reading frame. A library of mutagenized R genes can then be screened, for example, by transiently expressing the mutagenized R genes in the transgenic plant and by detecting necrosis as described herein, infra.

4.1.1.3. ISOLATION OF A PATHOGEN ELICITOR FROM FUNGI

Pathogen elicitors derived from fungi are typically found in the apoplastic space of a plant that is infected with a fungus. Thus, pathogen elicitors of fungi can best be isolated from apoplastic fluid of an infected plant. In addition, these pathogen elicitors may also be obtained from the pathogen itself (see, e.g., Van den Ackerveken et al, 1994, Mol. Gen. Genet. 243:277-285). The isolation of pathogen elicitors from the apoplastic space of plants can be carried out as described, see, e.g., Lauge et al, 1998, Proc. Natl. Acad. Sci. USA 95:9014-9018; Wubben et al, 1994, Mol. Plant Microbe Interact. 7:516-524; Huet et al, 1993, Phytochemistry 33:797-805; U.S. Patent No. 5,670,706, which describe the isolation of proteins from the apoplastic space of plants.

4.1.1.4. ISOLATION OF A PATHOGEN ELICITOR FROM NEMATODES

Plant pathogen elicitors derived from nematodes may be isolated from the nematode or from an infected plant. In a preferred embodiment, a pathogen elicitor is provided for the design of a corresponding R gene by devising a functional assay. For example, a T DNA plasmid library of R genes can be transferred into plants via the Agrobacterium rhizogenes "hairy root system" (Tepfer, 1990, Physiologia Plantarium 79:140-146). In this system, transformation by A. rhizogenes causes "hairy roots" to form with each root arising from an independent transformation event. These roots can be infected by plant pathogenic nematodes (for example, in tomato). A suspension of the pathogenic nematode of interest is added to the tissue culture plates on which the "hairy root" cultures are growing. R genes that recognize a pathogen elicitor of the nematode of interest provided this way will trigger a hypersensitive response. The positive R gene polynucleotide sequences can be obtained from the corresponding "hairy roots", for example, by PCR amplification from genomic DNA prepared from the root as appreciated by the skilled artisan.

4.1.2. CLONING AND SEQUENCING OF A

POLYNUCLEOTIDE SPECIFIC FOR A PATHOGEN ELICITOR

Once a pathogen elicitor polypeptide has been isolated, a polynucleotide encoding that pathogen elicitor may be isolated and characterized using techniques known in the art. A cDNA or genomic DNA specific for a pathogen elicitor protein or nucleic acid may be cloned and sequenced in a variety of ways, depending on the information regarding that pathogen elicitor that is available. Once a polynucleotide specific for the pathogen elicitor is isolated, it may be sequenced using methods known in the art, e.g. , dideoxy chain termination sequencing, see, e.g., Sambrook et al, supra.

The polynucleotides obtained using the methods of the present invention include polynucleotides having the DNA sequences presented herein, and additionally include any nucleotide sequence encoding a contiguous and functional pathogen-elicitor encoding open reading frame (ORF) that hybridizes to a complement of the DNA sequences presented herein under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 °C, and washing in O.lxSSC/0.1% SDS at 68°C (Ausubel F.M. et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3) and encodes a functionally equivalent gene product.

For oligonucleotide probes, highly stringent conditions may refer, e.g., to washing in 6XSSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55 °C (for 20-base oligos), and 60°C (for 23-base oligos).

Additionally contemplated are any nucleotide sequences that hybridize under moderately stringent conditions to the complement of the DNA sequences that encode an amino acid sequence that is encoded by a polynucleotide obtained using the methods of the invention and encodes a functionally equivalent pathogen elicitor product. Such moderately stringent conditions include, e.g., washing in 0.2XSSC/0.1% SDS at 42°C (Ausubel et al, 1989, supra).

Additionally contemplated are any nucleotide sequences that hybridize under low stringency conditions to the complement of the DNA sequences that encode an amino acid sequence encoded by a polynucleotide obtained using the methods of the invention. By way of example and not limitation, procedures using such conditions of low stringency are described in Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792.

4.1.2.1. HYBRIDIZATION CLONING A cDNA or genomic DNA specific for a pathogen elicitor may be cloned through screening a cDNA or genomic DNA library. Such a library may be prepared, for example, from messenger RNA or genomic DNA from the pathogen. For general background on molecular biology techniques and on how to prepare a cDNA library and a genomic library, see, e.g., Ausubel F.M. et al, supra; Sambrook et al, 1989, supra; and U.S. Patent No. 5,650,148.

The library may be screened with a nucleotide fragment specific for a part of the pathogen elicitor. For example, the protein sequence of the pathogen elicitor may be determined using techniques well known to those of skill in the art, such as via the Edman degradation technique. (See, e.g., Creighton, 1983, "Proteins: Structures and Molecular Principles", W.H. Freeman & Co., New York, pp. 34-49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen a cDNA library for the cDNA sequence encoding the pathogen elicitor.

Or, for example, two stretches of protein sequence specific for the pathogen elicitor may be determined. A set of degenerate oligonucleotides specific for each stretch is prepared and the oligonucleotides are used in a polymerase chain reaction ("PCR") amplification. Oligonucleotides are at least about 6 nucleotides long, more preferably at least about 10, more preferably at least about 15, more preferably at least about 20, more preferably at least about 30, more preferably at least about 40 nucleotides. The template in the PCR reaction would be, for example, a mixture of cDNA or genomic DNA that is known to contain or suspected to contain a DNA polynucleotide specific for the pathogen elicitor of interest. See, e.g., Kamoun et al, 1993, Mol. Plant Microbe Interact. 6:573-581, which describes cloning a pathogen elicitor gene using degenerate oligonucleotides in PCR. A cDNA template may be obtained in a variety of ways, for example, by isolating a mixture of different cDNA species from a cDNA library or, for example, by reverse transcribing total mRNA from a cell or organism known to (or suspected to) express the pathogen elicitor. For background on PCR, see, e.g., Ausubel, supra, and PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al, eds. Academic Press, Inc., New York.

In order to clone a full length cDNA or genomic DNA sequence from any species or to clone variant or heterologous forms of the pathogen elicitor molecules, labeled DNA probes made from nucleic acid fragments corresponding to any of the polynucleotides discussed herein or made using the methods of the invention may be used to screen a cDNA library or a genomic DNA library (for example, a phage library) as described in, e.g., Ausubel F.M. et al, supra; Sambrook et al, 1989, supra.

4.1.2.2. EXPRESSION CLONING

A cDNA specific for a pathogen elicitor of interest may also be obtained by screening an expression cDNA library with an antibody specific for the pathogen elicitor of interest.

An expression library can be constructed utilizing cDNA synthesized from RNA isolated from a tissue that expresses the pathogen elicitor, for example, the pathogen or an infected plant. In this manner, gene products made by the pathogen or the infected plant may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the pathogen elicitor. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor, New York.)

4.1.2.2.1. PRODUCTION OF ANTIBODIES

For the production of antibodies, various host animals may be immunized by injection with the pathogen elicitor, a pathogen elicitor peptide (e.g., one corresponding the a functional domain of a pathogen elicitor), truncated pathogen elicitor polypeptides (a pathogen elicitor in which one or more domains have been deleted), functional equivalents of the pathogen elicitor or mutants of the pathogen elicitor. Such host animals may include but are not limited to rabbits, mice, goats, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals. Antibodies useful for the present invention include monoclonal antibodies (see, e.g., Kohler et al, 1975, Nature 256:495-497; and U.S. Patent No. 4,376,110), chimeric antibodies (see, e.g., Morrison et al, 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al, 1984, Nature, 312:604-608; Takeda et al, 1985, Nature, 314:452-454), single chain antibodies (see, e.g., U.S. Patent 4,946,778; Bird, 1988, Science 242:423-426; Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al, 1989, Nature 334:544- 546), antibody fragments (see, e.g., Huse et al, 1989, Science, 246:1275-1281), anti- idiotypic antibodies or Fab fragments of such anti-idiotypes (see, e.g., Greenspan & Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438).

4.1.3. SYNTHESIS OF A PATHOGEN ELICITOR

A pathogen elicitor polypeptide may be synthesized using any method known in the art, for example, chemical synthesis or, more preferably, using recombinant DNA technology.

4.1.3.1. SYNTHESIS OF A PATHOGEN ELICITOR USING CHEMICAL SYNTHESIS

Pathogen elicitors, fragments thereof or fusion proteins thereof, can be chemically synthesized (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., New York).

4.1.3.2. SYNTHESIS OF A PATHOGEN ELICITOR

USING RECOMBINANT DNA TECHNOLOGY

Pathogen elicitors, fragments thereof or fusion proteins thereof, are advantageously produced by recombinant DNA technology using techniques well known in the art. Such methods can be used to construct expression vectors containing a pathogen elicitor nucleotide sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al, 1989, supra, and Ausubel et al, 1989, supra. Alternatively, RNA corresponding to all or a portion of a transcript encoded by a pathogen elicitor nucleotide sequence may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M.J. ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety. Any of host-expression vector system known in the art of biotechnology may be utilized to express the pathogen elicitor nucleotide sequence including, but not limited to, expression in bacteria, yeast, insect cells, mammalian cells, eukaryotic cells and plant cells. In these expression systems, any selection system may be used. Such selection may comprise growth on a selective medium (e.g., antibiotics, minimal media, etc.) or the use of an indicator (e.g., a dye, a fluorescent reagent, etc.). In cases where plant expression vectors are used, the expression of the pathogen elicitor coding sequence may be driven by any of a number of regulatory elements. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et αl., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et αl., 1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et αl., 1984, EMBO J. 3:1671-1680; Broglie et αl., 1984, Science 224:838-843); or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B (Gurley et αl., 1986, Mol. Cell. Biol. 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, New York, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9. As used herein, regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.

4.1.4. TRANSGENIC PLANTS EXPRESSING A PATHOGEN ELICITOR

A transgenic plant with the ability to express a plant pathogen elicitor polypeptide may be engineered by transforming a plant cell with a gene construct comprising a sequence encoding a plant pathogen elicitor protein or polypeptide. In one embodiment, a plant promoter is operably associated with a sequence encoding the desired plant pathogen elicitor protein or polypeptide. ("Operably associated" or "operably linked" is used herein to mean that transcription controlled by the "associated" or "operably linked" promoter produces a functional messenger RNA, whose translation produces the polypeptide.) In a preferred embodiment of the present invention, the associated promoter is a strong and non tissue- or developmental-specific plant promoter (e.g., a promoter that strongly expresses in many or all plant tissue types). Examples of such strong, "constitutive" promoters include, but are not limited to, the CaMV 35S promoter (Odell et αl., 1985, Nature 313:810-812), the T-DNA mannopine synthetase promoter, and their various derivatives. In another preferred embodiment, an inducible or repressible promoter is used to express the pathogen elicitor of interest in a plant, for example, a tet operator promoter as described in Weinmann et al, 1994, The Plant Journal 5:559-569; or a glucocorticoid-inducible promoter as described in McNellis et al, 1998, The Plant Journal 14:247-257; or an ethanol inducible promoter as described in Caddick et al, 1998, Nature Biotechnology 16:177-180. See, also, Gatz, 1995, Methods In Cell Biology 50:411-424, which describes inducible and repressible gene expression systems for plants.

In one embodiment of the invention, a pathogen elicitor is expressed in a plant so that the pathogen elicitor polypeptide will be localized in the apoplastic space. The pathogen elicitor may be directed to the apoplastic space, when expressed in a plant, by expressing the pathogen elicitor polypeptide as a fusion protein together with a peptide that acts as a signal or transporter so that an infective pathogen elicitor is localized in the apoplastic space of the transgenic plant. A variety of signal or transporter peptides can be used, for example, the PRlb signal sequence as described in Lund et al, 1992, Plant Molecular Biology 18:47-53; or the PR- la, b and c signal sequences as described in Pfitzner et al, 1987, Nucleic Acids Research 15:4449-4465. A fusion protein comprising a signal or transporter peptide and a pathogen elicitor polypeptide may be constructed by linking polynucleotides specific for each component to each other (e.g. , the polynucleotides are linked in frame) so that the desired fusion protein is made when the fusion polynucleotide is expressed in a transgenic plant. A skilled artisan would know how to construct a polynucleotide useful for expressing a pathogen elicitor in the apoplastic space of a transgenic plant.

In another embodiment of the present invention, it may be advantageous to engineer a plant with a gene construct comprising a sequence encoding a plant pathogen elicitor protein or polypeptide operably associated with a tissue- or developmental-specific promoter, such as, but not limited to, the CHS promoter, the PATATIN promoter, etc. In yet another embodiment of the present invention, it may be advantageous to transform a plant with a gene construct comprising a sequence encoding a plant pathogen elicitor protein or polypeptide operably linked to a modified or artificial promoter. Typically, such promoters, constructed by recombining structural elements of different promoters, have unique expression patterns and/or levels not found in natural promoters. See, e.g., Salina et al, 1992, Plant Cell 4:1485-1493, for examples of artificial promoters constructed from combining cis-regulatory elements with a promoter core.

In yet an additional embodiment of the present invention, the expression of a pathogen elicitor polynucleotide may be engineered by increasing the copy number of the gene encoding the desired protein or polypeptide using techniques known in the art. 4.1.4.1. TRANSFORMATION OF PLANTS AND PLANT CELLS

Plants and plant cells may be transformed using any method known in the art. In an embodiment of the present invention, Agrobacterium is employed to introduce the gene construct into plants. Such transformation preferably uses binary Agrobacterium T-DNA vectors (Bevan, 1984, Nuc. Acid Res. 12:8711-8721), and the co-cultivation procedure (Horsch et al, 1985, Science 227:1229-1231). Generally, the Agrobacterium transformation system is used to engineer dicotyledonous plants (Bevan et al, 1982, Ann. Rev. Genet 16:357-384; Rogers et al, 1986, Methods Enzymol. 118:627-641). The Agrobacterium transformation system may also be used to transform, as well as transfer, DNA to monocotyledonous plants and plant cells, (see Hernalsteen et al, 1984, EMBO J 3:3039-3041 ; Hooykass-Van Slogteren et al, 1984, Nature 311 :763-764; Grimsley et al, 1987, Nature 325:1677-179; Boulton et al, 1989, Plant Mol. Biol. 12:31-40.; and Gould et al, 1991, Plant Physiol. 95:426-434). In other embodiments, various alternative methods for introducing recombinant nucleic acid constructs into plants and plant cells may also be utilized. These other methods are particularly useful where the target is a monocotyledonous plant or plant cell. Alternative gene transfer and transformation methods include, but are not limited to, protoplast transformation through calcium-, polyethylene glycol (PEG)- or electroporation- mediated uptake of naked DNA (see Paszkowski et al, 1984, EMBO J 3:2717-2722, Potrykus et al. 1985, Molec. Gen. Genet. 199:169-177; Fromm et al, 1985, Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto, 1989, Nature 338:274-276) and electroporation of plant tissues (D'Halluin et al, 1992, Plant Cell 4:1495-1505). Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al, 1990, Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al, 1988, Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al, 1990, Plant Cell 2:603-618).

According to the present invention, a wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present invention and the various transformation methods mentioned above. In preferred embodiments, target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthamum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g., Arabidopsis).

4.1.4.2. SCREENING OF TRANSFORMED PLANTS

AND PLANT CELLS

According to the present invention, desired plants may be obtained by engineering one or more of the gene constructs expressing a pathogen elicitor as described herein into a variety of plant cell types, including but not limited to, protoplasts, tissue culture cells, tissue and organ explants, pollens, embryos, as well as whole plants. In an embodiment of the present invention, the engineered plant material is selected or screened for transformants (those that have incorporated or integrated the introduced gene construct(s)) following the approaches and methods described below. An isolated transformant may then be regenerated into a plant. Alternatively, the engineered plant material may be regenerated into a plant or plantlet before subjecting the derived plant or plantlet to selection or screening for the marker gene traits. Procedures for regenerating plants from plant cells, tissues or organs, either before or after selecting or screening for marker gene(s), are well known to those skilled in the art.

A transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection may be performed by growing the engineered plant material on media containing inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., the β-glucuronidase, luciferase, B or Cl genes) that may be present on the recombinant nucleic acid constructs of the present invention. Such selection and screening methodologies are well known to those skilled in the art.

Physical and biochemical methods may also be used to identify plant or plant cell transformants containing the gene constructs of the present invention. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, SI RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis (PAGE), Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins. Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of the recombinant construct in specific plant organs and tissues. The methods for doing all these assays are well known to those skilled in the art.

4.1.5. SCREENING PLANTS AND PLANT CELLS

Plants, or preferably plant cells in suspension culture, expressing a mutagenized R gene may be screened directly for recognition of a pathogen elicitor molecule, e.g., a preparation of apoplastic fluid from an infected plant. In this particular embodiment, any number of elicitor molecules produced by a given pathogen can be used to screen for and detect wild type or mutagenized R genes having the desired specificity to recognize any of the pathogen elicitor molecules. For instance, screening comprises injecting the elicitor into plant leaves and detecting a difference in the hypersensitive response as compared to a control plant leaf. In another aspect of this embodiment, Agrobacterium cells carrying a T- DNA construct expressing a mutagenized R gene is injected into the plant leaves concurrently with the injection of the elicitor molecule. In yet another aspect of this embodiment, plant cells in suspension culture that have been transformed with a construct expressing a mutagenized R gene can be screened by adding apoplastic fluid from an infected plant to the culture medium. In this aspect, recognition of the elicitor molecule by the mutagenized R gene can be detected by monitoring the accumulation of reactive oxygen species in the culture medium by various methods known to those of skill in the art, see, e.g., Piedras et al, 1998, Molecular Plant-Microbe Interactions 11(12):1155-1166.

4.1.6. LOCALIZATION OF A PATHOGEN ELICITOR IN AN INFECTED PLANT

A plant pathogen elicitor may be derived from a variety of pathogens, for example, a virus, a bacterium, a fungus, an insect or a nematode. A pathogen elicitor from a pathogen may be capable of inducing a hypersensitive response in the infected plant following the recognition of the pathogen elicitor by a plant polypeptide, i.e., an R gene product. However, such a pathogen elicitor may be localized in an infected plant in different locations. For example, pathogen elicitors derived from bacteria and viruses are often found in the cytoplasm of plant cells. Pathogen elicitors derived from fungal pathogens, for example, are often found in the apoplastic space of a plant and not within the cytoplasm of plant cells. The localization of a plant pathogen elicitor in an infected plant may determine the type of R gene product, infra, by which said pathogen elicitor is bound, and thus detected, in the plant and by which a hypersensitive response is triggered in the plant. Therefore, in order to engineer a plant capable of invoking a hypersensitive response to a pathogen elicitor of interest, it is necessary to consider the location in the plant in which the pathogen elicitor is found following infection by the pathogen. For example, a pathogen elicitor that is concentrated in the apoplastic space is preferably bound by an R gene product that is capable of recognizing an elicitor found in the apoplastic space, e.g., a transmembrane receptor found in the cell membrane of a plant cell. Even more preferably, such a plant cell is found in an area of the plant where the apoplastic space contains sufficiently high concentrations of the pathogen elicitor to recognize an R gene product and induce a hypersensitive response. Or, where the pathogen elicitor of interest is concentrated in the cytoplasmic space of one or more cells of an infected plant, an R gene product capable of recognizing that pathogen elicitor is preferably capable of recognizing such a pathogen elicitor in the cytoplasmic space, e.g., a cytoplasmic receptor.

The localization of a pathogen elicitor of interest in an infected plant may be determined by a variety of methods. For example, one may raise antibodies specific for the pathogen elicitor and run a Western blot with total protein from different compartments of a plant infected with a pathogen from which the pathogen elicitor of interest is derived. Plant compartments may be chosen as desired, for example, one may examine total protein from the apoplastic space and from all or may plant cells of different types. Or, for example, one may chose total protein from the apoplastic space from different parts of the plant, e.g., root, leaves, etc., and from different cell types of the plant. Where the pathogen elicitor of interest is a nucleic acid, one may isolate total nucleic acids of the appropriate kind, i.e., DNA or RNA, from different parts of the plant, supra, and run a Southern blot (for DNA) or Northern blot (for RNA). The localization of a pathogen elicitor of interest in an infected plant may also be determined by immunohistochemistry (for polypeptide pathogen elicitors) or in situ hybridization (for nucleic acid pathogen elicitors).

Any method that is known in the art for determining the localization of a pathogen elicitor in an infected plant may be used. It may also be necessary to determine the localization of a plant pathogen elicitor in an infected plant at different stages, i.e., at different time points following first exposure of the plant to the pathogen. The engineered R gene of the invention should be capable of detecting a pathogen elicitor in a location in which the pathogen elicitor is found for a time and at a concentration sufficient to elicit a hypersensitive response. 4.2. PLANT RESISTANCE GENES

A variety of plant resistance genes, or R genes, have been isolated and characterized. However, the available R genes do not suffice to render plants resistant to all pathogenic challenges. Moreover, as pathogens evolve, the pathogenic challenge to plants changes over time, thus, making it necessary to develop new R genes that can meet these new challenges.

R genes encode polypeptides that are believed in the art to act as receptors for plant pathogen elicitors derived from plant pathogens. Following recognition of the R gene- encoded receptor to a pathogen elicitor, a response is triggered in the plant, e.g., a hypersensitive response. R gene products may be distinguished by a variety of criteria, e.g. , their overall domain structure and their location in a plant cell (e.g., in the cytoplasm, in the cell membrane of a plant cell, attached to the cell membrane of a plant cell). Similarly, pathogen elicitors can also be distinguished by their structure and the location in an infected plant where they are found, see, supra. R genes encode polypeptides that can be specified based on the presence of certain conserved domains and the organization of those domains. See, e.g., Baker et al, 1997, Science 276:726-733, which describes the domain structures of R gene polypeptides. Domains found in R gene polypeptides include a leucine-rich repeat domain (LRR), a leucine zipper domain (LZ), a nucleotide binding site domain (NBS), a transmembrane domain (TM), a domain with homology to cytoplasmic domains of the Drosophila Toll gene and the mammalian interleukin-1 receptor (IL-1R) gene (TIR) and a domain with homology to serine-threonine protein kinases, e.g., mammalian Raf kinase, IRAK kinase, Drosophila Pelle kinase. The R gene polypeptides, thus, can be distinguished based on the presence and organization of conserved domains. For example, some R gene polypeptides have an LRR domain which is believed to mediate protein-protein interactions.

Various R gene specific polynucleotides have been cloned and sequenced from different plant species. Characterized R gene polynucleotides include sequences designated RPM 1, Genbank accession No. X87851 (Grant et al, 1995, Science 269:843-846); N, Genbank accession No. U15605 (Whitham et al, 1994, Cell 78:1101-1115; U.S. Patent No. 5,571,706); L6, Genbank accession No. U27081 (Lawrence et al, The Plant Cell 7:1195- 1206); L, LI, L2, L3, L4, L5, L7, L8, L9, L10, LI 1 and LH, Genbank accession Nos. AF093638, AF093639, AF093642, AF093643, AF093644, AF093645, AF093646, AF093647, AF093648, AF093640, AF093641 and AF093649 (Ellis et al, 1999, The Plant Cell 11:495-506); RPPl-WsA, RPPl-WsB and RPPl-WsC, Genbank accession Nos. AF098962, AF098963 and AF098964 (Botella et al, 1998, The Plant Cell 10:1847-1860); RPP5, Genbank accession No. U97106 (Parker et al, 1997, The Plant Cell 9:879-894; WO 96/31608); RGC2A, RGC2B, RGC2C, RGC2D, RGC2E, RGC2F, RGC2G, RGC2H, RGC2I, RGC2J, RGC2K, RGC2L, RGC2M, RGC2N, RGC2O, RGC2P, RGC2Q, RGC2S, RGC2T, RGC2U, RGC2V and RGC2W, Genbank Accession Nos. AF072268, AF072267, AF072269, AF072270, AF072276, AF072277, AF072278, AF072279, AF072280,

5 AF072271, AF072272, AF072281, AF072282, AF072273, AF072274, AF072283,

AF072284, AF072275, AF072285, AF072286, AF072287 and AF072288 (Meyers et al, 1998, The Plant Cell 10:1817-1832); RPS2, Genbank accession No. U12860 (Mindrinos et al, 1994, Cell 78:1089-1099; WO 95/28423) and U14158 (Bent et al, Science 265:1856- 1860); PIC11, PICl l-1, PIC12, PIC13, PIC14, PIC15, PIC16, PIC17, PIC18, PIC19,

10 PIC20, PIC21 and ssCS4, Genbank Accession Nos. AF056150, AF056151, AF056152, AF056153, AF056154, AF056155, AF056156, AF056157, AF056158, AF056159, AF056160, AF056161, AF056150 and AF052399 (Collins et al, 1998, Molecular Plant- Microbe Interactions 11:968-978); Prf, Genbank accession No. U65391 (Salmeron et al, 1996, Cell 86:123-133); M, Genbank accession Nos. U73916 and U76370 (Anderson et al,

15 1997, The Plant Cell 9:641-651); 12, Genbank accession Nos. AF118127 (Simons et al, 1998, The Plant Cell 10:1055-1068) and A60534 (WO 97/06259); I2C-1, 12C-2, 12C-3 and I2C-4, Genbank accession Nos. AF004878, AF004879, AF004880 and AF004881 (Ori et al, 1997, The Plant Cell 9:521-532); Pto, Genbank accession No. U02271 (Martin et al,

1993, Science 262:1432-1435; U.S. Patent No. 5,648,599); Xa21, Genbank accession No. 0 U37133 (Song et al, 1995, Science 270:1804-1806); HMl, Genbank accession No. L02540

(Johal et al, 1992, Science 258:985-987); Hsl^{pro l}, Genbank accession No. U79733 (Cai et al, 1997, Science 275:832-834); Cf-2.1, Cf-2.2, Genbank accession Nos. U42444, U42445 (Dixon et al, 1996, Cell 84:451-459; WO 96/30518); Cf-4, Genbank accession No. AJ002235 (Parniske et al, 1997, Cell 91:821-832; WO 96/35790); Cf-4A, Genbank 5 accession No. Y12640 (Takken et al, Plant J. 14:401-411); Cf-5, Genbank accession No. AF05993 (Dixon et al, 1998, The Plant Cell 10:1915-1926); Cf-4/9, Genbank accession No. AJ002237 (Parniske, Cell, supra); Cf-9, Genbank accession Nos. U15936 (Jones et al,

1994, Science 266:789-793; WO 95/18230) and AJ002236 (Parniske, Cell, supra); Hcr2- 0A, Hcr2-0B, Hcr2-2A, Hcr2-5B and Hcr2-5D, Genbank accession Nos. AF053994, 0 AF053995, AF053996, AF053997 and AF053998 (Dixon, The Plant Cell, supra); Hcr9,

Genbank accession Nos. AF119040 and AF119041 (Parniske et al, Mol. Plant Microbe

Interact. 12:93-102).

R genes were shown to be effective in diverse backgrounds. For example, the Cf-9

R gene from tomato was effective in tobacco and potato in conferring resistance to its 5 cognate pathogen elicitor (Hammond-Kosack et al, 1998, The Plant Cell 10:1251-1266).

Or, for example, the Pto gene from tomato was effective in tobacco plants (Rommens et al. , 1995, The Plant Cell 7:1537-1544). Also, the tobacco N gene effectively conferred resistance in transgenic tomato (Whitham et al, 1996, Proc. Natl. Acad. Sci. USA 93:8776- 8781). Thus, plant R genes are widely useful and may be expressed in different species without losing their capacity to provide resistance to their cognate pathogens. Thus, in accordance with the methods of the invention, plant resistance genes may be engineered and identified that are capable of enhancing the pathogen resistance of a plant which is of a different variety, species, genus or family, as compared to the plant from which the original R gene was obtained. The methods of the present invention also provide a means to compensate for loss of effectiveness of a given R gene (e.g., in terms of either elicitor recognitional specificity or signal transduction capability) resulting from the transfer of the R gene to a plant different from the source plant of the original R gene. That is, mutagenized R genes may be obtained, using the methods of the invention, which in a different host plant retain recognitional specificity for a given pathogen elicitor and which function to evoke the hypersensitive response so as to provide pathogen resistance, as seen in the source plant with the unmutagenized R gene.

R genes of the present invention are obtained through engineering and isolation of new R genes that are capable of rendering a plant in which these R genes are expressed less susceptible to infection by a pathogen of interest. The engineering and identifying of the R genes of the present invention, in a preferred embodiment, is carried out by subjecting one or more R gene specific polynucleotides to mutagenesis. In another preferred embodiment, such mutagenesis is done through DNA shuffling. In a preferred aspect, such mutagenesis is random or essentially random. In another preferred aspect, such mutagenesis is carried out on a part of one or more R gene cDNAs wherein that part corresponds to a domain of the R gene product. In a most preferred embodiment, the mutagenesis of R genes containing a LRR repeat is targeted to the LRR domain encoding fragment or fragments of the one or more R gene cDNAs.

4.2.1. CLONING AND SEQUENCING OF PLANT RESISTANCE GENES Plant R genes may be cloned using the techniques known in the art of biotechnology as described for the cloning of pathogen elicitor cDNAs and genes. For example, a polypeptide encoded by an R gene may be isolated using protein purification techniques known in the art, supra, and the protein may be partially sequenced. Such protein sequence may be used to design a pool of oligonucleotides for the screening of a library, supra. Or, for example, antibodies may be raised against the R gene product for the screening of an expression library, supra. A preferred method of cloning R genes that are homologous to known R genes is polymerase chain reaction with degenerate oligonucleotide primers. Such primers may be designed based on protein sequence that is conserved between more than one R gene. The sequence region between these degenerate primers can be amplified in a polymerase chain

5 reaction. For background on the polymerase chain reaction cloning approach using degenerate oligonucleotide primers, see, e.g., Aarts et al, 1998, Mol. Plant Microbe Interact. 11:251-258; Aarts et al, 1991, Plant Mol. Biol. 16:647-661; Botella et al, 1997, Plant J. 12:1197-1211; Collins et al, 1998, Mol. Plant Microbe Interact. 11:968-978; Leister et al, 1998, Proc. Natl. Acad. Sci. USA 95:370-375; Leister et al, 1996, Nat. Genet.

10 14:421-429; Yu et al, 1996, Proc. Natl. Acad. Sci. USA 93:11751-11756.

Full-length clones for R genes for which only a partial polynucleotide sequence is available may be obtained using methods known in the art of biotechnology, see, e.g., supra. For example, a partial polynucleotide sequence may be used to make a probe which can be used to screen a library, for example, a cDNA library or a genomic library, from an

15 organism suspected or known to have and or express an R gene of interest.

4.2.2. MUTAGENESIS OF PLANT RESISTANCE GENE cDNAs

R genes of the present invention are engineered to encode a polypeptide that is capable of reducing the pathogenic effect of a pathogen of interest in a plant. In a preferred 0 embodiment, an R gene of the present invention is capable of recognizing a pathogen elicitor of interest. In one aspect, one or more cDNAs or genomic DNAs specific for R genes are subjected to mutagenesis in order to generate a library of novel R gene polynucleotides, i.e., mutagenized R genes. The term "mutagenized", when used in reference to a polynucleotide, as used herein, means a polynucleotide with a nucleotide 5 sequence that has been altered in any way and to any extent, including, but not limited to, substitutions, deletions and insertions of single nucleotides or stretches of nucleotides of any length. The term also includes variants of a polynucleotide or engineered polynucleotides. In a further aspect, a library of novel R gene polynucleotides is screened to isolate an R gene specific for a pathogen of interest, i.e., an R gene encoding a 0 polypeptide that is capable of recognizing the pathogen elicitor of interest.

4.2.2.1. DNA SHUFFLING

R genes of the present invention may be made through mutagenesis of one or more R genes using DNA shuffling. For general background on DNA shuffling, see, e.g., 5 Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91 :10747-10751; Crameri et al, 1998, Nature 391:288-291; U.S. Patent Nos. 5,837,458; 5,830,721; 5,811,238; 5,605,793; WO 98/31837; WO 98/27230; WO 98/13487; WO 97/35966; WO 97/35957; WO 97/20078; WO 95/22625.

In a preferred aspect, DNA shuffling is carried out by providing polynucleotides corresponding to the R gene sequences that are to be mutagenized, for example, by polymerase chain reaction (PCR). The purified polynucleotides (e.g., cDNA) are treated with DNase I for partial digestion and the resulting shorter fragments (e.g., about 50 to 100 nucleotides in length) are isolated and subjected to an additional round of PCR amplification, preferably without the addition of further primers, i.e., only the shorter fragments are used as templates and primers in the second PCR reaction. The final PCR products are isolated, e.g., through agarose gel electrophoresis (i.e., providing size selection). This protocol can be repeated if desired.

4.2.2.2. SITE DIRECTED MUTAGENESIS

R genes of the present invention may also be made using site directed mutagenesis methods known in the art. See, e.g., U.S. Patent Nos. 5,789,166; 5,556,747; 5,354,670. One may also introduce mutations into a predetermined region of an R gene cDNA by using class IIS restriction endonucleases as described, e.g., in U.S. Patent No. 5,512,463 or using chimeric mutational vector as described in U.S. Patent No. 5,731,181 and WO 99/07865.

4.2.2.3. RANDOM MUTAGENESIS

Various systems for randomly mutagenizing a polynucleotide are known in the art of biotechnology. For example, polynucleotides may be mutagenized by using a immunoglobulin hypermutation system. See, e.g., U.S. Patent No. 5,885,827. Another method for performing random mutagenesis is the use of low fidelity PCR amplification to introduce mutations into a polynucleotide, which mutations increase in number with the number of PCR cycles that are carried out.

4.2.2.4. MUTAGENESIS USING A LIBRARY OF

RANDOM OLIGONUCLEOTIDES R genes may also be mutagenized by replacing a stretch of nucleotides in an R gene cDNA with an oligonucleotide of a random sequence. In another aspect, one may replace more than stretch of nucleotides in an R gene cDNA with a random oligonucleotide. This allows the generation of a library of R gene cDNAs which have maximum sequence divergens in one or more regions. Any method of replacing a stretch of nucleotides of a known sequence with a stretch of nucleotides with a random sequence may be used. For example, one may synthesize an oligonuceotide comprising three sequence stretches, a stretch of about 10 to 30 nucleotides at the 5' and the 3' end that is complementary to two sequences in an R gene cDNA and that are separated in the R gene cDNA by up to about 200 nucleotides, more preferably up to about 100 nucleotides. In the oligonucleotide, the two complementary stretches are linked to each other by a stretch of random sequence of about equal length at the corresponding sequence in the R gene cDNA. Thus, when using the oligonucleotide in a PCR assay, one may generate a library of R gene cDNAs with a random sequence stretch.

4.2.2.5. DOMAIN SWAP MUTAGENESIS R genes of the present invention may also be generated through mutagenesis by domain swap. For example, stretches of nucleotide sequence of one R gene cDNA that is located at a position that is corresponding to a stretch of nucleotide sequence in another R gene may be swapped. Such a relationship may be determined by comparing the overall domain arrangement and sequence similarities of two such stretches of nucleotide sequence in two, or more, R gene cDNAs. See, e.g., U.S. Patent No. 5,728,803.

4.3. EXPRESSION OF PLANT RESISTANCE GENE cDNAs

R gene cDNAs may be expressed as described for pathogen elicitors, supra. Such expression may be accomplished by any means known in the art and in any cell type. R genes of the invention may be expressed for any purpose, for example, in order to raise antibodies against the R gene product, to screen for an engineered R gene that encodes a polypeptide that is capable of recognizing a pathogen elicitor of interest, etc.

4.4. SCREENING OF PLANT RESISTANCE GENE PRODUCTS A library of engineered R genes is screened to isolate one or more R genes encoding a polypeptide capable of enhancing resistance of a plant to a pathogen of interest and a pathogen comprising a pathogen elicitor of interest. Any one of a variety of screening methods may be used, for example, screening in transgenic plants, screening in cultured cells, screening through use of an expression library, screening through binding of a pathogen elicitor of interest or an R gene product linked to a solid support, etc.

4.4.1. SCREENING IN TRANSGENIC PLANTS

A library of mutagenized R genes may be screened in transgenic plants. These transgenic plants may express one or more mutagenized R genes and/or one or more plant elicitors. 4.4.1.1. SCREENING IN TRANSGENIC PLANTS

EXPRESSING A PATHOGEN ELICITOR OF INTEREST

In order to screen a library of mutagenized R genes, the plant pathogen elicitor of interest may be expressed in a transgenic plant line and the library of mutagenized R genes may be screened using the plant line expressing the pathogen elicitor. In a most preferred embodiment, mutagenized R genes are screened by expressing the pathogen elicitor of interest in a transgenic plant and by expressing one or more mutagenized R genes in that transgenic plant, preferably by transient expression (e.g., by using Agrobacterium mediated gene transfer). For example, the pathogen elicitor may be expressed in a plant of the species which is to be rendered more resistant to the pathogen comprising the pathogen elicitor of interest. In a preferred aspect, the pathogen elicitor is expressed in a transgenic plant in an inducible or a constitutive manner. Any promoter that is capable of directing the expression of the pathogen elicitor of interest in the plant of interest may be used, for example, an inducible or repressible promoter may be used to express the pathogen elicitor

⁵ of interest in a plant, for example, a tet operator promoter as described in Weinmann et al. ,

1994, The Plant Journal 5:559-569; or a glucocorticoid-inducible promoter as described in

McNellis et al, 1998, The Plant Journal 14:247-257; or an ethanol inducible promoter as described in Caddick et al, 1998, Nature Biotechnology 16:177-180. See, also, Gatz, 1995,

Methods In Cell Biology 50:411-424, which describes inducible and repressible gene ^υ expression systems for plants. Examples of constitutive promoters include, but are not limited to, the CaMV 35S promoter (Odell et al, 1985, Nature 313:810-812), the T-DNA mannopine synthetase promoter, and their various derivatives.

In another aspect, the pathogen elicitor is expressed in a transgenic plant so that it is found, preferably exclusively, in the apoplastic space of the plant. The pathogen elicitor

^{Δ J} polypeptide is preferably expressed as a fusion protein comprising the pathogen elicitor and a signal sequence, thus, directing the deposition of the pathogen elicitor in the apoplastic space. Any signal sequence that is capable of directing the deposition of the pathogen elicitor in the apoplastic space in the plant of interest can be used, for example, the PRlb signal sequence as described in Lund et al, 1992, Plant Molecular Biology 18:47-53; or the

30 PR- la, b and c signal sequences as described in Pfitzner et al, 1987, Nucleic Acids

Research 15:4449-4465.

A transgenic plant expressing a pathogen elicitor of interest may be used to screen for an engineered R gene product capable of recognizing the pathogen elicitor. For example, one can shotgun clone the engineered R gene cDNAs into a binary T-DNA expression vector and transform Agrobacterium with the vector. A transgenic plant expressing the pathogen elicitor of interest is then transiently infected by injecting a suspension of Agrobacterium comprising one or more colonies from the R gene library. If the engineered R gene product encoded in an Agrobacterium colony is capable of recognizing the pathogen elicitor of interest, a hypersensitive response will be observed in the plant expressing the pathogen elicitor and infected with that Agrobacterium colony. Additionally, for example, the R gene polynucleotides may be shotgun cloned into a plasmid vector and transformed into E. coli. DNA prepared from the resulting E. coli colonies may be applied to microprojectile beads. The R gene sequences are then transiently expressed in plants expressing the elicitor after introducing the genes by microprojectile transformation (see, e.g., Klein et al, 1988, Proc. Nat. Acad. Sci. USA 85:4305-4309, which describes microprojectile transformation).

A library of mutagenized R genes can be screened using transgenic plants expressing the pathogen elicitor (i.e., elicitor-plant) in an efficient manner. For example, more than one mutagenized R gene may be transiently expressed in the elicitor-plant in order to increase the efficiency of the screening process. A variety of methods for transient gene expression in plants may be used including, but not limited to, Agrobacterium mediated transient expression (see, e.g., Scofield et al, 1996, Science 274:2063-2065) and microprojectile particle bombardment. For example, a liquid suspension made from individual Agrobacterium clones containing T-DNA plasmids expressing an R gene polynucleotide may be injected into a leaf panel of an elicitor-plant. Most dicots, for example, have at least 12 panels per leaf, thus allowing the screening of at least about 200 R gene polynucleotides at the 20-leaf-stage of the elicitor-plant.

The efficiency of screening of R gene clones in elicitor-plants can, however, be significantly increased by pooling R gene clones. For example, if a pool of 10 R gene clones is injected into each leaf panel as described above, at least about 2,000 R gene polynucleotides can be screened in a single elicitor-plant at the 20-leaf-stage. Or, if 100 R gene clones are pooled, at least about 20,000 R gene polynucleotides can be screened using a single 20-leaf elicitor-plant. When screening R gene polynucleotides in pools, it is preferred to screen the individual R gene polynucleotide clones from each pool that tested positive, e.g., for a hypersensitive response detected through the observation of necrosis on the elicitor-plant.

Other ways to increase the efficiency of screening large numbers of R gene polynucleotides in one or more elicitor-plants include the pooling of such polynucleotides using a matrix. For example, one may pool a group of N mutagenized R genes in an elicitor-plant (N being an integer that is greater than 1) so that N² mutagenized R genes may be screened in 2xN R gene polynucleotide pools. Such a screening approach may be carried out by arranging the mutagenized R genes in a two dimensional matrix, e.g., a matrix with 100 columns and 100 rows which can represent 10,000 mutagenized R gene polynucleotides through numbers in the places where columns and rows intersect. Then, one pools 100 mutagenized R gene polynucleotides found in one column or in one row of the matrix and injects each pool into an elicitor-plant, thus, 200 R gene polynucleotide pools are needed to screen 10,000 mutagenized R gene clones. If one of the 10,000 mutagenized R genes in this example is capable of recognizing the pathogen elicitor of interest, two of the pools would be positive in this example (e.g., display a necrotic reaction, thus indicating a hypersensitive response), i.e., one pool representing the column and one pool representing the row of the matrix in which the mutagenized R gene capable of recognizing the pathogen elicitor is found. Thus, the intersection of the column and the row which correspond to the two positive testing pools would identify the mutagenized R gene capable of recognizing the pathogen elicitor of interest without the need for further screening.

4.4.1.2. SCREENING IN TRANSGENIC PLANTS

EXPRESSING AN ENGINEERED RESISTANCE GENE PRODUCT

A transgenic plant expressing one or more mutagenized R genes is generated as described for transgenic plants expressing a pathogen elicitor, supra, by an expression construct comprising a mutagenized R gene polynucleotide and the control elements necessary for expressing that polynucleotide in the plant of interest. The transgenic plants, and thus the mutagenized R genes expressed in those plants, are screened by introducing the pathogen elicitor of interest into the transgenic plants. A pathogen elicitor may be introduced into a transgenic plant, for example, by infecting the plant with a pathogen which comprises the pathogen elicitor of interest. Or, for example, one may introduce a polynucleotide construct capable of transiently expressing the pathogen elicitor of interest in the transgenic plants expressing said mutagenized R genes. For example, one may transiently express the pathogen elicitor of interest in the transgenic plants, expressing said mutagenized R genes, by introducing a viral vector expressing the elicitor (see, Hammond- Kosack, 1994, Proc. Nat. Acad. Sci. USA 91:10445-10449).

4.4.1.3. SCREENING IN PLANTS BY

CONCURRENTLY EXPRESSING AN ENGINEERED RESISTANCE GENE PRODUCT AND A PATHOGEN ELICITOR A library of mutagenized R genes can also be screened by expressing a mutagenized

R gene from an expression construct that also contains an expression cassette (e.g., open reading frame and regulatory sequences) for the pathogen elicitor. Thus, each construct would include a polynucleotide specific for one mutagenized R gene and a polynucleotide sequence specific for the pathogen elicitor, with each polynucleotide being linked to its own regulatory sequences. One or more constructs can be transiently expressed in a plant (for example by using Agrobacterium gene transfer), thus leading to a hypersensitive reaction if the mutagenized R gene product recognizes the pathogen elicitor as both, the R gene and the elicitor, are coexpressed in the same plant cell.

One may increase the efficiency of this approach by expressing a large number of R gene-elicitor pairs in one plant, for example by arranging the different constructs in a matrix as described for mutagenized R genes, supra.

4.4.1.4. ASSAYING FOR NECROSIS

When screening an R gene library using transgenic plants expressing the pathogen elicitor and/or one or more R gene polynucleotide, a preferred way of screening for a desired R gene polynucleotide is to detect necrosis in the plants. Necrosis can be detected visually by observing the local collapse and/or discoloration of the affected plant tissue with a magnifying glass, a microscope or by eye. Such discoloration may change the appearance of the affected plant tissue to grey, yellow or brown (see, e.g., Scofield et al, 1996, Science 274:2063-2065). In another embodiment, necrosis can be observed by the abolition of the expression of a marker gene that is also carried on the transiently expressed DNA construct, e.g., a bacterial β-glucuronidase marker (see, e.g., Mindrinos et al, 1994, supra). In yet another embodiment, necrotic cell death can also be observed using histochemical staining reactions. For example, dead cells can be stained through their uptake of a dye, e.g., trypan blue dye. For example, plant tissue is boiled in a lactophenol-trypan blue solution (23% phenol, 25% glycerol, 25% lactic acid, 2.5 mg/niL typan blue and 2 volumes of ethanol) for 2 minutes, then it is destained in chloral hydrate 2.5 g/mL overnight. The dead cells take up the dye and are readily visible by their blue color. Thus, blue colored cells indicate necrosis and a hypersensitive response and, therefore, the presence of an R gene product that recognizes a pathogen elicitor of interest. See, e.g., Keogh et al, 1980, Trans. Br. Mycol. Soc. 74:329-333, which describes trypan blue staining.

Also, necrotic cell death correlates with various biochemical events in the plant, for example, the deposition of autofluorescent compounds including, e.g., callose and lignin. Callose and lignin can be visualized in order to detect necrosis and a hypersensitive response, for example, by clearing the plant tissue in lactophenol followed by two rinses, first in 50% ethanol and then in water. An ultraviolet epifluorescence microscope is used to visualize the autofluorescence of these compounds. Callose deposition can be detected by staining the cleared tissue for 1 hour in a solution containing 0.01 % (w/v) of aniline blue and 0.15 M K₂HPO₄ followed by measuring autofluorescence with an ultraviolet epifluorescence microscope. See, e.g., Dietrich et al, 1994, Cell 88:685-694, which describes aniline blue staining and epifluorescence microscopy.

4.4.1.5. ASSAYING VIA HR INDUCIBLE PROMOTER SYSTEMS

Another preferred way of screening for a desired R gene involves use of a promoter- marker system, where the promoter is inducible by the presence or activation of a hypersensitive response (HR inducible promoter). The HR inducible promoter-marker system may be introduced into a plant either concurrently or separately with the introduction into the plant of the pathogen elicitor of interest and the plant resistance gene(s) which gene may or may not be mutagenized. Functional interaction of the plant resistance gene with the pathogen elicitor results in a hypersensitive response, which induces the HR inducible promoter resulting in expression of the marker (e.g., transient expression). Introduction of the marker system may be carried out using known transformation methods, as explained above, with microprojectile bombardment being a preferred method. The expression of the marker provides a basis to select desirable plant resistance genes.

General information on HR inducible promoters can be found in Marco et al, 1990, Plant Mol. Biol. 15:145-154; Gopalan et al, 1996, Plant J. 10:591-600; and Dorey et al, 1998, Mol. Plant Microbe Interact. 11 : 1102-1109. (Preferred HR inducible promoters are Hsr203J, Pontier et al, 1994, Plant J. 5:507-521; Hra32, Jakobek et al, 1999, Mol. Plant Microbe Interact. 12(8):712-719; and Hin 1, Gopalan et al, 1996, Plant J. 10:591-600.

Markers which may be expressed under control of HR inducible promoters include a variety of markers, preferably screenable markers, most particularly visual markers (including fluorescent markers, e.g., fluorescent protein markers, and chemiluminescent markers, e.g., luciferase markers). Preferred visual markers include β-glucuronidase (GUS), Jefferson et al, 1987, EMBO J. 6:3901-3907; and green fluorescent protein (GFP), Cubitt et al, 1995, Trends Bio. Sci. 20:448-455.

4.4.2. SCREENING IN CULTURED CELLS

A library of engineered R genes may be screened in cultured cells to identify an R gene product capable of recognizing the pathogen elicitor of interest. For instance, screening may be accomplished through monitoring of chemical changes evidencing the presence of a functioning R gene, e.g., pH changes or production of reactive oxygen species such as peroxide. Piedras et al., 1998, Molecular Plant-Microbe Interactions, 11(12), 1155- 1166. Also, any method suitable for detecting protein-protein interactions may be employed for identifying the desired R genes. The engineered R genes and the pathogen elicitor of interest may be expressed in cultured cells of any kind, preferably plant cells, as described for pathogen elicitors, supra. The pathogen elicitor may also be provided in the form of the pathogen which comprises the pathogen elicitor.

Among the traditional methods which may be employed to identify interactions between the pathogen elicitor and R gene products are co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns. In these methods, cell lysates or proteins obtained from cell lysates containing the engineered R gene products of the invention and the pathogen elicitor of interest are used to identify R gene products in the lysate that interact with the pathogen elicitor of interest. For these assays, the engineered R gene products of the current invention may be used in full length, or in truncated or modified forms or as fusion-proteins. Similarly, the R gene component may be a complex of two or more of the peptides and proteins of the current invention.

Once isolated, such an engineered R gene product can be identified and can, in turn, be used, in conjunction with standard techniques, to identify proteins with which it interacts. For example, at least a portion of the amino acid sequence of an intracellular protein which interacts with an R gene product of the current invention, can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique. (See, e.g., Creighton, 1983, "Proteins: Structures and Molecular Principles", W.H. Freeman & Co., N.Y., pp.34-49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such intracellular proteins. Screening may be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known. (See, e.g. , Ausubel, supra, and PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al, eds. Academic Press, Inc., New York). One method that detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. One version of this system has been described (Chien et al, 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commercially available from Clontech (Palo Alto, CA).

Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one plasmid consists of nucleotides encoding the DNA-binding domain of a transcription activator protein fused to a nucleotide sequence encoding the pathogen elicitor of interest, a modified or truncated form or a fusion protein thereof, and the other plasmid consists of nucleotides encoding the transcription activator protein's activation domain fused to a cDNA encoding an engineered R gene product of the invention which has been recombined into this plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene; the DNA-binding domain hybrid cannot because it does not provide activation function, and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product. By way of example, and not by way of limitation, the pathogen elicitor of interest may be used as the bait gene product. Genomic or cDNA sequences specific for the engineered R gene products are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a bait gene product, i.e., the pathogen elicitor of interest, fused to the DNA- binding domain are cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For example, and not by way of limitation, a bait gene sequence specific for the pathogen elicitor of interest can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA- binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.

A cDNA library specific for engineered R gene products of the current invention can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4. This library can be co-transfected along with the bait gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene. Colonies which express HIS3 can be detected by their growth on petri dishes containing semi-solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait gene-interacting protein using techniques routinely practiced in the art.

4.4.3 SCREENING THROUGH IN VITRO BINDING ASSAYS In vitro systems may be designed to identify engineered R gene products capable of recognizing the pathogen elicitor of interest, fragments thereof, and variants thereof. The principle of the assays used to identify engineered R gene products that bind to the pathogen elicitor of interest involves preparing a reaction mixture of the engineered R gene products of the current invention and the pathogen elicitor of interest under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed from and/or detected in the reaction mixture. The engineered R gene products of the current invention used can vary depending upon the goal of the screening assay.

The screening assays can be conducted in a variety of ways. For example, one method of conducting such an assay involves anchoring the engineered R gene products, or a fragment or fusion protein thereof, or the pathogen elicitor of interest, or a fragment or fusion protein thereof, onto a solid phase and detecting complexes between the two components (i.e., an R gene product and the pathogen elicitor of interest) anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the engineered R gene products may be anchored onto a solid surface, and the pathogen elicitor of interest, which is not anchored, may be labeled, either directly or indirectly. In another embodiment, the pathogen elicitor of interest is anchored onto a solid surface.

In practice, microtiter plates may conveniently be utilized as the solid phase. The anchored component may be immobilized by non-covalent or covalent attachments. Non- covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the component to be immobilized may be used to anchor the component to the solid surface. The surfaces may be prepared in advance and stored. Another example of a solid support useful for the screening of engineered R gene products is a filter, e.g., a nylon filter. For example, an expression library may be generated using techniques known in the art, supra, to express the engineered R gene products. Preferably, the expression library is plated out on soft agar plates. Proteins expressed in the expression library may be bound to a filter paper which may then be screened with the pathogen elicitor of interest (or an anti-idiotype antibody raised against the pathogen elicitor of interest). In a preferred aspect, the pathogen elicitor is labeled or a labeled antibody to the pathogen elicitor is used to identify complexes of the pathogen elicitor and engineered R gene products. Spots on the filter paper that give a signal are then correlated to clones on the plates on which the expression library is grown. The clones are then further characterized, i.e., sequenced, etc.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one component of complexes formed, like, for example, the engineered R gene products (or a known fragment of a fusion polypeptide comprising the engineered R gene products) or the pathogen elicitor of interest to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.

In another embodiment, one can raise anti-idiotype antibodies to the pathogen elicitor of interest and screen for engineered R gene products that interact with, e.g., bind to, the anti-idiotype antibodies. R gene products identified this way will then be further examined for their ability to recognize the pathogen elicitor of interest and for their ability to trigger a hypersensitive response in a plant species of interest. Any variation of any of the screening methods may also be used.

4.5. TRANSGENIC PLANTS EXPRESSING AN ENGINEERED PLANT RESISTANCE POLYNUCLEOTIDE

Transgenic plants are generated that express an engineered R gene of the present invention. A transgenic plant expressing an engineered R gene of the invention is less susceptible to the pathogenic effects of the pathogen of interest, e.g., by recognizing the pathogen elicitor to which the R gene product was engineered to correspond. In a preferred aspect, a hypersensitive response is triggered in a transgenic plant of the present invention following infection of the plant with a pathogen which comprises the pathogen elicitor of interest. Transgenic plants may be made using any of the techniques known in the art as described for plant pathogen elicitor expressing transgenic plants, supra.

Transgenic plants expressing one or more R gene polynucleotides capable of rendering said plants more resistant to a pathogen of interest may be from any plant species, plant genus, plant family, plant order, plant class, plant division of the kingdom of plants. See, e.g., U.S. Patent Nos. 5,889,189; 5,869,720; 5,850,015; 5,824,842; PP10,742; PP 10,704; PP 10,682, which recite plant species, genuses, families, orders, classes and divisions in which the R genes isolated using the methods of the invention may be used. Examples of plants are monocots, dicots, crop plants (i.e., any plant species grown for purposes of agriculture, food production for animals including humans, plants that are typically grown in groups of more than about 10 plants in order to harvest for any reason the entire plant or a part of the plant, e.g., a fruit, a flower or a crop, e.g., grain, that the plants bear, etc.), trees (i.e., fruit trees, trees grown for wood production, trees grown for decoration, etc.), flowers of any kind (i.e., plants grown for purposes of decoration, for example, following their harvest), cactuses, etc.

Further examples of plants in which the R genes made using the methods of the invention may be expressed include viridiplantae, streptophyta, embryophyta, tracheophyta, euphyllophytes, spermatophyta, magnoliophyta, liliopsida, commelinidae, poales, poaceae, oryza, oryza sativa, zea, zea mays, hordeum, hordeum vulgare, triticum, triticum aestivum, eudicotyledons, core eudicots, asteridae, euasterids I, rosidae, eurosids II, brassicales, brassicaceae, arabidopsis, magnoliopsida, solananae, solanales, solanaceae, solanum, nicotiana.

Also included are, for example, crops of particular interest including Solanaceae, including processing and fresh market tomatoes, pepper and eggplant; leafy plants, including lettuce and spinach; Brassicas, including broccoli, brussels sprouts, calabrese, cale, cauliflower, red cabbage and white cabbage; cucurbits, including cucumber, melon, watermelon, zucchini and squash; large seeded plants, including peas, beans and sweetcorn; rooted plants, including carrots and onions; vegetatively propagated plants, including berries, grapes, banana, pineapple and rosaceous fruit and nut crops; and tropical crops, including mango and papaya.

Thus, the invention has use over a broad range of plants including, but not limited to, species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Panneserum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Titicum, Vicia, Vitis, Vigna, and Zea.

4.5.1. POLYNUCLEOTIDE CONSTRUCTS FOR

EXPRESSION OF ENGINEERED R GENE IN TRANSGENIC PLANTS

A polynucleotide construct capable of directing the expression of an engineered R gene product in a transgenic plant of interest is constructed using general recombinant DNA and cloning techniques known in the art of biotechnology, see, e.g., Sambrook et al, supra; Ausubel et al, supra. Such a polynucleotide construct typically comprises a polynucleotide sequence that encodes an engineered R gene product and a regulatory polynucleotide sequence. Regulatory sequences useful for the polynucleotide construct of the invention include, but are not limited to, a promoter, an enhancer, an intron, a splice donor, a splice acceptor, a polyadenylation sequence, a RNA stability regulating sequence, or an element of any one of the above (e.g., promoter elements including, but not limited to, a TATA box).

The polynucleotide construct comprises one or more regulatory elements capable of directing the expression of the engineered R gene product of the invention. In a preferred aspect, the regulatory elements are capable of directing expression in a plant species in which expression of the engineered R gene product is desired. In another preferred aspect, the regulatory elements are capable of directing expression in a cell type in which expression of the engineered R gene product is desired in the plant species of interest.

Regulatory elements useful for the polynucleotide construct of the present invention are known to those of skill in the art, for example, promoter and enhancer elements of genes known to be expressed in the cell type and plant species of interest. A promoter useful for expression of the engineered R gene product in a cell type of a plant species of interest may also be isolated using routine experimentation, for example, by isolating a promoter region of a gene known to be expressed in the desired fashion. For example, one may screen a genomic library with a cDNA probe specific for the 5' end of a messenger RNA known to be expressed in the cell type of interest of the plant species of interest. Such a 5' end cDNA probe should preferably be only about 100 base pairs to about 300 base pairs so that the clones identified in the genomic library are likely to include the 5' end of the gene possibly including the promoter region of the gene for which the probe is specific. The promoter region typically includes about 1,000 to about 2,000 base pairs upstream of the transcription initiation site. Thus, a promoter useful for the expression of the engineered R genes of the present invention is a polynucleotide from about 2,000 base pairs upstream to about 50 base pairs downstream of the transcription initiation site of a gene known to be expressed in the cell type of interest in the plant species of interest, or is a portion of the polynucleotide. In order to facilitate the proper processing of the engineered R gene product, it may be necessary to include a nucleotide stretch that encodes a peptide sequence necessary for such processing. For example, a peptide sequence which facilitates the entry of the R gene product into the endoplasmatic reticulum may be necessary, i.e., signal sequence.

4.6. ASSAYS FOR TESTING AN ENGINEERED RESISTANT PLANT LINE

Plant lines generated using methods of the present invention that express an engineered R gene product of the invention are more resistant to the pathogenic effects of a pathogen comprising a pathogen elicitor of interest when compared to a plant line of the same species that does not express the engineered R gene product of the invention (i.e., a wild-type plant). The increased resistance of a plant line generated using methods of the invention may be assayed for by any technique known to the skilled artisan. For example, one may infect a plant of the generated plant line and a plant of a wild-type plant line with a pathogen comprising the pathogen elicitor of interest. Following such infection, the plant of the generated plant line will have at least an approximately 20% higher probability of surviving infection than the wild-type plant, more preferably at least about 40%, more preferably at least about 60% and most preferably at least about 80%.

Another way of testing an R gene made using the methods of the invention is by testing for necrosis inducing activity, for example, as described in Mahe et αl., 1998, J. Peptide Res. 52:482-494. Thus, one can express an engineered R gene made with the methods of the invention in a transgenic plant and infect the transgenic plant with the pathogen of interest or, for example, by applying a defined amount of the isolated pathogen elicitor of interest to the plant. For example, when applying from about 10 μL to about 100 μL of a solution containing the pathogen elicitor at a concentration of from about 0.03 μM to about 30 μM to the transgenic plant expressing the engineered R gene, one would observe clear necrosis or severe spreading necrosis in the transgenic plant but not in a wild- type plant of the plant line from which the transgenic plant was derived.

Necrotic cell death can also be observed using histochemical staining reactions in addition to visual inspection, for example, as described Section 4.4.1.4., supra.

The following examples are provided to further illustrate the current invention but are not provided to in any way limit the scope of the current invention. 5. EXAMPLES

5.1. EXAMPLE: DIRECTED EVOLUTION OF A RESISTANCE

GENE TO ENHANCE RESISTANCE TO PHYTOPHTHORA INFESTANS IN POTATO

5.1.1. PHYTOPHTHORA INFESTANS ENCODED PATHOGEN ELICITORS

Phytophthora infestans is a major pathogen for solanaceous crops. Currently, there is a need for a more effective way to introduce genetic resistance to P. infestans in cultivated potato. Some genetic resistance has been identified in wild relatives, but the process of breeding such resistence into cultivated varieties is extremely slow.

Most members of another genus within the solanaceae family, Nicotiana, are resistant to many Phytophthora species. The mechanism of resistance to Phytophthora has been extensively examined in tobacco plant. It has been demonstrated that the expression of certain 1 OkDa extracellular proteins by Phytophthora is required for a tobacco plant to be resistant. These extracellular proteins were collectively termed elicitins. It is believed in the art that an elicitin serves as a ligand for a specific plant receptor, i.e., an R gene product. Upon recognition of an elicitin by its cognate R gene product, a chain of signal transduction events is triggered that culminates in the activation of hypersensitive cell death (i.e., a hypersensitive response) and in signaling to adjacent cells, activating defense mechanisms.

Three forms of elicitins, designated INF1, INF2a and INF2b, are expressed by P. infestans (Kamoun et al, 1997, Mol. Plant Microbe Interact. 10:13-20; Kamoun et al,

1997, Mol. Plant Microbe Interact. 10:1028-1030). INF1 is most closely related to other known elicitins, while INF2a and INF2b are unique in having additional C-terminal domains of 67 and 71 amino acids, respectively. INF1 was shown to be recognized by resistance functions in Nicotiana benthamiana but not in potato plants (Kamoun et al,

1998, The Plant Cell 10:1413-1426).

In the art, all three elicitors, INF1, INF2a and INF2b, are believed to be accessible on the outside of the P. infestans cells, thus, they can interact with an R gene product in a P. t« e5tα«-?-infected plant. Therefore, an R gene that can recognize one or more of these elicitors INF1, INF2a and INF2b can be engineered and identified using the methods described herein.

5.1.2. POLYNUCLEOTIDES ENCODING INF1. INF2a AND INF2b The sequences of INF 1, INF2a and INF2b are published under Genbank accession

Nos. U50844, AF004951 and AF004952 (Kamoun et al, 1997, Mol. Plant Microbe Interact. 10:13-20; Kamoun et al, 1997, Mol. Plant Microbe Interact. 10:1028-1030). The polynucleotide sequences of INF 1, INF2a and INF2b are used to design oligonucleotide primers for amplification of the open reading frames by polymerase chain reaction (PCR). Also, two restriction sites are introduced through design of the primers, i.e., a Nco I restriction site is introduced at the ATG initiation codons and an Xba I site just after the termination codons of each open reading frame. The coding sequences are amplified from P. infestans strain 88069 (Kamoun et al, 1997, Mol. Plant Microbe Interact. 10:1028-1030) by standard PCR techniques.

5.1.3. DNA CONSTRUCTS FOR THE EXPRESSION OF

PATHOGEN ELICITORS IN THE APOPLASTIC SPACE IN TRANSGENIC PLANTS

The LNFl , INF2a and INF2b PCR products are digested with Nco I and Xba I and gel purified. Plasmid SRS200 is digested with Nco I and Xba I restriction enzymes and gel purified. SRS200 is a pUC18 plasmid vector that carries the following DNA fragments encoding the following functional elements: a Cauliflower Mosaic Virus 35S promoter (CaMV35S), a Chlorophyll ab 5' untranslated leader (CabL), a sequence encoding the tobacco PR- la signal peptide and a nopaline synthase 3' termination sequence (NOS3'). The Nco I site in pSRS200 occurs at the 3' end of the PRla signal peptide DNA sequence and the Xba I site is just 5' to the NOS3' element. Standard methods are used to ligate the SRS200 and INFl, INF2a or INFb fragments together, transform the ligation products into E.coli and screen for the desired clones carrying INF coding sequences as in-frame fusions to the PRla signal peptide. The clone carrying INFl is designated SRS201, the INF2a clone is designated SRS202 and the INF2b clone is designated SRS203.

The three clones are digested with Eco RI and Hind III, releasing the fragments containing CaMV35S, CabL, PR-la signal sequence, INFl, INF2a or INFb and NOS3' . The fragments are gel purified. The binary T-DNA vector WTT2161 (generated by removing an Eco RI site from the pWTT2144 vector described in WO 97/01952, thus leaving a single Eco RI site that is in the multiple cloning site) is digested with Eco RI and Hind III whereby a 18 kb fragment is released, which is also gel purified. The purified fragments are ligated and transformed into E.coli using standard methods. The desired binary T-DNA vector clones are identified by standard techniques (e.g., restriction endonuclease digests to check whether expected fragments are released, sequencing, etc.). The T-DNA clones carrying INFl, INF2a or INF2b are designated SRS204, SRS205 and SRS206, respectively. Purified DNA for each of the 3 T-DNA clones is electroporated into Agrobacterium turnefaciens strain LBA4404 (Hoekema et al, 1983, Nature 303:179-180), by standard methods.

5.1.4. GENERATION OF TRANSGENIC POTATO PLANTS CARRYING THE SRS204, SRS205 OR SRS206 CONSTRUCTS

Standard protocols for Agrobacterium mediated transformation of potato are utilized to generate transgenic potato plants carrying the SRS204, SRS205 and SRS206 constructs. RNA is prepared from the transgenic plants and analyzed by RNA blot analysis with INFl, INF2a and INF2b specific radiolabelled probes to identify lines expressing the expected transgene derived messenger RNAs. Apoplastic fluid ("AF") is prepared from the plants that express the expected INFl transcript. The AF from INFl transgenics and untransformed control plants is injected into the leaves of N benthamiana plants. The cell death that forms at the injection site of IΝF1 carrying AF, but not control AF, indicates that the transgenic potato plants produce biologically active IΝF1 protein that is targeted to the apoplastic space.

5.1.5. R GENE POLYNUCLEOTIDES FOR IN VITRO MUTAGENESIS

It is believed in the art that elicitins are likely recognized by receptors that are located in the cell membrane of plant cells and that recognize their pathogen elicitor ligands on the extracellular surface of those plant cells. This is based, in part, on the fact that all known elicitin polynucleotide sequences predict polypeptides with hydrophobic signal peptides. Furthermore, injecting elicitin containing AF into tobacco leaves is sufficient to trigger hypersensitive cell death. Finally, experiments using viral vectors to transiently express elicitin in tobacco show that a signal peptide is essential to trigger hypersensitive cell death. Because of the evidence that eliciting recognition occurs extracellularly in Nicotiana species it is believed that the R-genes encoding proteins which are found, at least in part, on the extracellular surface will be best suited for being mutagenized to recognize elicitin. The Cladosporium resistance genes ("Cf genes") of tomato are thought to recognize extracelluar ligands. Additionally, they are well suited because they are known to function in potato, as is desired in this application, and a number of related Cf gene sequences have been described in the art that will aid the process of directed evolution.

Five Cf genes that mediate resistance against different races of Cladosporium have been cloned: Cf-4, Cf-5, Cf-9 and two closely related Cf-2 genes. Additionally, for each of the above Cf genes additional homologous sequences, called Hcr's for "Homologues of Cladosporium resistance", are known. Polynucleotide sequences for Cf genes are available: Cf-2.1, Cf-2.2, Genbank accession Nos. U42444, U42445 (Dixon et al, 1996, Cell 84:451- 459; WO 96/30518); Cf-4, Genbank accession No. AJ002235 (Parniske et al, 1997, Cell 91:821-832; WO 96/35790); Cf-4A, Genbank accession No. Y12640 (Takken et al, Plant J. 14:401-411); Cf-5, Genbank accession No. AF05993 (Dixon et α/.,1998, The Plant Cell 10:1915-1926); Cf-4/9, Genbank accession No. AJ002237 (Parniske, Cell, supra); Cf-9, Genbank accession Nos. U15936 (Jones et al, 1994, Science 266:789-793; WO 95/18230) and AJ002236 (Parniske, Cell, supra); Hcr2-0A, Hcr2-0B, Hcr2-2A, Hcr2-5B and Hcr2-5D, Genbank accession Nos. AF053994, AF053995, AF053996, AF053997 and AF053998 (Dixon, The Plant Cell, supra); Hcr9, Genbank accession Nos. AFl 19040 and AFl 19041 (Parniske et al, Mol. Plant Microbe Interact. 12:93-102).

5.1.6. IN VITRO MUTAGENESIS OF R GENE POLYNUCLEOTIDES DNA shuffling is used to mutagenize Cf gene polynucleotides (Stemmer, 1994,

Proc. Natl. Acad. Sci. USA 91:10747-10751; Crameri et al, 1998, Nature 391:288-291; U.S. Patent Nos. 5,837,458; 5,830,721; 5,811,238; 5,605,793). PCR primers are designed to amplify the full-length coding sequences of each of the Cf genes and Hcr's. The primers for the 5' ends of each of the Cf and Her genes contain 20 bp of common sequence at their 5 ' ends and incorporate a Nco I restriction site at the ATG initiation codon. The primers for the 3 ' end of the genes contain 20 bp of common sequence at the 5 ' end of the primer, and incorporate an Not I site after the Cf or Her termination codons.

PCR amplifications are performed with Pfu DNA polymerase (Stratagene Inc., La Jolla, California, USA) to minimize errors. Each Cf and Her gene from tomato of genotypes Moneymaker Cf-0, Cf-2, Cf-4, Cf-5 and Cf-9 (obtained from the Tomato Genetics Center, University of California at Davis, Davis, California, USA) is amplified. The PCR products are purified on agarose gels. Approximately 2 μg of purified PCR product are subjected to limited digestion with 0.001 units/μL of DNase I (Sigma, St. Louis, Missouri, USA) for 20 minutes in a 100 μL reaction of 50 mM Tris-HCl, pH 7.5, 1 mM MgCl₂. Fragments of 50-100 bp are purified from a 2% agarose gel. The purified fragments are resuspended in IX PCR buffer at a polynucleotide concentration of 10 ng/ L. The fragments are then added to a PCR reaction without adding any primers. PCR amplification is carried out in an MJ Research PTC- 150 thermocycler (Cambridge, Massachusets, USA) using the following program: 94° C for 60 seconds; 40 cycles at 94° C for 30 seconds, 50-55° C for 30 seconds, and 72° C for 30 seconds. The duration of the extension step at 72° C is increased by 5 seconds after each amplification cycle. A 40-fold dilution of the first PCR reaction is added to a second PCR reaction, see, Section 5.1.7, infra.

5.1.7. TRANSFERRING THE MUTAGENIZED R GENE

POLYNUCLEOTIDES INTO A T-DNA EXPRESSION VECTOR

A second round of PCR amplification is conducted in which primers are added that anneal to the common sequences carried on the 5' and 3' primers originally used to amplify the Cf and Her sequences from tomato DNA, see, Section 5.1.6, supra. The thermocycle program for the second PCR amplification is 20 cycles at 94° C for 30 seconds, 50° C for 30 seconds, and 72° C for 2 minutes. The products of the second PCR reaction are digested with Nco I and Not I and gel purified. The collection of mutagenized products are then shotgun cloned into a Nco I and Not I digested pSRS 210. pSRS210 is a derivative of the binary plasmid pWTT2161 described in Section 5.1.3, supra, and it is constructed by creating a Not I site, using site directed mutagenesis (see, Sambrook et al, supra), between the Nco I site and the Xba I site in pSRS200 described in Section 5.1.3, supra. Then, this Not I version of pSRS200 is digested with Eco RI and Hind III to release a 1.6 kb fragment carrying the CaMV35S promoter, the CabL5' leader and the NOS3' termination sequence. This 1.6 kg Eco RI - Hind III fragment is cloned into the pWTT2161 vector that is digested with Eco RI and Hind III. Thus, pSRS210 plasmid contains of a binary vector designed so that a CaMV35S promoter will express polynucleotide fragments corresponding to the sequence in the expression vector from the Nco I site to the Not I site. This vector also provides a NOS3' termination element located downstream of the Not I site. The pSRS210 vector also contains a nopaline synthase promoted neomycin phosphotransferase (NPT11) gene for antibiotic selection using, e.g., neomycin.

The ligation products are electroporated directly into Agrobacterium strain LBA4404. 50,000 transformants are selected (using neomycin) on petri plates filled with Minimal A Media (1 L of Minimal A Media contains 10.5g K₂HPO₄, 4.5g KH₂PO₄, l.Og (NH₄)₂SO₄, 0.5g Sodium Citrate, 15g Agar, 20g Sucrose, 0.12 MgSO₄) containing tefracycline at a concentration of l g/mL. Each transformant is picked from the T-Min tefracycline plates and transferred to a well in a 96 well microtiter dish in which each well is filled with 100 μL of T-Min media containing 1 g/mL tefracycline and 20 μM acetosyringone. The plates are incubated at 28 °C for 2 days when the culture in each well has reached saturated density. A Beckman Biomek robot (Beckman Coulter, Fullerton, California, USA) is used to create a mixed inoculum containing 12 cultures from each row of the microtiter plates. Thus, 8 mixed inocula are needed to represent all transformants on a 96 well plate and 4,167 pools represent the entire 50,000 clones initially selected, supra. The mixed inocula are prepared by removing 50 L of the content of each of the 12 wells to be pooled, and pipetting it into a tube designated for one of the mixed inocula. The cells are pelleted by centrifuging for 1 minute at 10,000 RPM in an Eppendorf microfuge. The mixture of cells are resuspended in infiltration media, i.e., 0.1X Murashige and Skoog salts (ICN Biomedicals Inc., Costa Mesa, California, USA) 0.1X B5 vitamins, 20mM MOPS pH 5.4, 1% (w/v) glucose, 2% sucrose, 200 mM acetosyringone.

5.1.8. SCREENING THE MUTAGENIZED R GENES BY

AGROBACTERIUM-MEOIATEO TRANSIENT EXPRESSION IN TRANSGENIC POTATO LINES EXPRESSING INFl, INF2a OR INF2b

Each of the mixed pools of Agrobacteria, see, Section 5.1.7., supra, is transferred to a sterile syringe. The mixed inoculum is injected, using a syringe without a needle, into the abaxial surface of a single leaf panel of transgenic potato plants expressing INFl, INf2a or INF2b. On average, twelve separate pools are injected into each leaf.

5.1.9. IDENTIFICATION OF AGROBACTERIUM

TRANSFORMANTS EXPRESSING MUTAGENIZED R GENES CAPABLE OF INDUCING NECROSIS IN TRANSGENIC POTATO LINES EXPRESSING INFl, INF2a OR INF2b

Five days following injection, each site of injection is inspected visually for evidence of necrosis. The pools of Agrobacterium that are observed to give rise to necrosis are tested again. Then, the individual clones of each necrosis-positive pool are tested individually to identify the desired R gene polynucleotides.

5.1.10. SCREENING FOR MUTAGENIZED R GENES THAT DO

NOT REQUIRE INFl. INF2a or INF2b TO CAUSE NECROSIS

Undesired products of the R gene shuffling process are clones that cause necrosis in plant tissue without the expression of the pathogen elicitor INFl, INF2a or INF2b. These sequences are identified by their ability to cause necrosis when expressed in potato leaves that do not express INfl, INf2a or LNf2b. 5.1.11. TESTING THE FUNCTION OF MUTAGENIZED R GENE

PRODUCTS IN STABLY TRANSFORMED POTATO PLANTS

The T-DNA binary vectors carrying evolved R-genes that give INFl, INf2a or INF2b dependent necrosis are transformed into potato plants by standard Agrobacterium-

^ mediated transformation protocols. A kanamycin resistant callus is regenerated into stably transformed plants (see, Newell et al, 1991, Plant Cell Rep. 10:30-34, which describes the stable transformation of potato plants). Plants expressing a mutagenized R gene transgene are identified by injecting AF made from plants that express the INFl, INF2a or IN2b. The active transgenics are the plants that develop necrosis at the site of injection of the AF containing the INF species that the evolved R-gene recognizes. The active plants are tested for resistance to a range of P. infestans isolates known to express INFl, LNf2a and INf2b (Kamoun et al, 1997, Mol. Plant Microbe Interact. 10:1028-1030, which describes P. infestans that express INFl, INf2a, INf2b). An inoculum of 10,000 sporangiospores per mL is sprayed onto the leaves of plants expressing evolved R-genes and control plants. The

^ plants are grown in diffuse light conditions, in 100% relative humidity, at 16°C, and in 12 hour photoperiod. After seven days the control plants show massive lesions and abundant sporulation of P. infestans. However, the transgenic plants expressing evolved R-genes have small necrotic spots on the inoculated leaves, but are otherwise significantly more vigorous and show little or no evidence of sporulation. The detection of necrotic spots on

90 the infected leaf and the lack of disease symptoms throughout the plant indicate an increased resistance of the plant.

5.2. EXAMPLE: DIRECTED EVOLUTION OF TOMATO

RESISTANCE GENES TO RECOGNIZE THE C. FULVUM AVR4 ELICITOR

25

5.2.1. CLADOSPORIUM FUL VUM ENCODED PATHOGEN ELICITORS

Two elicitors encoded by C. fulvum have been cloned: Avr4, which is recognized by Cf-4 resistance gene, and Avr9, which is recognized by Cf-9. The mature form of Avr4 is 86 amino acids long, while the Avr9 peptide, in its mature form, is 28 ammo acids long. Features they share in common are that they are low molecular weight, cysteine-rich peptides that are secreted by C. fulvum.

In this example DNA shuffling is carried out using Cf-2.1, Cf-2.2, Cf-5 and Cf-9 and the corresponding Her homologs, and a shuffled polynucleotide is selected that encodes ³⁵ a polypeptide with recognitional specificity for Avr4. 5.2.2. CONSTRUCTINGA POLYNUCLEOTIDE ENCODINGAVR4

The nucleotide sequence of mature Avr4 is published under Genbank Accession No: Y08356 (Joosten et al, 1997, The Plant Cell 9:1-13). The published sequence is used to design specific primers to amplify the sequence encoding the mature form of Avr4 from 5 Cladosporium fulvum (Joosten et al., 1997). Restriction sites for Nco I and Xba I are introduced by the oligos as described in Section 5.1.2, supra.

5.2.3. DNA CONSTRUCTS FOR EXPRESSION OF AVR4 IN THE APOPLASTIC SPACE OF TRANSGENIC PLANTS

¹⁰ The Avr4 PCR product is digested with Nco I and Xba I and cloned into Nco I-Xba

I digested pSRS200 as described in Section 5.1.3., supra. This clone is designated pSRS207. pSRS207 is digested with EcoRl and Hindlll and the fragment containing CaMV35S, CabL, PR-la signal sequence, mature Avr4, and NOS3' is ligated into EcoRl- Hindlll digestedpWTT2161 as described in Section 5.1.3., supra. The resulting clone is designated pSRS208. Purified DNA for pSRS208 is electroporated into Agrobacterium tumefaciens strain LBA4404 by standard methods.

5.2.4. GENERATION OF TRANSGENIC NICOTIANIA

BENTHAMIANA PLANTS CARRYING THE SRS208 CONSTRUCT

20

Nicotiana benthamiana is transformed with pSRS208 by standard Agrobacterium- mediated transformation (Horsch et α/.,1985, Science 227:1229-1231). Plants expressing Avr4 mRNA are identified as described in Section 5.1.4., supra, and expression of active

Avr4 elicitor is confirmed by preparing AP from these transgenics and injecting it into

^Δ 7^J5 tomato plants carrying the Cf-4 gene. Active Avr4 peptide is indicated by detection a hypersensitive response through necrosis at the sight of injection.

5.2.5. R GENE POLYNUCLEOTIDES FOR IN VITRO MUTAGENESIS

30

Cf R gene polynucleotides and Her homologs are amplified from tomato as described in Section 5.1.5., supra.

35 5.2.6. IN VITRO MUTAGENESIS OF R GENE POLYNUCLEOTIDES

DNA shuffling is carried out as described in Section 5.1.6., supra, except that Cf-4 encoding sequences are not utilized.

5.2.7. TRANSFERRING THE MUTAGENIZED R GENE POLYNUCLEOTIDES INTO A T-DNA EXPRESSION VECTOR

The products of the first PCR are reamplified as described in Section 5.1.7., supra. The collection of mutagenized products is shotgun cloned in pSRS210, and electroporated into Agrobacterium strain LBA4404 as described in Section 5.1.1., supra. The Agrobacterium transformants are transferred individually into a well of a 96 sample microtiter plate. Each Agrobacterium clone is resuspended separately in 1 ml of infiltration buffer (see, Section 5.1.7., supra, for composition).

5.2.8. SCREENING THE MUTAGENIZED R GENES BY AGROBACTERIUM-MEDIATED TRANSIENT EXPRESSION IN TRANSGENIC N BENTHAMIANA PLANTS EXPRESSING AVR4

Each of the resuspended Agrobacterium clones is transferred to a sterile syringe and pressure infiltrated into the abaxial surface of a leaf panel of the transgenic N benthamiana plants previously identified as expressing active Avr4 peptide.

5.2.9. IDENTIFICATION OF A GROBA CTERIUM TRANSFORMANTS EXPRESSING MUTAGENIZED R GENES CAPABLE OF INDUCING NECROSIS IN TRANSGENIC N BENTHAMIANA PLANTS EXPRESSING AVR4

Five days following injection, each site of infiltration is inspected visually for evidence of necrosis. The Agrobacterium clones that are observed to give rise to necrosis are tested again.

5.2.10. SCREENING FOR MUTAGENIZED R GENES THAT DO NOT REQUIRE AVR4 TO CAUSE NECROSIS

This screen is carried out as described in Section 5.1.10., supra. 5.2.11. TESTING THE FUNCTION OF MUTAGENIZED R GENES PRODUCTS IN STABLY TRANSFORMED TV. BENTHAMIANA AND TOMATO PLANTS

The T-DNA binary vectors carrying evolved R-genes that give Avr4 dependent necrosis are stably transformed into N benthamiana (Hjorsh et. al, supra) and L. esculentum, cv. Moneymaker Cf-0 (Yoder et al, supra). The desired recognitional specificity of the N. benthamiana transformations is demonstrated by injecting Avr4 containing AP into leaf panels and observing the resulting necrosis. No necrosis is observed with AP prepared from material not expressing Avr4. Recognitional specificity of the tomato transformants is demonstrated by infiltration of Avr4 containing AP.

Additionally the specificity is demonstrated by infection with C. fulvum expressing Avr4. The detection of necrotic spots on the infected leaf and the lack of disease symptoms throughout the plant indicate an increased resistance of the plant.

The present invention is not to be limited in scope by the specific exemplified embodiments described herein, which are intended as illustrations of single aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. All publications, patents and patent applications cited herein are incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of screening for a plant resistance gene capable of enhancing the resistance of a plant of interest when exposed to a pathogen elicitor of interest comprising: (a) introducing into the plant of interest the pathogen elicitor of interest;

(b) expressing in the plant of interest one or more mutagenized plant resistance gene polynucleotides; and

(c) identifying one or more of said mutagenized plant resistance gene polynucleotides capable of enhancing the resistance of said plant of interest to said pathogen of interest; wherein steps (a), (b) and (c) are carried out using the same plant; wherein steps (a) and (b) can be carried out in either order; and wherein step (c) is carried out following steps (a) and (b).

2. The method of Claim 1 wherein said introducing of step (a) comprises infection of said plant of interest by a pathogen comprising said pathogen elicitor of interest.

3. The method of Claim 1 wherein said introducing of step (a) comprises introducing into the plant of interest a polynucleotide capable of expressing said pathogen elicitor of interest in order to create a transgenic plant.

4. The method of Claim 1 wherein said identifying of step (c) comprises detecting necrosis in the plant of interest generated in steps (a) and (b), said identifying being carried out by a method selected from the group consisting of visual observation, histochemical staining, trypan blue staining, detection of the deposition of autofluorescent compounds, detection of callose deposition and detection of lignin deposition.

5. The method of Claim 4 wherein said plant resistance gene polynucleotides are mutagenized using DNA shuffling.

6. The method of Claim 5 wherein said pathogen elicitor is a polypeptide.

7. The method of Claim 6 wherein said pathogen elicitor is expressed in the apoplastic space of said transgenic plant.

8. The method of Claim 7 wherein said polynucleotide capable of expressing said pathogen elicitor is expressed in a pathogen selected from the group consisting of a bacterium, a virus, a fungus, an insect and a nematode.

9. The method of Claim 8 wherein said one or more of the mutagenized resistance gene polynucleotides are expressed transiently in said transgenic plant expressing the pathogen elicitor.

10. A method of generating a transgenic plant comprising introducing an expressible plant resistance gene polynucleotide into cells of a plant, wherein said plant resistance gene polynucleotide is screened for using a method according to Claim 1.

11. The method of Claim 10 wherein said plant resistance gene polynucleotide is generated using a method according to Claim 4.

12. The method of Claim 10 wherein said plant resistance gene polynucleotide is generated using a method according to Claim 5.

13. The method of Claim 10 wherein said plant resistance gene polynucleotide is generated using a method according to Claim 7.

14. A method of generating a plant resistance gene capable of enhancing the resistance of a plant of interest to a pathogen of interest comprising:

(a) constructing a library of two or more mutagenized plant resistance gene polynucleotides; and

(b) screening said plant resistance gene polynucleotides for their ability to enhance the resistance of a plant of interest to said pathogen of interest when at least one of said mutagenized plant resistance gene polynucleotides is expressed in said plant of interest.

15. The method of Claim 14 wherein said plant resistance gene polynucleotides are mutagenized using DNA shuffling.

16. The method of Claim 15 wherein said screening is carried out in one or more plants made transgenic for a polynucleotide capable of expressing a pathogen elicitor expressed by said pathogen.

17. The method of Claim 16 wherein said screening is carried out by expressing, in the transgenic plant or plants expressing the pathogen elicitor, one or more of the mutagenized plant resistance gene polynucleotides of step (a) in Claim 14.

18. The method of Claim 17 wherein said screening comprises detecting necrosis in the transgenic plant or plants, said detecting being carried out by a method selected from the group consisting of visual observation, histochemical staining, trypan blue staining, detection of the deposition of autofluorescent compounds, detection of callose deposition and detection of lignin deposition.

19. The method of Claim 18 wherein said pathogen elicitor is a polypeptide.

20. The method of Claim 19 wherein said pathogen elicitor is expressed in the apoplastic space of said transgenic plant or plants.

21. The method of Claim 16 wherein said polynucleotide capable of expressing said pathogen elicitor is expressed in a pathogen selected from the group consisting of a bacterium, a virus, a fungus, an insect and a nematode.

22. The method of Claim 17 wherein said one or more of the mutagenized resistance gene polynucleotides is expressed transiently in said transgenic plant or plants expressing the pathogen elicitor.

23. A method of generating a transgenic plant comprising introducing an expressible plant resistance gene polynucleotide into cells of a plant, wherein said plant resistance gene polynucleotide is generated using a method according to Claim 14.

24. The method of Claim 23 wherein said plant resistance gene polynucleotide is generated using a method according to Claim 15.

25. The method of Claim 23 wherein said plant resistance gene polynucleotide is generated using a method according to Claim 17.

26. The method of Claim 23 wherein said plant resistance gene polynucleotide is generated using a method according to Claim 18.

27. A method of generating a plant resistance gene capable of enhancing the resistance of a plant of interest to a pathogen of interest comprising:

(a) identifying one or more plant resistance gene polynucleotides; and

(b) screening said plant resistance gene polynucleotides for their ability to enhance the resistance of said plant of interest to said pathogen of interest when at least one of said plant resistance gene polynucleotides is expressed in said plant.

28. The method of Claim 27 wherein said screening is carried out in one or more plants made transgenic for a polynucleotide capable of expressing a pathogen elicitor expressed by said pathogen.

29. The method of Claim 28 wherein said screening is carried out by expressing, in the transgenic plant or plants expressing the pathogen elicitor, one or more of the resistance gene polynucleotides of step (a) in Claim 27.

30. The method of Claim 29 wherein said screening comprises detecting necrosis in the transgenic plant or plants, said detecting being carried out by a method selected from the group consisting of visual observation, histochemical staining, trypan blue staining, detection of the deposition of autofluorescent compounds, detection of callose deposition and detection of lignin deposition.

31. The method of Claim 30 wherein said pathogen elicitor is a polypeptide.

32. The method of Claim 31 wherein said pathogen elicitor is expressed in the apoplastic space of said transgenic plant or plants.

33. The method of Claim 28 wherein said polynucleotide capable of expressing said pathogen elicitor is expressed in a pathogen selected from the group consisting of a bacterium, a virus, a fungus, an insect and a nematode.

34. The method of Claim 29 wherein said one or more of the resistance gene polynucleotides is expressed transiently in said transgenic plant or plants expressing the pathogen elicitor.

35. A method of generating a transgenic plant comprising introducing an expressible plant resistance gene polynucleotide into cells of a plant, wherein said plant resistance gene polynucleotide is generated using a method according to Claim 27.

36. The method of Claim 35 wherein said plant resistance gene polynucleotide is generated using a method according to Claim 29.

37. The method of Claim 35 wherein said plant resistance gene polynucleotide is generated using a method according to Claim 30.

38. The method of Claim 1 further comprising introducing into the plant of interest a screenable marker gene under the control of a hypersensitive response-inducible promoter, wherein said identifying is carried out by detection of expression of the screenable marker gene.

39. The method of Claim 38 wherein said screeenable marker gene expresses a visual marker.

40. The method of Claim 39 wherein said visual marker is β-glucuronidase or green fluorescent protein.

41. The method of Claim 17 wherein said screening comprises detecting expression of a screenable marker gene, in which expression of said screenable marker gene is under the control of a hypersensitive response-inducible promoter.