KR20010041277A - Cultivar specificity gene from the rice pathogen magnaporthe grisea, and methods of use - Google Patents

Abstract

The present invention relates to a novel non-toxic gene from Magnaporthe grisea, a rice blast pathogen. The gene AVRI-CO39 confers strain specific nontoxicity to the Magnaporte Grisia strain carrying this gene. The present invention also relates to a method of improving the resistance of rice to rice pathogens using this gene and the product encoded by the gene.

Description

Variety specific genes from the rice pathogen Magnaporte Griscia and methods of using them {CULTIVAR SPECIFICITY GENE FROM THE RICE PATHOGEN MAGNAPORTHE GRISEA, AND METHODS OF USE}

Rice is a staple food for about two-thirds of the world's population. More than 90% of the world's rice is grown and consumed in developing countries. Rice blasts caused by the fungus Magnaporte Grisia are threatening to rice farming worldwide. The disease can reduce the yield of infected areas by 10-30%. Rice blasts continue to be a problem in rice growing areas in the southern United States. This is also a serious problem in California's rice growing areas.

Gene-for-gene hypothesis has been proposed to explain the very specific disease resistance / sensitivity relationships that often exist between species of plant pathogens and varieties of their host species. Genetic theories are known to be applicable to many host-pathogen interactions, including the interaction of Magnaporte Grisia, a rice blast fungus, with its host Oryza sativa. The ability to understand and manipulate these host-pathogen interactions is an important practical concern because Magnaporte Grisia can quickly overcome the new disease resistance of rice soon after its activity. Magnaporte Grisias also exist as a complex genus that contains many subspecific groups that differ in their host range but are not fertile. It is frequently found that some of these alternating hosts grow in close proximity to or in alternation with rice, and these different subspecific groups, because magnaportecria isolates that infect alternating hosts can also sometimes infect rice. It is of great interest to plant growers how evolutionarily they interact with each other.

Genetic gene resistance (also known as hypersensitivity resistance (HR) or rice specific resistance) depends on the specific recognition of pathogens that have invaded this plant. Many individual plant genes have been identified that regulate gene resistance. These genes are called resistance (R) genes. The function of a particular R gene depends on the genotype of the pathogen. When the expression of a pathogen gene causes the pathogen to produce a signal that triggers a strong defensive response in a plant with the corresponding R gene, the pathogen gene is called the Avr gene. This response was not observed in the absence of the Avr gene or the corresponding R gene in the plant. It should be noted that a single plant can have many R genes and a single pathogen can have many Avr genes. However, strong resistance occurs only when the Avr gene and its specific R gene match in host-pathogen interactions. In this example, resistance generally occurs as the activity of the HR response, where cells immediately after infection undergo planned necrosis to prevent further progression of pathogens into living plant tissue. Other features of the resistance response also include antimicrobial metabolites or pathogen-inhibiting enzymes, enhancement of plant cell walls at infected sites, and induction of signal transduction pathways that cause systemic acquired resistance (SAR) in plants.

The molecular basis of host-breed specificity and pathogenic variability in Magnaporte Grisia may begin to be identified using identification, mapping, and in some cases, cloning of specific Avr genes from pathogenic isolates of Magnaforte Grisia. It is only. For example, AVR2-YAMO (breed specificity) and PWL2 (host specificity) [Valent & Chumley, pp. 3.113-3.134 in Rice Blast Disease (R. Zeigler, S.A. Leong, P. Teng, Eds.), Wallingford: CAB International, 1994] all function as standard non-toxic genes by preventing infection of specific varieties or hosts. AVR2-YAMO encodes a 223-amino acid protein homologous to proteolytic enzymes, whereas PWL2 encodes a glycine-rich 145-amino acid polypeptide. Based on the predicted amino acid sequence of the protein, both can be secreted.

The fauna of both AVR2-YAMO and PWL2 appears to be widely distributed in other herbaceous plants infected with rice and Magnaporte greece isolates, so that Magnaporte greece isolates without rice infection are still present for rice. It may have a host or breed specific gene. In some cases, the AVR2-YAMO and PWL2 epithelial are functional and exhibit the same host or breed specificity as AVR2-YAMO or PWL2.

As another example of a potentially useful Avr gene, the cultivar specific gene AVR1-CO39, which determines nontoxicity on rice cultivar CO39, has been identified [Valent et al., Genetics 127: 87-101, 1991] Magnaforte Gricia Maps are located on chromosome 1 of Smith. Smith & Leong, Theor. Appl. Genet. 88: 901-908, 1994]. One site of chromosome 1 that appears to contain the AVR1-CO39 gene was isolated and cloned into a cosmid vector. Leong et al., Pp. 846-852 in Rice Genetics III, Proceedings of the Third Annual Rice Genetics Symposium, G.S. Khush, Ed., Island Harbor Press, Manila, 1996; To date, however, the gene itself has not been identified and characterized.

Ultimately, the availability of cloned varieties and host specific genes from Magnaporte Griscia and the corresponding R genes from rice provide a useful tool for manipulating and increasing resistance to this pathogen in cropland. Accordingly, it is an object of the present invention to provide a newly cloned Magnaporte Griscia variety specific gene, AVR1-CO39, and its functional moor for such use.

Summary of the Invention

According to one aspect of the present invention, the present invention provides AVR1-CO39, a nucleic acid isolated from Magnaportecria, which confers fungal plant pathogens containing the nucleic acid to rice cultivar CO39-specific nontoxicity. This nucleic acid is preferably sequence I.D. No. Include part or all of 1 or the sequence I.D. No. Hybridize with one or some or all of its complementary sequences.

According to another aspect of the invention, the invention provides a polypeptide encoded by some or all of the isolated nucleic acids of claim 1. Preferably, each of these polypeptides is represented by SEQ ID NO. It was selected from the group of polypeptides encoded by ORFs 1, 2, 3, 4, 5, 6, and 7 corresponding to 2, 3, 4, 5, 6, 7 and 8.

According to another aspect of the invention, the invention provides a transgenic engraftment bacterium, which is effective in conferring rice varieties CO39-specific nontoxicity to microorganisms expressing this gene by expressing the AVR1-CO39 gene site. . Preferably, this transgenic engraftment bacterium is SEQ ID NO. Express ORF3 of 1 or a functionally equivalent site thereof.

According to another aspect of the invention, the invention provides a method for improving the range of resistance of rice cultivar CO39 plants to pathogenic microorganisms. The method involves treating the plant with a engraftment bacterium that expresses a portion of AVR1-CO39 that is effective in triggering expression of the CO39-specific R gene in the plant.

According to another aspect of the invention, the invention provides a second method of improving the range of resistance of rice cultivar CO39 plants to pathogenic microorganisms. The method includes treating the plant with a protein extract comprising a polypeptide produced by the expression of AVR1-CO39 in an amount effective to trigger expression of the CO39-specific R gene in the plant.

These and other features and advantages of the present invention will be described in more detail in the description and examples set forth below.

The present invention relates to the field of disease resistance in plants. Specifically, the present invention provides a method for improving rice resistance to this pathogen by using a novel non-toxic gene from Magnaporte Grisia, a rice blast pathogen, and a product encoded by the gene.

I. Definition

Various terms relating to the biological molecules of the invention have been used above and also throughout the specification and claims. The terms "substantially the same", "percent similarity" and "percent identity" are defined in detail below.

For nucleic acids of the invention, the term "isolated nucleic acid" is often used. The term, when applied to DNA, refers to a DNA molecule isolated from a sequence that is simultaneously adjacent (in the 5 'and 3' directions) to the naturally occurring genome of the derived organism. For example, an “isolated nucleic acid” may include a DNA molecule inserted into a vector, such as a plasmid or viral vector, or a DNA molecule integrated into the genome DNA of a prokaryotic or eukaryotic cell. "Isolated nucleic acid molecule" may also include cDNA molecules.

For RNA molecules of the present invention, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term is present in its "substantially pure" form (the term "substantially pure" is defined below) sufficiently separated from the RNA molecule involved in its natural state (ie, cellular or tissue state). RNA molecules can also be referred to.

For proteins, the terms "isolated protein" or "isolated and purified protein" are often used herein. This term mainly refers to a protein produced by the expression of an isolated nucleic acid molecule of the invention. Alternatively, the term may refer to a protein that is sufficiently separated from other naturally related proteins and exists in "substantially pure" form.

The term "substantially pure" refers to a sample comprising at least 50-60% by weight of a compound of interest (eg, nucleic acid, oligonucleotide, protein, etc.). More preferably, the sample comprises at least 75%, most preferably 90-99%, by weight of the compound of interest. Purity was determined by appropriate methods (eg, chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.) for the compounds of interest.

For the antibodies of the invention, the term “immunologically specific” binds to one or more epitopes of one protein of interest, but does not recognize or bind another molecule in a sample comprising a mixed group of antigenic biological molecules. Say.

For oligonucleotides, the term “specifically hybridizes” refers to the association between two single-stranded nucleotide molecules having sequences that are sufficiently complementary to permit such hybridization under predetermined conditions commonly used in the art. Sometimes called "substantially complementary"). Specifically, the term refers to hybridization of oligonucleotides with substantially complementary sequences contained within single stranded DNA or RNA molecules of the invention and substantially excludes hybridization of single stranded nucleic acids and oligonucleotides of non-complementary sequences. .

The term “inoculated with pathogens” refers to the inoculation of plants with the pathogen.

The term "disease defense response" refers to a change in metabolism, biosynthetic activity or gene expression that enhances a plant's ability to inhibit the replication and spread of microbial pathogens (ie, resist microbial pathogens). Examples of plant disease defense responses include, but are not limited to, antimicrobial activity (also called phytoalexin) and induction of expression of defense (or defense-related) genes, for example, their products include peroxy Multiases, cell wall proteins, proteolytic enzyme inhibitors, hydrolases, disease-associated (PR) proteins and phytoalexin biosynthetic enzymes such as phenylalanine ammonia lyase and chalcone synthase. This protective response appears to be induced in plants by several signal transduction pathways, including secondary defense signaling molecules produced in plants. Agents that induce disease defense responses in plants include (1) microbial pathogens such as fungi, bacteria and viruses; (2) microbial components such as proteins and protein fragments and other protective reaction derivatives, small peptides, β-glucans, erycithin and harpins, cryptotoine and oligosaccharides; And (3) secondary defense signaling molecules produced by plants such as salicylic acid, H ₂ O ₂ , ethylene and jasmonate.

The term "promoter site" refers to the 5 'regulatory region of a gene.

The term “reporter gene” refers to a genetic sequence that is regulateably linked to a promoter site to form a transgene, and the expression of the reporter gene coding site is regulated by the promoter and the expression of the transgene is readily analyzed.

The term “selection marker gene” refers to a gene product that, when expressed, confers a selectable phenotype, eg, antibiotic resistance, on a transformed cell or plant.

The term "regulatoryly linked" means that the regulatory sequences necessary for the expression of the coding sequence are located in the DNA at an appropriate position relative to the coding sequence so that the coding sequence is expressed. This same definition sometimes applies to the arrangement of coding sequences and transcriptional regulatory elements (eg, promoters, amplifiers and termination elements) in expression vectors.

The term “DNA preparation” refers to the genetic sequence used to transform a plant and produce progeny transgenic plants. Such preparations can be administered to plants while in viral or plasmid vectors. It is also contemplated that other methods of delivery, such as Agrobacterium T-DNA mediated transformation and transformation using a biolistic process, are within the scope of the present invention. Transformed DNA can be prepared according to standard protocols as described in "Current Protocols in Molecular Biology", eds, Frederick M. Ausubel et al., John Wiley & Sons, 1998.

II. Description of AVR1-CO39 and its Encoded Peptides

In accordance with the present invention, the novel Magnaporte Grisia nontoxic gene was isolated and cloned. This gene was referred to herein as AVR1-CO39, which indicates its function as a gene that imparts breed specific interactions to rice cultivar CO39. The cloning of the AVR1-CO39 gene from Magnaporte Grisia strain 2539 and analysis of this gene are described in detail in Example 1. The gene contains four open reading frames, one of which (ORF3) appears to play a key role in giving breed-specific nontoxicity to the Magnaporte isolates that carry the gene. Mammals of the strain 2539 isolate AVR1CO39 gene have been identified in different arrangements of other Magnaporte isolates.

The genome clone of AVR1-CO39 from Magnaporte Grisia strain 2539 (typical AVR1-CO39 of the present invention) is described in detail herein and its nucleotide sequence is shown in Example 1 according to the sequence I.D. No. Described as 1. SEQ ID NO: I.D. No. 1 contains four open reading frames. It is believed that one or more of these open reading frames are involved in imparting non-toxicity to the breed CO39 by gene products expressed from the open reading frames or by retention of critical transcriptional or translational control sequences (see Example 1).

Although genome clones of AVR1-CO39 from Magnaporte Grisia isolate 2539 have been described and illustrated herein, the present invention is similar enough to be used in place of isolate 2539 AVR1-CO39 nucleic acid and protein for the purposes described below. It is intended to include nucleic acid sequences and proteins from Magnaporte isolates. These are sequence I.D. No. Variants on alleles of 1 and natural mutants include, but are not limited to, these are likely to be found in any given group of Magnaporte isolates. Since these variants are expected to have certain differences in nucleotide and nucleic acid sequences, the present invention provides a sequence I.D. No. At least about 60% (preferably 70%, more preferably at least 80%) to the nucleotide sequence and the coding site shown as 1 (and most preferably specifically comprising the coding site of SEQ ID NO. An isolated AVR1-CO39 nucleic acid molecule with sequence homology is provided. The invention also relates to the sequence I.D. No. 1 provides an isolated polypeptide product of the open reading frame, each of which is SEQ ID NO. No. Has at least about 60% (preferably 70% or 80% or more) sequence homology with an amino acid sequence of 2, 3, 4, 5, 6 or 7. Because natural sequence changes appear to be present between the AVR1-CO39 genes, those skilled in the art will maintain about 30-40% of the AVR1-CO39 gene and encoded polypeptide, while still maintaining the unique properties of the polypeptide. It would be expected to find nucleotide sequence changes. This expectation is partly due to the degeneracy of the genetic code as well as the known evolutionary consequences of conservative amino acid sequence changes that do not significantly change the properties of the encoded protein. Accordingly, such variants are considered to be substantially identical to each other and are included within the scope of the present invention.

For the purposes of the present invention, the term "substantially identical" refers to a nucleic acid sequence or amino acid sequence having a sequence change that does not substantially affect the properties of the protein (ie, the structure and / or biological activity of the protein). The term “substantially identical,” in particular with respect to nucleic acid sequences, is intended to refer to conserved sequences that control coding sites and expression and mainly degenerates codons encoding the same amino acids or encodes replacement amino acids conserved in the encoded polypeptide. To change the codon. With respect to the amino acid sequence, the term “substantially identical” generally refers to conservative substitutions and / or changes in the site of the polypeptide that do not affect the structure or function. In this specification, the terms "percent identity" and "percent similarity" are used in the comparison between amino acid sequences. These terms are intended to be defined as in the UWGCG sequence analysis program available from the University of Wisconsin (Devereaux et al., Nucl. Acids Res. 12: 387-397, 1984).

The following description describes the general procedure involved in the practice of the present invention. Reference to particular materials is for illustrative purposes only and is not intended to limit the invention. Unless otherwise specified, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989) (hereinafter referred to as “Sambrook et al.”) Or Ausubel et al. (Eds) Current Protocols in Molecular Biology, The general cloning method as described in John Wiley & Sons (1998) (hereinafter referred to as "Ausubel et al.") Was used.

A. Preparation of AVR1-CO39 Nucleic Acid Molecules Encoding, Encoded Polypeptides and Antibodies Specific to the Polypeptides

1. Nucleic Acid Molecules

The AVR1-CO39 nucleic acid molecules of the invention can be prepared by two general methods: (1) synthesis from appropriate nucleotide triphosphates, or (2) separation from biological sources. Both methods use protocols well known in the art.

SEQ ID NO: I.D. No. The availability of nucleotide sequence information, such as genome isolates with 1, enables the production of the isolated nucleic acid molecules of the present invention by oligonucleotide synthesis. Synthetic oligonucleotides can be prepared by phosphoramadite performed on an Applied Biosystems 38A DNA synthesizer or similar equipment. The preparation obtained can be purified according to methods known in the art such as high performance liquid chromatography (HPLC). Long double stranded polynucleotides, such as the DNA molecules of the present invention, must be synthesized in several steps due to the size limitations inherent in current oligonucleotide synthesis methods. Thus, for example, a 1.05 kb double stranded molecule can be synthesized as several smaller fragments with appropriate complementarity. The complementary fragments thus produced can be annealed with each fragment having a sticky end suitable for attachment of adjacent fragments. Adjacent fragments can be joined by annealing the sticky ends in the presence of DNA ligase to make an entire 1.05 kb double stranded molecule. The synthetic DNA molecule thus prepared can then be cloned into an appropriate vector and amplified.

AVR1-CO39 genes can also be isolated from appropriate biological sources using methods known in the art. In one embodiment, the genome clone is isolated from Magnaporte Grisia strain 2539 cosmid library. In another embodiment, cDNA clones comprising one or more open reading frames of the genome AVR1-CO39 locus can be isolated.

According to the invention, the sequence I.D. No. Nucleic acids having an appropriate level of sequence homology with some or all of the coding region of 1 can be identified using hybridization and appropriately stringent wash conditions. For example, hybridization can be performed using 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 μg of denatured and fragmented salmon sperm, according to the method described in Sambrook et al. It can be carried out using a hybridization solution comprising DNA, 0.05% sodium pyrophosphate and at least 50% formamide. Hybridization was performed at 37-42 ° C. for at least 6 hours. Following hybridization, the filters were washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes to 1 hour at 37 ° C. in 2 × SSC and 0.1% SDS; (4) 2 h at 45 ° C. to 55 ° C. in 2 × SSC and 0.1% SDS, changing solution every 30 minutes. Alternatively, the Amasino hybridization protocol (Anal. Biochem. 152: 304-307) is preferred for use in the present invention and is described in more detail in Example 1.

One universal formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules with specific sequence homology is as follows: Sambrook et al., 1989):

T _m = 81.5 ° C. + 16.6 Log [Na ⁺ ] +0.41 (% G + C) -0.63 (% formamide) -600 / # bp at double strand

As an example of this formula, the T _m obtained using [Na ⁺ ] = [0.368] and 50% formamide, 42% GC content and average 200 base size probe is 57 ° C. A 1 ° C. to 1.5 ° C. decrease in Tm of the DNA double strand reduces homology by 1%. Thus, objects with sequence identity of at least about 75% can be observed using a hybridization temperature of 42 ° C.

Nucleic acids of the invention can be maintained as DNA in any suitable cloning vector. In a preferred embodiment, the clone is maintained in a plasmid cloning / expression vector such as pGEM-T [Promega Biotech, Madison, WI] or pBluescript [Stratagene, La Jolla, CA], both vectors being suitable E. coli hosts. Proliferate in cells.

AVR1-CO39 nucleic acid molecules of the present invention include cDNA, genome DNA, RNA, and fragments thereof, which may be single stranded or double stranded. Thus, the present invention provides the sequence I.D. No. Provided are oligonucleotides (sense or antisense strand of DNA or RNA) having a sequence that can hybridize with at least one sequence of a nucleic acid molecule of the invention, such as a selected fragment of cDNA having 1. Such oligonucleotides may be used to detect positive or negative regulation of the expression of AVR1-CO39 in or prior to translation of mRNA into or retrieval of the AVR1-CO39 gene or mRNA in test samples of fungal isolates, for example by PCR amplification. It is useful as a probe for.

2. Protein

The AVR1-CO39 genome isolates described herein comprise four open reading frames (ORF's 1-4), each of which infers their deduced amino acid sequences. No. It is described herein as 2 to 5. Any one of these polypeptides can be prepared by various methods according to known methods. If produced in situ, the polypeptide can be purified from a suitable source, such as a fungal isolate.

Alternatively, the availability of nucleic acid molecules encoding this polypeptide allows for the production of proteins using in vitro expression methods known in the art. For example, the cDNA or gene can be cloned into an appropriate in vitro transcription vector such as pSP64 or pSP65 for in vitro transcription and then cell-free translation can be performed in a suitable cell-free translation system such as wheat seed or rabbit reticulocytes. . In vitro transcription and translation systems are commercially available, for example, from Promega Biotech, Madison, Wisconsin or BRL, Rockville, Maryland.

According to a preferred embodiment, expression in an appropriate prokaryotic or eukaryotic system can produce higher amounts of polypeptide encoded in AVR1-CO39. For example, the sequence I.D. No. Some or all of the DNA molecules, such as cDNA with 1, are inserted into plasmid vectors suitable for expression in bacterial cells (such as Escherichia coli) or yeast cells (such as Saccharomyces cerevisiae) or insect cells. It can be inserted into baculovirus for expression in E. coli. These vectors contain regulatory elements essential for the expression of DNA in the host cell, and are positioned in such a way as to permit expression of the DNA in the host cell. Sequence, transcription initiation sequence, and, optionally, amplifier sequence.

AVR1-CO39 polypeptides generated by gene expression in recombinant prokaryotic or recombinant eukaryotic systems can be purified according to methods known in the art. In a preferred embodiment, a commercial expression / secretion system can be used, whereby the recombinant protein is easily purified from the surrounding medium by expressing it and then secreting it from the host cell. If no expression / secretion vector is used, an alternative method is to purify the recombinant protein by affinity separation methods such as immunological interaction with antibodies that specifically bind the recombinant protein. Such methods are commonly used by those skilled in the art.

The AVR1-CO39 encoded polypeptides of the invention prepared by the above methods can be analyzed according to standard methods. Example 1 describes a method for analyzing the ability to impart functional activity, ie nontoxicity.

The present invention also provides an antibody capable of immunospecifically binding to a polypeptide of the present invention. Polyclonal antibodies or monoclonal antibodies to any peptide encoded by ORFs of AVR1-CO39 can be prepared according to standard methods. Monoclonal antibodies, after standard protocols, are used in quillers ( ) And Milstein. In a preferred embodiment, antibodies have been prepared that immunospecifically react with several epitopes of AVR1-CO39 encoded polypeptide.

Polyclonal antibodies or monoclonal antibodies that immunospecifically interact with one or more polypeptides encoded by AVR1-CO39 may be used to identify and purify such proteins. For example, antibodies can be used for affinity isolation of proteins with their immunospecific interaction. Antibodies may also be used to immunoprecipitate proteins from samples containing mixtures of proteins and other biological molecules. Other uses of antibodies are described below.

B. Use of AVR1-CO39 Nucleic Acids, Encoded Proteins, and Antibodies

The potential of recombinant genetic engineering methods to improve disease resistance of agriculturally important plants has received considerable attention in recent years. Protocols for the stable introduction of genes into plants as well as for increasing gene expression are currently available. The present invention provides a nucleic acid molecule capable of improving the ability of a plant to resist the attack of pathogens upon its stable introduction into a recipient plant or constitutive microorganism. In addition, the AVR1-CO39 encoded protein of the present invention may be directly applied to plants to induce disease defense responses.

AVR1-CO39 Nucleic Acids

AVR1-CO39 nucleic acids (genome clones or cDNAs) can be used for various purposes in accordance with the present invention. DNA, RNA or fragments thereof can be used as probes to detect the presence and / or expression of the AVR1-CO39 gene. Methods in which AVR1-CO39 nucleic acids can be used as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) Northern hybridization; And (4) combined amplification reactions such as polymerase chain reaction (PCR). The AVR1-CO39 nucleic acid of the present invention may also be used as a probe for identifying molar animals from other Magnaporte isolates. As described above, AVR1-CO39 nucleic acids are also used to help produce large quantities of substantially pure AVR1-CO39 proteins or selected portions thereof.

Perhaps more important, however, is the use of the AVR1-CO39 nucleic acid to broaden the range of resistance of rice varieties with CO39 resistance genes against pathogens, but not Magnaportecria isolates with AVR1-CO39 non-toxic genes. For example, in one embodiment of the invention, the AVR1-CO39 coding site is a heterologous promoter, preferably a promoter that is generally induced by a pathogen (ie, inducible by attack by a wide range of pathogens) or a wound. Is operably linked with an inducible promoter. Such promoters include, but are not limited to:

a) promoter of a gene encoding lipoxygenase (preferably from a plant, most preferably from rice [Peng et al., J. Biol. Chem. 269: 3755-3761, 1994; Peng et al., Abstract presented at the general meeting of the International Program on Rice Biotechnology, Malacca, Malaysia, Sept. 15-19, 1997]);

b) promoters of genes encoding peroxidase (preferably from plants, most preferably from rice (Chittoor et al., Mol. Plant-Microbe Interactions 10: 861-871, 1997) ;

c) A promoter of a gene encoding hydroxymethylglutaryl-CoA reductase (preferably from a plant, most preferably from rice [Nelson et al., Plant Mol. Biol. 25: 401 -412, 1994);

d) a promoter of a gene encoding phenylalanine ammonia lyase (preferably from rice [Lam et al., Abstract of the general meeting of the International Program on Rice Biotechnology, Malacca, Malaysia, Sept. 15-19, 1997];

e) a promoter of a gene encoding glutathione-S-transferase (preferably from a plant, most preferably from rice, or alternatively from a potato, a PRP1 promoter);

f) Promoters from pollen specific genes, such as corn Zmg13, which appears to be expressed in rice transgenic pollen containing corn genes, (Aldemita et al., Abstract of the general meeting of the International Program on Rice Biotechnology, Malacca , Malaysia, Sept. 15-19, 1997);

g) a promoter from a gene encoding chitinase (preferably from a plant, most preferably from rice (Zhu & Lamb, Mol. Gen. Genet. 226: 289-296, 1991);

h) Promoters from genes initially derived (within 5 hours post-inoculation) in the interaction of Magnaforte Griscia with rice (Bhargava &Hamer; Abstract B-10, 8th International Congress Molecular Plant Microbe Interactions, Knoxville, TN July 14-19, 1996);

i) Promoters from plant (preferably rice) virus genes, which are described in Hammond-Kosack et al., Mol. Plant-Microbe Interactions 8: 181-185 (1994), which are all contained on bacterial plasmids or plant viral vectors;

j) promoters from genes associated with plant (preferably rice) respiratory bursts (Groom et al., Plant J. 10 (3): 515-522, 1996); And

k) promoters from plant (preferably rice) anthocyanin pathway genes, see Reddy, pp 341-352 in Rice Genetics III, supra; Reddy et al., Abstract of the general meeting of the Internatinal Program on Rice Biotechnology, Malacca, Malaysia, Sept. 15-19, 1997].

The chimeric genes were then used to transform rice varieties that already have the appropriate R gene. Transplanted plants upon injury or attack by plant pathogens will be induced to produce the AVR1-CO39 gene product, which triggers the R gene defense response. In such embodiments, care should be taken not to use such promoters, as the use of promoters induced by necrosis can be fatal to plants by causing unlimited hypersensitivity reactions. Kim et al., Proc. Natl. Acad. Sci. USA 91: 10445-10449, 1994].

In a preferred embodiment, the coding site of AVR1-CO39 (preferably the coding site corresponding to ORF3) was inserted into an expression vector in a microorganism that proliferates on rice plants. A suspension of this recombinant microorganism was sprayed onto rice varieties with the appropriate R gene. When pathogens attack, two levels of protection can occur: (1) Gene products produced by recombinant entrepreneurs trigger interactions on plant surfaces that prevent further infiltration by pathogens (eg, fungi) Conidia give rise to adhering groups, but not penetrating pegs); (2) The gene product produced by the recombinant engraftment is transferred to the plant tissue at the wound site, where it interacts with the corresponding R gene product to induce an internal disease defense response. Thus, such pretreatment usually confers resistance to Magna Forte isolates (and, presumably, other plant pathogens), which are toxic to these varieties. This aspect is described in more detail in Example 3 below.

With regard to the use of engraftment bacteria, it should be mentioned that bacterial expression, phage expression and delivery systems such as those sold by Invitrogen will be particularly useful. Bacterial systems express protein hybrids with filins so that foreign proteins are exposed outside of the bacteria. Phage systems also express hybrid proteins with enveloped components and expose these foreign proteins to the outside.

The AVR1-CO39 gene can also be used as a tool to identify and isolate the corresponding R gene. Thus, a method similar to that described for isolation of tomato CF-9 gene for resistance to Cladosporium fulvum (Jones et al., Science 266: 789-193, 1994), AVR1 R gene in rice corresponding to -CO39 can be isolated by transposon tagging: (1) AVR1-CO39 was transformed into susceptible rice line and constitutively expressed in susceptible rice line; (2) transgenic lines were crossbred with resistant lines with identifiable transposons; (3) killing live plants of F1 progeny that constitutively express both the Avr gene and the corresponding R gene, thereby simplifying screening for live F1 progeny; (4) Living F1 progeny must survive by blocking AVR1-CO39 transgene or the corresponding R gene, presumably by transposon. Thus, transposons can be isolated, along with the genes they block.

2. AVR1-CO39 Proteins and Antibodies

Purified gene products of AVR1-CO39, or fragments thereof, can be used to generate polyclonal or monoclonal antibodies, which are also AVR1-CO39 polypeptides in transformed microbial phenotypes, transgenic plants, or other biological materials. It can also function as a sensitive detection reagent for the presence and accumulation of Polyclonal or monoclonal antibodies immunologically specific for the AVR1-CO39 polypeptide can be used in various assays to detect and quantify proteins. Such assays include, but are not limited to: (1) flow cytometry; (2) immunochemical localization of proteins expressed in cells or tissues; And (3) immunoblot analysis of extracts from various cells and tissues (eg, dot blots, western blots). Additionally, as described above, antibodies can be used for purification of AVR1-CO39 polypeptides (eg, affinity column purification, immunoprecipitation).

In a preferred embodiment, the purified AVR1-CO39 polypeptide (most preferably from ORF3) is used as a pretreatment or cotreatment to confer a wide range of pathogen resistance to rice varieties carrying the CO39 R gene. Thus, in a manner similar to the use of AVR1-CO39 expressing engraftment microorganisms described above, peptide solutions were applied to plants and inoculation with subsequent or co-occurring wounds or pathogenic microorganisms brought the peptide into contact with the R gene product, thereby protecting Stimulates the reaction The inventors of the present invention experimentally present the possibilities of this approach, as described in detail in Example 4 below.

The following specific examples are provided to illustrate aspects of the present invention. They are by no means intended to limit the scope of the invention.

Example 1

Cloning and Analysis of AVR1-CO39

Chromosomal fragments thought to contain the varieties specific gene AVR1-CO39 were isolated from Magnaporte Grisia strain 2539 after chromosomal walking (Leong et al., 1996) using a map-based cloning approach. In this Example the inventors of the present invention describe the identification, cloning and analysis of the AVR1-CO39 gene.

Way:

Hybridization Protocol. The hybridization method was modified from Amasino (1986) (Acceleration of Nucleic Acid Hybridization Rate by Polyethylene Glycol, Analytical Biochemistry 152: 304-307). While the hybridization buffer maybe prepared according to the protocol Shino, it sikyeotgo except for PEG and NaCl decreased the concentration of _{NaHPO 4: 0.125M NaHPO 4, 7} % SDS, 50% formamide, 1.0 mM EDTA, pH 7.2. High stringency conditions were used (42 ° C., 16 hours). Washing after hybridization was done: rinsing once with 2X SSC at room temperature; Wash once in 2 × SSC for 10 min at 65 ° C .; One wash in 2 × SSC for 15 minutes at 65 ° C., one final wash for 15 minutes at 65 ° C. in 0.1 × SSC, 0.1% SDS. Final wash conditions were more stringent than hybridization conditions such that T _m was 68. Thus, greater than 95% homology will be needed to maintain the hybrids. The phosphate containing buffer was not used after hybridization described in Amashino (1986).

Chromosome walking method. The full genome DNA library of Magnaporte Grisia strain 2539, consisting of 5,194 clones, was constructed using cosmid vectors pMLF1 (Leong et al., 1994, supra) and pMLF2 (An et al., Gene 176: 93-96). , 1996). Clones were used individually as templates in the populations as colony blots as well as in the group subjected to restriction enzyme treatment to electrophoresis and blotting. The latter blot was initially used to identify candidate populations containing hybridization clones. Colonies blots derived from these populations were then screened. Steps were performed using the final clone prepared from the insert DNA by treating the cosmid clone with restriction enzyme ApaI, which did not cleave the vector and recyclized the plasmid by binding. This method produces a derivative comprising DNA from each end of the insert (An et al., 1996 supra). Separation of both ends of this insert from the vector is accomplished by treatment with restriction enzymes ApaI and NotI. The necessary final clones were then identified by its inability to hybridize with previous cosmids in walking.

Transformation of Toxic Strain Guy11 Using Cosmid in the AVR1-CO39 Locus: Cosmids from within the genetic gap with AVR1-CO39 are described in LEUNG et al. (1990) using the transformation protocol described in Guy11. The method was modified as follows: The protoplasts were incubated in complete medium (CM) + sorbitol and then poured into 100 ml of dissolved CM + 20% sucrose agar medium. This agar medium was then poured onto four Petri plates. When the agar medium solidified (1 hour), 15 ml of 1.5% moisture agar medium containing 800 μg hygromycin B per ml (300 μg final concentration per ml) was overlaid.

Generation of frameshift mutations within the open reading frame at the AVR1-CO39 locus. The first two clones were made by cloning the 1.05 kb fragment into pBSKS II ⁺ (pBSCO39). Then J. mutant 1.05 kb fragments. It was cloned into pCB1004, a hyg ^r vector from J. Sweigard (Dupont). Plasmids were linearized by restriction enzyme NotI treatment and transformed into Guy11 protoplasts.

Initial frameshift mutations were generated in ORF 2 and ORF 3 by the following restriction enzyme treatment and recombination:

Frameshift in ORF2: The AccI site at nucleotide (nt) position 499 was cleaved and the 2 nt 3 'overhang was removed using T4 DNA polymerase. This site was then recombined to remove 4 bp or to have a net frameshift of -2. The nucleotide sequence was changed from 5 'CTAGACAGTCTACCTCTCTGCCA 3' (SEQ ID NO: 9) to 5 'CTAGACAGTACCTCTCTGCCA 3' (SEQ ID NO: 10).

Frameshift at ORF 2 and ORF 3: The PflMI site at nt position 641 was cleaved and the 3 nt 3 ′ overhang was removed using T4 polymerase. Klenow-filled HindIII fragments containing streptomycin resistance genes from pHP45Ω (Prentki and Kritsch, Gene 29: 303, 1984) were coupled to the blunt-ended PflMI fragment. The conserved HindIII site was then restriction enzyme treated and recombined. Accordingly, 3 nt of the PflMI site was replaced with 4 nt from the HindIII site. This resulted in a net frame shift of +1. The nucleotide sequence was changed from 5 ′ CCAGCAGCCAATGCTTGGAAAGATTG 3 ′ (SEQ ID NO: 11) to 5 ′ CCAGCAGCCAAAGCTTTGGAAAGAT TG 3 ′ (SEQ ID NO: 12).

In the ΔAccI preparation, the peptide contained only 19 amino acids (aa) in its original sequence and was cleaved after 36 aa. The original ORF2 peptide is 77 aa. In the ΔPfl preparation, the ORF2 peptide sequence remained almost unchanged except at the terminal 10 aa, with the result that the peptide was 17 aa longer. On the other hand, ORF3 holds only 20 aa from its N-terminus and ends after 31 aa.

Frameshift in ORF1 was generated by "Quick Change" site mutagenesis (Stratagene) using primers designed to introduce extra G nucleotides after ATG:

P1: CAACGTACTAGAAATGGAGTAATAAGTACC (SEQ ID NO: 13)

P2: GGTACTTATTAGTCCATTTCTAGTACGTTG (SEQ ID NO: 14)

Mutagenes basically completely destroyed the ORF.

Generation of ATG Mutations in an Open Reading Frame at the AVR1-CO39 Locus: A 1.05 kb fragment containing AVR1-CO39 was cloned into pCB1004 and the following mutant preparations were made using QuickChange mutagenesis (Stratagene):

ΔORF1 (ATG-> TTT): The initiation codon of ORF1 was removed by quick change mutagenesis using primers with mutant ATG sequences.

ΔORF3 (ATG-> TTT): The initiation codon of ORF3 was removed by quick change mutagenesis using primers with mutant ATG sequences.

The following clones were made but the phenotype was not yet determined by testing the mutant alleles by transformation.

ΔORF2 (ATG-> TTT): The initiation codon of ORF3 was removed by quick change mutagenesis using primers with mutant ATG sequences.

result:

As mentioned above, genes that confer breed specific interactions with rice varieties CO39 were isolated from Magnaportecria strain 2539 using a map-based cloning method followed by a 20 step chromosomal walk. Guy11, a commonly toxic strain on CO39, was rendered nontoxic by subcloning and transforming the AVR1-CO39 locus to a 1.05 kb site. The nucleotide sequence of this 1.05 kb region of chromosome 1 is described below as SEQ ID NO: 1:

DNA sequencing revealed four small open reading frames of length 45, 77, 89 and 69 amino acids (ORF1, ORF2, ORF3, ORF4, respectively, as shown on SEQ ID NO. 1 above). The amino acid sequences encoded by the four open reading frames are described below as SEQ ID NOs 2, 3, 4 and 5, respectively. Three different open reading frames were also identified (ORF5, 6 and 7 described below as SEQ ID NOs 6, 7 and 8, respectively).

The sequence around the ATG of ORF3 matched four of the five conserved bases found at the translational initiation site of the fungus and identified two putative cleavage sites for removal of the hydrophobic amino terminus and signal peptide interrupted by lysine at position 2 It is included. The fourth open reading frame was identified on the opposite strand. However, only two surrounding ATG sequences matched the fungal translation initiation site consensus sequence.

Certain site mutations in ORF1, ORF2 and ORF3 were generated to assess the role of these ORFs in imparting nontoxicity. The translation initiation codon of each ORF was converted from ATG to TTT. In ORF1 and ORF3, these mutations resulted in loss of nontoxicity. Frameshift mutations in ORF1 and ORF3 also resulted in loss of nontoxicity. In contrast, the frameshift mutation of ORF2 was not. Taken together, these data suggest a role for ORF1 and ORF3 in contributing non-toxicity to Magnaporte Grisia strain 2539 on rice cultivar CO39.

The absence of any putative TATA element as well as the splicing site and lasso sequence near the upstream of ATG of ORF1 may suggest that ORF1 overlaps with sequences critical for promoting transcription of AVR1-CO39.

Example 2

Distribution of AVR1-CO39 Mammals in Various Isolates of Magnaporte Grisia

The distribution of AVR1-CO39 mammals was investigated by probing a large sample of host specific form of Magnaporte Grisia using fragments of AVR1-CO39 DNA using hybridization conditions as described in Example 1. The results of these investigations suggest that isolates that infect rice, Digitaria and wheat are usually deficient in Avr-CO39's mole. However, the moron of this gene is commonly found in isolates of Ellutine (Setaria) infections. Further analysis of the AVR1-CO39 locus from the toxic rice isolate Guy11 suggests that at least 20 kb of DNA comprising the AVR1-CO39 locus of isolate 2539 is absent.

Example 3

Improved resistance to Magnaporte gricia infection in rice plants sprayed with bacterial phenotypes expressing ORF3 of AVR1-CO39

ORF3 of AVR1-CO39 described in Example 1 was transferred into a pET expression vector in Escherichia coli. Suspensions containing the transformed Escherichia coli were sprayed onto the leaves of rice plants with the corresponding R gene for AVR1-CO39. This plant was then inoculated with Magnaporte Grisia isolate Guy11, which is a toxic strain for the tested plant variety. As a control, plants were sprayed with E. coli suspension without ORF-3 encoding plasmids and then inoculated with isolate Guy11.

Inoculated plants pretreated with ORF3-expressing Escherichia coli had reduced damage size and number compared to inoculated control plants pretreated with E. coli lacking the ORF3-expressing plasmid. These data support ORF3's role in the nontoxicity of Magnaporte Grisia.

Example 4

Improved resistance to Magnaporte gricia infection in rice plants sprayed with proteins encoded by ORF3 of AVR1-CO39

ORF3 of AVR1-CO39 described in Example 1 was transferred into a pET expression vector in Escherichia coli. The effect of protein extracts from IPTG-derived E. coli cells with either the pET vector alone (control) or with the pET-ORF3 preparation was tested for toxicity. Cell protein extracts were concentrated by ammonium sulfate precipitation. Variety CO39 was inoculated with the concentrated Magnaporte Grisia strain Guy11 with concentrated protein extract to yield 5 × 10 ⁵ conidia and 20 mg total protein extract in 10 ml sterile water.

Plants inoculated co-treated with an ORF3-comprising protein extract reduced damage size and number compared to control plants co-treated and inoculated with protein extracts lacking ORF3. These data further support ORF3's role in imparting non-toxicity to Magnaporte Gricia.

While certain preferred embodiments of the invention have been described above and specifically illustrated, it is not intended to limit the invention to these embodiments. As set forth in the claims below, various modifications are possible herein without departing from the scope and spirit of the invention.

SEQUENCE LISTING

<110> Sally A. Leong

Mark L. Farman

<120> Cultivar SPecificity Gene from the Rice

Pathogen Magna Porthe grisea, and Methods of Use

<130> WARF P98067WO

<140> US 09 / 257,585

<141> 1999-02-25

<150> US 60 / 075,925

<151> 1998-02-25

<160> 14

<170> FastSEQ for Windows Version 3.0

<210> 1

<211> 1047

<212> DNA

<213> MagnaPorthe grisea

<400> 1

gatctgtaaa ttacatatat ttattttgcc gcattttgct aaccgcctat tctttttaaa 60

attttaacga ttaagaacgc aattcaattt tgcgttctac acaaattaac aattcgtcca 120

aaagaggtat ttaagcgaag atttggcatt tttttaatcc atttttaaaa aaatacatct 180

gctttaaccc acctttgcca agggtacccg gctagcatag ccttggttac caaaaacggc 240

taaagctgtc gatctatact acattcggcg ctctgaacaa ctaagcaaca gcgaggagat 300

cacaccctaa atcatgctgc tagtaatgcg atataatggc caaacaacgt actagaaatg 360

actaataagt acccagtcaa gtcaacttgc tgtagtatta tatttaacga agcgtccatt 420

tactgccagg gcaagtttat caatgggacc agtgttctcc ctcctctgga caactcagtt 480

ctttgcaaac gctagacagt ctacctctct gccaccattt ttacttttca aaaatttact 540

ccttgccgct actgaaactt ctacaattga aagagcccac aatgaaagtc caagctacat 600

tcgccaccct tatcgccctt gcggcttact ttccagcagc caatgcttgg aaagattgca 660

tcatccaacg ttataaagac ggcgatgtca acaacatata tactgccaat aggaacgaag 720

agataactat tgaggaatat aaagtcttcg ttaatgaggc ctgccatccc tacccagtta 780

tacttcccga cagatcggtc ctttctggcg attttacatc agcttacgct gacgacgatg 840

agtcttgttg atcaataaga gtccaggttg aaaaattcgc caccatggta atagagggtt 900

atttatctcg gaatagcagc cgtgtgtgca attatcacgg ctgttcctct gcgataggga 960

tattagaagc aggacaaatt tacggcaata gcaaccaatt gtccttgtct atggattcgc 1020

ccgtcgaatg gaggcgacgg cggatcc 1047

<210> 2

<211> 45

<212> PRT

<213> MagnaPorthe grisea

<400> 2

Met Thr Asn Lys Tyr Pro Val Lys Ser Thr Cys Cys Ser Ile Ile Phe

1 5 10 15

Asn Glu Ala Ser Ile Tyr Cys Gln Gly Lys Phe Ile Asn Gly Thr Ser

20 25 30

Val Leu Pro Pro Leu AsP Asn Ser Val Leu Cys Lys Arg

35 40 45

<210> 3

<211> 77

<212> PRT

<213> MagnaPorthe grisea

<400> 3

Met Gly Pro Val Phe Ser Leu Leu TrP Thr Thr Gln Phe Phe Ala Asn

1 5 10 15

Ala Arg Gln Ser Thr Ser Leu Pro Pro Phe Leu Leu Phe Lys Asn Leu

20 25 30

Leu Leu Ala Ala Thr Glu Thr Ser Thr Ile Glu Arg Ala His Asn Glu

35 40 45

Ser Pro Ser Tyr Ile Arg His Pro Tyr Arg Pro Cys Gly Leu Leu Ser

50 55 60

Ser Ser Gln Cys Leu Glu Arg Leu His His Pro Thr Leu

65 70 75

<210> 4

<211> 89

<212> PRT

<213> MagnaPorthe grisea

<400> 4

Met Lys Val Gln Ala Thr Phe Ala Thr Leu Ile Ala Leu Ala Ala Tyr

1 5 10 15

Phe Pro Ala Ala Asn Ala TrP Lys AsP Cys Ile Gln Arg Tyr Lys

20 25 30

AsP Gly AsP Val Asn Asn Ile Tyr Thr Ala Asn Arg Asn Glu Glu Ile

35 40 45

Thr Ile Glu Glu Tyr Lys Val Phe Val Asn Glu Ala Cys His Pro Tyr

50 55 60

Pro Val Ile Leu Pro AsP Arg Ser Val Leu Ser Gly AsP Phe Thr Ser

65 70 75 80

Ala Tyr Ala AsP AsP AsP Glu Ser Cys

85

<210> 5

<211> 68

<212> PRT

<213> MagnaPorthe grisea

<400> 5

Met Ala Gly Leu Ile Asn Glu AsP Phe Ile Phe Leu Asn Ser Tyr Leu

1 5 10 15

Phe Val Pro Ile Gly Ser Ile Tyr Val Val AsP Ile Ala Val Phe Ile

20 25 30

Thr Leu AsP AsP Ala Ile Phe Pro Ser Ile Gly Cys TrP Lys Val Ser

35 40 45

Arg Lys Gly AsP Lys Gly Gly Glu Cys Ser Leu AsP Phe His Cys Gly

50 55 60

Leu Phe Gln Leu

65

<210> 6

<211> 35

<212> PRT

<213> MagnaPorthe grisea

<400> 6

Met AsP Ala Ser Leu Asn Ile Ile Leu Gln Gln Val AsP Leu Thr Gly

1 5 10 15

Tyr Leu Leu Val Ile Ser Ser Thr Leu Phe Gly His Tyr Ile Ala Leu

20 25 30

Leu Ala Ala

35

<210> 7

<211> 34

<212> PRT

<213> MagnaPorthe grisea

<400> 7

Met Arg Pro Ala Ile Pro Thr Gln Leu Tyr Phe Pro Thr AsP Arg Ser

1 5 10 15

Phe Leu Ala Ile Leu His Gln Leu Thr Leu Thr Thr Met Ser Leu Val

20 25 30

AsP Gln

<210> 8

<211> 54

<212> PRT

<213> MagnaPorthe grisea

<400> 8

Met Val Ile Glu Gly Tyr Leu Ser Arg Asn Ser Ser Arg Val Cys Asn

1 5 10 15

Tyr His Gly Cys Ser Ser Ala Ile Gly Ile Leu Glu Ala Gly Gln Ile

20 25 30

Tyr Gly Asn Ser Asn Gln Leu Ser Leu Ser Met AsP Ser Pro Val Glu

35 40 45

TrP Arg Arg Arg Arg Ile

50

<210> 9

<211> 23

<212> DNA

<213> MagnaPorthe grisea

<400> 9

ctagacagtc tacctctctg cca 23

<210> 10

<211> 21

<212> DNA

<213> MagnaPorthe grisea

<400> 10

ctagacagta cctctctgcc a 21

<210> 11

<211> 26

<212> DNA

<213> MagnaPorthe grisea

<400> 11

ccagcagcca atgcttggaa agattg 26

<210> 12

<211> 27

<212> DNA

<213> MagnaPorthe grisea

<400> 12

ccagcagcca aagctttgga aagattg 27

<210> 13

<211> 30

<212> DNA

<213> MagnaPorthe grisea

<400> 13

caacgtacta gaaatggagt aataagtacc 30

<210> 14

<211> 30

<212> DNA

<213> MagnaPorthe grisea

<400> 14

ggtacttatt agtccatttc tagtacgttg 30

Claims (29)

A nucleic acid molecule isolated from Magnaporthe grisea, which confers rice varieties CO39-specific nontoxic against fungal plant pathogens having the nucleic acid. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is AVR1-CO39. The nucleic acid molecule of claim 2 having a sequence comprising some or all of SEQ ID NO: 1. The group of claim 1 wherein SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 A nucleic acid molecule characterized by encoding a polypeptide having a characteristic of a polypeptide comprising a sequence selected from. The group of claim 4 wherein SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 A nucleic acid molecule characterized by encoding a polypeptide having a sequence selected from. A recombinant DNA molecule comprising the nucleic acid molecule of claim 1, wherein the recombinant DNA molecule is operably linked to a vector for the transformed cell. Cells transformed using the recombinant DNA molecule of claim 6. 8. The cell of claim 7, wherein the cell is selected from the group consisting of bacterial cells, fungal cells, insect cells and plant cells. 10. The transformed cell of claim 8 which is an engraftment bacterial cell. A transgenic plant regenerated from the transformed cells of claim 8. An isolated nucleic acid molecule having a sequence selected from the group consisting of the following sequences: a) part or all of SEQ ID NO: 1; b) allelic variants of some or all of SEQ ID NO: 1; c) a natural mutant of part or all of SEQ ID NO: 1; d) a sequence that hybridizes with some or all of SEQ ID NO: 1 or its complementary strand and encodes a polypeptide substantially identical to the polypeptide encoded by SEQ ID NO: 1; And e) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 A sequence encoding part or all of a polypeptide having An oligonucleotide of about 10 to about 100 nucleotides in length that hybridizes specifically to a portion of the nucleic acid molecule of claim 11. A recombinant DNA molecule comprising the nucleic acid molecule of claim 11, wherein the recombinant DNA molecule is operably linked with the vector for transformed cells. A cell transformed using the recombinant DNA molecule of claim 13. The cell of claim 14, wherein the cell is selected from the group consisting of bacterial cells, yeast cells, and plant cells. The cell of claim 15 which is an engraftmentary bacterial cell. Transgenic plant regenerated from the cell of claim 15. A polypeptide encoded by the nucleic acid molecule of claim 11. An antibody immunologically specific for the polypeptide of claim 18. A protein encoded by an isolated nucleic acid molecule from Magnaporte Grisia, which confers rice cultivar CO39-specific nontoxicity against fungal plant pathogens containing such nucleic acid. The protein of claim 20 encoded by the AVR1-CO39 gene. 22. The protein of claim 21, encoded by ORF3 of AVR1-CO39. The group of claim 20 wherein SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 A protein characterized by having an amino acid sequence selected from. An antibody immunologically specific for the protein of claim 20. A transgenic engraftment bacterium expressing a portion of AVR1-CO39 effective to confer rice cultivars CO39-specific nontoxicity against microorganisms expressing the AVR1-CO39 gene. The transgenic engraftment bacterium according to claim 24, which expresses or is functionally equivalent to ORF3 of SEQ ID NO: 1. Enhancing the range of resistance of rice cultivar CO39 to pathogenic microorganisms, including treating plants with polypeptides produced by expression of AVR1-CO39 in an amount effective to trigger expression of CO39-specific R genes in plants. How to let. 28. The method of claim 27, comprising treating the plant with a solution comprising the polypeptide. 28. The method of claim 27, comprising treating the plant with a germ bacterium expressing a portion of the AVR1-CO39 gene that produces a polypeptide effective for triggering expression of a CO39-specific R gene in the plant. Way.