WO2001002574A1

WO2001002574A1 - Negative regulators of systemic acquired resistance

Info

Publication number: WO2001002574A1
Application number: PCT/US2000/018270
Authority: WO
Inventors: Xinnian Dong; Xin Li
Original assignee: Duke University
Priority date: 1999-07-02
Filing date: 2000-06-30
Publication date: 2001-01-11
Also published as: AU6066300A; WO2001002574A9

Abstract

The invention generally features a non-naturally occurring plant (e.g. a transgenic plant) having decreased expression of an endogenous negative regulator polypeptide of systemic acquired resistance (SAR), wherein the decreased expression renders the non-naturally occurring plant resistant to a plant pathogen.

Description

NEGATIVE REGULATORS OF SYSTEMIC ACQUIRED RESISTANCE

Statement as to Federally Sponsored Research

This invention was made in part with Government funding and the Government therefore has certain rights in the invention. In particular, portions of the invention disclosed herein were funded by the United States Department of Agriculture and the National Science Foundation.

Background of the Invention

This application claims benefit of U.S. provisional application 60/142,214, filed on July 2, 1999.

Systemic acquired resistance ("SAR") is a general plant defense response that can be triggered after a local infection by a necrotizing pathogen. This response is long lasting and effective against a variety of pathogens including fungi, bacteria, and viruses (Ryals et al., Plant Cell 8: 1809-1819, 1996). Induction of SAR is correlated with up-regulation of several well-characterized pathogenesis-related ( "PR") genes (Van Loon and Van Kammen, Virology 40:199-211, 1970; Ward et al., Plant Cell 3: 1085-1094, 1991; Yalpani et al., Plant Cell 3:809-818, 1991; Uknes et al., Plant Cell 4:645-656, 1992). In particular, PR-1 and β-l,3-glucanase ("BGL2," also known as PR-2) have been widely used as molecular markers for SAR (Uknes et al., Plant Cell 4:645-656, 1992; Bowling et al., Plant Cell 6:1845-1857, 1994; Cao et al., Plant Cell 6:1583-1592, 1994). Convincing evidence has shown that salicylic acid ("SA") is a necessary and sufficient signal for SAR induction. The onset of SAR is accompanied by an increase in the endogenous levels of SA (Malamy et al., Science 250:1002-1004, 1990; Metraux et al., Science 250: 1004-1006, 1990; Rasmussen et al., Plant Physiol. 97: 1342-1347, 1991) and can also be brought on by exogenous application of SA (White, Virology 99:410-412, 1979) or its analogs such as 2,6-dichloroisonicotinic acid ("INA"; Metraux et al., In: Advances in Molecular Genetics of Plant-Microbe Interactions, Hennecke, H. and Verma D.P.S., eds., Kluwer Academic Publishers, Dordrecht, The Netherlands, Vol. 1, pp. 432-439. 1991) and benzo(l,2,3)-thiadiazole-7-carbothioic acid S-methyl ester ("BTH"; Gδrlach et al., Plant Cell 8:629-643, 1996). Furthermore, transgenic plants expressing the SA-degrading enzyme salicylate hydroxylase are unable to accumulate SA and are compromised in SAR (Gaffney et al, Science 261 :754-756, 1993).

Several genetic screens have been conducted in Arabidopsis to identify the regulatory components of the signaling pathway leading to SAR. Among the SAR compromised mutants, nprls (also known as nimls) are the only mutants identified that affect a function downstream of the SA signal (Cao et al., Plant Cell 6:1583- 1592, 1994; Delaney et al, Proc. Natl. Acad. Sci. USA 92:6602-6606, 1995;

Glazebrook et al, Genetics 143:973-982, 1996; Ryals et al., Plant Cell 9:425-439, 1997; Shah et al., Mol. Plant Microbe Interact. 10:69-78, 1997). The nprl mutants are impaired in the ability to accumulate PR mRNA or to mount an SAR response in the presence of an SAR inducer. The NPR1 gene encodes a novel protein containing at least four ankyrin repeats (Cao et al., Cell 88:57-63, 1997; Ryals et al., Plant Cell 9:425-439, 1997), which are found in proteins of diverse functions (Bork, Proteins 17:363-374, 1993), including IkB and Cactus that regulate animal immune responses (Thanos and Maniatis, Cell 80:529-532, 1995; Lemaitre et al., Cell 86:973-983, 1996). The functional importance of these ankyrin repeats in NPR1 has been clearly demonstrated by the isolation of several nprl mutants that contain lesions in the ankyrin repeat consensus (Cao et al., Cell 88:57-63, 1997; Ryals et al., Plant Cell 9:425-439, 1997). Specifically, in the nprl-1 mutant, where a highly conserved histidine (residue 334) in the third ankyrin repeat is changed to a tyrosine, the SA- or INA-induced expression of a reporter gene BGL2-GUS and the endogenous PR genes is abolished (Cao et al., Cell 88:57-63, 1997).

Despite such progress, a need exists in the art for identifying other important genetic signaling components of SAR. Such components provide novel targets for developing not only crops having resistance to a broad spectrum of pathogens, but also for developing new crop protection methodologies. Summary of the Invention The present invention capitalizes on the discovery of a negative regulator polypeptide of systemic acquired resistance (SAR). Manipulation of the expression of this SAR component facilitates the production of plants having broad spectrum disease resistance.

Accordingly, the invention generally features a non-naturally occurring plant having decreased expression of an endogenous negative regulator polypeptide of SAR, wherein the decreased expression renders the non-naturally occurring plant resistant to a plant pathogen. Preferably, the non-naturally occurring plant is a transgenic plant or a mutant plant. In preferred embodiments, such a transgenic plant includes a transgene that, when expressed in the transgenic plant, silences gene expression of the endogenous negative regulator polypeptide of SAR.

In still other preferred embodiments, the transgenic plant includes a transgene that expresses an antisense molecule of the negative regulator polypeptide of SAR; a transgene that, when expressed in the transgenic plant, co-suppresses expression of the negative regulator polypeptide of SAR (such as a mutated form of the negative regulator polypeptide); or a transgene that encodes a dominant negative gene product; or any combination thereof.

In other preferred embodiments, the plant of the invention includes a point mutation, a deletion, or an insertion, or any combination thereof, in a gene that encodes the negative regulator polypeptide of SAR. Decreased expression of the negative regulator polypeptide of SAR in the plant of the invention may occur at the transcriptional, translational, post-transcriptional, or post-translational levels of gene expression. In another aspect, the invention features an isolated nucleic acid molecule (for example, an isolated DNA molecule such as cDNA) including a sequence encoding a negative regulator polypeptide of SAR. In preferred embodiments, the isolated nucleic acid molecule includes a negative regulator polypeptide of SAR that is substantially identical to the amino acid sequence shown in Fig. 3A. An exemplary isolated nucleic acid molecule of the invention is shown in SEQ ID NO: 1. In even another embodiment, the negative regulator polypeptide of SAR has at least 30% identity to the amino acid sequence shown in Fig. 3A. Preferably, such a polypeptide is leucine-rich or has substantial identity to the N domain of a retinoblastoma protein or both. Such a polypeptide is shown in SEQ ID NO:2. In other preferred embodiments, the isolated nucleic acid molecule of the invention includes a sequence that encodes a polypeptide that, when expressed in a cell of a plant, represses transcription or is substantially localized to the nucleus or both.

In yet other embodiments, the isolated nucleic acid molecule including a sequence encoding a negative regulator polypeptide of SAR hybridizes specifically to a nucleic acid molecule including the sequence of Fig. 3 A or 3B.

In still other embodiments, the invention features the isolated nucleic acid molecule operably linked to an expression control region.

In related aspects, the invention features an expression vector including the isolated nucleic acid molecule of the invention and a cell (for example, a prokaryotic cell, such as Agrobacterium or E. coli, or a eukaryotic cell, such as a mammalian, insect, yeast, or plant cell) including the isolated nucleic acid molecule or vector.

In yet another aspect, the invention features a transgenic plant or transgenic plant component including a nucleic acid molecule of the invention, wherein the nucleic acid molecule is expressed in the transgenic plant or the transgenic plant component. Preferably, the transgenic plant or transgenic plant component is an angiosperm (for example, a monocot or dicot). In preferred embodiments, the transgenic plant or transgenic plant component is a cruciferous plant. The invention further includes a seed produced by the transgenic plant or transgenic plant component, or progeny thereof.

In still other related aspects, the invention features an isolated DNA molecule encoding an antisense RNA of a negative regulator polypeptide of SAR; an expression vector including the DNA molecule encoding the antisense RNA of a negative regulator of SAR; or a transgenic plant or transgenic plant component (or a seed or a cell thereof) that harbors the vector or the isolated DNA molecule encoding the antisense RNA molecule.

In another aspect, the invention features a substantially pure negative regulator polypeptide of SAR. In preferred embodiments, the polypeptide includes an amino acid sequence that is substantially identical to the amino acid sequence of Fig. 3A or to the N domain of a retinoblastoma protein of Fig. 3C. In still other preferred embodiments, the polypeptide represses transcription, is substantially localized to the nucleus, or is leucine-rich, or has any combination of these characteristics. An exemplary negative regulator polypeptide is shown in SEQ ID NO:2.

In another aspect, the invention features a method of producing a negative regulator polypeptide of SAR, including the steps of: (a) providing a cell transformed with an isolated nucleic acid molecule of the invention; (b) culturing the transformed cell under conditions for expressing the isolated nucleic acid molecule; and (c) recovering the negative regulator polypeptide of SAR. The invention further includes a recombinant negative regulator polypeptide of SAR produced according to this method.

In another aspect, the invention features a substantially pure antibody (e.g. a polyclonal or monoclonal antibody) that specifically recognizes and binds to a negative regulator polypeptide of SAR. Preferably, such an antibody recognizes and binds to a recombinant negative regulator polypeptide of SAR. In yet another aspect, the invention features methods of isolating a negative regulator gene of SAR or a fragment thereof. One such method includes the steps of: (a) contacting the nucleic acid molecule of Fig. 3 A or a portion thereof with a nucleic acid preparation from a plant cell under hybridization conditions providing detection of nucleic acid sequences having at least 30% or greater sequence identity to the nucleic acid sequence of Fig. 3 A; and (b) isolating the hybridizing nucleic acid sequences.

A second method of the invention includes the steps of: (a) providing a sample of plant cell DNA; (b) providing a pair of oligonucleotides having sequence identity to a region of the nucleic acid molecule of Fig. 3A; (c) contacting the pair of oligonucleotides with the plant cell DNA under conditions suitable for polymerase chain reaction-mediated DNA amplification; and (d) isolating the amplified negative regulator gene of SAR or fragment thereof. In preferred embodiments, the amplification step is carried out using a sample of cDNA prepared from a plant cell; and the negative regulator gene of SAR encodes a polypeptide which is at least 30% identical to the amino acid sequence of Fig. 3A.

Further, the invention features several gene silencing methods for conferring disease resistance on a plant. Such methods include, without limitation, co- suppression and antisense technologies, expression of altered or mutated forms of the negative regulator polypeptide, as well as the expression of dominant negative forms of the negative regulator. Silencing the expression of the negative regulator provides a plant or plant component of the invention with broad spectrum disease resistance to plant pathogens including, but not limited to, those described herein.

In yet another aspect, the invention features a method for conferring disease resistance on a transgenic plant cell. The method includes reducing the level of an endogenous negative regulator polypeptide of SAR in a transgenic plant cell, a transgenic plant, or a transgenic plant component. In preferred embodiments, methods for reducing the level of the endogenous negative regulator polypeptide of SAR include expressing a transgene encoding an antisense nucleic acid molecule of a negative regulator gene of SAR in the transgenic plant cell. In other preferred embodiments, reducing the level of the endogenous negative regulator polypeptide of SAR includes co-suppression of the endogenous negative regulator gene of SAR in the transgenic plant cell. Still another method for reducing the level of the endogenous negative regulator polypeptide of SAR includes expressing a dominant negative gene product (for example, a mutated form of the endogenous negative regulator polypeptide of SAR) in the transgenic plant cell, the transgenic plant, or the transgenic plant component. And in still other preferred embodiments, the expression of altered or mutant forms of the negative regulators polypeptide may be used to confer disease resistance. Plants of the invention may be regenerated from any of these plant cells according to standard methods known in the art. In yet another aspect, the invention features a method for identifying a compound that modulates the expression of a gene encoding a negative regulator polypeptide of SAR. The method includes the steps of: (a) providing a cell (for example, a prokaryotic cell, such as E. coli, or a eukaryotic cell, such as a mammalian, yeast, insect cell, or plant cell) including a gene encoding a negative regulator polypeptide of SAR; (b) applying to the cell a candidate compound; and (c) measuring expression of the gene encoding the negative regulator polypeptide of SAR, an increase or decrease in expression relative to an untreated control sample being an indication that the compound modulates expression of the negative regulator polypeptide of SAR. In preferred embodiments, the gene encodes a negative regulator polypeptide of SAR that is substantially identical to the amino acid sequence shown in Fig. 3A. In other preferred embodiments, the compound decreases the expression of the gene that encodes the negative regulator polypeptide of SAR. In yet other preferred embodiments, the compound increases the expression of the gene that encodes the negative regulator polypeptide of SAR. Compounds identified using these methods are useful as crop protectants.

In another aspect, the invention features a method for identifying a compound that modulates the activity of a negative regulator polypeptide of SAR in a plant or plant component. In general, the method includes the steps of: (a) providing a plant or plant component expressing an isolated DNA molecule encoding a negative regulator polypeptide of SAR; (b) applying to the plant or plant component a candidate compound; and (c) measuring the activity of the negative regulator polypeptide of SAR, an increase or decrease in activity relative to an untreated control sample being an indication that the compound modulates activity of the negative regulator polypeptide of SAR. In preferred embodiments, the gene encodes a negative regulator polypeptide of SAR that is substantially identically to the amino acid sequence shown in Fig. 3A. In another preferred embodiment, the compound decreases the activity of the negative regulator polypeptide of SAR. In another embodiment, the compound increases the activity of the negative regulator polypeptide of SAR. In another aspect, the invention features a method for increasing the resistance of a plant or plant component to a pathogen, involving applying a compound to the plant or plant component that decreases the expression of a gene encoding a negative regulator polypeptide of SAR. In another aspect, the invention features a method for increasing the resistance of a plant or plant component to a pathogen, involving applying a compound to the plant or plant component that decreases activity of a negative regulator polypeptide of SAR.

In yet another aspect, the invention features a method for increasing the resistance of a plant or plant component to a pathogen, involving applying a chemical regulator of SAR to a plant or plant component that has decreased expression of a negative regulator polypeptide of SAR. In preferred embodiments, the chemical inducer of SAR is S A, INA, or BTH, or any combination thereof.

By "negative regulator polypeptide of SAR" is meant a polypeptide which represses a plant's systemic acquired resistance. An exemplary negative regulator polypeptide of SAR, SNI1, from Arabidopsis is described herein. Additional negative regulator polypeptides may be identified and isolated from any plant species, especially agronomically important crop plants, using any of the sequences disclosed herein in combination with conventional methods known in the art. By "gene silencing" is meant a decrease in the level of gene expression (for example, expression of a gene encoding a negative regulator polypeptide of SAR (for example, SNI1)) by at least 30-50%, preferably by at least 50-80%, and more preferably by at least 80-95% or greater relative to the level in a control plant (for example, a wild-type plant). Reduction of such expression levels may be accomplished by employing standard methods which are known in the art including, without limitation, antisense and co-suppression technologies, expression of a dominant negative gene product, or through the generation of mutated genes using standard mutagenesis techniques. Levels of negative regulator polypeptide or transcript are monitored according to any standard technique including, but not limited to, northern blotting, RNase protection, or immunoblotting. By "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).

By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 20%, preferably 50%, more preferably 80%, and most preferably 90%, or even 95% or greater identity to a reference amino acid sequence (for example, the amino acid sequence shown in Fig. 3A or to an N domain (e.g., the N domain of retinoblastoma shown in Fig. 3C)) or nucleic acid sequence (for example, the nucleic acid sequences shown in Fig. 3A or Fig. 3B). For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids or greater. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides of greater.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705, BLAST, FASTA, PILEUP/PRETTYBOX programs, or other publicly available sequence analysis programs, for example those found at http://www.expasy.ch/tools). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. By a "substantially pure polypeptide" is meant a negative regulator polypeptide of SAR (for example, an SNI polypeptide such as SNI1) that has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a negative regulator polypeptide of SAR. A substantially pure negative regulator polypeptide of SAR may be obtained, for example, by extraction from a natural source (for example, a plant cell); by expression of a recombinant nucleic acid encoding a negative regulator polypeptide of SAR; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

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

By "derived from" is meant isolated from or having the sequence of a naturally-occurring sequence (for example, a cDNA, genomic DNA, synthetic DNA, or combination thereof). By "specifically hybridizes" is meant that a nucleic acid sequence is capable of hybridizing to a DNA sequence at least under low stringency conditions as described herein, and preferably under high stringency conditions, also as described herein. These conditions are well known in the art and may be altered in order to identify or detect identical or related polynucleotide sequences. By "transformed cell" is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule, for example, a DNA molecule encoding a negative regulator polypeptide of SAR.

By "positioned for expression" is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription (i.e., facilitates the production of an RNA molecule) and, if a protein product is desired, translation of the sequence (i.e., facilitates the production of, for example, a negative regulator polypeptide of SAR or a recombinant protein).

By "reporter gene" is meant a gene whose expression may be assayed; such genes include, without limitation, β-glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), β- galactosidase, herbicide resistant genes, and antibiotic resistance genes.

By "expression control region" is meant any minimal sequence sufficient to direct transcription. Included in the invention are promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-, tissue-, or organ-specific gene expression, or elements that are inducible by external signals or agents (for example, light-, pathogen-, wound-, stress-, or hormone-inducible elements or chemical inducers such as SA or INA); such elements may be located in the 5' or 3' regions of the native gene or engineered into a transgene construct.

By "operably linked" is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s).

By "plant cell" is meant any self-propagating cell bounded by a semi- permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

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

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

By "transgene" is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell.

Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene having sequence identity to an endogenous gene of the organism.

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

By "pathogen" is meant an organism whose infection of viable plant tissue elicits a disease response in the plant tissue. Such pathogens include, without limitation, bacteria, mycoplasmas, fungi, oomycetes, insects, nematodes, viruses, and viroids. Examples of such plant diseases caused by these pathogens are described in

Chapters 11-16 of Agrios, Plant Pathology, 3rd ed., Academic Press, Inc., New York,

1988.

Examples of bacterial pathogens include, without limitation, Erwinia (for example, E. carotovora), Pseudomonas (for example, P. syringae), and Xanthomonas

(for example, X. campepestris and X. oryzae).

Examples of fungal or fungal-like disease-causing pathogens include, without limitation, Alternaria (for example, A. brassicola and A.solanϊ), Ascochyta (for example, A. pisi), Botrytis (for example, B. cinerea), Cercospora (for example, C. kikuchii and C. zaea-maydis), Colletotrichum sp. (for example, C. lindemuthianum),

Diplodia (for example, D. maydis), Erysiphe (for example, E. graminis f.sp. graminis and E. graminis f.sp. hordei), Fusarium (for example, F. nivale and F. oxysporum, F. graminearum, F. solani, F. monilforme, and F. roseum), Gaeumanomyces (for example, G. graminis f.sp. tritici), Helminthosporium (for example, H. turcicum, H. carbonum, and H. maydis), Macrophomina (for example, M. phaseolina and Maganaporthe grisea), Nectria (for example, N. heamatocacca), Peronospora (for example, P. manshurica, P. tabacina), Phoma (for example, P. betae), Phymatotrichum (for example, P. omnivorum), Phytophthora (for example, P. cinnamomi, P. cactorum, P. phaseoli, P. parasitica, P. citrophthora, P. megasperma f.sp. sojae, and P. infestans), Plasmopara (for example, P. viticola), Podosphaera (for example, P. leucotricha), Puccinia (for example, P. sorghi, P. striiformis, P. graminis f.sp. tritici, P. asparagi, P. recondita, and P. arachidis), Puthium (for example, P. aphanidermatum), Pyrenophora (for example, P. tritici-repentens), Pyricularia (for example, P. oryzea), Pythium (for example, P. ultimum), Rhizoctonia (for example, R. solani and R. cerealis), Scerotium (for example, S. rolfsii), Sclerotinia (for example, S. sclerotiorum), Septoria (for example, S. lycopersici, S. glycines, S. nodorum and S. tritici), Thielaviopsis (for example, T. basicola), Uncinula (for example, U. necator), Venturia (for example, V. inaequalis), and Verticillium (for example, V. dahliae and V. albo-atrum). Examples of pathogenic nematodes include, without limitation, root-knot nematodes (for example, Meloidogyne sp. such as M. incognita, M. arenaria, M. chitwoodi, M. hapla, M. javanica, M. graminocola, M. microtyla, M. graminis, and M. naasi), cyst nematodes (for example, Heterodera sp. such as H. schachtii, H. glycines, H. sacchari, H. oryzae, H. avenae, H. cajani, H. elachista, H. goettingiana, H. graminis, H. mediterranea, H. mothi, H. sorghi, and H. zeae, or, for example,

Globodera sp. such as G. rostochiensis and G. pallida), root-attacking nematodes (for example, Rotylenchulus reniformis, Tylenchuylus semipenetrans, Pratylenchus brachyurus, Radopholus citrophilus, Radopholus similis, Xiphinema americanum, Xiphinema rivesi, Paratrichodorus minor, Heterorhabditis heliothidis, and Bursaphelenchus xylophilus), and above-ground nematodes (for example, Anguina funesta, Anguina tritici, Ditylenchus dipsaci, Ditylenchus myceliphagus, and Aphenlenchoides besseyi).

Examples of viral pathogens include, without limitation, tobacco mosaic virus (TMV), tobacco necrosis virus (TNV), potato leaf roll virus, potato virus X, potato virus Y, tomato spotted wilt virus, and tomato ring spot virus. By "increased level of resistance" is meant a level of resistance to a disease- causing pathogen in a non-naturally occurring plant (or cell or seed thereof) which is greater than the level of resistance in a control plant (for example, a non-transgenic plant or wild-type). In preferred embodiments, the level of resistance in a non- naturally occurring transgenic plant of the invention is at least 5% to 20% (and preferably 30% or 40%) greater than the resistance exhibited by a control plant. In other preferred embodiments, the level of resistance to a disease-causing pathogen is 50% greater, 60% greater, and more preferably even 75% or 90% greater than a control plant; with up to 100% or greater above the level of resistance as compared to a control plant being most preferred. The level of resistance is measured using conventional methods. For example, the level of resistance to a pathogen may be determined by comparing physical features and characteristics (for example, plant height and weight) or by comparing disease symptoms (for example, delayed lesion development, reduced lesion size, leaf wilting and curling, water-soaked spots, amount of pathogen growth, and discoloration of cells) of the non-naturally occurring plant (e.g., a transgenic plant).

By "detectably-labeled" is meant any direct or indirect means for marking and identifying the presence of a molecule, for example, an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule or a fragment thereof. Methods for detectably-labeling a molecule are well known in the art and include, without limitation, radioactive labeling (for example, with an isotope such as ³²P or ³⁵S) and nonradioactive labeling (for example, fluorescence of chemiluminescent labeling, for example, fluorescein labeling).

As discussed above, a gene encoding a negative regulator polypeptide of SAR has been identified. Accordingly, the invention provides a number of important advances and advantages for the protection of plants against pathogens. For example, by reducing the expression of such negative regulators of SAR in a plant, the invention facilitates an effective and economical means for in-plant protection against plant pathogens. Such protection against pathogens reduces or minimizes the need for traditional chemical practices (for example, application of fungicides, bactericides, nematicides, insecticides, or viricides) that are typically used by farmers for controlling the spread of plant pathogens and providing protection against disease- causing pathogens. In addition, because plants having decreased expression of a negative regulator of SAR are less vulnerable to pathogens and their diseases, the invention further provides for increased production efficiency, as well as for improvements in quality and yield of crop plants and ornamentals. Thus, the invention contributes to the production of high quality and high yield agricultural products, for example, fruits, ornamentals, vegetables, cereals and field crops having reduced spots, blemishes, and blotches that are caused by pathogens; agricultural products with increased shelf-life and reduced handling costs; and high quality and yield crops for agricultural (for example, cereal and field crops), industrial (for example, oilseeds), and commercial (for example, fiber crops) purposes. Furthermore, because the invention reduces the necessity for chemical protection against plant pathogens, the invention benefits the environment where the crops are grown. Genetically-improved seeds and other plant products that are produced using plants expressing the genes described herein also render farming possible in areas previously unsuitable for agricultural production.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Detailed Description of the Invention

The drawings will first be described. Drawings

FIGURE 1A shows the expression of the BGL2-GUS reporter gene in wild type (WT), nprl-1, snil nprl-1, and snil NPR1 plants. Two-week-old seedlings grown on MS (Murashige-Skoog) media in the presence or absence of INA (0.1 mM) were stained for GUS activity according to the methods described by Jefferson et al. (EMBO J. 6:3901-3907, 1987). FIGURE IB shows an RNA blot analysis of PR-1 gene expression in wild type (WT), nprl-1, snil nprl-1, and snil NPRl plants. RNA samples were prepared from 2- week-old seedlings grown on MS media or MS with 0.1 mM INA. For each sample, 10 μg of total RNA was loaded. The probe for PR-1 was made as described previously by Bowling et al. (Plant Cell 9:1573-1584, 1997). As loading controls, the 18S rRNA bands detected by ethidium bromide staining were used.

FIGURE IC shows the induction of BGL2-GUS by different concentrations of INA in wild type (WT), nprl-1, snil nprl-1, and snil NPRl plants. Ten 2-week-old seedlings grown on MS media containing 0, 0.002, 0.01, and 0.1 mM INA were collected and GUS activity was measured according to the methods described by

Jefferson et al. (EMBO J. 6:3901-3907, 1987). The values represent the average of 3 replicates ± SE. GUS activity is given as absolute fluorescence units per minute per microgram of protein.

FIGURE ID shows the morphological phenotypes of wild type (WT), snil nprl-1, and snil NPRl plants. The plants were grown on soil for 4 weeks before the photograph was taken.

FIGURE IE shows the growth of Psm ES4326 in wild type (WT), nprl-1, snil nprl-1, and snil NPRl plants. Four-week-old soil-grown plants were treated with 0.65 mM INA 2 days prior to infection by dipping the plants into a Psm ES4326 suspension (OD600 = 0.2) in 10 mM MgCl₂ and 0.01% Silwet L-77 as described by Bowling et al. (Plant Cell 6:1845-1857, 1994). Eight leaves were excised for each phenotype, treatment, and time point (0, 1, 2, and 3 days), rinsed with H₂O, weighed, and bacteria were extracted and plated according to methods described by Bowling et al. (Plant Cell 6:1845-1857, 1994). Error bars represent 95% confidence limits of log-transformed data from four replicates (Sokal and Rohlf, Biometry 2^nd Edition, W.H. Freeman and Company, N.Y., 1981). "cfu" represents colony-forming units.

FIGURE IF shows the average disease rating of P. parasitica Noco2 infection in wild type (WT), nprl-1, snil nprl-1, and snil NPRl plants. Two-week-old seedlings were treated with 0.65 mM INA 2 days prior to infection with a spore suspension (3 x 10⁴ spores/ml). The disease symptoms were scored 7 days after the infection with respect to the number of conidiophores observed on each seedling (30 seedlings were examined for each phenotype and treatment). The scales are defined as follows: 0, no conidiophores on the plants; 1, no more than 5 conidiophores per infected leaf; 2, 6-20 conidiophores on a few infected leaves; 3, 6-20 conidiophores on most infected leaves; 4, 5 or more conidiophores on all infected leaves; 5, 20 or more conidiophores on all infected leaves. Average disease ratings were calculated by summing the scores of all 30 seedlings and dividing by 30. The error bars represent standard errors.

FIGURE 2A is a schematic of the map of the snil locus on chromosome IV The cosmids that complemented the snil mutation are labeled by a "*" and the number of independent transformants tested is shown in parentheses.

FIGURE 2B shows the complementation of snil in PR gene expression. Using protocols described in Figure 1, GUS staining was performed on nprl-1, snil nprl-1, and snil nprl-1 transformed with the complementing cosmid 6-22 while RNA blot analysis was carried out in snil nprl-1 and snil NPRl transformed with the complementing cosmid 4.

FIGURE 2C shows the complementation of snil in response to pathogen infection. Using the protocols described in Figure 1, nprl-1, snil nprl-1, and snil nprl transformed with cosmid 4 were infected by Psm ES4326 and P. parasitica Noco2.

FIGURE 3 A shows the SNI1 cDNA sequence (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO: 2). The 11 nucleotides that are deleted in the mRNA as a result of the snil mutation are underlined.

FIGURE 3B shows the SNI1 genomic DNA sequence (SEQ ID NO:3). Exons are highlighted and start and stop codons are underlined.

FIGURE 3C shows sequence homology between SNI1 and Rb. Using a BLAST search of the GenBank database, SNI1 (SEQ ID NO:4) was found to share 22% amino acid sequence identity and 42% amino acid sequence similarity with the mouse retinoblastoma (Rb) protein (SEQ ID NO:5) in the N domain (Hensey et al., J. Biol. Chem. 269: 1380-1387, 1994) with an E value of 1.1. FIGURE 4 shows the nuclear localization of SNI1-GFP. The 35S-SNI1-GFP DNA preparation (20 μg) was delivered into onion epidermal cells using particle bombardment. After 12 hours of incubation, the GFP fluorescence was observed using a fluorescence microscope with an excitation wavelength of 488 nm. The nuclei in the cells were visible in the bright-field images (data not shown). The 35S-GFP construct was used as a control.

FIGURE 5 is a schematic showing a model for the regulation of SAR by SNIl and NPRl. In wild type (SNIl NPRl), SNIl is a repressor of R genes (a blocked line) and, therefore, a negative regulator of SAR. Induction of PR gene expression (an arrow) and SAR requires both activation of a positive regulator, presumably a TF by SA and derepression (a blocked line) of SNIl by the SA-activated NPRl. In a SNIl nprl-1 mutant, the compromised NPRl function (dashed oval) results in a failure to derepress SNIl and a lack of inducible PR gene expression and resistance. In a snil nprl double mutant, the function of SNIl is compromised (dashed rectangle) and the repression by SNIl is released even in the absence of an SAR inducer, leading to a background level expression of RR genes (a dashed arrow). Activation of the TF is still required for a full-scale induction of RR gene expression and SAR. In a snil NPRl single mutant, the lack of SNIl function results in a background level of RR gene expression. FIGURE 6 shows the enhanced sensitivity of snil to INA, a chemical inducer of SAR, and the enhanced resistance response to R. parasitica Noco infection.

Genetic Dissection of SAR

To further dissect the SAR pathway, a suppressor screen in the nprl-1 background was conducted. In particular, mutants that restore the inducible RR gene expression in the nprl-1 background have been identified. These mutants most likely harbor mutations in genes that function downstream or parallel to NPRl in the SAR pathway. The isolation and characterization of snil (suppressor of nprl, inducible 1), a recessive mutant that restores wild type levels of inducible RR gene expression and disease resistance is described below. SNIl represents a new class of SAR regulatory components. The SNIl gene was cloned using a map-based approach and found to encode a novel, leucine-rich, nuclear protein, which shares sequence homology with the animal tumor suppressor retinoblastoma (Rb) (Bernards et al., Proc. Natl. Acad. Sci. USA 86:6474-6478, 1989). The sequence data also revealed that the snil mutation causes mis-splicing of the mRNA resulting in a frameshift in the amino terminal region of the protein. Based on the genetic and molecular characterization of snil, a working model is proposed for SNIl as a negative regulator of SAR.

There now follows a description of the cloning and characterization of a gene encoding a negative regulator of SAR. In addition, a description for isolating additional negative regulators and methods of engineering disease resistant plants is provided. These examples are provided for the purpose of illustrating the invention, and should not be construed as limiting.

Identification of the snil Mutant in the nprl-1 Background

Seeds of nprl-1 (40,000), containing the BGL2-GUS reporter gene, were mutagenized using 0.3% ethyl-methanesulfonate (EMS) as described by Bowling et al. (Plant Cell 6: 1845-1857, 1994), and suppressors of the nprl-1 mutation were screened in the M₂ population for restored expression of the SAR-responsive BGL2-GUS in the presence of the SAR inducer, INA (0.1 mM) as follows. Two-week-old M₂ seedlings grown on Murashige-Skoog (MS) medium (Murashige and Skoog, Physiol. Plant 15:473-497, 1962) containing 0.1 mM INA (MS-INA) were tested for GUS activity using 4-methylumbelliferyl β-D-glucuronide (MUG) as substrate (Bowling et al., Plant Cell 6:1845-1857, 1994). Those seedlings that showed positive GUS activity were transferred to soil to set seeds. The same screen was then performed on the M₃ progeny. The presence of the nprl-1 mutation in the suppressor mutants was confirmed by the restriction digestion polymorphism generated by the nprl-1 mutation. More specifically, PCR primers (F4: 5'GAAGCTATTGGATAGATG3' (SEQ ID NO:6) and R5: 5'GTTGAGCAAGTGCAACT3' (SEQ ID NO:7)) were used to amplify a 770 bp genomic fragment containing the nprl-1 locus. In nprl-1, an Nlalll restriction digestion site is abolished.

Of 7,000 M₂ plants examined, 13 showed an increase in BGL2-GUS expression after induction by INA and were designated sni (suppressor of nprl-1, inducible) mutants. In particular, the snil mutant exhibited sporadic, weak GUS staining under uninduced conditions but consistent, strong GUS staining after induction by INA (Figure 1 A). GUS activity in snil was detected most strongly in the veins of both leaves and roots whereas in wild type, uniform GUS staining was detected in leaves while no GUS staining was seen in roots.

To confirm the effects of sni mutations on the expression of endogenous RR genes and rule out possible mutants of the BGL2-GUS reporter gene, standard RNA blot analysis was performed on all sni mutants to identify those which recovered inducible PR-1 gene expression in the nprl-1 background. Among these mutants, only snil expresses the RR-7 gene at levels similar to the wild-type under INA induction (Figure IB). Consistent with BGL2-GUS reporter gene expression (Figure 1 A), a higher background expression of PR-1 was detected in the snil mutant without induction. Because of its interesting phenotypes, snil was chosen for in-depth analyses.

Genetic Characterization of snil

The snil nprl-1 double mutant (BGL2-GUS) was backcrossed with nprl-1 (BGL2-GUS) and the resulting F, progeny lost the inducible BGL2-GUS expression. These F, plants were then allowed to self-fertilize, and the F₂ plants were assayed for the presence or the absence of INA-induced GUS expression using 5-bromo-4-chloro-3-indolyl glucuronide as substrate (Jefferson et al., EMBO J. 6:3901-3907, 1987). The obtained segregation ratio for GUS staining was used to determine the Mendelian characteristics of snil. Of 112 F₂ plants examined, 27 had strong GUS staining while the rest had no staining, demonstrating that the snil phenotype is recessive and results from a single nuclear mutation (χ² = 0.047; P>0.5; Sokal and Rohlf, Biometry 2^nd Edition, W.H Freeman and Company, New York, 1981). Complementation tests between snil and other recessive sni mutants showed that snil is not allelic to other sni mutants. In addition, morphological phenotypes of the F₂ population were found to cosegregate with the snil mutation, namely reduced plant size and pointed first pair of true leaves.

The snil nprl-1 double mutant was also crossed with wild-type plants containing the BGL2-GUS reporter gene, and the F, progeny lost the background expression of BGL2-GUS under uninduced conditions but showed wild-type GUS staining after an INA treatment. The F₂ plants were also examined for segregation of inducible BGL2-GUS expression to determine whether the snil phenotype is caused by a reversion of nprl-1 or by a second-site mutation. If snil is linked to nprl-1, all F₂progeny should have shown inducible BGL2-GUS expression. However, of 181 plants tested, 148 showed strong GUS staining while 33 displayed no detectable GUS staining after INA treatment, indicating that snil is not linked to nprl-1 (χ² = 0.032; P>0.5; Sokal and Rohlf, supra). From the same F₂ population, snil NPRl was also identified using the reduced-plant- size and the pointed-first-true-leaf phenotypes of snil to detect snil homozygotes and the PCR-Nlalll digestion method to detect NPRl homozygotes.

In the wild-type NPRl background, the snil mutant (snil NPRl) expressed RR-7 and BGL2-GUS to a higher level than in snil nprl under induced conditions (Figures IB and IC). As seen in snil nprl plants, ectopic GUS staining was detected in roots in snil NPRl plants. In leaves, INA induction resulted in uniform GUS staining. This pattern is different from that observed in the sni nprl-1 double mutant where GUS staining was detected predominantly in the vascular tissues. This difference in the INA-induced GUS expression between snil nprl-1 and snil NPRl implied that NPRl function is still required for high level RR gene induction in nonvascular tissues in the snil mutant. Introduction of snil into other nprl mutant alleles showed that expression of snil phenotypes is not specific to the nprl-1 mutant allele (data not shown).

Both snil nprl and snil NPRl plants are smaller than wild type (Figure ID). In snil seedlings, the emerging first pair of true leaves are much narrower than those of the wild type (data not shown). These phenotypes were found to cosegregate with snil and shown later by complementation analysis to be caused by snil . In snil mutant plants, no macroscopic or microscopic lesions were detected by trypan blue staining even after an INA treatment (data not shown), indicating that cell death is not involved in causing the snil phenotypes.

The Effects of snil on SA Accumulation and Sensitivity

The endogenous levels of S A in snil NPRl were measured under non-inducing conditions and after infection by an SAR-inducing pathogen Pseudomonas syringae pv. maculicola ES4326/αvrRpt2 (Psm ES4326/αvrRpt2) as follows. Tissues were harvested from 4-week-old, soil-grown plants, ground in liquid N₂, and weighed. SA extraction was performed using a modified protocol derived from Raskin et al. (Proc. Natl. Acad. Sci. USA 86:2214-2218, 1989). 3 ml of methanol (90%) was added to the ground tissues and the resulting mixture was vortexed and sonicated for 20 minutes. After centrifugation at 7,000 rpm for 10 minutes the supernatant was collected, the pellet was re-extracted with 2 ml of methanol (100%) and re-centrifuged at 7,000 rpm for another 10 minutes. The supernatants from both extractions were combined and air-dried in a water bath (60°C). The dried samples were resuspended in 2.5 ml 5% trichloroacetic acid (TCA), vortexed, sonicated for 5 minutes, and centrifuged at 7,000 rpm for 10 minutes. The supernatants were collected and extracted in 100/99/1 (vol) ethylacetate/cyclopentane/isopropanol by vigorous vortexing for 10 minutes. The top organic phase was removed and air-dried in a water bath (60 °C). The dried extract was resuspended in 250 μl of mobile phase (0.2 M KAc, 0.5 mM EDTA, pH 5), vortexed, and sonicated for 5 minutes, and spun through a 0.22 μm nylon filter (Costar). To measure the amount of extracted SA, the samples were separated through a 100 x 4.6 sperisorb DDS2 column (Keystone Scientific Inc.) with a particle size of 3 μm and a pore size of 80 angstroms at a mobile-phase flow rate of 1 ml minutes. Fluorescent detection was performed on an HPLC spectrofluorescence detector equipped with a Xenon-mercury arc lamp at an excitation/emission wavelength of 295/405 nm. This procedure had a 25% recovery rate as determined by extracting known amounts of S A. The results showed that both the base-line and the induced levels of SA found in snil (0.4 ± 0.004 μg/g and 1.3 ± 0.45μg/g, respectively) are comparable to those of the wild type (0.2 ± 0.008 μg/g and 1.7 ± 0.69 μg/g, respectively). The sensitivity of snil NPRl and snil nprl-1 mutants to SA and INA was measured by a quantitative assay of the BGL2-GUS reporter gene expression. As shown in Figure IC, wild type displayed the highest level of BGL2-GUS expression at 0.1 mM INA, a concentration normally used for SAR induction while nprl-1 remained nonresponsive to INA. However, in the snil NPRl and snil nprl-1 mutants, maximum levels of BGL2-GUS expression were reached at a concentration (0.01 mM) ten times lower than in the wild type. Similar results were obtained when SA was used as the inducer (data not shown). Comparing snil NPRl and snil nprl-1, higher levels of BGL2-GUS were detected for sni NPRl, indicating that a functional NPRl is required for maximum induction of the reporter gene.

Resistance of snil to Pathogens

The effect of snil on pathogen resistance in the nprl-1 or NPRl background was tested using a bacterial pathogen Psm ES4326 which causes leaf spots and an oomycete pathogen Peronospora parasitica (P. parasitica) Noco2 which results in downy mildew on uninduced wild type plants as follows. Infection of wild type and mutant Arabidopsis with Psm ES4326 and P. parasitica Noco2 was carried out as previously described by Bowling et al. (Plant Cell 6: 1845-1857, 1994). For the Psm ES4326 infection, 4 samples (8 leaves/sample) were taken for each genotype, treatment, and time point to determine the in planta growth of the bacteria and the 95% confidence limits of the log-transformed data were calculated as described by Sokal and Rohlf (supra). For the P. parasitica Noco2 infected plants, disease rating was determined for each plant according to Cao et al. (Proc. Natl. Acad. Sci. USA 95:6531-6536, 1998). For each genotype and treatment, 30 plants were examined, and an average disease rating was calculated. After INA induction, wild type plants became resistant to these two pathogens while nprl-1 remained susceptible (Figures IE and IF). In snil nprl-1, the snil mutation restored the induced resistance. In snil NPRl, the effect of snil on resistance was insignificant under the infection conditions used, i.e., the pattern of resistance in snil NPRl is indistinguishable from that of the wild type. Although in both snil nprl-1 and snil NPRl background expression of RR genes is elevated, these mutants still required further induction to become resistant to Psm ES4326 and R. parasitica Noco2 at the inoculants used in these experiments.

Map-Based Cloning of SNIl The snil gene was mapped by crossing snil nprl-1, which is in the Columbia ecotype (Col-0, carrying the BGL2-GUS reporter gene), to the SNIl NPRl Landsberg ecotype (La-er, also carrying the BGL2-GUS reporter gene). The F₂ progeny homozygous for snil were identified in two-week-old plate-grown seedlings using the morphological phenotypes associated with snil. The snil homozygosity of those progeny critical for determining the map position of snil was later confirmed in the F₃ generation using the snil -specific BGL2-GUS reporter gene expression pattern. The chromosomal position of snil was determined using various CAPS (cleaved amplified golymorphic sequences) markers described by Konieczny and Ausubel (Plant J. 4:403-410, 1993). Fifty-eight F₂ progeny homozygous for snil were used to determine the crude map position of snil. Using CAPS markers SC5 and AG on chromosome IV, 2 and 0 heterozygotes were detected, respectively, indicating that SC5 is ~ 1.7 cM on the centromeric side of snil and AG is closely linked to snil (Figure 2A).

For fine mapping, CAPS markers g4539 and g3883-1.4 (http://genome-www.stanford.edu/arabidopsis/aboutcaps.html) were used to examine 718 F₂ progeny homozygous for snil. Twenty-three heterozygotes were discovered using g4539 (~1.6 cM on the centromeric side of snil) and 22 with g3883-1.4 (~ 1.5 cM on the telomeric side of snil). Because the heterozygotes found by these two markers were mutually exclusive, it was determined that snil is flanked by these two markers. No heterozygotes were discovered using the marker ch42 located between g4539 and g3883-1.4, indicating that ch42 is closely linked to snil. Because the ch42 sequence was found in the middle of a bacterial artificial chromosome (BAC) clone, F28J12, which had been completely sequenced by the ESSAII project (http://www.mips.biochem.mpg.de/proj/thal/), two CAPS markers, designated XL3 and XL7, were generated on both sides of ch42 according to the sequence information.

XL3 was amplified using primers 5'CTGGCATCCGTGAAAC3'(SEQ ID NO:8) and 5'GCAGGACTTGATGTATCC3' (SEQ ID NO:9) and the polymorphism between Col and La-er was detected by Ndel restriction digestion. XL7 was amplified using primers 5'CCATCCAAAGGCGATAAC3' (SEQ ID NO: 10) and 5'CCAAACTACTACCGGATG3' (SEQ ID NO: 11) and the polymorphism was revealed by Ddel digestion. These markers were used to narrow down the interval containing snil to a 43.3 kb region through the detection of one mutually exclusive recombinant for each marker, XL7 and XL3.

Because this 43.3 kb region is contained in the BAC clone F28J12, two cosmid libraries were generated from F28J12 using the binary vector pCLD04541 (carrying the kanamycin resistance selection) or pSLJ75516 (carrying the herbicide glufosinate ammonium or "basta" resistance selection). F28J12 was obtained from the Arabidopsis Biological Resource Center at the Ohio State University, and the BAC DNA was prepared according to the protocol provided with the clone. To subclone the BAC, 2 μg of the DNA was partially digested with the Taql restriction endonuclease for 5 minutes at 60 °C, and ligated into the Clal site of binary vector pCLD04541 or pSLJ75516. The ligated products were then packaged using Gigapack XL (Stratagene) and used to infect E. coli (DH5α).

By probing the libraries with PCR fragments derived from the interval between XL7 and XL3, cosmid contigs spanning the snil region were produced (Figure 2A). These PCR fragments were generated using the following primers: XL4 (F: 5'GTGATGGTGAGGGCTTC3' (SEQ ID NO: 12); R: 5'CGTCGGGATCTACAGG3' (SEQ ID NO: 13)), XL7, ch42, XL11 (F: 5'GATGGCAATTGCTGGAG3' (SEQ ID NO: 14); R: 5'CTAATGGGATGCGACTC3' (SEQ ID NO: 15)) and XL1 (F: 5'GATGAGATGTGCTGAG3' (SEQ ID NO: 16); R: 5'CATCGATTTCGCCGTTC3' (SEQ ID NO: 17)). The positive clones were then analyzed by restriction digestion using Xhol and Hindlll. Because the region was completely sequenced, a contig was easily constructed from both libraries by analyzing the restriction patterns of the clones. The contig made with pCLD04541 was transformed (Clough and Bent, Plant J. 16:735-743, 1998) into a snil line (snil nprl-1) that had lost the BGL2-GUS reporter gene and was therefore kanamycin sensitive. The resulting transformants from pCLD04541 clones were selected on MS medium containing 50 mg/ml kanamycin. The contig made with pSLJ75516 was transformed into snil nprl-1 and transformants were selected by spraying ten-day-old soil-grown plants with 0.04% basta (in 0.01% Silwet L-77; Union Carbide, Danbury, CT).

Complementation of snil was determined first by the restoration of wild type morphology and then by the loss of inducible expression of the BGL2-GUS reporter gene or the endogenous PR-1 gene. Segregation of these phenotypes in the T₂ generation was monitored to distinguish true complementation from contamination by nprl-1. PCR was also performed to confirm the presence of the cosmid clone in the complementing transformants using the vector-specific primers. Complementation of the snil mutation was observed when cosmid clones 4, 7, and 6-22 were transformed into snil nprl-1 mutants. In these transformants, which are snil nprl-1 ::SNI1, expression of PR-1 and the BGL2-GUS reporter was completely inhibited, as seen in the nprl-1 mutant (Figure 2B). The transformants also displayed susceptibility to both Psm ES4326 and P. parasitica Noco2 even after an INA induction (Figure 2C). When the cosmids containing SNIl was transformed into the snil NPRl single mutant, the background expression of RR genes was repressed; the plants behaved like wild type, showing inducible RR gene expression (Figure 2B). The T₂ progeny of the complementing lines all segregated for the SNIl and snil phenotypes, which corresponded to the presence and the absence of the selective markers, respectively. This indicated that these are true complementing transformants rather than contaminants from either nprl-1 or wild type. Transgenic plants containing cosmids other than clones 4, 7, and 6-22 showed the snil mutant phenotypes.

Sequence Analysis of SNIl The 15 kb genomic region in the snil mutant corresponding to the insert of the complementing cosmid 4 (Figure 2A) was PCR amplified and sequenced using an ABI automatic sequencer. The snil mutation was identified by aligning the obtained sequence with that of the wild type. In this entire 15 kb region, only one mutation (G to A) was found. The PCR primers used for sequencing were then combined to perform RT-PCR (GeneAmp Kit; Perkin Elmer) and the cDNA sequence in the region flanking the snil locus was determined. The partial cDNA sequence was then used as a probe to screen a cDNA library made in λ ZAPII containing 1-3 kb inserts (Kieber et al., Cell 72:427-441, 1993). From the 10⁶ plaques screened, one cDNA clone was isolated. The cDNA clone was shown to contain almost full length SNIl, missing only 11 nucleotides of the 5' end which was determined by RT-PCR and sequencing. A full length cDNA clone was generated by PCR, put under the control of the constitutive 35S promoter of cauliflower mosaic virus, transformed into snil, and shown to complement the snil mutation. By comparing the genomic and the cDNA sequences, introns were identified in the SNIl gene. The G to A mutation in snil was found to be in a 3' intron acceptor site and the effect of this mutation on splicing was detected by RT-PCR of the mutant mRNA and sequencing analysis.

Sequence data were analyzed with different programs available through the internet (http://www.expasy.ch/tools).

Sequence analysis predicted that SNIl encodes a novel protein of 432 amino acids (Figure 3A) and molecular weight of 48.8 kDa. Hydropathy plot analysis showed that SNIl is a soluble protein with no obvious transmembrane domains. A survey of the amino acid sequence showed that SNIl is a leucine-rich protein (12.7%). A database search for homologs yielded one EST clone isolated from cotton (accession number: AI054954), implying that SNIl may be conserved in plants. Even though no substantial homology was found between SNIl and any known proteins, a short stretch of homology was discovered with the mouse Retinoblastoma (Rb) protein (Bernards et al., Proc. Natl. Acad. Sci. USA 86:6474-6478, 1989) (Figure 3C), a tumor suppressor that represses the transcription regulated by transcription factors, such as E2F (Nevins, Science 258:424-429, 1992).

Comparison of the cDNA sequence and the genomic sequence revealed that the SNIl gene consists of 15 exons (Figure 3B). The G to A mutation in snil occurred in the 3' intron acceptor site at the junction between the fifth intron and the sixth exon. RT-PCR and sequencing analysis of the snil mutant showed that the mutation caused a 11-nucleotide deletion in the cDNA, resulting in a frameshift early in the coding region (Figure 3 A, amino acid residue 140).

Expression and Subcellular Localization of SNIl

Standard RNA blot analysis using Arabidopsis poly A⁺ RNA detected a very faint band of approximately 1500 nucleotides in both uninduced and induced samples (data not shown). In addition, transgenic Arabidopsis lines were generated carrying a SNIl promoter fusion to the GUS reporter gene. Very weak GUS staining was detected mainly in the veins. INA treatment seemed to have no effect on the staining pattern (data not shown).

To determine the subcellular localization of SNIl, the green fluorescent protein (GFP) gene was fused to either the 5' or the 3' end of SNIl as follows. The GFP gene carried by the plasmid pRT2ΔN-mGFP (Stacey et al., Plant Cell 11:349- 363, 1999) was fused to either the 5' or the 3' end of the SNIl gene by inserting the SNIl coding sequence into the Bglll and Ncol sites of the plasmid, respectively. The resulting plasmids were purified using a Qiagen Midiprep Kit (Valencia, CA), and 20 μg of DNA was bombarded into onion epidermal cells according to a protocol by von Arrnim and Deng (Cell 79: 1035-1045, 1994). GFP was then observed using a fluorescence microscope. The resulting fusion genes were driven by the constitutive 35S promoter of the cauliflower mosaic virus and the resulting fusion proteins had a MW of 75 kDa, which is above the size exclusion limit (40-60 kDa) for passive diffusion of protein through the nuclear pores (Raikhel, Plant Physiol. 100:1627-1632, 1992). When either 35S-GFP-SNI1 or 35S-SNI1-GFP was delivered into onion epidermal cells by particle bombardment, GFP fluorescence was observed predominantly in the nuclei (Figure 4). Weak fluorescence was also detected in the cytoplasm. When 35S-GFP was transiently expressed, the smaller GFP protein (26 kDa) was found to be evenly distributed throughout the cells.

Generation of anti-SNI polyclonal antibodies

A polyclonal anti-SNI peptide antibody was raised against the synthetic peptide CFSDPHEGDSISE (SEQ ID NO: 18), corresponding to amino acids 381-392 of the SNI polypeptide (Fig. 3A), with the addition of a N-terminal cysteine for later purification. The peptide was synthesized and purified according to standard methods. The purified peptide was next coupled to the carrier molecule, KLH (keyhole limpet hemocyanin). Antibody was obtained by immunizing a white female New Zealand rabbit every 4 weeks with the peptide/KLH conjugate emulsified in Freund's adjuvant according to standard methods. Blood samples were subsequently obtained two weeks after injection of the conjugate and serum was screened by standard western blot analysis. The synthetic peptide without KLH was reduced using Pierce Reduce- Imm Column coupled to a sulfoLink column (Pierce) and was used to purify the antibody according to the manufacturer's instructions. Using standard western blotting techniques, the polyclonal antibody was found to bind specifically to a fragment of SNIl produced in E. coli and plant extracts.

SNIl as a Negative Regulator of SAR

To identify additional regulators of SAR, a screen for suppressors of nprl-1 was performed. The recessive mutant snil was further characterized and shown to restore inducible RR gene expression and pathogen resistance in the nprl-1 background. The snil mutant could only be identified in an nprl mutant background because the snil NPRl single mutant is almost indistinguishable from the wild type with respect to either RR gene expression or disease resistance. The recessive, loss-of-function snil represents a new class of S AR-related mutants, distinct from the previously identified, dominant mutants such as cprό and ssil which constitutively express high levels of RR-7 in the nprl background in an SA-dependent fashion (Clarke et al., Plant Cell 10:557-569, 1998) or lesion-dependent fashion (Shah et al., Plant Cell 11:191-206, 1999).

The characteristics of the snil mutant and the nuclear localization of the SNIl-GFP fusion protein indicate that the wild type SNIl is a repressor of RR gene expression and SAR, which most likely functions downstream of NPRl. The lack of induction of SAR in nprl mutants could be explained if a functional, SA-activated NPRl is required to release the repression of RR gene transcription by SNIl. The snil mutation, which causes a frameshift early in the coding region, abolishes SNIl repression of the RR genes and eliminates the requirement for NPRl. Release of SNIl repression by the mutation also results in elevated background RR gene expression in leaves and in roots, and reduced threshold levels of SA and INA that are required to induce RR gene expression.

SNIl appears to represent a novel repressor of transcription, which directly binds to a specific DNA sequence and inhibits the transcriptional machinery.

Alternatively, SNIl may bind to DNA indirectly through interaction with a DNA binding protein because no apparent DNA binding domain has been found in SNIl. In support of this hypothesis, a cis-element, which contains the consensus of a binding site for the plant-specific transcription factor WRKY family, was found in the RR-7 promoter and shown to negatively regulate the expression of the gene (Lebel et al., Plant J. 16:223-233, 1998). Mutations in this element result in an expression pattern of RR-7 similar to that observed in the snil mutant, i.e., an elevated background expression and an enhanced response to INA induction. It is equally plausible that SNIl affects transcription by sequestering a transcriptional activator. The stretch of sequence homology detected between SNIl and the tumor suppressor gene Rb suggests such a possibility because Rb negatively regulates gene expression by interacting with transcription factors such as E2F (Nevins, Science 258:424-429, 1992). The biological function of the homologous region in Rb, known as the N domain (Hensey et al., J. Biol. Chem. 269:1380-1387, 1994), is still unclear even though it is highly conserved among Rb homologs isolated from many organisms. Cell culture and in vitro experiments have indicated that this domain is dispensable for Rb function (Jacks et al., Nature 359:295-300, 1992; Lee et al., Nature 359:288- 294, 1992, Genes Dev. 8:2008-2021 1994; Fung et al., Oncogene 8:2659-2672, 1993). However, deletion mutants of this domain failed to rescue Rb mutant mice, suggesting that this domain may in fact be of functional importance (Riley et al., Mol. Cell. Biol. 17:7342-7352, 1997).

The inducible, rather than constitutive, RR gene expression and disease resistance observed in snil nprl-1 suggests the requirement of a separate activation event in inducing RR gene expression and SAR in addition to the regulations of NPRl and SNIl. This parallel activation event only became evident when both the NPRl and SNIl functions were abolished. SA is required for this induction event because expression of the SA-degrading salicylate hydroxylase gene (nahG) in the snil nprl mutant prevents the induction of the RR genes by exogenous application of SA (data not shown). This activation event probably does not require the function of NPRl but may be facilitated by NPRl because the RR gene induction that occurs in snil nprl-1 is less dramatic than in snil NPRl. Indeed, in a yeast two-hybrid screen, NPRl was found to bind specifically to a subclass of bZIP transcription factors. These bZIP transcription factors may be the positive regulators of the RR-7 gene that are activated by SA, in parallel to NPRl, to induce RR gene expression and SAR. In support of this, mutations in the binding site for the bZIP transcription factors in the RR-7 promoter abolish the inducibility of RR-7 by SA and INA (Lebel et al., Plant J. 16:223-233, 1998). The consequence of the NPRl-bZIP transcription factor interaction has yet to be revealed. Binding of NPRl to the bZIP transcription factors may facilitate the activation of these transcription factors or recruit NPRl to its functional location. Taking all the data together, a working model to explain the signal transduction pathway leading to the activation of RR gene expression and illustrate the roles of SNIl and NPRl in the induction of SAR is shown in Figure 5. In wild type plants, induction of RR genes and SAR by S A or INA may require two separate events: inactivation of SNIl by S A through a function of NPRl and activation of a positive regulator, presumably the bZIP transcription factor, by SA through a mechanism that does not require NPRl but may be facilitated by NPRl. In an nprl mutant, where SNIl repression cannot be removed due to the lack of NPRl activity, induction of SAR is blocked. However, in the snil nprl-1 double mutant, where RR gene expression is no longer repressed due to the snil mutation, the requirement for NPRl is eliminated and background levels of RR gene expression are observed under uninduced conditions. The lack of SNIl repression may also explain the elevated basal expression of RR genes in the snil NPRl single mutant. When a wild type SNIl gene was transformed into snil NPRl and snil nprl-1, this background expression of RR genes was repressed and the phenotype of the transformants reverted back to wild type and nprl-1, respectively.

Identification and cloning of SNIl provides a basis to further unravel the regulatory mechanism of RR gene expression and SAR. The data described herein suggests that induction of SAR involves both activation and derepression events. The significance of SNIl repression is underlined by the leakiness in RR gene regulation observed in snil mutants under noninducing conditions and by the reduced plant size of these mutants. Identification of snil also suggests a possible means of engineering plants with enhanced disease resistance. In particular, disease resistance analysis has shown that disruption of SNIl function resulted in enhanced sensitivity to chemical inducers of SAR such as SA and INA, as well as resulting in an enhanced resistance response to pathogens such as Peronospora parasitica (Figure 6).

Microarray Gene Expression Monitoring and Discovery

The cells, plants and plant components, and methods described herein may also be used in combination with standard microarray technologies (for example, cDNA microarrays or DNA chips) for monitoring the expression of genes controlled by or associated with negative regulators of SAR. In particular, microarray expression monitoring provides a basis for understanding how SNIl integrates into the SAR signal transduction pathway. Moreover, such technology enables the identification of novel genes associated with the SAR pathway. For example, target genes that regulate or are regulated by SNIl may be identified by correlating changes in gene expression with specific changes in cells having either reduced or increased levels of the SAR negative regulator protein. Genes exhibiting altered expression levels in such cells are identified and subsequently serve as specific targets whose expression is modified to generate transgenic plants having increased resistance to a pathogen.

In general, microarray technology utilizes organized DNA libraries of interest (for example, a cDNA library) whose members are arrayed on a solid support (e.g., a glass slide). Such cDNAs are amplified by polymerase chain reaction, purified, and small quantities are deposited onto known locations of a microchip according to standard methods such as high-speed robotics. Microarrays of cDNAs accordingly provide expression information for each gene represented on the microchip. Arrayed sequences, in turn, serve as targets for hybridization to cDNA probes prepared from RNA samples of plant cells or tissues of interest. Multi-color fluorescence labeling of cDNA probes further provides a means facilitating comparative differential expression analysis of a variety of plant tissues under different physiological and environmental conditions. Typically, a gene whose expression is increased or decreased in an appropriate genetic background is considered as a useful target involved in plant disease resistance.

Exemplary microarray methods for differential and quantitative monitoring of gene expression and for identifying novel genes are provided in Schena et al. (Science 270:467-470, 1995), Schena et al. (Proc. Natl. Acad. Sci. USA 93:10614-10619, 1996), Heuller et al. (Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997), and Case- Green et al. (Curr. Opin. Chem. Biol. 2:404-410, 1998). In one particular example, microarrays may be used to examine gene expression in plant tissue (e.g., a leaf or root) having reduced levels of a negative regulator of SAR. For example, plants having reduced SNIl levels (such as plants expressing a dominant-negative SNIl or that express antisense SNIl RNA) are resistant to pathogens, whereas in wild type plants, SNIl is a repressor of RR genes, and, therefore, a negative regulator of SAR. Total mRNA from leaves of S A-induced wild-type Arabidopsis (control) and a sni mutant test plant is fluorescently labeled using a single round of reverse transcriptase. Fluorescently labeled mRNA is then hybridized to a microarray containing target DNA, such as expressed sequence tags from an Arabidopsis cDNA library. The array is washed under high stringency conditions and scanned with a confocal laser scanning device to detect hybridization via emission of the fluorescently labeled DNA. Comparative expression analysis of control and test plants are determined, and array elements displaying altered fluorescence ratios are selected for further analysis. For example, genes highly induced in the test plants as compared to wild- type plants are selected and characterized by sequencing. Such genes may then be used to engineer disease resistant plants.

Alternatively, microarrays may be used to examine gene expression in plant tissue having increased levels of a negative regulator of SAR (for example, plants overexpressing wild-type SNIl or plants expressing wild-type SNIl protein in a snil nprl genetic background). In such plants, SAR is repressed, abolishing induced RR gene expression and resistance. Accordingly, these plants have increased susceptibility to their pathogens and provide a means for identifying novel target genes that regulate negative regulators of SAR such as SNIl. In one working example, total mRNA from roots of INA-induced wild-type

Arabidopsis (control) and test plants expressing wild- type SNIl in an snil nprl background is fluorescently labeled and hybridized to a microarray as described above. The array is washed and analyzed using a confocal laser scanning device. Comparative expression data between control and test plants is determined, and array elements displaying altered fluorescence ratios are then selected for further analysis. For example, genes having decreased expression in the test plants as compared to wild type are then selected and characterized by sequencing.

Isolation of Other Genes Encoding Negative Regulator Polypeptides of SAR

Any plant cell can serve as the nucleic acid source for the molecular cloning of a gene encoding a negative regulator polypeptide of SAR. Isolation of such a negative regulator gene involves the isolation of those DNA sequences which encode a protein exhibiting negative regulator polypeptide of SAR-associated structures, properties, or activities, for example, those described herein for SNIl. Based on the negative regulator genes and polypeptides described herein, the isolation of additional sequences coding for negative regulators of SAR is made possible using standard strategies and techniques that are well known in the art.

In one particular example, the negative regulator sequences described herein (for example, those described for SNIl) may be used, together with conventional screening methods of nucleic acid hybridization screening. Such hybridization techniques and screening procedures are well known to those skilled in the art and are described, for example, in Benton and Davis, Science 196:180-182, 1977; Grunstein and Hogness, Proc. Natl. Acad. Sci., USA 72:3961-3695, 1975; Ausubel et al., 1999, Current Protocols in Molecular Biology, Wiley Interscience, New York; Berger and Kimmel (supra); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. In one particular example, all or part of the SNIl cDNA (described herein) may be used as a probe to screen a recombinant plant DNA library for genes having sequence identity to a gene encoding a negative regulator polypeptide of SAR. Hybridizing sequences are detected by plaque or colony hybridization according to the methods described below. Alternatively, using all or a portion of the amino acid sequence of a negative regulator polypeptide of SAR (for example, SNIl), one may readily design specific oligonucleotide probes, including degenerate oligonucleotide probes (i.e., a mixture of all possible coding sequences for a given amino acid sequence). These oligonucleotides may be based upon the sequence of either DNA strand and any appropriate portion of the negative regulator polypeptide gene sequence. General methods for designing and preparing such probes are provided, for example, in Ausubel et al. (supra), and Berger and Kimmel, Guide to Molecular Cloning Techniques, 1987, Academic Press, New York. These oligonucleotides are useful for gene isolation, either through their use as probes capable of hybridizing to complementary sequences or as primers for various amplification techniques, for example, polymerase chain reaction (PCR) cloning strategies. If desired, a combination of different oligonucleotide probes may be used for the screening of a recombinant DNA library. The oligonucleotides may be detectably-labeled using methods known in the art and used to probe filter replicas from a recombinant DNA library. Recombinant DNA libraries are prepared according to methods well known in the art, for example, as described in Ausubel et al. (supra), or they may be obtained from commercial sources.

In one particular example of this approach, gene sequences having greater than 80% identity to SNIl are detected or isolated using high stringency conditions. High stringency conditions may include hybridization at about 42 °C and about 50% formamide, 0.1 mg/ml sheared salmon sperm DNA, 1% SDS, 2X SSC, 10% Dextran sulfate, a first wash at about 65 °C, about 2X SSC, and 1% SDS, followed by a second wash at about 65 °C and about 0.1X SSC. Alternatively, high stringency conditions may include hybridization at about 42 °C and about 50% formamide, 0.1 mg/ml sheared salmon sperm DNA, 0.5% SDS, 5X SSPE, IX Denhardt's, followed by two washes at room temperature and 2X SSC, 0.1% SDS, and two washes at between 55- 60°C and 0.2X SSC, 0.1% SDS.

In another approach, low stringency hybridization conditions for detecting negative regulator genes having about 30% or greater sequence identity to SNIl described herein include, for example, hybridization at about 42 °C and 0.1 mg/ml sheared salmon sperm DNA, 1% SDS, 2X SSC, and 10% Dextran sulfate (in the absence of formamide), and a wash at about 37°C and 6X SSC, about 1% SDS. Alternatively, the low stringency hybridization may be carried out at about 42 °C and 40% formamide, 0.1 mg/ml sheared salmon sperm DNA, 0.5% SDS, 5X SSPE, IX Denhardt's, followed by two washes at room temperature and 2X SSC, 0.1% SDS and two washes at room temperature and 0.5X SSC, 0.1% SDS. These stringency conditions are exemplary; other appropriate conditions may be determined by those skilled in the art. As discussed above, oligonucleotides may also be used as primers in amplification cloning strategies, for example, using PCR. PCR methods are well known in the art and are described, for example, in PCR Technology, Erlich, ed., Stockton Press, London, 1989; PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc., New York, 1990; and Ausubel et al. (supra). Primers encoding specific structural features of a negative regulator polypeptide of SAR, for example, the N domain motif, are useful for isolating genes having similar structural domains. Primers are optionally designed to allow cloning of the amplified product into a suitable vector, for example, by including appropriate restriction sites at the 5' and 3' ends of the amplified fragment (as described herein). If desired, negative regulator gene sequences may be isolated using the PCR "RACE" technique, or Rapid Amplification of cDNA Ends (see, for example, Innis et al. (supra)). By this method, oligonucleotide primers based on an negative regulator gene sequence (for example, SNIl) are oriented in the 3' and 5' directions and are used to generate overlapping PCR fragments. These overlapping 3'- and 5 '-end RACE products are combined to produce an intact full-length cDNA. This method is described in Innis et al. (supra); and Frohman et al., Proc. Natl. Acad. Sci. USA 85:8998- 9002, 1988.

Alternatively, any plant cDNA or cDNA expression library may be screened by functional complementation of a plant having a mutation in a gene encoding a negative regulator polypeptide of SAR (for example, a snil mutant) according to standard methods described herein.

Confirmation of a sequence's relatedness to the negative regulator polypeptide of SAR gene family may be accomplished by a variety of conventional methods including, but not limited to, functional complementation assays and sequence comparison of the gene and its expressed product. In addition, the activity of the gene product may be evaluated according to any of the techniques described herein, for example, the functional or immunological properties of its encoded product.

In addition, the SNIl sequence disclosed herein provides a basis for searching databases such as Genbank to identify SNIl homologs.

Once a gene encoding a negative regulator polypeptide of SAR is identified, it is cloned according to standard methods and used for the construction of plant expression vectors as described below.

Polypeptide Expression

Polypeptides of the invention may be expressed and produced by transformation of a suitable host cell with all or part of a gene encoding a negative regulator polypeptide of SAR (for example, the SNIl cDNA described herein) in a suitable expression vehicle or with a plasmid construct engineered for increasing the expression of the encoded polypeptide in vivo.

Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. The polypeptide may be produced in a prokaryotic host, for example, E. coli, or in a eukaryotic host, for example, Saccharomyces cerevisiae, mammalian cells (for example, COS 1 or NIH 3T3 cells), or any of a number of plant cells or whole plant including, without limitation, algae, tree species, ornamental species, temperate fruit species, tropical fruit species, vegetable species, legume species, crucifer species, monocots, dicots, or in any plant of commercial or agricultural significance. Particular examples of suitable plant hosts include, but are not limited to, conifers, petunia, tomato, potato, pepper, tobacco, Arabidopsis, lettuce, sunflower, oilseed rape, flax, cotton, grape, citrus, sugarbeet, celery, soybean, alfalfa, Medicago, lotus, Vigna, cucumber, carrot, eggplant, cauliflower, horseradish, morning glory, poplar, walnut, apple, grape, asparagus, cassava, rice, maize, millet, onion, barley, orchard grass, oat, rye, and wheat. Such cells are available from a wide range of sources including the American Type Culture Collection (Rockland, MD); or from any of a number seed companies, for example, W. Atlee Burpee Seed Co. (Warminster, PA), Park Seed Co. (Greenwood, SC), Johnny Seed Co. (Albion, ME), or Northrup King Seeds (HarstviUe, SC). Descriptions and sources of useful host cells are also found in Vasil I.K., Cell Culture and Somatic Cell Genetics of Plants, Vol I, II, III Laboratory Procedures and Their Applications Academic Press, New York, 1984; Dixon, R.A., Plant Cell Culture- A Practical Approach, IRL Press, Oxford University, 1985; Green et al., Plant Tissue and Cell Culture, Academic Press, New York, 1987. For prokaryotic expression, DNA encoding a negative regulator polypeptide of

SAR is carried on a vector operably linked to control signals capable of effecting expression in the prokaryotic host. If desired, the coding sequence may contain, at its 5' end, a sequence encoding any of the known signal sequences capable of effecting secretion of the expressed protein into the periplasmic space of the host cell, thereby facilitating recovery of the protein and subsequent purification. Prokaryotes most frequently used are various strains of E. coli; however, other microbial strains may also be used. Plasmid vectors are used which contain replication origins, selectable markers, and control sequences derived from a species compatible with the microbial host. Examples of such vectors are found in Pouwels et al. (supra) or Ausubel et al. (supra). Commonly used prokaryotic control sequences (also referred to as

"regulatory elements") are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences. Promoters commonly used to direct protein expression include the beta-lactamase (penicillinase), the lactose (lac) (Chang et al., Nature 198:1056, 1977), the tryptophan (Tip) (Goeddel et al., Nucl. Acids Res. 8:4057-4074, 1980), and the tac promoter systems, as well as the lambda-derived P_L promoter and N-gene ribosome binding site (Simatake et al., Nature 292:128-132, 1981).

One particular bacterial expression system for polypeptide production is the E. coli pET expression system (Novagen, Inc., Madison, WI). According to this expression system, DNA encoding a negative regulator polypeptide (for example, S7V77) is inserted into a pET vector in an orientation designed to allow expression. Since the negative regulator gene is under the control of the T7 regulatory signals, expression of the gene is induced by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains which express T7 RNA polymerase in response to IPTG induction. Once produced, the recombinant negative regulator polypeptide is then isolated according to standard methods known in the art, for example.

Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system which is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S- transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX-2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa. For eukaryotic expression, the method of transformation or transfection and the choice of vehicle for expression of the negative regulator polypeptide of SAR will depend on the host system selected. Transformation and transfection methods are described, for example, in Ausubel et al. (supra); Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990; Kindle, K., Proc. Natl. Acad. Sci. USA 87:1228, 1990; Potrykus, I., Gene Transfer to Plants: A Laboratory Manual, Springer- Verlag, 1995; and BioRad (Hercules, CA) Technical Bulletin #1687 (Biolistic Particle Delivery Systems). Expression vehicles may be chosen from those provided, for example, in Cloning Vectors: A Laboratory Manual (P.H. Pouwels et al., 1985, Supp. 1987); Clontech Molecular Biology Catalog (Catalog 1992/93 Tools for the Molecular Biologist, Palo Alto, CA); and the references cited above. Other expression constructs are described by Fraley et al. (U.S. Pat. No. 5,352,605).

Construction of Plant Transgenes

Most preferably, a negative regulator polypeptide of SAR (for example, SNIl) is produced by a stably-transfected plant cell line, a transiently-transfected plant cell line, or by a transgenic plant. A number of vectors suitable for stable or extrachromosomal transfection of plant cells or for the establishment of transgenic plants are available to the public; such vectors are described in Pouwels et al. (supra), Weissbach and Weissbach (supra), and Gelvin et al. (supra). Methods for constructing such cell lines are described in, for example, Weissbach and Weissbach (supra), and Gelvin et al. (supra).

Typically, plant expression vectors include (1) a cloned plant gene under the transcriptional control of 5' and 3' regulatory sequences and (2) a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region (for example, one conferring inducible or constitutive, pathogen- or wound-induced, environmentally- or developmentally-regulated, or cell- or tissue- specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal. Once the desired nucleic acid sequence is obtained as described above, it may be manipulated in a variety of ways known in the art. For example, where the sequence involves non-coding flanking regions, the flanking regions may, if desired, be subjected to mutagenesis or deleted.

The negative regulator of SAR sequence may, if desired, be combined with other DNA sequences in a variety of ways. For example, the DNA sequence of the invention may be employed with all or part of the gene sequences normally associated with the expression of a negative regulator polypeptide. In its component parts, a DNA sequence encoding a negative regulator polypeptide of SAR is combined in a DNA construct having a transcription initiation control region capable of promoting transcription and translation in a host cell.

In general, the constructs will involve regulatory regions functional in plants which provide for modified production of a negative regulator polypeptide of SAR as discussed herein. The open reading frame coding for the negative regulator polypeptide of SAR or functional fragment thereof will be joined at its 5' end to a transcription initiation regulatory region such as the sequence naturally found in the 5' upstream region of the negative regulator structural gene. Numerous other transcription initiation regions are available which provide for constitutive or inducible regulation. For applications where developmental, cell, tissue, hormonal, or environmental expression is desired, appropriate 5' upstream non-coding regions are obtained from other genes, for example, from genes regulated during meristem development, seed development, embryo development, or leaf development.

Regulatory transcript termination regions may also be provided in DNA constructs of this invention as well. Transcript termination regions may be provided by the DNA sequence encoding the negative regulator polypeptide of SAR or any convenient transcription termination region derived from a different gene source. The transcript termination region will contain preferably at least 1-3 kb of sequence 3' to the structural gene from which the termination region is derived. Plant expression constructs having a negative regulator polypeptide of SAR as the DNA sequence of interest for expression (in either the sense or antisense orientation) may be employed with a wide variety of plant life. Such genetically-engineered plants are useful for a variety of industrial and agricultural applications as discussed infra, including the generation of disease resistant plants. Importantly, this invention is applicable to dicotyledons and monocotyledons, and will be readily applicable to any new or improved transformation or regeneration method.

The expression constructs include at least one promoter operably linked to at least one negative regulator polypeptide of SAR gene. An example of a useful plant promoter according to the invention is a caulimovirus promoter, for example, a cauliflower mosaic virus (CaMV) promoter. These promoters confer high levels of expression in most plant tissues, and the activity of these promoters is not dependent on virally encoded proteins. CaMV is a source for both the 35S and 19S promoters. Examples of plant expression constructs using these promoters are found in Fraley et al., U.S. Pat. No. 5,352,605. In most tissues of transgenic plants, the CaMV 35S promoter is a strong promoter (see, for example, Odell et al., Nature 313:810-812, 1985). The CaMV promoter is also highly active in monocots (see, for example, Dekeyser et al., Plant Cell 2:591-602, 1990). Moreover, activity of this promoter can be further increased (i.e., between 2-10 fold) by duplication of the CaMV 35S promoter (see for example, Fang et al., Plant Cell 1:141-150, 1989 or McPherson and Kay, U.S. Pat. No. 5,378,142).

Other useful plant promoters include, without limitation, the nopaline synthase (NOS) promoter (Rogers and Fraley, U.S. Pat. No. 5,034,322), the octopine synthase promoter (Frornm et al., Plant Cell 1:977-984, 1989), figwort mosiac virus (FMV) promoter (Rogers, U.S. Pat. No. 5,378,619), the rice actin promoter (Wu and McElroy, WO 91/09948), and the ubiquitin promoter system (Quail et al., U.S. Pat. 5,614,399).

Exemplary monocot promoters include, without limitation, commelina yellow mottle virus promoter, sugar cane badna virus promoter, rice tungro bacilliform virus promoter, maize streak virus element, and wheat dwarf virus promoter. For certain applications, it may be desirable to produce the negative regulator polypeptide of SAR in an appropriate tissue, at an appropriate level, or at an appropriate developmental time. For this purpose, there are an assortment of gene promoters, each with its own distinct characteristics embodied in its regulatory sequences, shown to be regulated in response to inducible signals such as the environment, hormones, and/or developmental cues. These include, without limitation, gene promoters that are responsible for heat-regulated gene expression, light-regulated gene expression (for example, the pea rbcS-3A; the maize rbcS promoter; the chlorophyll a/b-binding protein gene found in pea; or the Arabssu promoter), hormone-regulated gene expression (for example, the abscisic acid (ABA) responsive sequences from the Em gene of wheat; the ABA-inducible HVAl and HVA22, and rd29A promoters of barley and Arabidopsis; and wound-induced gene expression (for example, of wunl), organ-specific gene expression (for example, of the tuber-specific storage protein gene; the 23-kDa zein gene from maize described by; or the French bean β-phaseolin gene), or pathogen-inducible promoters (for example, the PR-l, prp-l, or β-1,3 glucanase promoters, the fungal-inducible wirla promoter of wheat, and the nematode-inducible promoters, TobRB7-5A and Hmg-1, of tobacco and parsley, respectively).

Plant expression vectors may also optionally include RNA processing signals, e.g, introns, which have been shown to be important for efficient RNA synthesis and accumulation (Callis et al., Genes and Dev. 1: 1183-1200, 1987). The location of the RNA splice sequences can dramatically influence the level of transgene expression in plants. In view of this fact, an intron may be positioned upstream or downstream of a negative regulator polypeptide of SAR-encoding sequence in the transgene to modulate levels of gene expression. In addition to the aforementioned 5' regulatory control sequences, the expression vectors may also include regulatory control regions which are generally present in the 3' regions of plant genes. For example, the 3' terminator region may be included in the expression vector to increase stability of the mRNA. One such terminator region may be derived from the PI-II terminator region of potato. In addition, other commonly used terminators are derived from the octopine or nopaline synthase signals.

The plant expression vector also typically contains a dominant selectable marker gene used to identify those cells that have become transformed. Useful selectable genes for plant systems include genes encoding antibiotic resistance genes, for example, those encoding resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin, or spectinomycin. Genes required for photosynthesis may also be used as selectable markers in photosynthetic-deficient strains. Finally, genes encoding herbicide resistance may be used as selectable markers; useful herbicide resistance genes include the bar gene encoding the enzyme phosphinothricin acetyltransferase and conferring resistance to the broad spectrum herbicide Basta® (Hoechst Marion Rousel, Frankfurt, Germany).

Efficient use of selectable markers is facilitated by a determination of the susceptibility of a plant cell to a particular selectable agent and a determination of the concentration of this agent which effectively kills most, if not all, of the transformed cells. Some useful concentrations of antibiotics for tobacco transformation include, for example, 20-100 μg/ml (kanamycin), 20-50 μg/ml (hygromycin), or 5-10 μg/ml (bleomycin). A useful strategy for selection of transformants for herbicide resistance is described, for example, by Vasil et al., supra.

In addition, if desired, the plant expression construct may contain a modified or fully-synthetic structural negative regulator polypeptide of SAR coding sequence which has been changed to enhance the performance of the gene in plants. Methods for constructing such a modified or synthetic gene are described in Fischoff and Perlak, U.S. Pat. No. 5,500,365.

It should be readily apparent to one skilled in the art of molecular biology, especially in the field of plant molecular biology, that the level of gene expression is dependent, not only on the combination of promoters, RNA processing signals, and terminator elements, but also on how these elements are used to increase the levels of selectable marker gene expression.

Plant Transformation Upon construction of the plant expression vector, several standard methods are available for introduction of the vector into a plant host, thereby generating a transgenic plant. These methods include (1) Agrøbαcter wm-mediated transformation (A. tumefaciens or A. rhizogenes) (see, for example, Lichtenstein and Fuller In: Genetic Engineering, vol 6, PWJ Rigby, ed, London, Academic Press, 1987; and Lichtenstein, C.P., and Draper, J,. In: DNA Cloning, Vol II, D.M. Glover, ed, Oxford, IRI Press, 1985)), (2) the particle delivery system, (3) microinjection protocols, (4) polyethylene glycol (PEG) procedures, (5) liposome-mediated DNA uptake, (6) electroporation protocols, (7) the vortexing method, or (8) the so-called whiskers methodology (see, for example, Coffee et al., U.S. Pat. 5,302,523). The method of transformation is not critical to the invention. Any method which provides for efficient transformation may be employed. As newer methods are available to transform crops or other host cells, they may be directly applied. Suitable plants for use in the practice of the invention include, but are not limited to, sugar cane, wheat, rice, maize, sugar beet, potato, barley, manioc, sweet potato, soybean, sorghum, cassava, banana, grape, oats, tomato, millet, coconut, orange, rye, cabbage, apple, watermelon, canola, cotton, carrot, garlic, onion, pepper, strawberry, yam, peanut, onion, bean, pea, mango, citrus plants, walnuts, and sunflower.

The following is an example outlining one particular technique, an Agrobacterium-mediated plant transformation. By this technique, the general process for manipulating genes to be transferred into the genome of plant cells is carried out in two phases. First, cloning and DNA modification steps are carried out in E. coli, and the plasmid containing the gene construct of interest is transferred by conjugation or electroporation into Agrobαcterium. Second, the resulting Agrobαcterium strain is used to transform plant cells. Thus, for the generalized plant expression vector, the plasmid contains an origin of replication that allows it to replicate in Agrobαcterium and a high copy number origin of replication functional in E. coli. This permits facile production and testing of transgenes in E. coli prior to transfer to Agrobαcterium for subsequent introduction into plants. Resistance genes can be carried on the vector, one for selection in bacteria, for example, streptomycin, and another that will function in plants, for example, a gene encoding kanamycin resistance or herbicide resistance. Also present on the vector are restriction endonuclease sites for the addition of one or more transgenes and directional T-DNA border sequences which, when recognized by the transfer functions of Agrobαcterium, delimit the DNA region that will be transferred to the plant.

In another example, plant cells may be transformed by shooting into the cell tungsten microprojectiles on which cloned DNA is precipitated. In the Biolistic Apparatus (Bio-Rad) used for the shooting, a gunpowder charge (22 caliber Power Piston Tool Charge) or an air-driven blast drives a plastic macroprojectile through a gun barrel. An aliquot of a suspension of tungsten particles on which DNA has been precipitated is placed on the front of the plastic macroprojectile. The latter is fired at an acrylic stopping plate that has a hole through it that is too small for the macroprojectile to pass through. As a result, the plastic macroprojectile smashes against the stopping plate, and the tungsten microprojectiles continue toward their target through the hole in the plate. For the instant invention the target can be any plant cell, tissue, seed, or embryo. The DNA introduced into the cell on the microprojectiles becomes integrated into either the nucleus or the chloroplast.

In general, transfer and expression of transgenes in plant cells are now routine practices to those skilled in the art, and have become major tools to carry out gene expression studies in plants and to produce improved plant varieties of agricultural or commercial interest.

Transgenic Plant Regeneration

Plant cells transformed with a plant expression vector can be regenerated, for example, from single cells, callus tissue, or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant; such techniques are described, for example, in Vasil supra; Green et al, supra; Weissbach and Weissbach, supra; and Gelvin et al., supra.

In one particular example, an expression construct containing a cloned negative regulator polypeptide of SAR under the control of the 35S CaMV promoter and the nopaline synthase terminator and carrying a selectable marker (for example, kanamycin resistance) is transformed into Agrobacterium. Transformation of leaf discs (for example, of tobacco or potato leaf discs) with vector-containing Agrobacterium is carried out as described by Horsch et al. (Science 227:1229-1231, 1985). Putative transformants are selected after a few weeks (for example, 3 to 5 weeks) on plant tissue culture media containing kanamycin (for example 100 μg/ml). Kanamycin-resistant shoots are then placed on plant tissue culture media without hormones for root initiation. Kanamycin-resistant plants are then selected for greenhouse growth. If desired, seeds from self-fertilized transgenic plants can then be sowed in a soil-less medium and grown in a greenhouse. Kanamycin-resistant progeny are selected by sowing surfaced sterilized seeds on hormone-free kanamycin- containing media. Analysis for the integration of the transgene is accomplished by standard techniques (see, for example, Ausubel et al. supra; Gelvin et al. supra). Transgenic plants expressing the selectable marker are then screened for transmission of the transgene DNA by standard immunoblot and DNA detection techniques. Each positive transgenic plant and its transgenic progeny are unique in comparison to other transgenic plants established with the same transgene. Integration of the transgene DNA into the plant genomic DNA is in most cases random, and the site of integration can affect the levels and the tissue and developmental patterns of transgene expression. Consequently, a number of transgenic lines are usually screened for each transgene to identify and select plants with the most appropriate expression profiles.

Transgenic lines are evaluated for levels of transgene expression. Expression at the RNA level is determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis are employed and include PCR amplification assays using oligonucleotide primers designed to amplify only transgene RNA templates and solution hybridization assays using transgene-specific probes (see, for example, Ausubel et al., supra). The RNA-positive plants are then analyzed for protein expression by western immunoblot analysis using negative regulator polypeptide specific antibodies (see, for example, Ausubel et al., supra). In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using transgene-specific nucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. In addition, if desired, once the recombinant negative polypeptide is expressed in any cell or in a transgenic plant (for example, as described above), it may be isolated, for example, using affinity chromatography. In one example, an anti-SNIl polypeptide antibody (for example, produced as described in Ausubel et al., supra, or by any standard technique) may be attached to a column and used to isolate the polypeptide. Lysis and fractionation of SNIl -producing cells prior to affinity chromatography may be performed by standard methods (see, for example, Ausubel et al., supra). Once isolated, the recombinant protein can, if desired, be further purified, for example, by high performance liquid chromatography (see, for example, Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, eds., Work and Burdon, Elsevier, 1980).

Polypeptides of the invention, particularly short polypeptide fragments, can also be produced by chemical synthesis (for example, by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, IL).

Engineering Disease Resistance As is discussed above, plants having decreased expression of a negative regulator polypeptide (for example, SNIl) are useful, for example, for activating a plant's SAR defense pathways that confer anti-pathogenic properties. Plants having decreased expression of such SAR negative regulator polypeptides are generated according to standard gene silencing methods including, without limitation, co- suppression and antisense methodologies, expression of dominant negative gene products, and creation of plants having mutated genes encoding a negative regulator polypeptide of SAR.

Co-Suppression

One preferred method of silencing gene expression is co-suppression (also referred to as sense suppression). This technique, which involves introduction of a nucleic acid configured in the sense orientation, has been shown to effectively block the transcription of target genes (see for example, Napoli et al., Plant Cell, 2:279-289, 1990 and Jorgensen et al., U.S. Patent No. 5,034,323).

Generally, sense suppression involves transcription of the introduced sequence. However, co-suppression may also occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous gene to be repressed. The introduced sequence generally will be substantially identical to the endogenous gene targeted for repression. Such identity is typically greater than about 50%, but higher identities (for example, 80% or even 95% are preferred because they result in more effective repression. The effect of co-suppression may also be applied to other proteins within a similar family of genes exhibiting homology or substantial homology. Segments from a gene from one plant can be used directly, for example, to inhibit expression of homologous genes in different plant species.

In sense suppression, the introduced sequence, requiring less than absolute identity, need not be full length, relative to either the primary transcription product or to fully processed mRNA. A higher identity in a shorter than full length sequence compensates for a longer, sequence of lesser identity. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments may be equally effective. Squences of at least 50 base pairs are preferred, with introduced sequences of greater length being more preferred (see, for example, those methods described by Jorgensen et al., U.S. Patent No. 5,034,323). In one working example, constitutive sense expression of the SNIl gene of

Arabidopsis or a SNIl homolog may be expressed in Russet Burbank potato to control Phytophthora infestans infection. In one particular approach, a plant expression vector is constructed that contains a SNIl cDNA sequence expressed under the control of the enhanced CaMV 35S promoter as described by McPherson and Kay (U.S. Patent 5,359,142). This expression vector is then used to transform Russet Burbank according to the methods described in Fischhoff et al. (U.S. Patent 5,500,365). To assess resistance to fungal infection, transformed Russet Burbank and appropriate controls are grown to approximately eight-weeks-old, and leaves (for example, the second or third from the top of the plant) are inoculated with a mycelial suspension of P. infestans. Plugs of R. infestans mycelia are inoculated on each side of the leaf midvein. Plants are subsequently incubated in a growth chamber at 27 °C with constant fluorescent light.

Leaves of transformed Russet Burbank and control plants are then evaluated for resistance to R. infestans infection according to conventional experimental methods. For this evaluation, the number of lesions per leaf and percentage of leaf area infected are recorded every twenty-four hours for seven days after inoculation. From these data, levels of resistance to R. infestans are determined. Transformed potato plants that have an increased level of resistance to R. infestans relative to control plants are taken as being useful in the invention. Alternatively, to assess resistance at the whole plant level, transformed and control plants are transplanted to potting soil containing an inoculum of R. infestans. Plants are then evaluated for symptoms of fungal infection (for example, wilting or decayed leaves) over a period of time lasting from several days to weeks. Again, transformed potato plants expressing the SNIl gene that co-suppress endogenous gene expression having an increased level of resistance to the fungal pathogen, R. infestans, relative to control plants are taken as being useful in the invention.

In another working example, expression of the SNIl gene of Arabidopsis or a SNIl homolog in tomato is used to control bacterial infection, for example, to Pseudomonas syringae. Specifically, a plant expression vector is constructed that contains the <S7V77 sequence, in sense orientation, which is expressed under the control of the enhanced CaMV 35S promoter as described by McPherson and Kay, supra. This expression vector is then used to transform tomato plants according to the methods described in Fischhoff et al., supra. To assess resistance to bacterial infection, transformed tomato plants and appropriate controls are grown, and their leaves are inoculated with a suspension of R. syringae according to standard methods, for example, those described herein. Plants are subsequently incubated in a growth chamber, and the inoculated leaves are subsequently analyzed for signs of disease resistance according to standard methods. For example, the number of chlorotic lesions per leaf and percentage of leaf area infected are recorded and evaluated after inoculation. From a statistical analysis of these data, levels of resistance to R. syringae are determined. Transformed tomato plants having an increased level of resistance to R. syringae relative to control plants are taken as being useful in the invention. Antisense Suppression

In antisense technology, a nucleic acid segment from the desired plant gene is cloned and operably linked to an expression control region such that the antisense strand of RNA is synthesized. The construct is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been shown that antisense RNA inhibits gene expression.

The nucleic acid segment to be introduced in antisense suppression is generally substantially identical to at least a portion of the endogenous gene or genes to be repressed, but need not be identical. The vectors of the present invention therefore can be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homology to the target gene. Segments from a gene from one plant can be used, for example, directly to inhibit expression of homologous genes in different plant species.

The introduced sequence also need not be full length relative to either the primary transcription product or to fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Moreover, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective. In general, such an antisense sequence will usually be at least 15 base pairs, preferably about 15-200 base pairs, and more preferably 200-2,000 base pairs in length or greater. The antisense sequence may be complementary to all or a portion of the gene to be suppressed (for example, SNIl), and, as appreciated by those skilled in the art, the particular site or sites to which the antisense sequence binds as well as the length of the antisense sequence will vary, depending upon the degree of inhibition desired and the uniqueness of the antisense sequence. A transcriptional construct expressing a plant negative regulator antisense nucleotide sequence includes, in the direction of transcription, a promoter, the sequence coding for the antisense RNA on the sense strand, and a transcriptional termination region. Antisense sequences may be constructed and expressed as described, for example, in van der Krol et al. (Gene 72: 45-50, 1988); Rodermel et al. (Cell 55: 673-681, 1988); Mol et al. (FEBS Lett. 268: 427-430, 1990); Weigel and Nilsson (Nature 377: 495-500, 1995); Cheung et al., (Cell 82: 383-393, 1995); and Shewmaker et al. (U.S. Pat. No. 5,107,065).

In one working example, antisense expression of the SNIl gene of Arabidopsis or a SNIl homolog is used to control fungal diseases, for example, the infection of tissue by Magnaporthe grisea, the cause of rice blast. In one particular approach, a plant expression vector is constructed that contains the cDNA sequence of SNIl homolog in antisense orientation that is constitutively expressed under the control of the rice actin promoter described by Wu et al. (WO 91/09948). This expression vector is then used to transform rice plants according to conventional methods, for example, using the methods described in Hiei et al. (Plant Journal 6:271-282, 1994). To assess resistance to fungal infection, transformed rice plants expressing antisense sequences and appropriate controls are grown, and their leaves are inoculated with a mycelial suspension of M. grisea according to standard methods. Plants are subsequently incubated in a growth chamber and the inoculated leaves are subsequently analyzed for disease resistance according to standard methods. For example, the number of lesions per leaf and percentage of leaf area infected are recorded and evaluated after inoculation. From a statistical analysis of these data, levels of resistance to M. grisea are determined. Transformed rice plants that express antisense SNIl RNA having an increased level of resistance to M. grisea relative to control plants are taken as being useful in the invention.

Dominant Negatives

Transgenic plants expressing a transgene encoding a dominant negative gene product of a negative regulator polypeptide of SAR are assayed in artificial environments or in the field to demonstrate that the transgene confers disease resistance on the transgenic plant. Dominant negative transgenes are constructed according to methods known in the art. Typically, a dominant negative gene encodes a mutant negative regulator polypeptide of SAR (for example, a mutated SNIl) which, when overexpressed, disrupts the activity of the wild type negative regulator polypeptide. Transgenic plants having an increased ability to grow in the presence of a plant pathogen, as compared to non-transgenic plants, are useful in the invention.

Mutants

Plants having decreased expression of a negative regulator polypeptide of SAR are also generated using standard mutagenesis methodologies. Such mutagenesis methods include, without limitation, treatment of seeds with ethyl methylsulfate or fast neutron irradiation, as well as T-DNA insertion methodologies. Expression of a negative regulator polypeptide of SAR and disease resistance phenotypes in mutated and non-mutated lines are evaluated according to standard procedures (for example, those described herein). When compared to non-mutated plants, mutated plants having decreased expression of a gene encoding a negative regulator polypeptide of SAR (for example, a SNIl homolog) exhibit increased disease resistance relative to their non-mutated counterparts.

Identification and Application of Molecules that Modulate Negative Regulator Polypeptide Expression Isolation of the SNIl cDNA also facilitates the identification of molecules that increase or decrease SNIl expression. According to one approach, candidate molecules are added at varying concentrations to a culture medium of cells (for example, prokaryotic cells such as E. coli or eukaryotic cells such as yeast, mammalian, insect, or plant cells) expressing 5N77 mRNA. SNIl expression is then measured, for example, by standard northern blot analysis (Ausubel et al, supra) using a SNIl cDNA (or cDNA fragment) as a hybridization probe. The level of SNIl expression in the presence of the candidate molecule is compared to the level measured for the same cells in the same culture medium but in the absence of the candidate molecule. If desired, the effect of candidate modulators on expression may, in the alternative, be measured at the level of SNIl protein production using the same general approach and standard immunological detection techniques, such as western blotting or immunoprecipitation with a S 7- specific antibody. Candidate modulators may be purified (or substantially purified) molecules or may be one component of a mixture of compounds. In a mixed compound assay, SNIl expression is tested against progressively smaller subsets of the candidate compound pool (for example, produced by standard purification techniques, for example, HPLC) until a single compound or minimal compound mixture is demonstrated to modulate S7V77 gene expression.

A molecule that promotes a decrease in SNIl expression is considered particularly useful in the invention; such a molecule may be used, for example, as a chemical regulator to increase a plant's resistance to a pathogen. Modulators found to be effective at the level of SNIl expression or activity may be confirmed as useful in planta and, if successful, may be used as anti-pathogen compounds in the field.

For agricultural uses, the molecules, compounds, or agents identified using the methods disclosed herein may be used as chemicals applied as sprays or dusts on the foliage of plants. Typically, such agents are to be administered on the surface of the plant in advance of the pathogen in order to prevent infection. Seeds, bulbs, roots, and tubers are also treated to prevent pathogenic attack after planting by controlling pathogens carried on them or existing in the soil at the planting site. Soil to be planted with vegetables, ornamentals, shrubs, or trees can also be treated with chemical fumigants for control of a variety of microbial pathogens. Treatment is preferably done several days or weeks before planting. The chemicals can be applied by either a mechanized route, for example, a tractor or with hand applications. The molecules, compounds, or agents may also be applied to plants in combination with another molecule which affords some benefit to the plant.

Enhanced Sensitivity to Chemical Regulators of SAR

Because sni plants have been found to have enhanced sensitivity to exogenously applied INA as well as enhanced disease resistance relative to wild type and nprl plants (Figure 6), the invention also includes methods of increasing the resistance of a plant having decreased expression of a negative regulator polypeptide of SAR through the exogenous application of chemical regulators of SAR. By "enhanced sensitivity" is meant that the developmental, physiological, or molecular processes that are typically regulated or controlled during SAR exhibit increased or elevated responses to the presence of an exogenously applied chemical regulator relative to those responses in a wild-type plant.

Analysis of a plant's sensitivity to a chemical regulator is accomplished using a wide variety of bioassays. These assays include, but are not limited to, evaluating and monitoring gene expression (for example, of RR genes) and resistance to pathogen attack and disease. By comparing phenotypes of wild-type plants and candidate plants (for example, a plant having decreased expression of a negative regulator polypeptide of SAR such as sni), one is readily able to determine whether such a candidate plant has an increased sensitivity to a chemical regulator of SAR. Thus, in plants exhibiting an increased sensitivity to an SAR chemical regulator, the aforementioned RR-inducible genes have an increased, elevated, or heightened level of chemical-mediated expression. For example, when such chemical regulators are applied to plant tissue, typically to the leaves of whole plants, levels of RR mRNA and proteins develop in the plant tissue which are increased relative to wild-type levels. Exemplary chemical regulators useful in the methods of the invention include chemicals known to be inducers of PR proteins in plants, or close derivatives thereof. These include benzoic acid, salicylic acid, BTH, INA, and derivatives and analogs thereof. In addition, plants having enhanced sensitivity to a chemical regulator of SAR require the application of lower concentrations of such regulators for promoting increased levels of disease resistance than control plants.

Interacting Polypeptides The isolation of sequences encoding a negative regulator polypeptide of SAR also facilitates the identification of polypeptides which interact with other polypeptides involved in SAR. Such polypeptide-encoding sequences are isolated by any standard two hybrid system (see, for example, Fields et al., Nature 340:245-246, 1989; Yang et al., Science 257:680-682, 1992; Zervos et al., Cell 72:223-232, 1993). For example, all or a part of the SNIl sequence may be fused to a DNA binding domain (such as the GAL4 or Lex A DNA binding domain). After establishing that this fusion protein does not itself activate expression of a reporter gene bearing appropriate DNA binding sites (for example, a lacZ or LEU2 reporter gene), this fusion protein is used as an interaction target. Candidate interacting proteins fused to an activation domain (for example, an acidic activation domain) are then co-expressed with the SNIl fusion in host cells, and interacting proteins are identified by their ability to contact the S7V77 sequence and stimulate reporter gene expression. SNI1- interacting proteins identified using this screening method provide good candidates for proteins that are involved in the systemic acquired resistance signal transduction pathway.

Antibodies

The polypeptides described herein (or imunogenic fragments or analogs) may be used to raise antibodies useful in the invention; such polypeptides may be produced by recombinant or peptide synthetic techniques (see, for example, Solid Phase Peptide Synthesis, 2nd ed., 1984, Pierce Chemical Co., Rockford, IL; Ausubel et al., supra). The peptides may be coupled to a carrier protein, such as KLH as described in Ausubel et al, supra. The KLH-peptide is mixed with Freund's adjuvant and injected into guinea pigs, rats, or preferably rabbits. Antibodies may be purified by peptide antigen affinity chromatography.

Monoclonal antibodies may be prepared using any of the polypeptides described above and standard hybridoma technology (see, for example, Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981 ; Ausubel et al., supra; Harlow and Lane, Antibodies- A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. 1988).

Once produced, polyclonal or monoclonal antibodies are tested for specific recognition of an antigen by western blot or immunoprecipitation analysis (by the methods described in Ausubel et al., supra). Antibodies which specifically recognize SAR negative regulator polypeptides (for example, SNIl) are considered to be useful in the invention; such antibodies may be used, for example, in an immunoassay to monitor the level of such a polypeptide produced by a plant.

Use

The invention described herein is useful for a variety of agricultural and commercial purposes including, but not limited to, improving systemic acquired resistance against plant pathogens, increasing crop yields, improving crop and ornamental quality, and reducing agricultural production costs. In particular, silencing or decreasing the expression of a negative regulator polypeptide of SAR (for example, S7V77) in a plant cell provides a method for activating systemic acquired resistance to plant pathogens and can be used to protect plants from pathogen infestation that reduces plant productivity and viability.

The invention also provides for broad-spectrum pathogen resistance by facilitating the natural mechanism of host resistance. For example, sense and antisense transgenes or both can be expressed in plant cells at sufficiently high levels to silence the expression of a negative regulator polypeptide (for example, SNIl) thereby turning on a systemic resistance plant defense response constitutively in the absence of signals from the acquired pathogen. The level of expression associated with such a plant defense response may be determined by measuring the levels of defense response gene expression as described herein or according to any conventional method. If desired, the transgenes are expressed by a controllable promoter such as a tissue-specific promoter, cell-type specific promoter, or by a promoter that is induced by an external signal or agent such as a pathogen- or wound- inducible control element, thus limiting the temporal or tissue expression or both of a systemic acquired resistance defense response. If desired, the genes designed to silence the expression of a negative regulator polypeptide of SAR may also be expressed in roots, leaves, or fruits, or at a site of a plant that is susceptible to pathogen penetration and infection. The invention is also useful for controlling plant disease by enhancing a plant's SAR defense mechanisms. In particular, the invention is useful for combating diseases known to be inhibited by plant SAR defense mechanisms. These include, without limitation, viral diseases caused by TMV and TNV, bacterial diseases caused by Pseudomonas and Xanthomonas, and fungal diseases caused by Erysiphe,

Peronospora, Phytophthora, Colletotrichum, and Magnaporthe grisea. In particular exemplary approaches, constitutive or inducible expression of a gene encoding a negative regulator polypeptide of SAR (for example, in sense or antisense orientation) in a transgenic plant is useful for controlling powdery mildew of wheat caused by Erysiphe, bacterial leaf spot of pepper caused by Xanthomonas campestris, bacterial wilt and bacterial spot of tomato caused by Pseudomonas syringae and Xanthomonas campestris, and bacterial blights of citrus and walnut caused by Xanthomonas campestris.

Other Embodiments The invention further includes analogs of any naturally-occurring plant negative regulator polypeptide of SAR (for example, SNIl). Analogs can differ from the naturally-occurring polypeptide by amino acid sequence differences, by post- translational modifications, or by both. Analogs of the invention will generally exhibit at least 40%, more preferably 50%, and most preferably 60% or even having 70%, 80%, or 90% identity with all or part of a naturally-occurring plant negative regulator polypeptide amino acid sequence. The length of sequence comparison is at least 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably more than 35 amino acid residues. Modifications include in vivo and in vitro chemical derivatization of polypeptides, for example, acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring negative regulator polypeptide (for example, SNIl) by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethyl methylsulfate or by site-specific mutagenesis as described in Sambrook et al., supra, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, for example, D-amino acids or non-naturally occurring or synthetic amino acids, for example, β or γ amino acids.

In addition to full-length polypeptides, the invention also includes negative regulator polypeptide fragments. As used herein, the term "fragment," means at least 20 contiguous amino acids, preferably at least 30 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 or more contiguous amino acids. Fragments of negative regulator polypeptides (for example, SNIl) can be generated by methods known to those skilled in the art or may result from normal protein processing (for example, removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events). In preferred embodiments, a negative regulator polypeptide fragment includes an N domain motif (for example, such as a domain that is substantially identical to the N domain of the retinoblastoma protein (SEQ ID NO:5) shown in Fig. 3C) as described herein.

Furthermore, the invention includes nucleotide sequences that facilitate specific detection of a gene encoding a negative regulator polypeptide of SAR. Thus, the nucleic acid sequences described herein or portions thereof may be used as probes to hybridize to nucleotide sequences from other plants (for example, dicots, monocots, gymnosperms, and algae) by standard hybridization techniques under conventional conditions. Sequences that hybridize to a negative regulator polypeptide coding sequence or its complement and that encode a negative regulator polypeptide are considered useful in the invention. As used herein, the term "fragment," as applied to nucleic acid sequences, means at least 5 contiguous nucleotides, preferably at least 10 contiguous nucleotides, more preferably at least 20 to 30 contiguous nucleotides, and most preferably at least 40 to 80 or more contiguous nucleotides. Fragments of negative regulator polypeptide nucleic acid sequences (for example, SNIl) can be generated by methods known to those skilled in the art.

All publications and patent applications mentioned in this specification are herein incorporated by reference.

Other embodiments are with the claims.

What is claimed:

Claims

1. A non-naturally occurring plant having decreased expression of an endogenous negative regulator polypeptide of systemic acquired resistance (SAR), wherein said decreased expression renders said non-naturally occurring plant resistant to a plant pathogen.

2. The non-naturally occurring plant of claim 1, wherein said plant is a transgenic plant.

3. The non-naturally occurring plant of claim 2, wherein said transgenic plant comprises a transgene that, when expressed in said transgenic plant, silences gene expression of said endogenous negative regulator polypeptide of SAR; a transgene that expresses an antisense molecule of said negative regulator polypeptide of SAR; or a transgene that, when expressed in said transgenic plant, co-suppresses expression of said negative regulator polypeptide of SAR or both.

4. The non-naturally occurring plant of claim 2, wherein said transgenic plant comprises a transgene that encodes a dominant negative gene product.

5. The non-naturally occurring plant of claim 4, wherein said dominant negative gene product comprises a gene that encodes a mutated form of said negative regulator polypeptide of SAR.

6. The non-naturally occurring plant of claim 1, wherein said plant comprises a point mutation, a deletion, or an insertion in a gene that encodes said negative regulator polypeptide of SAR.

7. The non-naturally occurring plant of claim 1, wherein said decreased expression of said negative regulator polypeptide of SAR occurs at the transcriptional level, the translational level, or post-translational level.

8. The non-naturally occurring plant of claim 1, wherein said negative regulator polypeptide of SAR has at least 30% identity to the amino acid sequence shown in Fig. 3A.

9. An isolated nucleic acid molecule comprising a sequence encoding a negative regulator polypeptide of SAR.

10. The isolated nucleic acid molecule of claim 9, wherein said sequence encodes a negative regulator polypeptide of SAR having at least 30% identity to the amino acid sequence shown in Fig. 3A.

11. The isolated nucleic acid molecule of claim 9, wherein said sequence encodes a negative regulator polypeptide of SAR that is leucine-rich; has at least 20% sequence identity to the N domain of a retinoblastoma protein; that represses transcription, or that is substantially localized to the nucleus.

12. The nucleic acid molecule of claim 9, wherein said nucleic acid molecule is cDNA.

13. The isolated nucleic acid molecule of claim 9, wherein said isolated nucleic acid molecule hybridizes specifically to a nucleic acid molecule comprising the sequence of Fig. 3A or 3B.

14. The isolated nucleic acid molecule of claim 9, wherein said isolated nucleic acid molecule is operably linked to a promoter functional in a plant cell.

15. An expression vector comprising the isolated nucleic acid molecule of claim 9, said vector being capable of directing expression of the polypeptide encoded by said isolated nucleic acid molecule.

16. A cell comprising the isolated nucleic acid molecule of claim 9.

17. The cell of claim 16, wherein said cell is a plant cell.

18. The cell of claim 16, wherein said cell is a bacterial cell.

19. The cell of claim 16, wherein said bacterial cell is Agrobacterium.

20. A transgenic plant or transgenic plant component comprising the nucleic acid molecule of claim 9, wherein said nucleic acid molecule is expressed in said transgenic plant or said transgenic plant component.

21. The transgenic plant or transgenic plant component of claim 20, wherein said transgenic plant or transgenic plant component is from an angiosperm, a dicot, a monocot, or a crucifer.

22. A seed from the transgenic plant or transgenic plant component of claim 20.

23. An isolated DNA molecule encoding an antisense RNA of a negative regulator polypeptide of SAR.

24. An expression vector comprising the DNA molecule of claim 23.

25. A transgenic plant or transgenic plant component comprising the vector of claim 24.

26. A seed from the transgenic plant or transgenic plant component of claim

25.

27. A cell from the transgenic plant or transgenic plant component of claim

25.

28. A substantially pure negative regulator polypeptide of SAR.

29. The substantially pure polypeptide of claim 28, said polypeptide comprising an amino acid sequence having at least 30% identity to the amino acid sequence of Fig. 3 A or having at least 20% identity to the N domain of a retinoblastoma protein.

30. The substantially pure polypeptide of claim 28, wherein said polypeptide comprises an amino acid sequence that is leucine rich, represses transcription, or is predominately localized to the nucleus.

31. A method of producing a negative regulator polypeptide of SAR, said method comprising the steps of:

(a) providing a cell transformed with the isolated nucleic acid molecule of claim 9;

(b) culturing said transformed cell under conditions for expressing said isolated nucleic acid molecule; and

(c) recovering said negative regulator polypeptide of SAR.

32. A recombinant negative regulator polypeptide of SAR produced according to the method of claim 31.

33. A substantially pure antibody that specifically recognizes and binds to a negative regulator polypeptide of SAR.

34. The antibody of claim 33, wherein said antibody recognizes and binds to a recombinant negative regulator polypeptide of SAR.

35. A method of isolating a negative regulator gene of SAR or fragment thereof, said method comprising the steps of:

(a) contacting the nucleic acid molecule of Fig. 3 A or a portion thereof with a nucleic acid preparation from a plant cell under hybridization conditions providing detection of nucleic acid sequences having at least 30% or greater sequence identity to the nucleic acid sequence of Fig. 3A; and

(b) isolating said hybridizing nucleic acid sequences.

36. A method of isolating a negative regulator gene of SAR or fragment thereof, said method comprising the steps of:

(a) providing a sample of plant cell DNA;

(b) providing a pair of oligonucleotides having sequence identity to a region of the nucleic acid molecule of Fig. 3A;

(c) contacting the pair of oligonucleotides with said plant cell DNA under conditions suitable for polymerase chain reaction-mediated DNA amplification; and

(d) isolating the amplified negative regulator gene of SAR or fragment thereof.

37. The method of claim 36, wherein said amplification step is carried out using a sample of cDNA prepared from a plant cell.

38. The method of claim 36, wherein said negative regulator gene of SAR encodes a polypeptide which is at least 30% identical to the amino acid sequence of Fig. 3A.

39. A process for conferring disease resistance on a transgenic plant or transgenic plant component, said method comprising the steps of:

(a) introducing into plant cells a transgene encoding a negative regulator polypeptide of SAR operably linked to a promoter functional in said plant cells to yield transformed plant cells; and

(b) regenerating a transgenic plant or transgenic plant component from said transformed plant cells, wherein the negative regulator polypeptide of SAR is expressed in the cells of said transgenic plant or transgenic plant component, thereby conferring disease resistance on said transgenic plant or transgenic plant component.

40. The method of claim 39, wherein expression of said transgene cosuppresses the expression of an endogenous negative regulator of SAR.

41. The method of claim 39, wherein said transgene encoding said negative regulator polypeptide of SAR is constitutively or inducibly expressed.

42. The method of claim 39, wherein said transgene encoding said negative regulator polypeptide of SAR is expressed in a tissue-specific, cell-specific, or organ- specific manner.

43. A process for conferring disease resistance on a transgenic plant or transgenic plant component, said method comprising the steps of:

(a) introducing into plant cells a transgene encoding an antisense coding sequence of a negative regulator polypeptide of SAR operably linked to a promoter functional in said plant cells to yield transformed plant cells; and

(b) regenerating a transgenic plant or transgenic plant component from said transformed plant cells, wherein the antisense coding sequence of the negative regulator polypeptide of SAR is expressed in the cells of said transgenic plant or transgenic plant component, thereby conferring disease resistance on said transgenic plant or transgenic plant component.

44. The method of claim 43, wherein said transgene encoding an antisense coding sequence of a negative regulator polypeptide of SAR is constitutively or is inducibly expressed.

45. The method of claim 43, wherein said transgene encoding an antisense coding sequence of a negative regulator polypeptide of SAR is expressed in a tissue- specific, cell-specific, or organ-specific manner.

46. A process for conferring disease resistance on a transgenic plant or transgenic plant component, said method comprising the steps of:

(a) introducing into plant cells a transgene encoding a dominant negative gene product of a negative regulator polypeptide of SAR operably linked to a promoter functional in said plant cells to yield transformed plant cells; and

(b) regenerating a transgenic plant or transgenic plant component from said transformed plant cells, wherein said dominant negative gene product of said negative regulator of SAR is expressed in the cells of said transgenic plant or transgenic plant component, thereby conferring disease resistance on said transgenic plant or transgenic plant component.

47. The method of claim 46, wherein said transgene encoding said dominant negative gene product is constitutively or is inducibly expressed.

48. The method of claim 46, wherein said transgene encoding said dominant negative gene product of a negative regulator polypeptide of SAR is expressed in a tissue-specific, cell-specific, or organ- specific manner.

49. A method for conferring disease resistance on a transgenic plant cell, said method comprising reducing the level of an endogenous negative regulator polypeptide of SAR in a transgenic plant cell.

50. The method of claim 49, wherein said transgenic plant cell is from a monocot, a dicot, or a gymnosperm.

51. The method of claim 49, wherein reducing the level of said endogenous negative regulator polypeptide of SAR comprises expressing a transgene encoding an antisense nucleic acid molecule of a negative regulator gene of SAR in said transgenic plant cell.

52. The method of claim 49, wherein reducing the level of said endogenous negative regulator polypeptide of SAR comprises co-suppression of said endogenous negative regulator gene of SAR in said transgenic plant cell.

53. The method of claim 49, wherein reducing the level of said endogenous negative regulator polypeptide of SAR comprises expressing a dominant negative gene product in said transgenic plant cell.

54. The method of claim 53, wherein said dominant negative gene product is a mutated form of said endogenous negative regulator polypeptide of SAR.

55. A method for identifying a compound which modulates the expression of a gene encoding a negative regulator polypeptide of SAR in a cell, said method comprising the steps of:

(a) providing a cell comprising a gene encoding a negative regulator polypeptide of SAR;

(b) applying to said cell a candidate compound; and

(c) measuring expression of said gene encoding said negative regulator polypeptide of SAR, an increase or decrease in expression relative to an untreated control sample being an indication that said compound modulates expression of said negative regulator polypeptide of SAR.

56. The method according to claim 55, wherein said gene encodes a negative regulator polypeptide of SAR having at least 30% identity to the amino acid sequence shown in Fig. 3A.

57. The method of claim 55, wherein said compound decreases the expression of said gene that encodes said negative regulator polypeptide of SAR.

58. The method of claim 55, wherein said compound increases the expression of said gene that encode said negative regulator polypeptide of SAR.

59. A method for identifying a compound which modulates the activity of a negative regulator polypeptide of SAR in a cell, said method comprising the steps of:

(a) providing a cell expressing a gene encoding a negative regulator polypeptide of SAR;

(b) applying to said cell a candidate compound; and

(c) measuring the activity of said negative regulator polypeptide of SAR, an increase or decrease in activity relative to an untreated control sample being an indication that said compound modulates activity of said negative regulator polypeptide of SAR.

60. The method according to claim 59, wherein said gene encodes a negative regulator polypeptide of SAR having at least 30% identity to the amino acid sequence shown in Fig. 3A.

61. The method of claim 59, wherein said compound decreases said activity of said negative regulator polypeptide of SAR.

62. The method of claim 59, wherein said compound increases said activity of said negative regulator polypeptide of SAR.

63. A method for increasing the resistance of a plant or plant component to a pathogen, said method comprising applying a compound to said plant or plant component that decreases the expression of a gene encoding a negative regulator polypeptide of SAR.

64. A method for increasing the resistance of a plant or plant component to a pathogen, said method comprising applying a compound to said plant or plant component that decreases activity of a negative regulator polypeptide of SAR.

65. A method for increasing the resistance of a plant or plant component to a pathogen, said method comprising applying a chemical regulator of SAR to a plant or plant component that has decreased expression of a negative regulator polypeptide of SAR.

66. The method of claim 65, wherein said chemical inducer of SAR is SA.

67. The method of claim 65, wherein said chemical inducer of SAR is INA.

68. The method of claim 65, wherein said chemical inducer of SAR is BTH.