WO2003016551A2 - Novel salicylic acid-binding protein encoding nucleic acid, sabp2, and methods of use thereof - Google Patents

Abstract

Novel nucleic acid molecules encoding SA-binding proteins involved in SA-mediated disease resistance responses are disclosed. Methods of use of the nucleic acid molecules and proteins of the invention are also provided.

Description

NOVEL SALICYLIC ACID-BINDING PROTEIN ENCODING NUCLEIC ACID, SABP2, AND METHODS OF USE THEREOF

Daniel F. Klessig and Dhirendra Kumar

This application claims priority to US provisional Application, 60/312,863 filed August 16, 2001, the entire contents of which are incorporated by reference herein.

Pursuant to 35 U.S.C. §202 (c), it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Science Foundation, Grant No. MCB-0196046 and IBN-0110272.

FIELD OF THE INVENTION

This invention relates to proteins involved in signal transduction pathways in higher plants . More specifically, the invention provides a novel nucleic acid molecule encoding a protein involved in stress and disease resistance pathways in multicellular plants. The biological molecules of the invention may be used to advantage to (i) create transgenic plants, (ii) identify compounds that induce or enhance disease resistance in plants, and (iii) elucidate the molecular mechanisms responsible for modulation of salicylic acid-mediated disease resistance in plants.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application by author name and year of publication in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. Several patent documents are also referenced to facilitate the practice of the present invention. The disclosure of each of these publications and patent documents is incorporated by reference herein.

Plants can respond to infection by microbial pathogens through the activation of a variety of defense responses. At the sites of infection, a hypersensitive response (HR) is often initiated. The hallmark of this response is the formation of necrotic lesions, a process that is likely due to programmed host cell death. In addition, associated with the HR is the restriction of growth and spread of pathogens. Frequently, defense responses are also activated in tissues distal to the sites of infection according to a phenomenon known as systemic acquired resistance (SAR) . Development of SAR results in an enhanced and long-lasting resistance to secondary challenge by the same or even unrelated pathogens. Associated both with HR (and associated local resistance) and with SAR is the expression of pathogenesis-related (PR) genes, several of whose products have been shown to have antimicrobial activity (for review, see Klessig and Malamy, 1994; Hammond-Kosach and Jones, 1996; Durner et al . , 1997; Dempsey et al . , 1999) .

A mounting body of evidence indicates that salicylic acid (SA) plays a key role in the activation of certain defense responses in a number of dicotyledonous species. For example, rises in endogenous SA levels correlate with the induction of PR genes and development of resistance in tobacco and cucumber (Malamy et al . , 1990 and 1992, Metraux et al . , 1990; Rasmussen et al . , 1991). In addition, several mutants of Arabidopsis (e.g., cpr's, lsd's, acd's) have been isolated which constitutively express PR genes and show enhanced resistance. They also demonstrate elevated levels of SA (Bowling et al . , 1994; Dietrich et al . , 1994; Greenberg et al . , 1994; Yoshioka et al., 2001). Conversely, Arabidopsis mutants defective in SA signal transduction (e.g., nprl, niml, sail) exhibit enhanced susceptibility to pathogens (Cao et al., 1994; Delaney et al . , 1995; Shah et al . , 1997). Exogenously applied SA also induces PR gene expression and enhanced resistance in tobacco (White, 1979; Antoniw and White, 1980) and a variety of other plants (for review, see Klessig and Malamy, 1994; Durner et al . , 1997) . Furthermore, transgenic Arabidopsis and tobacco plants that express the bacterial salicylate hydroxylase (nahG) gene, whose product converts SA into biologically inactive catechol, fail to develop SAR and show increased susceptibility to primary infections by both virulent and avirulent pathogens (Gaffney et al., 1993; Delaney et al., 1994). During the past several years, attempts to elucidate the mechanisms of SA action in plant disease resistance have been made by identifying the cellular components with which SA interacts. Initial studies led to the identification of a SA-binding protein, SABP, that was later shown to be a catalase (CAT) (Chen and Klessig,

1991; Chen et al . , 1993a). Further analysis demonstrated that SA inhibited tobacco catalase activity in suspension cells and in crude leaf extracts. SA also inhibited the purified enzyme (Chen et al., 1993b; Conrath et al . , 1995; Durner and Klessig, 1996) . Thus, it was proposed that increases in SA after pathogen infection might inhibit catalase activity, producing elevated levels of H₂0₂ that could activate certain defense responses, including PR gene expression. Supporting this hypothesis was the observation that prooxidants induced PR-1 gene expression (Chen et al . , 1993b), while antioxidants suppressed the SA-mediated expression of PR-1 genes (Conrath et al., 1995; Wendehenne et al . , 1998). In addition, the other major H₂0₂-scavenging enzyme, ascorbate peroxidase (APX) , was subsequently shown to be inhibited by SA (Durner and Klessig, 1995) .

In contrast, several other studies have questioned the role of H₂0₂ and the SA-mediated inhibition of CAT and APX during the activation of defense responses. No detectable increases in H₂0₂ levels were found during the establishment of SAR (Neuenschwander et al . , 1995) and significant reductions in CAT activity were not observed in tobacco infected with Pseudomonas syringae or in leaf discs pretreated with SA (Bi et al . , 1995) . In addition, H₂0₂ and H₂0₂-inducing chemicals were unable to induce PR-1 gene expression in NahG transgenic plants (Bi et al . , 1995; Neuenschwander et al . , 1995). Moreover, high concentrations of H₂0₂ (150 mM - 1000 mM) were shown to induce SA accumulation (Neuenschwander et al., 1995; Leon et al., 1995; Summermatter et al . , 1995). Finally, transgenic plants having significantly lower CAT activity via transformation with CAT antisense or cosuppressing sense constructs, did not exhibit constitutive PR-1 gene expression unless there was concurrent development of necrosis (Chamnonpol et al . , 1996; Takahashi et al . , 1997) . From these results, it appears that H₂0₂ acts upstream of SA in the signal transduction cascade rather than, or in addition to, acting downstream of SA.

One possible explanation for these conflicting findings is that SA and H₂0₂ are involved in a self- amplifying feedback loop (Van Camp, 1998; Draper, 1997; Shirasu et al., 1997). An alternative mechanism through which SA-mediated inhibition of CAT may signal defense responses is via the generation of SA free radicals, which could then activate a lipid peroxidation pathway (Durner and Klessig, 1996, Anderson et al . , 1998). However, this possibility remains a topic of debate (Tenhaken and Rύbel, 1997) .

Taken together, these studies suggest that the activation of defense responses is mediated through the interaction of SA with other cellular factors, rather than, or in addition to interactions with CAT and APX. Thus, the isolation and identification of additional cellular factors associated with SA action in plant disease resistance is essential for elucidating the molecular mechanisms directing SA-mediated defense responses .

SUMMARY OF THE INVENTION In accordance with the present invention, a novel nucleic acid molecule, SABP2, is provided. SABP2 encodes the novel high-affinity salicylic acid-binding protein 2 (SABP2) which is involved in the SA-mediated signal transduction pathway leading to defense responses to pathogen infection in higher plants .

In one aspect of the invention, an isolated nucleic acid molecule comprising an SABP2-encoding nucleic acid sequence is provided. In a preferred embodiment, the nucleic acid molecule is isolated from a plant and has the SABP2-encoding nucleic acid sequence of SEQ ID NO: 1. The nucleic acid molecules of the invention may be inserted into an expression vector, such as a plasmid or viral vector, and may further be transformed into a host cell or plants. Exemplary host cells and plants include tobacco, Arabidopsis, rice, maize, wheat, tomato, potato, barley, canola, bacteria, yeast, insect and mammalian cells .

According to another aspect of the invention, an isolated SABP2 polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 1 is provided. In a preferred embodiment, the SABP2 polypeptide comprises the amino acid sequence of SEQ ID NOS: 2 and has enzymatic activity. In another embodiment of the invention, antibodies immunologicaliy specific for at least one epitope of an SABP2 polypeptide are also provided. According to yet another aspect of the invention, methods for identifying agents which modulate SABP2 enzymatic activity and expression are provided. The methods comprise introducing SABP2-encoding nucleic acid molecules into a host cell, treating the cells or resulting plant with agents suspected of modulating SABP2 enzymatic activity and expression and assaying SABP2 activity and expression levels in the presence and absence of the agent. According to a preferred aspect of the invention, a method is provided to enhance resistance of a plant to plant pathogens or other disease causing agents comprising overexpressing a SABP2-encoding nucleic acid molecule having the sequence of SEQ ID NO: 1. In another embodiment of the invention, SABP2 expression is enhanced by the addition of an agent that enhances SABP2 expression in a host cell.

According to yet another aspect of the invention, a method is provided to inhibit SABP2 function in a plant. This method comprises introducing a mutated SABP2- encoding nucleic acid molecule into a plant which encodes a non-functional SABP2 protein. In a preferred embodiment, the SABP2-encoding nucleic acid molecule is an antisense molecule of SEQ ID NO: 1. Alternatively, methods for inhibition of SABP2 expression comprise introduction of nucleic acid constructs effective to cause dsRNA mediated gene silencing of the SABP2 gene (Smith et al . , 2000; Wesley et al . , 2001).

In accordance with another aspect of the invention, an in vitro screening method for identifying compounds having binding affinity for SABP2 is provided. This method comprises providing a predetermined amount of SABP2 bound by a detectably labeled agent having binding affinity for SABP2, treating SABP2 with at least one test compound suspected of having binding affinity for SABP , measuring displacement of the detectably labeled agent in the presence and absence of the test compound, thereby identifying test compounds with binding affinity for SABP2. In a particularly preferred embodiment of the invention, the test compounds are salicylic acid analogues .

In another embodiment of the invention, an in vitro screening method for identifying compounds which modulate SABP2 enzymatic activity thereby increasing disease resistance in plants is provided. This method comprises providing a predetermined amount of SABP2 in the presence labeled substrate, treating the SABP2 with at least one compound suspected of modulating SABP2 enzymatic activity, and measuring conversion of substrate to product in the presence and absence of the test compound, thereby identifying test compounds which modulate SABP2 activity.

According to another aspect of the present invention, transgenic plants comprising SEQ ID NO: 1 are provided. The transgenic plants of the invention exhibit enhanced disease resistance. Knock-out plants wherein SABP2 expression has been eliminated are also provided. Also within the scope of the invention are transgenic plants comprising variants of SEQ ID NO : 1 encoding proteins having altered SABP2 function.

In a further embodiment of the invention, the screening methods provided above are performed employing nucleic acids, proteins and antibodies encoded by the SABP2 homologs and functional orthologs identified in Arabidopsis (e.g., AtSB2L5 and AtSB2 9) and other species. GenBank Accession numbers of exemplary nucleic acid sequences are provided in Table IV. These functionally equivalent homologs to SABP2 may also be used to modulate disease resistance in higher plants. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the resolution of the protein composition from pooled protein fractions containing the highest levels of SA-binding activity from each step in the purification of SABP2 on a 12.5% SDS-polyacrylamide gel. The right lane shows the resolution of an aliquot of fraction #16 of the second Mono Q column which contained the highest amount of SA-binding activity. The molecular size markers are in the left-most lane.

Figure 2 shows a graph depicting the elution profile of SA-binding activity from the Superdex G75 HR 10/30 gel filtration FPLC column. The position of the molecular mass markers in kiloDaltons are indicated by the arrows. The SA-binding activity of each fraction was determined using [³H-SA] .

Figure 3 shows the resolution of three proteins (28- 32 kDa) on a 12.5% SDS-polyacrylamide gel that co-purify with SA-binding activity. The molecular size markers are in lane M. Lanes 14-19 are fractions eluted from the second mono Q column. The relative SA-binding activity is given in arbitrary units above each fraction.

Figure 4 shows the nucleotide and deduced amino acid sequences of a SABP2 clone isolated from leaves of tobacco (SEQ ID NOS: 1 and 2) . The five peptide sequences obtained by microsequencing are underlined in the deduced SABP2 amino acid sequence.

Figure 5 shows the amino acid sequence homology of SABP2 to hydroxynitrile lyases and eukaryotic esterases . Aligned from top to bottom are the deduced amino acid sequence of SABP2 (SEQ ID NO: 2), alpha-hydroxynitrile lyase (S-acetone-cyanohydrin lyase) from Manihot esculenfca (SEQ ID NO: 3), S-acetone-cyanohydrin lyase from Hevea brasiliensis (SEQ ID NO: 4) , a putative S- acetone-cyanohydrin lyase from Arabidopsis thaliana (SEQ ID NO: 5) and an esterase from rice, PIR7B (SEQ ID NO: 6) . The residues of the catalytic triad are indicated by triangles and the lipase signature sequence of SABP2 is boxed.

Figure 6 shows an in gel lipase assay of purified recombinant His6-SABP2 fractionated on an SDS-PAGE and then reacted with the synthetic lipase/esterase substrate, MUF butyrate. The fluorescence at the position of SABP2 in the gel is due to the release of MUF by SABP2 ' s lipase/esterase activity.

Figures 7A and 7B show blots of SABP2 induction at the mRNA [7A] and protein [7B] levels by TMV infection of resistant Xanthi nc [NN] , but not susceptible Xanthi [nn] , Nicotiana tabacum. Antibody against SABP2 was used in the immunoblot shown in 7B.

Figures 8A-8D show that silencing of SABP2 suppresses disease resistance (as measured by TMV lesion size) and also suppresses PR-1 induction by SA and by TMV. Figure 8A - SABP2 expression was monitored by RT- PCR using beta-tubulin as an internal control. Figure 8B - 24 hours post treatment with SA; PR-1 expression with SA and TMV was monitored by northern analysis, using EtBr stained rRNA as control for equal loading (not shown) . Figure 8C - Lesion size in mm. Figure 8D - Analysis of 36 hours post infection time point confirmed suppression of PR-1 induction by TMV in SABP2-silenced lines (data not shown due to space limitations) .

Figure 9 shows the relationship of Arabidopsis thaliana SABP2.-l.ike (AtSB2L) proteins to each other and to tobacco SABP2 (TSABP2)

DETAILED DESCRIPTION OF THE INVENTION SA is a key component in the signal transduction pathway (s) leading to activation of certain defense responses in plants after pathogen attack. Previous studies have identified several proteins, including catalase and ascorbate peroxidase, through which the SA signal might act. A novel nucleic acid molecule encoding the SA-binding protein 2, SABP2 , is described herein. SABP2 was identified through the use of a high specific activity ligand, [³H] -SA (15-30 Ci/mmole) . SABP2 , which is soluble, differs significantly from a previously- described SABP, which was subsequently shown to be a catalase. For example, SABP2 is much less abundant in leaf tissue than the catalase, and has an apparent native mass (M_r) of between about 20 and 40 kDa, as compared with the molecular mass of catalase, which is approximately 240 kDa. The SABP2 from tobacco reversibly binds SA with an apparent K_d of approximately 90 nM, an affinity that is 100-150 fold higher than that between SA and tobacco catalase. Strikingly, SABP2 , isolated from plant extracts exhibits an even greater affinity (approximately 15-fold) for the synthetic functional SA analog, benzothiadiazole (BTH) , which is a more effective inducer than SA of PR gene expression and disease resistance in monocots as well as dicots (Gδrlach, et al . , 1996; Lawton et al . , 1996; Morris et al, 1998; Benhamou and Belanger, 1998) .

SAPB2 preferentially binds SA and active analogs that induce PR gene expression and enhanced disease resistance. Exemplary analogs include without limitation 5-chlorosalicylic acid (5-CSA) and 2 , 6-dihydroxybenzoic acid 2,6-DHBA). SABP2 shows only minimal binding to inactive analogs such as 4-hydroxybenzoic acid (4-HBA) . See Table IV. Unexpectedly, the recombinant, purified SABP2 did not bind BTH. This likely reflects the fact that synthetic BTH is an S-methyl ester which is converted by plant esterases into the active acid form (Kaji, M. , et al., 1997; Ishii, H. , et al . , 1999; Pillonel, C. 2001). Presumably esterases in the partially purified SABP2 preparations convert BTH to its active form before it is bound by SABP2 , while such esterases are not present in the purified SABP2 produced in E. coli .

The novel SABP2 encoding nucleic acid molecules of the invention may be used to advantage for expression of SABP2 in large quantities. They may also be used to generate transgenic plants having altered expression levels of SABP2. Such transgenic plants may be genetically engineered to overexpress or underexpress SABP2. Alternatively, knockout plants may be created wherein endogenous SABP2 gene expression is abolished. In yet another aspect, plant expression vectors can be generated wherein expression of SABP2 is under the control of an inducible promoter.

In yet another embodiment of the invention, methods are provided for identifying agents or compounds which have binding affinity for SABP2. Such agents may be used in methods for controlled modulation of plant defense responses. Exemplary agents may alter SABP2 enzymatic activity and/or bind tightly to SABP2 and compete for SA binding . Although the SABP2 encoding nucleic acid from tobacco is described and exemplified herein, this invention is intended to encompass nucleic acid molecules from other plant species that are sufficiently similar to be used interchangeably with tobacco SABP2 encoding nucleic acids for the purposes described below. For example, Arabidopsis, like tobacco, have a SABP2-like activity (Du and Klessig, 1997) . Arabidopsis contains 18 full-length homologs of SABP2 , two of which (AtSB2L5 and AtSB2L9) bind SA (Table IV) and appear to be functional equivalents (orthologs) of SABP2. Thus, using the primers described herein, one may successfully isolate and utilize SABP2 homologs and possible orthologs from other plant species in the methods described herein.

Finally, given the homology of human nucleic acids encoding proteins having function comparable to SABP2 , identification and characterization of such molecules is encompassed within the present invention. For example, lecithin (phosphatidylcholine) cholesterol acetyl transferase (LCAT, GenBank Accession No. X04981; SEQ ID NO: 31) , an enzyme found in mammals, including humans, may also be regulated by SA and analogs thereof.

Exemplary methods for assessing the activity of molecules such as LCAT include SA-binding assays, such as the assay set forth in Example II herein. Additionally, SA or aspirin's effect on the enzymatic activity of human LCAT can be determined by pre-treating purified LCAT protein or extracts containing LCAT with O.OOlmM- ImM of SA or aspirin for 30-180 minutes at 22-37° C before adding this pre-treated protein to the standard assay for LCAT esterase activity described by Nakayama et al . , 1984. The concentration of SA or aspirin in the pre- treatment will be maintained in the enzyme assay reaction.

It is known that aspirin (acetyl SA: ASA) and SA reduces the risk of heart disease and stroke. It is also believed that this protective effect of aspirin/SA is due to its ability to inhibit the aggregation of neutrophils and platelets (Weissman et al . , 1991; Abramson et al . , 1985) . However, it is also likely that aspirin/SA exerts its protective effects by stimulating the activity of LCAT. Our finding that the critical SA-binding protein for plant disease resistance responses, namely SABP2 , shares both esterase/lipase activity and sequence homology with LCAT, as well as membership in the same super family of /β fold hydrolases suggests that aspirin/SA binds LCATs. Furthermore, SA and aspirin were shown to elevate LCAT activity and reduce serum cholesterol in rats (Nakayama et al . 1984) while over expression of LCAT in rabbits (Hoeg et al . , 1996) or overexpression of the combination of LCAT and cholesterol ester transfer protein in mice (Foeger et al . , 1999) were shown to protect these transgenic mammals against atherosclerosis. Therefore, we suspect that aspirin/SA bind mammalian (e.g. human) LCATs and thereby enhance their enzymatic activity. This enhanced activity reduces the risk of atherosclerosis and associated heart disease and strokes. Thus, in accordance with the present invention, LCAT encoding sequences will also be assessed for SA binding function and enzymatic activity.

I. Definitions

The following definitions are provided to facilitate an understanding of the present invention:

The term "pathogen-inoculated" refers to the inoculation of a plant with a pathogen.

The term "disease defense response" refers to a change in metabolism, biosynthetic activity or gene expression that enhances a plant's ability to suppress the replication and spread of a microbial pathogen (i.e., to resist the microbial pathogen) . Examples of plant disease defense responses include, but are not limited to, production of low molecular weight compounds with antimicrobial activity (referred to as phytoalexins) and induction of expression of defense (or defense-related) genes, whose products include, for example, peroxidases, cell wall proteins, proteinase inhibitors, hydrolytic enzymes, pathogenesis-related (PR) proteins and phytoalexin biosynthetic enzymes, such as phenylalanine ammonia lyase and chalcone synthase (Dempsey and Klessig, 1995; Dempsey et al . , 1999). Such defense responses appear to be induced in plants by several signal transduction pathways involving secondary defense signaling molecules produced in plants. Certain of these defense response pathways are SA dependent, while others are partially SA dependent and still others are SA independent. Agents that induce disease defense responses in plants include, but are not limited to: (1) microbial pathogens, such as fungi, oomycetes, bacteria and viruses; (2) microbial components and other defense response elicitors, such as proteins and protein fragments, small peptides, β-glucans, elicitins, harpins and oligosaccharides; and (3) secondary defense signaling molecules produced by the plant, such as SA, H₂0₂, ethylene and jasmonates. The terms "defense-related genes" and "defense- related proteins" refer to genes or their encoded proteins whose expression or synthesis is associated with or induced after infection with a pathogen to which the plant is usually resistant. "Transgenic plants" refers to a plant whose genome has been altered by the introduction of at least one heterologous nucleic acid molecule.

"Nucleic acid" or a "nucleic acid molecule" as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5 ' to 3 ' direction. With reference to nucleic acids of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.

When applied to RNA, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues) . An "isolated nucleic acid" (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

The terms "percent similarity" , "percent identity" and "percent homology" when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program. The term "substantially pure" refers to a preparation comprising at least 50-60% by weight of a given material (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-95% by weight of the given compound. Purity is measured by methods appropriate for the given compound (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like) .

A "replicon" is any genetic element, for example, a plasmid, cosmid, bacmid, plastid, phage or virus, that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded.

A "vector" is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element .

An "expression operon" refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.

The term "oligonucleotide, " as used herein refers to sequences, primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.

The phrase "specifically hybridize" refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-deter ined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence. The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be "substantially" complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize" or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically. The term "primer" as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as appropriate temperature and pH, the primer may be extended at its 3 ' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3 ' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5 ' end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product. Polymerase chain reaction (PCR) has been described in US Patents 4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which are incorporated by reference herein. The term "promoter region" refers to the 5' regulatory regions of a gene (e.g., CaMV 35S promoters and/or tetracycline repressor/operator gene promoters) . As used herein, the terms "reporter, " "reporter system", "reporter gene," or "reporter gene product" shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radio immunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like.

The terms "transform", "transfect", "transduce", shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion and the like.

The introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism. In bacterial, yeast, plant and mammalian cells, for example, the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid. Alternatively, the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist in the recipient cell or host organism only transiently.

The term "selectable marker gene" refers to a gene that when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell or plant. The term "operably linked" means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.

The term "DNA construct" refers to a genetic sequence used to transform plants and generate progeny transgenic plants. These constructs may be administered to plants in a viral or plasmid vector. Other methods of delivery such as Agrobacterium T-DNA mediated transformation and transformation using the biolistic process are also contemplated to be within the scope of the present invention. The transforming DNA may be prepared according to standard protocols such as those set forth in "Current Protocols in Molecular Biology", eds. Frederick M. Ausubel et al., John Wiley & Sons, 1995.

The phrase "double-stranded RNA mediated gene silencing" refers to a process whereby target gene expression is suppressed in a plant cell via the introduction of nucleic acid constructs encoding molecules which form double-stranded RNA structures with target gene encoding mRNA which are then degraded. The term "co-suppression" refers to a process whereby expression of a gene, which has been transformed into a cell or plant (transgene) , causes silencing of the expression of endogenous genes that share sequence identity with the transgene. Silencing of the transgene also occurs.

Amino acid residues are identified in the present application according to the three-letter or one-letter abbreviations in the following Table:

TABLE I:

3-letter 1-letter

Amino Acid Abbreviation Abbreviation

L-Alanine Ala A

L-Arginine Arg R

L-Asparagine Asn N

L-Aspartic Acid Asp D

L-Cysteine Cys C

L-Glutamine Gin Q

L-Glutamic Acid Glu E

Glycine Gly G

L-Histidine His H

L-Isoleucine He I

L-Leucine Leu L

L-Methionine Met M

L-Phenylalanine Phe F

L-Proline Pro P

L-Serine Ser S

L-Threonine Thr T

L-Tryptophan Trp W

L-Tyrosine Tyr Y

L-Valine Val V

L-Lysine Lys K

Amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue, provided the desired properties of the polypeptide are retained. All amino-acid residue sequences represented herein conform to the conventional left-to-right amino-terminus to carboxy-terminus orientation.

The term "isolated protein" or "isolated and purified protein" is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure" form. "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

"Mature protein" or "mature polypeptide" shall mean a polypeptide possessing the sequence of the polypeptide after any processing events that normally occur to the polypeptide during the course of its genesis, such as proteolytic processing from a polyprotein precursor. In designating the sequence or boundaries of a mature protein, the first amino acid of the mature protein sequence is designated as amino acid residue 1. As used herein, any amino acid residues associated with a mature protein not naturally found associated with that protein that precedes amino acid 1 are designated amino acid -1, -2, -3 and so on. For recombinant expression systems, a methionine initiator codon is often utilized for purposes of efficient translation. This methionine residue in the resulting polypeptide, as used herein, would be positioned at -1 relative to the mature SABP2 protein sequence.

A low molecular weight "peptide analog" shall mean a natural or mutant (mutated) analog of a protein, comprising a linear or discontinuous series of fragments of that protein and which may have one or more amino acids replaced with other amino acids and which has altered, enhanced or diminished biological activity when compared with the parent or nonmutated protein. The present invention also includes active portions, fragments, derivatives and functional or non-functional mimetics of SABP2-related polypeptides, or proteins of the invention. An "active portion" of such a polypeptide means a peptide that is less than the full length polypeptide, but which retains measurable biological activity.

A "fragment" or "portion" of an SABP2-related polypeptide means a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to thirteen contiguous amino acids and, most preferably, at least about twenty to thirty or more contiguous amino acids. Fragments of the SABP2-related polypeptide sequence, antigenic determinants, or epitopes are useful for eliciting immune responses to a portion of the SABP2-related protein amino acid sequence for the effective production of immunospecific anti-SABP2 antibodies.

Different "variants" of the SABP2-related polypeptides exist in nature. These variants may be alleles characterized by differences in the nucleotide sequences of the gene coding for the protein, or may involve different RNA processing or post-translational modifications. The skilled person can produce variants having single or multiple amino acid substitutions, deletions, additions or replacements. These variants may include but are not limited to: (a) variants in which one or more amino acids residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the SABP2-related polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. Other SABP2-related polypeptides of the invention include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non-conserved positions. In another embodiment, amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques are known to the person having ordinary skill in the art. To the extent such allelic variations, analogues, fragments, derivatives, mutants, and modifications, including alternative nucleic acid processing forms and alternative post-translational modification forms, result in derivatives of the SABP2- related polypeptide that retain any of the biological properties of the SABP2-related polypeptide, they are included within the scope of this invention.

The term "functional" as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose. The phrase "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

The term "tag," "tag sequence" or "protein tag" refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the detection or isolation, of that sequence. Thus, for example, a homopolymer nucleic acid sequence or a nucleic acid sequence complementary to a capture oligonucleotide may be added to a primer or probe sequence to facilitate the subsequent isolation of an extension product or hybridized product. In the case of protein tags, histidine residues (e.g., 4 to 8 consecutive histidine residues) may be added to either the amino- or carboxy-terminus of a protein to facilitate protein isolation by chelating metal chromatography. Alternatively, amino acid sequences, peptides, proteins or fusion partners representing epitopes or binding determinants reactive with specific antibody molecules or other molecules (e.g., flag epitope, c-myc epitope, transmembrane epitope of the influenza A virus hemaglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione

S-transferase, and the like) may be added to proteins to facilitate protein isolation by procedures such as affinity or immunoaffinity chromatography. Chemical tag moieties include such molecules as biotin, which may be added to either nucleic acids or proteins and facilitates isolation or detection by interaction with avidin reagents, and the like. Numerous other tag moieties are known to, and can be envisioned by the trained artisan, and are contemplated to be within the scope of this definition. A "clone" or "clonal cell population" is a population of cells derived from a single cell or common ancestor by mitosis.

A "cell line" is a clone of a primary cell or cell population that is capable of stable growth in vi tro for many generations .

An "immune response" signifies any reaction produced by an antigen, such as a viral or plant antigen, in a host having a functioning immune system. Immune responses may be either humoral in nature, that is, involve production of immunoglobulins or antibodies, or cellular in nature, involving various types of B and T lymphocytes, dendritic cells, macrophages, antigen presenting cells and the like, or both. Immune responses may also involve the production or elaboration of various effector molecules such as cytokines, lymphokines and the like. Immune responses may be measured both in in vi tro and in various cellular or animal systems.

An "antibody" or "antibody molecule" is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen, such as epitopes of an SABP2 binding protein. The term includes polyclonal, monoclonal, chimeric, and bispecific antibodies. As used herein, antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule such as those portions known in the art as Fab, Fab', F(ab')2 and F(v).

The following description sets forth the general procedures involved in practicing the present invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. Unless otherwise specified, general biochemical and molecular biological procedures, such as those set forth in Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory (1989) (hereinafter "Sambrook et al . " ) or Ausubel et al . (eds) Current Protocols in Molecular Biology. John Wiley & Sons (1997) (hereinafter "Ausubel et al . " ) are used.

II. Preparation of SABP2 Encoding Nucleic Acid Molecules:

Nucleic acid molecules of the invention encoding SABP2 polypeptides may be prepared by two general methods: (1) synthesis from appropriate nucleotide triphosphates, or (2) isolation from biological sources. Both methods utilize protocols well known in the art. The availability of nucleotide sequence information, such as the DNA sequences encoding SABP2 , enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramidite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be used directly or purified according to methods known in the art, such as high performance liquid chromatography (HPLC) .

Specific probes for identifying such sequences as the SABP2 encoding sequence may be between 15 and 40 nucleotides in length. For probes longer than those described above, the additional contiguous nucleotides are provided within SEQ ID NO: 1.

Additionally, cDNA or genomic clones having homology with SABP2 may be isolated from other species using oligonucleotide probes corresponding to predetermined sequences within the SABP2 nucleic acids of the invention. Such homologous sequences encoding SABP2 may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed, according to the method of Sambrook et al . , Molecular Cloning. Cold Spring Harbor Laboratory (1989), using a hybridization solution comprising: 5X SSC, 5X Denhardt ' s reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al . , 1989) is as follows:

T_m = 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex

As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57°C. The T_m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 °C.

The nucleic acid molecules described herein include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded. Thus, nucleic acids are provided having sequences capable of hybridizing with at least one sequence of a nucleic acid sequence, such as selected segments of the sequences encoding SABP2. Also contemplated in the scope of the present invention are methods of use for oligonucleotide probes which specifically hybridize with the DNA from the sequences encoding SABP2 under high stringency conditions. Primers capable of specifically amplifying the sequences encoding SABP2 are also provided. As mentioned previously, such oligonucleotides are useful as primers for detecting, isolating and amplifying sequences encoding SABP2. Antisense nucleic acid molecules which may be targeted to translation initiation sites and/or splice sites to inhibit the expression of the SABP2 gene or production of its encoded protein are also provided. Such antisense molecules are typically between 15 and 30 nucleotides in length and often span the translational start site of SABP2 mRNA molecules. Antisense constructs may also be generated which contain the entire SABP2 cDNA in reverse orientation. Alternatively, the SABP2 gene may be silenced using a construct that contains both sense and complementary antisense sequences separated by an intron sequence. This "intron-spliced hairpin RNA" approach results in total silencing of the targeted gene by forming double stranded RNA (dsRNA) structures which are degraded by the host cell machinery (Smith et al., 2000; Wesley et al . , 2001).

Also provided in accordance with the present invention are transgenic plants containing the aforementioned SABP2-encoding nucleic acids, or fragments or derivatives thereof. Such transgenic plants and their utility in disease resistance are described in greater detail below.

III. Preparation of SABP2 Proteins and Antibodies:

The SABP2 protein of the present invention may be prepared in a variety of ways, according to known methods. The protein may be purified from appropriate sources, e.g., plant cells or tissues as described in detail in Example 1. Example 1 describes the isolation of SABP2 from tobacco leaves, followed by purification by ammonium sulfate fractionation, ion-exchange, hydrophobic-interaction, and gel filtration chromatography .

Once nucleic acid molecules encoding SABP2 have been obtained, the SABP2 protein can be produced using in vitro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vi tro transcription vector, such a pSP64 or pSP65 vector for in vi tro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocytes . In vi tro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin or BRL, Rockville, Maryland.

According to a preferred embodiment, larger quantities of SABP2 may be produced by expression in a suitable procaryotic or eucaryotic system. For example, part or all of a DNA molecule may be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E. coli ) or a yeast cell (such as Saccharomyces cerevisiae) , or into a baculovirus vector for expression in an insect cell. Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, translation control sequences and, optionally, enhancer sequences .

The SABP2 protein produced by gene expression in a recombinant procaryotic or eucaryotic system may be purified according to methods known in the art. In a preferred embodiment, the recombinant protein contains several (e.g., 6-8) histidine residues on the amino or carboxyl termini, which allows the protein to be affinity purified on a nickel column. If histidine tag-vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein. Such methods are commonly used by skilled practitioners. SABP2 protein, prepared by the aforementioned methods, may be analyzed according to standard procedures. Methods for analyzing the physical characteristics and biological activity of SABP2 are set forth in United States Patent No. 6,136,552, the disclosure of which is incorporated by reference herein. The present invention also provides antibodies capable of immunospecifically binding to proteins of the invention. Polyclonal or monoclonal antibodies directed toward SABP2 may be prepared according to standard methods . Monoclonal antibodies may be prepared according to general methods of Kohler and Milstein, following standard protocols. In a preferred embodiment, antibodies are prepared, which react immunospecifically with various epitopes of SABP2. Polyclonal or monoclonal antibodies that immunospecifically interact with SABP2 may be utilized for identifying and purifying such proteins. For example, antibodies may be utilized for affinity separation of proteins with which they immunospecifically interact. Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules.

IV. Uses of SABP2 Nucleic Acid Molecules and Proteins: A. Nucleic Acids Encoding SABP2-related Proteins

Nucleic acids encoding SABP2 proteins may be used for a variety of purposes in accordance with the present invention. DNA, RNA, or fragments thereof encoding SABP2 proteins may be used as probes to detect the presence of and/or expression of such genes. Methods in which nucleic acids encoding SABP2 proteins may be utilized as probes for such assays include, but are not limited to: (1) in si tu hybridization; (2) Southern hybridization (3) Northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR) . The nucleic acids of the invention may also be utilized as probes to identify related genes from other plant species, animals and microbes. As is well known in the art, hybridization stringencies may be adjusted to allow hybridization of nucleic acid probes with complementary sequences of varying degrees of homology. Thus, nucleic acids encoding SABP2 proteins may be used to advantage to identify and characterize other genes of varying degrees of relation to the genes of the invention thereby enabling further characterization of the molecular mechanisms controlling SA-mediated disease response in higher plants. Additionally, the nucleic acids of the invention may be used to identify genes encoding proteins that interact with SABP2 (e.g., by the "interaction trap" technique), which should further accelerate identification of the molecular components involved in the SA-mediated disease response. A new yeast two-hybrid screen (Cytotrap) available from Stratagene, which is based on the SOS-Ras signaling pathway, is complementary to the Gal4 or LexA interaction trap system which assays for protein-protein interactions in the nucleus. In contrast, the Stratagene Cytotrap system monitors interaction in the cytoplasm, hence its name. This system, which appears to have several advantages, may be the preferred screen for

SABP2-interacting proteins, particularly if SABP2 is tethered to the plasma membrane via myristoylation.

Nucleic acid molecules, or fragments thereof, encoding SABP2 genes, for example, may also be utilized to control the production of SABP2 proteins, thereby regulating the amount of protein available to participate in the induction or maintenance of disease resistance in plants. As mentioned above, antisense oligonucleotides corresponding to essential processing sites in SABP2- related mRNA molecules or other gene silencing approaches may be utilized to inhibit SABP2 protein production in targeted cells. Yet another approach entails the use of double-stranded RNA mediated gene silencing. Alterations in the physiological amount of SABP2 proteins may dramatically affect the activity of other protein factors involved in the induction or maintenance of disease resistance.

The nucleic acid molecules of the invention may also be used to advantage to identify mutations in SABP2 encoding nucleic acids from plant samples. Nucleic acids may be isolated from plant samples and contacted with the sequences of the invention under conditions where hybridization occurs between sequences of sufficient complementarity. Such duplexes may then be assessed for the presence of mismatched DNA. Mismatches may be due to the presence of a point mutation, insertion or deletion of nucleotide molecules. Detection of such mismatches may be performed using methods well known to those of skill in the art. Nucleic acids encoding the SABP2 proteins of the invention may also be introduced into host cells. In a preferred embodiment, plant cells are provided which comprise an SABP2 protein encoding nucleic acid such as SEQ ID NO: 1 or a variant thereof. Host cells contemplated for use include, but are not limited to, tobacco, Arabidopsis, rice, maize, wheat, tomato, potato, barley, canola, bacteria, yeast, insect and other animal cells including human cells. The nucleic acids may be operably linked to appropriate regulatory expression elements suitable for the particular host cell to be utilized. Methods for introducing nucleic acids into host cells are well known in the art. Such methods include, but are not limited to, transfection, transformation, calcium phosphate precipitation, electroporation, lipofection and biolistic methods.

The host cells described above or extracts prepared from them containing SABP2 may be used as screening tools to identify compounds which modulate SABP2 protein activity. Modulation of SABP2 activity, for example, may be assessed by measuring alterations in SABP2-SA binding activities or SABP2 enzymatic activities in the presence of a test compound. Test compounds may also be assessed for the induction and/or suppression of expression of genes regulated by SABP2 proteins . The availability of SABP2 protein encoding nucleic acids enables the production of plant species carrying part or all of an SABP2-related gene or mutated sequences thereof, in single or amplified copies. Transgenic plants comprising any one of the SABP2-related sequences described herein are contemplated for use in the present invention. Such plants provide an in vivo model for examining SA-mediated defense responses, and may be particularly useful in elucidating the molecular mechanisms that modulate SA-mediated defense responses. Such plants may also facilitate the identification of other endogenously expressed gene products which play a role in SA-mediated defense responses. Methods of introducing transgenes and making knockouts in plants are known to those of skill in the art. The alterations to the SABP2-related genes envisioned herein include modifications, deletions, and substitutions. Such modifications, deletions or substitutions can result in an SABP2 having altered characteristics or functions. Alternatively, modifications and deletions can render the naturally occurring gene nonfunctional, producing a "knock out" plant. In this context, a "targeted gene" or "knock-out" is a DNA sequence introduced into the plant by way of human intervention, including but not limited to, the methods described herein. The targeted genes of the invention include DNA sequences which are designed to specifically alter SA-mediated defense responses. Substitutions of a naturally occurring gene for a gene from a second species results in a plant which produces an SABP2-related gene from the second species.

Substitution of the naturally occurring gene for a gene having a mutation results in a plant with a mutated SABP2 protein. A transgenic plant carrying a SABP2 gene is generated by direct replacement of the first plant's SABP2 gene with the second SABP2 gene. These transgenic plants are valuable for use in in vivo assays for elucidation of molecular mechanisms associated with local and systemic defense response systems.

Methods of use for the transgenic plants of the invention are also provided herein. Knockout plants of the invention may be treated with agents that induce local or systemic plants defense responses. Such plants provide a biological system for assessing the protective role played by SABP2. For example, agents which modulate the action or expression of SABP2 proteins may be screened in studies using SABP2 knock out plants.

In another embodiment of the invention, transgenic plants are provided that have enhanced disease resistance. Such transgenic plants may have altered SABP2 activity due to the overexpression or underexpression of SABP2-related genes.

As described above, the SABP2-related protein- encoding nucleic acids are also used to advantage to produce large quantities of substantially pure proteins, or selected portions thereof. B. SABP2 Proteins and Antibodies

SA is a physiologically active compound in plants, animals and microbes. SABP2 almost certainly plays a role in one or more of the physiological effects mediated by SA in plants (see Cutt and Klessig, 1992). SABP2 , encoded by the nucleic acid molecules of the invention, can be used in biochemical assays for screening for novel, biologically active analogues of SA. As used herein, the term "SA analogue" is intended broadly to refer to functional analogues, instead of or in addition to structural analogues of SA, which can substitute for SA to induce SA-mediated disease defense responses in plants, or other SA-mediated responses (e.g., induction of the alternative oxidase gene or flowering, as described below) .

As one example, assays for SA analogues may be based on the ability of analogues to bind SABP2 or to compete with SA for binding to SABP2. A good correlation between binding to SABP2 (measured by competition with labeled SA for binding to SABP2 ) and biological activity of seven SA analogues for induction of defense responses to microbial pathogens has been demonstrated, as described in U.S. Patent No. 6,136,552. One such previously identified agent, BTH, a commercially available synthetic activator of plant defense responses that is more active than SA in inducing defense responses such as PR-1 gene expression, binds SABP2 in partially purified plant extracts about 15 fold more avidly than SA (Du and Klessig, 1997) . Since SABP2 is part of the signal transduction pathway leading to disease resistance, it may be possible to enhance induction of disease resistance using analogues of SA which are not readily metabolized by the plants. It has previously been demonstrated that SA is rapidly conjugated to glucose to form SA β-glucoside (Malamy et al . , 1992) . This glucoside is not active for induction of disease resistance. Thus a derivative, or analogue of SA that is not readily metabolized, yet binds SABP2 and stimulates the disease resistance response, would be a superior inducer of resistance.

SABP2 likely plays a role in the numerous biological processes shown to be affected by the addition of exogenous SA, including plant disease resistance, thermogenesis, floral development, and senescence (Morris et al . , 2000) . Thus, these processes may be affected by altering expression levels and/or characteristics of SABP2. Current technologies of genetic engineering make both readily available. In plants, altering the level of SABP2 within the organism can be readily achieved by making transgenic plants that express SABP2 gene under a strong constitutive or inducible promoter in the sense orientation to overproduce SABP2 or in an antisense orientation or intron-spliced hairpin RNA approach to disrupt expression of endogenous SABP2 gene(s). For example, if SA and SABP2 are both involved in flower induction, then it is feasible to inhibit flowering by blocking expression of the endogenous SABP2 gene using antisense technology or by blocking function of the endogenous SABP2 by production of a dominant negative mutant form of SABP2. In addition, if SA is a signal for this (or other) process (es), but the signal is not mediated by SABP2 , then overexpression of SABP2 gene could also block this process, since SABP2 may act to sequester the SA signal, given it's high affinity for SA. Two complementary approaches can be employed to regulate SABP2 gene expression levels. The first approach involves potato virus X (PVX)- or tobacco rattle virus (TRV) -based gene silencing, pioneered by David Baulcombe's group (Ratcliff, F. et al . , 1997; Ruiz, M.T. et al . , 1998) . The second involves construction of transgenic tobacco in which the SABP2 gene is overexpressed (sense constructs) or underexpressed (dsRNA-mediated gene silencing) . Underexpression is most effectively accomplished with constructs encoding intron-spliced RNAs with a "loopless" hairpin structure (dsRNA) that then induces gene silencing (Smith et al . , 2000; Wesley et al . , 2001).

The PVX-based (or TRV-based) gene silencing approach, also called virus-induced gene silencing (VIGS) , has several advantages. First, it is very rapid as construction of transgenic plants is not required. Second, since VIGS can be done on adult plants, it eliminates the potential problem that the silenced gene may be required for normal plant development and/or regeneration, in which case construction of the appropriate transgenic plants might not be feasible. Since VIGS is more effective in Nicotiana benthamiana, most of the silencing experiments are being done in this host or N.b. transgenic lines expressing the N gene of N. tabacum or Pto of tomato.

The transgenic approach is complementary to VIGS; although slow, it allows one to look at the effect of overexpression as well as underexpression. Moreover, in addition to facilitating more complete silencing of SABP2 and its potential ortholog(s), because the lines are stably transformed, they can be readily propagated and utilized for many generations in large number. Furthermore, some of the defense responses to be tested (e.g. ion fluxes, cell death) are more readily carried out in suspension culture which can be easily prepared from the transgenic plants.

In yet another embodiment of the invention, new response systems may be developed in plants, animals and microbes. Introduction of the SABP2 gene under control of an appropriate promoter should facilitate its expression in organisms or tissues in which SABP2 is not normally expressed. These organisms or tissues could then become responsive to SA that is either generated endogenously or applied exogenously. For example, it is possible to genetically engineer the synthesis of SABP2 in specific types of plant tissue (or animal tissue) by using tissue-specific promoters to drive (control) the expression of the SABP2 gene. Some of the tissues may not normally express an endogenous copy of the SABP2 gene or may express the gene at a very low level . Increasing synthesis of SABP2 in this way may affect the host tissue by making the tissue more responsive to SA, without affecting other tissues in which the engineered gene is not expressed. For example, if the amount of SABP2 is a limiting factor in a physiological process such as flower development, then an enhanced production of SABP2 in tissue or cells responsible for flower development could result in greater flower production (and subsequent seed production) , without affecting other physiological processes. In another example, the gene encoding SABP2 can be introduced under appropriate control elements into an organism together with a second gene under the control of a promoter which contains a SA responsive element (SARE) that makes it inducible by SA. Such an SARE element from the PR-2d and PR-1 genes has recently been characterized (Shah and Klessig, 1996; Lebel et al . , 1998) . The expression of this second gene should then be inducible by application of exogenously applied SA. This simple strategy should be feasible if the SA-SABP2 complex directly activates the SARE-containing promoter. However, if there are other components downstream of SABP2 in the signal transduction pathway, then their presence in the tissue or organism of interest will also be necessary for the system to work. Since SA is relatively innocuous in many systems, particularly animals, and several genes in plants (from which SAREs would be obtained) are highly induced by SA (>100x) , this would be an excellent system for inducible high level expression of foreign genes in transformed cells, tissues, or organisms. There is precedence for transfer of inducible gene expression systems between very divergent organisms. For example, the GAL4 system found in yeast has been shown to function in both plants and animals (Ma et al, 1988; Kakidani and Ptashne, 1988) . In another embodiment of the invention, it may be advantageous to alter the binding properties of SABP2 through genetic engineering so that it recognizes and responds to novel SA analogues. For example, an SA-like pathway could be developed including analogues of SA and modified complementary SABP2. This system would parallel the naturally occurring SA signal transduction pathway but will be based on discrete and non-competitively binding analogues. In this way, the normal SA-based cellular functions of a plant will continue undisturbed. However, an increase in newly introduced functions may be induced. The engineered plant, containing the modified complementary SABP2 and other downstream mechanisms necessary for SA-induced expression, may be activated by the application of the non-competitive SA analogue. In this embodiment, plant functions would be influenced by two discrete signal transduction systems.

The following examples are provided to illustrate an embodiment of the invention. They are not intended to limit the scope of the invention in any way.

EXAMPLE I PURIFICATION AND CLONING OF SABP2-ENCODING NUCLEIC

ACID MOLECULES In order to elucidate the molecular mechanism(s) that modulate SA-mediated defense responses in plants, identify novel defense-inducing molecules and construct transgenic plants with enhanced disease/stress resistance, proteins associated with SA binding activity were identified. One such protein, SABP2 , has been purified and cloned as described herein below.

I. Materials and Methods:

The following protocols are provided to facilitate the practice of the present invention.

Leaf extraction: Tobacco {Nicotiana tabacum cv Xanthi nc [NN] ) plants were grown in a greenhouse at 22 °C during a 16 hour light period. 7.5 kg of fully expanded leaves from 7-8 week old plants were harvested, de-ribbed, weighed and rapidly frozen in liquid nitrogen. The tissue was stored at -80°C until further use. [All subsequent steps were carried out at 4°C unless specified otherwise.] The frozen leaves (in batches of 1.5 kg each) were ground into a fine powder using a pestle mortar and homogenized in a mixer grinder with 3 vol (w/v) of Buffer A (20 mM sodium citrate, 5 mM MgS0₄, 1 mM EDTA, pH 6.3, 14 mM β-mercaptoethanol supplemented with 0.1 mM phenylmethylsulfonylflouride (PMSF) , 1 mM benzamidine-HCl and 1.5% (w/w) polyvinylpolypyrrolidone (PVPP) . The homogenate was filtered through 2 layers of muslin cloth and 2 layers of miracloth (Calbiochem) . The filtrate was centrifuged at 11,000 g in a GS-3 rotor (Sorvall) for 30 minutes, and the supernatant was collected and used for further purification.

[³H] Salicylic acid-binding assay: The protein extracts/ fractions were mixed with 0.1 μM [³H] -SA (23.6 Ci/mmol) (custom synthesized by NEN Life Science Products) in Buffer A to bring the total volume of the reaction mixture to 150 μl . The extracts were then incubated on ice for 60 minutes. Spin columns were prepared by packing Sephadex G-25 fine (Pharmacia) equilibrated with Buffer A into 1 ml disposable syringe barrels. The G-25 was packed to 1 ml and equilibrated by centrifuging at lOOOg for 4 minutes in a H-1000B rotor (Sorvall) at 4°C. The entire binding mixture was loaded onto the column and centrifuged under similar conditions. Bound [³H]-SA which is present in the flow through, was mixed with 4 ml scintillation cocktail (Ecolite, ICN) and measured in a liquid scintillation counter. Appropriate controls were also assayed. Since it was found that the chloride interferes with SA binding, care was taken to avoid its use in any form in the purification process.

Protein determination: Identification of protein in the fractions was determined using Bradford reagent (BioRad) . BSA was used as standard protein.

Ammonium sulfate fractionation: Powdered ammonium sulfate [(NH₄)₂S0₄] was slowly added to a final concentration of 50% saturation to a continuously stirring sample of leaf extract supernatant. The slurry was incubated for an additional 30 minutes after the final addition of (NH₄)₂S0₄ followed by centrifugation at ll,000g in a GS-3 rotor (Sorvall) for 40 minutes at 4°C. The supernatant was brought to 75% saturation by slowly adding additional (NH₄)₂S0₄. After complete dissolution of the (NH₄)₂S0₄, the slurry was incubated for additional 30 minutes followed by centrifugation at ll,000g for 30 minutes. The pellet was resuspended in a minimum volume of Buffer A and retained for further purification.

Sephadex G-100 chromatography: Sephadex G-100 was equilibrated in Buffer A and packed in to a 500 ml (C26/100, Pharmacia) column. The resuspended (NH₄)₂S0₄ pellet was loaded on to this Sephadex G-100 column and eluted overnight in Buffer A. Fractions of 7 ml were collected and assayed for SA-binding activity. The fractions with high [³H]-SA binding activity were pooled together and precipitated with 75% (NH₄)₂S0₄ saturation. Further chromatographic steps were carried out using Fast Protein Liquid Chromatography system (FPLC, Pharmacia) .

Q Sepharose anion exchange chromatography: The Sephadex G-100 purified (NH₄)₂S0₄ pellet from two, 1.5 kg preparations were resuspended in Buffer B (10 mM Bicine, pH 8.5, 14 mM β-mercaptoethanol, 0.1 mM PMSF, and 1 mM benzamidine-HCl) and desalted on a desalting column (HiPrep, 26/10, Pharmacia) equilibrated in Buffer B. The desalted protein preparation was loaded at flow rate of 1 ml/min on to a 25 ml Q Sepharose Fast Flow column

(XK16/20, Pharmacia) pre-equilibrated with Buffer B. The loaded column was washed with 400 ml of 15 mM (NH₄)₂S0₄ in Buffer B. The bound proteins were eluted at a flow rate of 0.5ml/min with a 125 ml x 125 ml linear gradient of 15-180mM (NH₄)₂S0₄ in Buffer B. Fractions of 4 ml were collected and only the fractions with high [³H] -SA binding activity were pooled. Solid (NH₄)₂S0₄ was slowly added to a concentration of 1 M to the pooled fractions for further purification by Butyl Sepharose chromatography.

Butyl Sepharose: The total pooled protein fractions (from 7.5 kg) from Q Sepharose chromatography containing 1 M(NH₄)₂S0₄ was applied at a flow rate of 0.5ml/min to a 15 ml Butyl Sepharose 4 Fast Flow column (XK16/20, Pharmacia) pre-equilibrated with 1.2 M (NH₄)₂S0₄ in Buffer B. After washing with 150 ml of 1 M (NH₄)₂S0₄ in Buffer B, the bound proteins were fractionated with a 75 ml x 75 ml linear gradient of decreasing (NH₄)₂S0₄ (1 to 0 M) in Buffer B. The 4 ml fractions were collected and assayed for [³H]-SA binding. The fractions with high binding activity were pooled and precipitated with 75% (NH₄)₂S0₄ saturation. The pellet was resuspended in Buffer B and desalted on a desalting column (HiPrep, 26x10, Pharmacia) .

Mono Q anion exchange: The desalted protein preparations from Butyl Sepharose were applied to a Mono Q HR column (5/5, Pharmacia) at a flow rate of 0.25ml/min. After washing with 20 ml of 15 mM (NH₄)₂S0₄ in Buffer B, the bound proteins were eluted with a 10 ml x 10 ml linear gradient of 15-180mM (NH₄)₂S0₄ in Buffer B at a flow rate of 0.25ml/min. Fractions of 0.5 ml were collected and fractions containing high [³H] -SA binding were pooled. The protein solution was precipitated with 75% (NH₄)₂S0₄ saturation and desalted using a HiTrap desalting column (5ml, Pharmacia) equilibrated with Buffer C (Buffer B + 150 mM [NH₄]₂S0₄) .

Superdex-75: The desalted protein preparation from the previous mono Q purification step was loaded at a flow rate of 0.25ml/min on to a Superdex 75 column (HR 10/30, Pharmacia), pre-equilibrated with Buffer C. The column was eluted with 25 ml Buffer C. Fractions of 0.25 ml were collected and the fractions containing high [³H] -SA binding activity were pooled. The pooled fractions were precipitated with 75% (NH )₂S0₄ and desalted using a HiTrap desalting column (5ml, Pharmacia) in Buffer B.

Mono Q anion exchange: The desalted protein preparations from the Superdex 75 purification step were applied to a second mono Q column. After washing with 20 ml of 15 mM (NH₄)₂S0₄ in Buffer B, the bound proteins were eluted with a 10 ml x 10 ml linear gradient of 15-180 mM (NH₄)₂S0₄ in Buffer B. Fractions of 0.5 ml were collected and assayed for [³H]-SA binding activity. Preparative Polyacrylamide Gel Electrophoresis: The fractions with high binding activity were desalted individually with NAP-5 columns (Pharmacia) pre- equilibrated with 5 mM Bicine, pH 8.5. The samples were concentrated by speed-vac, and then electrophoresed on a 12.5% SDS-polyacrylamide gel (16 cm x 20 cm, BioRad) as described by Laemmli (1970). After staining with 0.1% coomassie blue R-250 and destaining, the protein bands which co-purified with SA-binding activity, were cut out and sequenced at the Cornell University Protein Sequencing Facility.

Protein Sequencing: The protein samples were subjected to in-gel digestion with trypsin, and the tryptic fragments were eluted and subjected to matrix-assisted laser desorption ionization (MALDI) mass spectrometry or further purification by reverse phase HPLC . Several HPLC peaks were subjected to sequencing by Edman degradation.

Estimation of Apparent Molecular Mass : The apparent molecular mass of the native SABP2 protein was estimated by means of gel-filtration chromatography on a Superdex 75 column. The purified SABP2 (0.2 ml) from a mono Q column was loaded onto the Superdex 75 column at a flow rate of 0.25 ml/min and eluted at the same flow rate with Buffer C. The column was calibrated with the following standards: ovalbumin (43kDa) , chymotrypsinogen A (25kDa) and ribonuclease A (13.7kDa) (Pharmacia).

Cloning: Based on the amino acid sequence from the SABP2 protein, several degenerate oligonucleotides, both in forward and reverse directions were custom synthesized (Invitrogen) using the preferred codon usage for tobacco. Total RNA was isolated from the young tobacco leaves using Trizol (Invitrogen) following the manufacturer's instructions. The total RNA was treated with RNase-free DNase (Promega) to remove any contaminating genomic DNA. This purified sample of RNA was used to reverse transcribe cDNA using Superscript II Reverse

Transcriptase (Invitrogen) and universal oligo-dT₍₁₄₎ with an adapter sequence.

A combination of degenerate primers (5'- ACWCARTTYTTRCCHTAYGG-3' (SEQ ID NO: 7; where W is A or T; R is A or G; Y is C or T; and H is A, C or T) encoding the sequence TQFLPYG (SEQ ID NO: 8) of peptides pk32, 34 and 36 and universal adapter primer (5'- GACTCGAGTCGACATCGA-3' ; SEQ ID NO: 9) were used to PCR amplify part of the SABP2 cDNA. A small fraction of the PCR amplification products were used as a template for a second round of PCR amplification using the same set of primers. The amplified products were fractionated on a 1.5% agarose gel using 1 x TAE buffer. The DNA bands were visualized by ethidium bromide staining. The amplified DNA bands were excised out of the gel and the DNA was purified using a gel extraction kit (Qiagen) . The purified DNA was then ligated into a PCR cloning vector pGEMT (Promega) . DNA was prepared from the clones containing the inserts. The purified DNA was sequenced by cycle sequencing using the BigDye Terminator and analyzed on a Perkin Elmer Automatic DNA sequencer.

To isolate the 5' half of the SABP2 gene, 5' RACE ready cDNA was prepared from the total RNA isolated from tobacco leaves using the SMART RACE cDNA amplification kit (Clontech) . PCR amplification was first performed with the SABP2 specific primer, E-6

(5' -AGAGATCAGTTGTATTTATG-3 ' ; SEQ ID NO : 10) and the Universal Primer Mix (Clontech) using the 5' RACE ready cDNA as template. A second PCR amplification was performed using the E-6 primer and the Nested Universal Primer (NUP, Clontech) . The amplified products were analyzed on a 1.2% agarose gel and purified as described earlier. The fragments were then cloned into pGEMT vectors and sequenced. All PCR amplifications were carried out using the Advantage 2 PCR kit (Clontech) following manufacturer's instructions.

II. Results:

A. SABP2 Purification: The initial attempts to purify SABP2 from tobacco leaves indicated that it is present in very low amounts. Therefore, a large amount of tissue was used as the starting material for isolation of sufficient amounts of purified SABP2 for amino acid sequencing. The soluble proteins from 7.5 kg of tobacco leaves were fractionated with ammonium sulfate, and the 50-75% saturation (NH₄)₂S0₄ fractions were further purified using the following purification scheme involving Sephadex G-100 gel filtration (desalting and size fractionation) , Q Sepharose (anion exchanger) , Butyl Sepharose (hydrophobic interaction) , mono Q (strong anion exchanger) , Superdex 75 (size fractionation) and a second mono Q. This scheme lead to an approximately 24,000 fold purification of SA- binding activity (Table II). Analysis of the pooled fractions containing the SA-binding activity from the various chromatographic steps by SDS-PAGE shows the purification of SABP2 (Figure 1) .

TABLE II: PURIFICATION OF SABP2 FROM TOBACCO LEAVES

Fractionation Total Percent Total Specific Purification steps activity (%⁾ protein activity (fold)

(dpm) (mg) (dpm mg^"1)

Crude extract 7509343 100 31626.250 237 1.0

50-75% ( H ₂S0₄ 1892720 25 4723.252 400 1.7

Sephadex G-100 1385625 18.4 2216.825 625 2.6

Q Sepharose 1023300 13.6 56.925 17976 75.8

Butyl Sepharose 123840 1.6 4.398 28158 118.8

Mono Q 1386700 18.4 0.368 3762930 15877.3

Superdex 75 55460 0.7 0.013 4160420 17554.5

Mono Q 45515 0.6 0.008 5689375 24005.8

The presence of SA-binding proteins was monitored using a very efficient [³H]-SA binding assay. Extraction of the leaf proteins at low pH (6.3) helped in precipitating out many proteins, while the SABP2 remained soluble. Most of the non-specific SA-binding activity was removed in the 0-50% (NH₄)₂S0₄ fractions, and the 50- 75% fraction contained most of the specific SA-binding activity (as determined by competition with unlabeled SA) . Most of the high molecular weight SA-binding activity co-purifying with the carbonic anhydrase activity (e.g., SABP3 , Slaymaker et al . 2002) was in the early fractions collected from the Sephadex G-100 column. The later fractions, which contained SABP2 but did not contain any detectable carbonic anhydrase activity, were pooled and retained for further purification. Washing of the Q Sepharose column with 400 ml of

Buffer B containing 15 mM (NH₄)₂S0₄ removed protein lacking SA-binding activity. The presence of high concentrations of (NH₄)₂S0₄ in the protein fractions did not interfere with the [³H]-SA binding assay. Analysis of the SDS-polyacrylamide gel electrophoresis of the fractions from the second mono Q column indicated that three proteins (28, 30 and 32 kDa) co-purified with [³H] -SA binding activity (Figure 3) . The amount of the 28 kDa protein most closely tracked with level of SA binding activity, which strongly suggested that this protein was SABP2. The native molecular mass of the SABP2 was estimated to be -28 to 30 kDa by chromatography on the Superdex 75 column (Figure 2) .

This estimate was in good agreement with the -28 kDa weight estimated by SDS-PAGE. These results indicated that SABP2 is a monomeric protein. The sequences from the three proteins (28, 30 and 32 kDa) were determined and are provided in Table III, sequence identification numbers 11-27 are shown in parentheses.

TABLE III: Amino Acid Sequences From 28, 30 and 32 kDa Proteins

Protein Peptide Designation Peak# Sequence

28kDa Peptide # 1 pk22a VTALDLAASGTDLR (11) pk23 XTALDLAASGTD (12) pk55^* VXALDLAASGIDLR (13)

Peptide # 2 pk36 TPAENW/ILDTQFLPYG (14) pk34 TPAENTLDTQF / ELM / P YG (15) pk32 XPAENWLDTQFLPY (16)

Peptide # 3 pk29 YPEe/nPL/gTS/tMF (17)

Peptide # 4 pk22b hYALFMEDLHK(lδ) Peptide # 5 pk30^* AQ/KYFTDER (19) pkX YFQDER (20)

30kDa Peptide pklβa LVPVDVSIDP (21) Peptide pklβb i/yhfyiyplns (22) Peptide pkl7 F/s/aLYDFVD/fG/fHK (23) Peptide pk25a YVTPENNLYF (24)

Peptide # pk25b i/sdyhis/qf /ieel (25)

32kDa Peptide # 1 pkll NSIQPDFYANK (26) Peptide # 2 pk26 DIDGVPETLDLR(27)

* Sequences obtained from SABP2 purified and sequenced using a different purification scheme

"x" means any amino acid.

"/" means the amino acid at that position is either the amino acid shown before or after the ' / ' Amino acids in lower case means that data was only clear enough to make a tentative assignment.

The 28 kDa protein is SABP2 because (1) it most closely co-purified with the SA-binding and (2) in an earlier purification scheme the 28 kDa protein co- purified with SA binding activity, while the presence of the 30 kDa and 32 kDa proteins were not evident in those fractions. In addition, partial amino acid sequence analysis of tryptic peptides of the 28 kDa protein obtained in the earlier purification scheme indicated that it is the same 28 kDa protein obtained by the purification scheme described above. Further, expression of the cloned cDNA in E. coli produces a protein of the correct molecular weight and very high SA specific binding activity, with correct specificity for SA analogs (see Table IV) .

B. Features of the SABP2 cDNA clone:

PCR amplification was performed using a degenerate primer (5 ' -ACWCARTTYTTRCCHTAYGG-3 ' ;where W is A or T; R is A or G; Y is C or T; and H is A, C or T; SEQ ID NO : 7) encoding the sequence TQFLPYG (SEQ ID NO : 8) of peptides pk32, 34 and 36 and universal adapter primer (5'- GACTCGAGTCGACATCGA-3' ; SEQ ID NO : 9). The template used for PCR was the cDNA reverse transcribed from total RNA isolated from tobacco leaves. Agarose gel analysis of the PCR products did not result in any discrete bands. Thus, a second round of PCR amplification was performed using a small fraction of the PCR amplification product as the template with the same set of primers. This resulted in amplification of a ~650bp fragment which was gel purified and cloned into a pGEMT cloning vector. The clone, sequenced using plasmid specific primers, was 639 bp long with a complete 3' UTR and a polyA tail. Analysis of the deduced amino acid sequence indicated that the clone encoded four of the five peptides (peptides #2-#5 of the 28 kDa protein in Table III) identified by partial amino acid sequence determination of SABP2. For several of these peptides, the match to the deduced amino acid sequence is close, but not exact. This may be due to sequencing errors or may indicate that SABP2 is encoded by a gene family with the difference between peptide sequence and deduced amino acid sequence resulting from them coming from different family members.

The DNA sequence did not show significant homology to any known sequence in the NCBI database. However, the corresponding protein exhibited significant homology to several known plant proteins (Figure 5) . This comparison also suggested that the cDNA clone corresponds to the C- terminal half (127 residues) of a 28-30 kDa protein.

In order to isolate the missing 5 ' half of the SABP2 gene, 5' RACE was carried out using tobacco cDNA as the template. The first round of 5' RACE resulted in the amplification of a very faint DNA band identified by agarose gel analysis. PCR amplification was then repeated using the E-6 (5 ' -AGAGATCAGTTGTATTTATG-3 ' ; SEQ ID NO: 10) primer, a Nested Universal Primer (Clontech) and a small fraction of the first PCR product as the template. This second round of PCR amplification yielded a -850 bp fragment which was further cloned and sequenced.

Analysis of the sequence confirmed that the amplified product from 5 ' RACE contained the missing 5' portion of the SABP2 gene as well as the portion of the gene 5 ' to the E-6 primer that was present in the original 3' end clone. Further analysis of the 5' RACE product, together with the original 3' end clone of the SABP2 gene, revealed that the SABP2 mRNA is at least 1079 nucleotides in length and contains a large open reading frame that encodes a 260 amino acid protein with a calculated molecular mass of 29.3 kDa and an estimated pi of 5.45.

The sequences corresponding to all five peptides obtained from the sequencing of the tryptic fragments of the purified SABP2 protein are underlined in the deduced amino acid sequence presented in Figure 4. This result indicates that the clone corresponds to the purified SABP2 protein. The alignment of the deduced amino acid sequence of the open reading frame along with the homologous proteins (Figure 5) indicates that the clone encompasses the full length SABP2 cDNA.

C. Proof that the 28 KDa protein is SABP2

To rigorously establish that the 28KDa protein from plant extract was SABP2 , the cloned cDNA was expressed in E. coli as His₅-tagged fusion protein and the recombinant protein was purified on a Ni column and tested for SA- binding activity (Table IV) . The recombinant protein exhibited very high ³H-SA binding activity, and this binding was inhibited effectively by addition of a large molar excess of unlabeled SA or it's biologically active (for induction of PR genes or enhanced disease resistance) analogs but not it's inactive analogs (e.g. 4-HBA) .

TABLE IV

TOBACCO SABP2 AND TWO ARABIDOPSIS HOMOLOGS PRODUCED IN E.

COLI HAVE SA-BINDING ACTIVITY WHICH IS SPECIFIC FOR SA

AND ITS BIOLOGICALLY ACTIVE ANALOGS

ASSAY Bound [ΗjSA Bound [³H]SA Bound [³H]SA Bound[³H]SA Biological Activity

SABP2 AtSB2L9* AISB2L5* AtSB2Ll* of competitor

No competitor 140206** 16486 12927 70 ._

ImM unlabeled SA 160 36 121 42 active

ImM unlabeled ₅-CSA 260 42 64 38 active

ImM unlabeled 2,6-DHBA 924 1072 1742 36 active

1 mM unlabeled 4-HBA 95634 3620 4757 65 inactive *AtSB2 l is Genbank Accession NM_127926 ( SEQ ID NO : 32 ) ; AtSB2L5 is Genbank

Accession NM_121068 ( SEQ ID NO : 36 ) ; AtSB2L9 is Genbank Accession NM_119878

(SEQ ID NO: 40) .

**Radioactivity is measured in dpm.

III. Discussion:

SABP2 is a member of the super family of alpha/beta fold hydrolases. This gene has not previously been identified in tobacco. However, Arabidopsis, which has SABP2-like activity (Du and Klessig, 1997), contains a large gene family with 18 full-length members that encode proteins with 32%-57% identity and 46-71% similarity to the tobacco SABP2. We referred to them as A._fc.SABP2 like (AtSB2L) with AtSB2Ll having the highest percentage identity over the entire 260 a. a. of tobacco SABP2 and AtSB2Ll8 the lowest. See Example IV. This Arabidopsis family of hypothetical/putative proteins shows high homology to alpha-hydroxynitrile lyases/ (S) -acetone- cyanohydrin lyases (hnl or acl) isolated from cassava Manihot esculenta and rubber plants (Hevea brasilienses) (Figure 5) . This enzyme, together with a β- glycosidase, releases hydrogen cyanide (HCN) through a process termed cyanogenesis, from cyanogenic glycosides (Hickel et al . , 1996). HCN acts as a repellent to herbivores including insects (Kakes, 1990; Poulton, 1990) . HCN may also play a role in resistance to pathogens (Hickel et al . , 1996; Murphy et al . , 1999). Chivasa and Carr (1998) have also shown that KCN induces resistance to tobacco mosaic virus (TMV) in tobacco. KCN also induces expression of an alternative oxidase, which Carr and coworkers suggest plays a role in resistance to viruses (movement and/or replication) such as TMV, but not to bacterial or fungal pathogens (Chivasa et al., 1997; Murphy et al . , 1999).

In contrast to some plants, which release large quantities of HCN from their cyanogenic glycoside reserves upon predation (Conn, 1981), most plants, including Arabidopsis contain little or no cyanogenic glycosides (Waspi, et al.,1998). Perhaps in these low or non-cyanogenic plant species, SABP2/AtSB2Ls releases small quantities of HCN from previously undetected small reserves, which are sufficient to activate a very local response (s), perhaps through induction of an alternative oxidase .

Alternatively, SABP2 may not possess hnl activity, but may have another function. Rice, for example, which lacks readily detectable levels of cyanogenic glycosides (Waspi, et al . , 1998), contains a gene termed Pirlh whose expression is induced following infection with the avirulent Pseudomonas syringae pv. syringae . Infection with avirulent P. s. s. also induces acquired resistance to the rice blast fungus, Pyricularia oryzae (Smith and Metraux, 1991) . Pirlh is homologous (35% identity, 56% similarity) to the cassava and rubber plant hnl (and to SABP2; 42% identity, 61% similarity), but has no hnl activity. Rather it exhibits esterase activity towards naphthol AS-esters (Waspi, et al . , 1998).

Interestingly, Pirlb contains a conserved catalytic triad of serine, aspartate and histidine and a lipase signature sequence (Waspi, et al . , 1998). The N-terminal regions of lipase/esterases, like Pirlb, contain the serine residue of the catalytic triad and the lipase signature sequence while its aspartate and histidine reside in the C-terminal half. The serine, aspartate and histidine residues of the triad are also present and appropriately positioned within SABP2. SABP2 also contains the lipase signature sequence. This protein appears to have a functional esterase/lipase, based on it's ability to to release MUF ( -Methyl umbelliferone) from MUF butyrate (Figure 6) . There is considerable evidence for the involvement of lipids and lipases in cellular signaling. For example, diacylglycerol activates protein kinase C which then modulates many Ca²⁺-dependent cellular processes (Niskizuka, 1986) .

Moreover, two additional Arabidopsis genes encoding lipase-like proteins, EDS1 and PAD4 , when mutated, result in increased susceptibility to pathogens. The products of both genes are thought to function upstream of SA (Falk et. al., 1999; Zhou et . al . , 1998) and may form part of a signal-amplification loop with SA (Jirage et . al . , 1999). However, lipase activity has not yet been demonstrated for either protein (Falk et . al . , 1999; Jirage et. al . , 1999). SABP2 also has strong homology to several esterases such as the ethylene-induced esterase from Ci trus sinensis (58% identity and 72% homology, Zhong et al . , 2001) and polyneurideine aldehyde esterase from Rauvolfia serpentina (57% identity and 75% homology) (Dogru et al . , 2000).

In addition to SABP2 ' s activity on MUF butyrate, sequence comparisons and structural analyses suggest that this protein is a lipase. Supporting this hypothesis

SABP2 ' s N-terminal sequence contains a domain (a. a. 15- 127) present in mammalian lecithin (phosphatidylcholine) cholesterol acyltransferase (LCATs) and similar proteins found in mammals, yeast, C. elegans and Arabidopsis . In mammals, LCAT, which has a role in cholesterol transport and metabolism, converts phosphatidylcholine and cholesterol to lysophosphatidylcholine and cholesteryl esters in a sequential reaction on the surface of high density lipoproteins (Jones, A., 2000). LCAT can also use additional acyl donors besides phosphatidylcholine, such as phosphatidylethanolamine, and other acyl acceptors in addition to cholesterol including water. Thus, based on its homology to LCAT and other alpha/beta fold hydrolases, SABP2 may be a phospholipase involved in lipid metabolism and signaling in plants. Taken together, these sequence analyses strongly implicate SABP2 as a critical signaling component in SA- mediated disease resistance, perhaps by acting as either a esterase/lipase .

EXAMPLE II ANALYSIS OF SABP2 GENE COPY NUMBER, EXPRESSION PATTERNS

AND ENZYMATIC ACTIVITY

Determination of SABP2 distribution and expression patterns provides valuable insight into the mechanisms and nature of plant pathogen resistance. Accordingly, experiments were performed to assess the effects of altering SABP2 expression levels on pathogen resistance. The following materials and methods are provided to facilitate the practice of Example II. Distribution and copy number can be determined by Southern analysis and expression of SABP2 can be determined by Northern analysis, according to the methods of Sambrook et al . , 1989.

SABP2 Gene Expression, Distribution, and Copy Number

Southern hybridization can be utilized to determine the distribution and copy number of SABP2 in different plant species. Homologs in humans, mouse, Drosophila and S. cerevisiae may also be identified using sequence information in the cDNA clone set forth herein. Database analysis already indicates that homologs are present in a diverse group of plants including Arabidopsis, rice, cassava and rubber tree. The cassava and rubber tree proteins are ACLs that may play a role in cyanogenesis while the rice protein, termed PIR7b, does not have lyase activity but is an esterase. As mentioned previously, expression of Pir7b is induced, in parallel with acquired resistance to the rice blast fungus Pyricularia oryzae, by infection with avirulent P.s. pv syringae (Waspi, U. et al. , 1998) .

Southern blot analysis done at both low and high stringency identified only 2-4 bands with restriction enzymes that do not have recognition sequences in the

SABP2 coding region. Since tobacco is amphidiploid, this result suggests that SABP2 is encoded by a single gene (but two alleles) or by a very small gene family. Slight variation in the deduced a. a. sequence of the cloned SABP2 from the a. a. sequence obtained from the purified protein for three of the five tryptic peptides also suggests the presence of at least two genes or alleles of a single gene. In contrast, Arabidopsis contains 18 AtSBP2Ls, most of which are expressed and two have been shown to have SA-binding activity (Table IV) .

Expression studies have been initiated and already indicate that the tobacco SABP2 and one of its putative Arabidopsis ortholog, AtSB2L9, are induced in plants resisting infection by viral [tobacco mosaic virus (TMV) or turnip crinkle virus (TCV) ] or bacterial (P. s.) pathogens. See Figure 7. These studies can be extended to other plant-pathogen interactions, e.g. P. s. pv tabaci, and P.s. pv tomato with or without AvrPto in tomato or Pto-transformed Nicotiana benthamiana, for SABP2 and P. s. and Peronospora parasitica for AtSB2L5, AtSB2L9, and other putative orthologs in Arabidopsis. While TMV induction of SABP2 expression is SA dependent, SA (or BTH) treatment alone is insufficient for this induction, suggesting the involvement of another factor. A likely candidate is ethylene since its synthesis is induced by TMV (de Laat, A.M.M. et al . , 1982) and ethylene has recently been shown to induce an esterase in citrus which is highly homologous to SABP2 and the AtSB2Ls (Zhong, G.Y. et al . , 2001). Therefore, induction of SABP2 and its Arabidopsis ortholog (s) by ethylene can be tested in a similar fashion. A general picture of the temporal and spatial expression of the two (or more) tobacco SABP2 genes/alleles is obtained by cloning the second gene/allele and designing member/allele-specific probes (usually corresponding to the divergent 3'UTR). In situ hybridization can then be used to determine a a more refined expression pattern.

Localization A better understanding of SABP2 ' s function is facilitated by determing its subcellular location. Subcellular location can be determined by immunofluorescence microscopy, which has been used extensively, for example, to determine the subcellular locations of other proteins, including the adenovirus DBP (Voelkerding, K.V. et al . , 1986) and tobacco PR-1 proteins (Carr, J.P. et al., 1989; Dixon, D.C. et al . , 1991) . An alternative and complementary approach is subcellular fractionation followed by western analysis using anti-SABP2 antibodies. Subcellular fractionation techniques used to identify SABP3 (Slaymaker, D.H., et al., 2002) and SABP4 and characterize NO-sensitive aconitases (Navarre, R. , et al . , 2000), can be readily modified for western analysis of SABP2. High affinity antibodies (Ab) have been prepared in rabbits to native and SDS-denatured SABP2. The strongest Ab is to the native SABP2 and is highly specific, reacting only with SABP2 in western analysis of total protein extracts from mock- or TMV-infected tobacco (see Fig. 7B) . Thus, this Ab is very suitable for immunofluorescence microscopy.

Initial localization is done in TMV-infected plants or in a transgenic line overexpressing SABP2. Alternatively, analysis may be conducted of plants overexpressing SABP2 tagged at its C-terminus with the HA epitope, for which highly specific, high affinity Ab are commercially available. Others have used a similar approach to localize bacterial avr factors/effectors to the plant plasma membrane (PM) (Nimchuk, Z. et al . , 2000

Shan, L., et al . , 2000). Interestingly, SABP2 possesses a myristoylation site near its N-terminus (12GACHGG17; SEQ ID NO:28), in addition to potential glycosylation (114NSSF117 ; SEQ ID NO: 50) and PKC and CK2 phosphorylation sites. Since myristoylation frequently directs proteins to the plasma membrane and many receptors and other signaling components are associated with the plasma membrane, SABP2 ' s putative myristoylation sequence will be mutated to determine if it is necessary for fatty acylation, membrane association or function of SABP2. AtSB2L5 and AtSB2L9, SAPB2 related genes in Arabidopsis, also have myristoylation sites near their N-terminus.

Envzmatic activity

SABP2 and its ortholog (s) appear to possess enzymatic activity. A comparison of amino acid sequence and/or enzyme activity (s) for SABP2 and its Arabidopsis ortholog (s) (which have SA-binding activity and whose silencing suppresses certain defense responses) will facilitate the identification of SABP2 ' s biochemical activity that is relevant to SA-mediated defense responses .

SABP2 has esterase/lipase activity based on its ability to release MUF from MUF butyrate (Fig. 6) while AtSB2L5 has the lipase signature sequence (GXSXG) (SEQ ID NO: 29 , and AtSB2L9 has the modified signature sequence AXSXG (SEQ ID NO: 30) found in some lipases (Taniyama et al . , 1999) . In preliminary experiments no effect of SA on this activity was observed. Our inability to detect an effect of SA on the lipase/esterase activity likely reflects the poor conditions for - SA binding in the gel assay. An alternative explaination is that while this low level of activity on the synthetic substrate is unresponsive to SA, SABP2 ' s activity on it's natural substrate would presumably be much higher, and SA responsive.

To further assess this putative enzymatic activity, a set of model substrates will be employed in a systematic approach to identify the enzyme activity (s) of SABP2 and its Arabidopsis ortholog(s). This set has previously been employed by Waspi et al . (Waspi, U. et al., 1998) and Baudouin et al (Baudouin et al . , 1997) to characterize two other α/β fold hydrolases, Pir7b of rice and Hsr203J of tobacco, the first of which is closely related by sequence to SABP2 and the AtSB2Ls. Like SABP2 they contain the catalytic triad and lipase signature sequence and are induced at the transcription level during defense responses to pathogens. Pir7b's preferred substrate is naphthol AS-esters while Hsr203J prefers short-chain dinitrophenyl acyl esters. For esterase activity the following potential substrates, p-nitrophenyl butyrate, acetylcholine, 2-naphthol butyrate/acetate and 2-naphthol AS-acetate will be employed. Amidase activity will be monitored with acetanilide and nitro-acetanilide, while epoxide hydrolase activity analysis will utilize 9R-10S-epoxystearic acid. Protease activity will be assayed with casein as the substrate.

The data indicate that SABP2 (and its Arabidopsis ortholog[s]) is a lipase/esterase, based on its activity on MUF-butyrate. SABP2 ' s esterase/lipase activity, and potential for SA to alter this activity suggests a link between SA and lipids/fatty acids. While the nature of this connection is presently unclear, it is intriguing that not only do two other proteins involved in disease resistance (EDS1 and PAD4) have a putative lipase activity and interface with SA (perhaps through a positive feedback loop) , but several more recent reports provide further evidence for a role of lipids/FAs in SA-mediated defense signaling. Our own work on the Arabidopsis mutant ssi2 suggests that oleic acid or its derivative suppresses SA-mediated defense responses such as PR-1 induction and resistance to P.s. and P. parasitica, while in contrast it acts in conjunction with JA to induce PDF1.2 expression and resistance to Botrytis cinerea (Kachroo et al . , 2001) . Lamb and colleagues (Maldonado et al . , 2002) recently described another Arabidopsis mutant dirl which is defective in induction of resistance. DIR1 is a putative lipid transfer protein, which is required for the production or transmission of the mobile signal that moves from infected tissue through the phloem to induce SAR in uninoculated parts of the plant . EXAMPLE III

SABP2 IS IMPORTANT FOR RESISTANCE TO PATHOGENS The present inventors have demonstrated that SABP2 silencing in plants gives rise to decreased resistance to TMV and also represses induction of the PR-1 gene by TMV infection or by SA treatment.

In our initial transgenic tobacco (Xanthi nc [NN] and Xanthi [nn] ) lines, SABP2 is being expressed behind the powerful constitutive CaMV 35S promoter in the sense orientation or as an intron-containing dsRNA. The results obtained from our experiments with 16 transgenic lines with the dsRNA-SABP2 construct and 4 lines with the empty vector construct are described hereinbelow. RT-PCR analyses indicated that SABP2 expression was suppressed between -75% and -100% (almost no detectable RNA) in these 16 lines (Fig. 8) . These 16 lines develop TMV lesions which on average were 41% larger than on the empty vector lines (Fig. 8) , as predicted if SABP2 plays a positive role in resisting TMV infection. Note that in the SA-deficient, NahG transgenic tobacco lines TMV lesion size was on average 23% larger (Gaffney, T. et al., 1993). Thus, suppression of SABP2 expression appears to be at least as disruptive to TMV resistance as destruction of the SA signal . Since Gaffney and colleagues observed a much larger negative effect of NahG on TMV-induced SAR than on local resistance, this type of SAR experiment will also be performed. Primary infection experiments and systemic spread will also be assessed as soon as the T2 generation of our SABP2 silenced/suppressed lines are available. We also have lines with intermediate levels of suppression which could be used to test whether reduction in lesion size (SAR) is ^•proportional to suppression of SABP2 levels.

In parallel, expression of SA-inducible PR genes, in particular PR-1, can be monitored in these plants. This can be done before and after infection as well as after treatment with varying concentrations of SA or BTH. If SABP2 participates in SA-induced PR-1 expression, then SABP2 suppressed plants should be less responsive to SA and BTH while SABP2-overexpressers might exhibit increased responsiveness to these compounds or even express PR-1 in the absence of exogenous SA or BTH.

Consistent with such a positive role, the data presented herein indicate that reduction of SABP2 expression in the dsRNA transgenic tobacco lines suppresses induction of PR-1 by SA and by TMV, as shown in Figure 8 for five of the best silenced lines, thus again implicating SABP2 in several defense responses .

EXAMPLE IV IDENTIFICATION AND CHARACTERIZATION OF ARABIDOPSIS SB2LS The extensive collection of mutants affecting disease resistance and SA-mediated defense responses and the availability of its genomic sequence make Arabidopsis an ideal plant to study disease resistance. Moreover, Arabidopsis contains an SABP2-like activity (Du, H. et al . , 1997) and has a large gene family (18 full-length members) which encode proteins with 32-57% identity and 46-71% similarity to SABP2 , two of which have already been shown to have specific SA-binding activity (see Table IV) , one of which is induced by infection. Note that while at least 15 are expressed (ESTs available) and have sequences suggesting they are esterases, lipases or lyases, none have a known function. Thus, characterization of this family to determine which has SA-binding activity and of these, what affect does silencing (knock out (KO) or dsRNA mediated) have on the plant's phenotype, particularly with respect to disease resistance, will help further our understanding of the disease resistance pathway.

Two parallel types of experimentation will be performed initially. First, we will continue to clone, express, and analyze the SA-binding activity of the select members of this family. A dendrogram of SB2Ls (Fig. 9) suggests that they can be grouped into five subfamilies. To date several members of subfamily I and II, the subfamilies most closely related to tobacco SABP2 , have been tested for SA-binding activity. All members of subfamilies I and II and select members of the more distant subfamilies III-V can also be analyzed.

In parallel two approaches will be used to obtain transgenic or mutant Arabidopsis with altered expression of one or more members of the AtSB2L family. If a limited number have SA-binding activity, then T-DNA derived KO or activation-tagged mutants for these family members will be obtained from the UW Arabidopsis KO Facility (α and β populations) and from the Salk Institute Genomic Analysis Lab's- libraries. We have previously obtained KOs of several family members of the TGA family of transcription factors from the UW facility and five AtSB2L KO lines have already been obtained from the Salk Institute collection that correspond to two SA-binding AtSB2Ls, AtSB2L5 (subfamily I) and AtSB2L9 (subfamily II), another member of subfamily II (AtSB2L7) and two members of subfamily V (AtSB2Ll4 and 15) . These five KO lines can initially be screened for altered defense responses to avirulent P. s. (e.g. HR, pathogen growth) or to SA treatment (e.g. induction of PR-1, PR-2, and PR-5), as well as SA-binding activity for AtSB2L7, AtSB2Ll4 and

AtSB2Ll5. (If, as anticipated, one or more of these three latter KOs do not have SA-binding activity, it will serve as a good negative control) . A secondary screen of the more interesting KOs would include resistance to an avirulent pathovar of P. parasitica and expression of other defense genes such as GSTs, PR-3, PR-4 and defensin (PDF1.2) .

Knockout of a single AtSB2L may not alter defense response because of possible redundancy. If there are only a small number of AtSB2Ls with SA-binding activity, then one approach will be to cross the different KO lines (assuming they are available) to generate lines with 2, 3, or more of the SA-binding AtSB2Ls silenced. This approach is less likely to be successful if a significant number (e.g. >5) of the AtSB2Ls bind SA. The better approach then will be dsRNA-mediated gene silencing in stable transgenics as outlined above for SABP2. Because of sequence divergence among the family members, particularly at the DNA level, silencing of all members with one construct is not possible. Initially and in parallel with initial screening of the readily available KOs, we will silence all members of subfamily II with one construct and most subfamily I members using sequences that are highly conserved within that subfamily. Effective silencing requires a minimum of 50 nucleotides with 80% or more identity. In subfamily II there is a 65 nucleotide region with 80% identity among all five members located between 678 and 742, with the A of the initiation codon of AtSB2L9 designated 1. No such conserved region is present in all four subfamily I members; however, there is a 153n region with 79% identity for AtSB2Ll, 2, and 8 located between 3 and 155 with the A of AtSB2Ll ' s ATG as 1. This sequence will be used to silence these three members in the Salk line in which the fourth member, AtSB2L5, is knocked out. Also once we determine the number of AtSB2Ls that bind SA, careful analysis of sequences of these genes may identify a highly conserved region against which the dsRNA could be targeted for silencing of all, or at least most, of the SA-binding AtSB2Ls. Construction and characterization of transgenic lines that overexpress one of the SA-binding AtSB2Ls may also be informative and will be done for a limited number of these AtSB2Ls, particularly if silencing of that AtSB2L alters defense responses. The expression of SA-binding AtSB2Ls will also be monitored to see if they are induced by infection with avirulent or virulent pathogens (e.g. TCV, P. s., P. parasitica) or treatment with SA (or BTH) .

This large family of AtSB2Ls may provide an opportunity to identify a region of these proteins which is required for SA binding. Comparison of the a. a. • sequences of these AtSB2Ls with SA-binding activity (and SABP2) versus those of non-binding homologues may reveal a region (and possibly even critical a.a.'s) that is likely involved in SA binding. This will be confirmed by swapping of this putative domain between closely related binding and non-binding homologues, preferably within the same subfamily. Site-directed mutagenesis could then be used to confirm or dispel the involvement of a.a.'s predicted to be important based on sequence analysis.

Once the enzymatic activity involved in transmitting the SA signal by SABP2 and its Arabidopsis ortholog (s) has been defined, then the effects of these mutations on it, as well as SA binding, will be determined. The Arabidopsis thaliana SABP2,-.like gene family members are referred to herein as follows: AtSB2Ll, Genbank Accession No NM_127926 (SEQ ID NO:32), AtSB2L2 Genbank Accession No NM_127924 (SEQ ID NO:33), AtSB2L3 Genbank Accession No NM_127925 (SEQ ID NO:34), AtSB2L4 Genbank Accession No NM_127922 (SEQ ID NO:35), AtSB2L5, Genbank Accession No NM_121068 (SEQ ID NO:36), AtSB2L6 Genbank Accession No NM_127919 (SEQ ID NO:37), AtSB2L7 Locus At2g23560 Accession No NM_127920 (SEQ ID NO:38), AtSB2L8 Genbank Accession No NM_127923 (SEQ ID NO:39), AtSB2L9 Genbank Accession No NM_119878 (SEQ ID NO:40), AtSB2LlO Genbank Accession No NM_114904 (SEQ ID NO: 41), AtSB2Lll Genbank Accession No NM_113902 (SEQ ID NO:42), AtSB2Ll2 Genbank Accession No NM_117058 (SEQ ID NO:43), AtSB2L13 Genbank Accession No NM_102400 (SEQ ID NO:44), AtSB2Ll4 Genbank Accession No NM_103121 (SEQ ID NO:45), AtSB2L15 Genbank Accession No NM_105591 (SEQ ID NO:46), AtSB2Ll6 Genbank Accession No NM_117770 (SEQ ID NO:47), AtSB2Ll7 Genbank Accession No NM_111924 (SEQ ID NO:48), and AtSB2Ll8 Genbank Accession No NM_125216 (SEQ ID NO: 49) .

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While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. An isolated nucleic acid molecule comprising an SABP2-encoding nucleic acid sequence of SEQ ID NO: 1.

2. A cDNA produced by reverse transcription of an mRNA encoded by the nucleic acid molecule of claim 1.

3. The nucleic acid molecule of claim 1 isolated from tobacco.

4. An RNA molecule encoded by the SABP2-encoding nucleic acid molecule of claim 1.

5. An expression vector comprising the nucleic acid molecule of claim 1.

6. An expression vector of claim 5 wherein said vector is selected from the group of vectors consisting of plasmid, cosmid, baculovirus, bacteria, yeast and viral vectors.

7. A host cell transformed with an expression vector of claim 5.

8. A host cell of claim 7, wherein said host cell is selected from the group consisting of tobacco, Arabidopsis, rice, maize, wheat, soybean, tomato, potato, barley, canola, bacteria, yeast, insect and mammalian cells.

9. A host cell of claim 7, wherein said host cell is a tobacco cell.

10. A host cell of claim 7, wherein said host cell is an Arabidopsis thaliana cell.

11. An isolated SABP2 polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 1.

12. An isolated nucleic acid molecule encoding a protein sequence of SEQ ID NO: 2.

13. The isolated polypeptide of claim 11, which encodes an enzyme with a function selected from the group consisting of lyases, lipases, esterases and lecithin (phosphatidylcholine) cholesterol acyltransferases .

14. The plant enzyme of claim 13, wherein loss of function of the enzyme in a plant results in altered resistance of the plant to plant pathogens or other disease-causing agents.

15. An antibody immunologically specific for at least one epitope of an SABP2 polypeptide of SEQ ID NO: 2.

16. A method for identifying agents which modulate SABP2 enzymatic activity in a host cell, comprising the steps of: a) introducing an SAPB2-encoding nucleic acid into said host cell; b) treating said host cell with agents suspected of modulating SABP2 enzymatic activity; and c) assaying SABP2 enzymatic activity in the presence and absence of said agent in said host cell or extracts thereof.

17. A method for identifying agents which modulate SABP2 expression in a host cell, comprising the steps of: a) introducing an SAPB2-encoding nucleic acid into said host cell; b) treating said host cell with agents suspected of modulating SABP2 expression in said host cell; and c) assaying SABP2 expression in the presence and absence of said agent in said host cell or extracts thereof .

18. A method to enhance resistance of a plant to plant pathogens or other disease causing agents comprising overexpressing an SABP2-encoding nucleic acid molecule of SEQ ID NO: 1 in a plant cell.

19. The method of claim 18, wherein SABP2 expression is further enhanced by addition of an agent identified by the method of claim 17.

20. A method to inhibit SABP2 function in a plant, said method comprising the introduction of a mutated SABP2-encoding nucleic acid into said plant, said mutated SABP2-encoding nucleic acid encoding a non-functional SABP2 protein.

21. The method of claim 20, wherein said mutated SABP2-encoding nucleic acid encodes an antisense molecule of SEQ ID NO: 1.

22. The method of claim 20, wherein said mutated SABP-2 encoding nucleic acid comprises an intron- containing double stranded RNA segment of SEQ ID NO: 1.

23. A method to inhibit SABP2 expression in a plant, said method comprising the introduction of a mutated SABP2-encoding nucleic acid into said plant, said mutated SABP2-encoding nucleic acid inhibiting expression of SABP2.

24. The method of claim 23, wherein said mutated SABP2-encoding nucleic acid encodes an antisense molecule of SEQ ID NO: 1.

25. The method of claim 23, wherein said mutated

SABP-2 encoding nucleic acid comprises an intron- containing double stranded RNA segment of SEQ ID NO: 1.

26. A screening method for identifying test compounds having binding affinity for SABP2 , comprising the steps of: a) providing a predetermined amount of SABP2 bound by a detectably labeled agent having binding affinity for SABP2 ; b) treating said SABP2 with at least one test compound suspected of having binding affinity for SABP2 ; c) measuring displacement of said detectably labeled agent in the presence and absence of said test compound, thereby identifying test compounds with binding affinity for SABP2.

27. A method as claimed in claim 26, wherein said agent is detectably labeled salicylic acid.

28. A method as claimed in claim 26, wherein said test compound is a salicylic acid analogue.

29. A method as claimed in claim 26, wherein said SABP2 is affixed to a solid support.

30. A screening method for identifying SABP2 test compounds which modulate SABP2 enzymatic activity thereby increasing disease resistance in plants, comprising the steps of: a) providing a predetermined amount of SABP2 in the presence of an SABP2 substrate, SABP2 enzymatic activity catalyzing formation of a reaction product, at least one or said substrate and said product being detectable; b) treating said SABP2 with at least one test compound suspected of modulating SABP2 enzymatic activity; and c) measuring conversion of substrate to product in the presence and absence of said test compound, thereby identifying test compounds which modulate SABP2 activity.

31. A method as claimed in claim 30, wherein said SABP2 is affixed to a solid support.

32. A transgenic plant comprising SEQ ID NO : 1, said plant having increased resistance to disease.

33. The transgenic plant of claim 32, wherein said plant is fertile.

34. A fertile plant produced by the method of claim

18.

35. A fertile plant produced by the method of claim

20

36. A fertile plant produced by the method of claim

21

37. A fertile plant produced by the method of claim

22.

38. A transgenic knock-out plant wherein SABP2 expression has been significantly reduced relative to wild-type non-transgenic plants.

39. The plant of claim 38, which is fertile.

40. An isolated SABP2 nucleic acid molecule comprising a sequence selected from the group consisting of: a) SEQ ID NO: 1 ; b) a sequence which specifically hybridizes with SEQ ID NO: 1; c) a sequence encoding a polypeptide of SEQ ID NO : 2 ; d) a complement of SEQ ID NO: 1; and e) a natural allelic variant of a sequence selected from the group consisting of a) , b) , c) and d) .

41. A method for identifying agents which modulate SABP2-homolog enzymatic activity in a host cell, comprising the steps of: a) introducing an SAPB2 homolog-encoding nucleic acid into said host cell; b) treating said host cell with agents suspected of modulating SABP2 homolog enzymatic activity; and d) assaying SABP2 homolog enzymatic activity in the presence and absence of said agent in said host cell or extracts thereof.

42. A method for identifying agents which modulate SABP2 homolog expression in a host cell, comprising the steps of: a) introducing an SAPB2 homolog-encoding nucleic acid into said host cell; b) treating said host cell with agents suspected of modulating SABP2 homolog expression in said host cell; and b) assaying SABP2 homolog expression in the presence and absence of said agent in said host cell.

43. A method to enhance resistance of a plant to plant pathogens or other disease causing agents comprising overexpressing a SABP-2 homolog encoding nucleic acid molecule selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 40 in a plant cell.

44. The method of claim 43, wherein SABP2 expression is further enhanced by addition of an agent identified by the method of claim 42.

45. A method to inhibit SABP2 homolog function in a plant, said method comprising the introduction of a mutated SABP2 homolog-encoding nucleic acid into said plant, said mutated SABP2 homolog-encoding nucleic acid encoding a non-functional homologous SABP2 protein.

46. The method of claim 45, wherein said mutated

SABP2-encoding nucleic acid encodes an antisense molecule selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 40.

47. The method of claim 45, wherein said mutated

SABP2 homolog-encoding nucleic acid comprises an intron- containing double stranded RNA segment selected from the group consisting of SEQ ID NOS: 36 and 40.

48. A method to inhibit SABP2 homolog expression in a plant, said method comprising the introduction of a mutated SABP2 homolog-encoding nucleic acid into said plant, said mutated SABP2 homolog-encoding nucleic acid encoding a non-functional, homologous SABP2 protein.

49. The method of claim 48, wherein said mutated SABP2-encoding nucleic acid encodes an antisense molecule selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 40.

50. The method of claim 48, wherein said mutated SABP-2 encoding nucleic acid comprises an intron- containing double stranded RNA segment selected from the group consisting of SEQ ID NO: 36 and 40.

51. A screening method for identifying test compounds having binding affinity for a SABP2 homolog, comprising the steps of: a) providing a predetermined amount of SABP2 homolog bound by a detectably labeled agent having binding affinity for said SABP2 homolog; b) treating said SABP2 homolog with at least one test compound suspected of having binding affinity for said SABP2 homolog; c) measuring displacement of said detectably labeled agent in the presence and absence of said test compound, thereby identifying test compounds with binding affinity for said SABP2 homolog.

52. A method as claimed in claim 51, wherein said agent is detectably labeled salicylic acid.

53. A method as claimed in claim 51, wherein said test compound is a salicylic acid analogue.

54. A method as claimed in claim 51, wherein said SABP2 homolog is affixed to a solid support.

55. A method as claimed in claim 51, wherein said SABP2 homolog is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 31-49.

56. A screening method for identifying test compounds which modulate SABP2 homolog enzymatic activity thereby increasing disease resistance in plants, comprising the steps of: a) providing a predetermined amount of SABP2 homolog in the presence of an SABP2 homolog substrate, said SABP2 homolog enzymatic activity catalyzing formation of a reaction product, at least one of said substrate and said product being detectable; b) treating said SABP2 homolog with at least one test compound suspected of modulating SABP2 homolog enzymatic activity; and c) measuring conversion of substrate to product in the presence and absence of said test compound, thereby identifying test compounds which modulate SABP2 homolog activity.

57. A method as claimed in claim 56, wherein said SABP2 homolog is affixed to a solid support.

58. A method as claimed in claim 56 wherein said SABP2 homolog is encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 31-49.

59. A transgenic plant comprising a heterologous encoding nucleic acid selected from the group consisting of SEQ ID NOS: 36 and 40, said plant having increased resistance to disease.

60. The transgenic plant of claim 59, wherein said plant is fertile.

61. The transgenic plant of claim 59 wherein said heterologous encoding nucleic acid is an intron- containing double stranded RNA segment of a sequence selected from the group consisting of SEQ ID NOS: 36 and 40.

62. A transgenic knock-out plant wherein SABP2 homolog expression has been significantly reduced relative to wild-type non-transgenic plants.

63. The plant of claim 61, which is fertile.

64. A method for screening functional homologs of

SABP2 to identify orthologs thereof, comprising: a) providing a predetermined amount of said putative SABP2 homolog; b) determining level of binding affinity for salicylic acid in the presence and absence of analogs thereof; c) determining level of esterase/lipase activity of said homolog; d) determining level of esterase/lipase activity in the presence and absence of salicylic acid; e) altering expression of said putative SABP2 homolog is a transgenic plant and determining whether such alteration modifies defense responses to pathogen infection; and d) those homologs having SA binding affinity which is displaced by SA analogs, possessing esterase/lipase activity which is altered by salicylic acid and which, upon introduction into a host plant, modify defense responses to pathogen infection being identified as functional orthologs of SABP2.

65. The method of claim 64, wherein said putative SABP2 homologs are encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 31-49.

66. The method of claim 64, wherein said putative SABP2 homologs are encoded by a nucleic acid selected from the group consisting of SEQ ID NOS: 36 and 40.