WO1998037755A1 - Plant pathogen response gene - Google Patents

Abstract

DNA molecules encoding a family of zinc-finger DNA binding domains, which appears to function to monitor levels of a superoxide-dependent signal and negatively regulates a plant cell death pathway, including wild-type LSD1, LOL1 and LOL2, and proteins which physically interact with LSD1, indicating a function with LSD1 of controlling plant cells' response to pathogens.

Description

PLANT PATHOGEN RESPONSE GENE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 60/039,063 filed February 28, 1997.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to a novel DNA molecule that encodes a novel polypeptide, LSDl, which has an effect in regulating the initial response of plants to pathogens and the subsequent spread of plant cell death engendered by infection, the protein encoded by the gene, and transgenic plants comprising the DNA molecule. This invention also relates to novel DNA molecules encoding LSDl related proteins LOLl and LOL2. In addition, it relates to novel DNA molecules encoding proteins which directly interact with LSDl .

Description of the Related Art Controlled induction of cell death occurs during both normal plant development and as the rapid, localized response to pathogen infection known as the hypersensitive response (HR) (Stakman, 1915; Goodman and Novacky, 1994; Dangl et al., 1996). The HR is a feature of most, but not all, disease resistance reactions in plants. The disclosure of these publications and all others cited herein, as well as of the priority application, is incorporated herein by reference.

Genetic control of disease resistance reactions is of two broad classes. The first is determined by specific interactions between particular alleles of pathogen avr (avirulence) gene loci and an allele of the corresponding plant disease resistance (R ) locus. When these alleles are present in both host and pathogen, the result is disease resistance in the plant, and the interaction is said to be "incompatible". If either the plant R allele or the cognate pathogen avr gene are absent or inactive, disease results and the interaction in said to be "compatible" (reviewed by Flor, 1971; Crute, 1985; Keen, 1990; Pryor and Ellis, 1993). A great deal of progress has been made recently in understanding the molecular structure of R genes and their predicted products (reviewed by Dangl, 1995; Staskawicz et al, 1995; Bent, 1996). These molecules function to recognize avr dependent signals and trigger the plant cell to begin the chain of signal transduction events culminating in a halt of pathogen growth. The simplest mechanistic interpretation of allele-specific disease resistance is that the R gene product recognizes the avr gene product directly. Although no direct avr-R protein interaction has been shown in planta, expression of avr genes in plant cells can be sufficient to trigger the HR in a ^-dependent manner, and avr-R protein-protein interactions can occur in yeast two-hybrid systems (Gopalan et al., 1996; Scofield et al., 1996; Tang et al, 1996). The second mode of genetic control of disease resistance is termed "non-host" resistance and describes in essence those interactions which lack genetic variability in either host or pathogen such that no virulent pathogen and no susceptible host line have been identified. While it is not beyond reason to assume that traditional "non-host" interactions are simply a series of allele specific recognition events occurring simultaneously (Whalen et al., 1988; Kobayashi et al., 1989; Valent et al., 1990), it is also possible that this mode of resistance is mechanistically distinct from that mediated by allele-specific interactions. Pathogen ligands (termed elicitors) which mediate several key non-host interactions have been isolated, although their corresponding plant receptors have not (Cosio, et al. 1992; Nϋrnberger et al, 1994). Subsequent to pathogen recognition by either of these two systems, the plant cell deploys a battery of inducible defense responses. Chief among the earliest events are

+ + calcium influx, K -H exchange leading to alkalinization of the extracellular space, and an oxidative burst (reviewed in Godiard et al., 1994; Hammond-Kosack and Jones, 1996). The latter is potentially mediated by a plasma membrane NADPH oxidase analogous to that used by mammalian neutrophils (Low and Merida, 1996), although other models exist (Bolwell et al., 1995). Parts of this cascade are linearly regulated in at least some systems:

2+ blocking of Ca influx blocks anion channel activity, the oxidative burst and downstream events including cell death; blocking anion channels effects only ROI production and

2+ defense gene activation, but not Ca influx (Nϋrnberger et al., 1994; Levine et al., 1996; May et al., 1996).

Consequent production of reactive oxygen intermediates (ROI) occurs with kinetics and magnitude suggesting a key role in either pathogen elimination, subsequent signaling of downstream effector functions, or both (reviewed by Baker and Orlandi, 1995; Low and Merida, 1996). H2O2 can have a key role in resistance responses, and cell wall strengthening (Brisson et al., 1994; Levine et al., 1994; Levine et al., 1996), and superoxide produced as the proximal ROI in the burst has also been implicated in initiating HR (Doke, 1983; Jabs et al., 1996). Transcription and translation of plant genes are required for HR. These signals are thought to culminate in transcriptional activation of a variety of plant genes, HR, and the production of both local and systemic signals that protect the plant from further infection. It is unclear whether these effector functions are controlled by linear, interdigitating, or bifurcating signal pathways.

Cell death during the HR may be a direct consequence of ROI toxicity, or it may be a secondary consequence of signals derived from ROI. It is not known whether HR is required to halt pathogen growth. Nonetheless, HR is correlated with the onset of systemic acquired resistance (SAR) to secondary infection in distal tissue (reviewed by Ryals et al., 1996). In at least tobacco and Arabidopsis, enzymatic blocking of salicylic acid (SA) accumulation subsequent to infection alters disease resistance responses, and SA in distal tissues is required for SAR (Gaffney et al., 1993; Delaney et al., 1994; Vernooij et al., 1994). SA accumulates following the oxidative burst to high levels locally at infection sites. The biochemical properties of SA as an inhibitor of a variety of enzymes suggest a model whereby SA or a radical derived from it poisons the infected cell, causing its death (Enyedi et al., 1992; Malamy et al., 1992; Chen et al., 1994; Durner and Klessig, 1995; Rueffler et al., 1995). Recent descriptions of the morphology of cell death during infection suggest, in at least some cases, parallels with animal apoptosis (Mittler et al., 1995; Kosslak et al., 1996; Levine et al., 1996; Ryerson and Heath, 1996; Wang et al, 1996a; reviewed by Dangl et al., 1996). A molecular understanding of both the signaling events that control the onset of this specialized plant cell death and the mechanisms by which these cells die will hasten approaches to manipulate cell death to protect plants from disease.

A number of researchers have isolated mutants in Arabidopsis which exhibit constitutive onset of HR-like cell death in the absence of pathogen (Greenberg and Ausubel, 1993; Dietrich et al., 1994; Greenberg et al, 1994). These mutants resemble a variety of mutants in crop species isolated since the 1920s and broadly categorized as "lesion mimic mutations" (Langford, 1948; Kiyosawa, 1970; Walbot et al., 1983; Johal et al., 1994). A series of non-allelic mutations was isolated which expressed histochemical and molecular markers associated with disease resistance responses. These mutants subdivide the lesion mimic class into a "lesions simulating disease resistance" or Isd phenotype (Dietrich et al., 1994). These mutants also exhibited heightened resistance to otherwise virulent bacterial and oomycete pathogens when lesions were present, demonstrating that these cell death phenotypes can trigger pathogen non-specific resistance resembling SAR. Similar "accelerated cell death" or acd mutants have been described by Greenberg and Ausubel (Greenberg et al., 1994). Greenberg and Ausubel (1993) additionally isolated a mutant which though expressing an acd phenotype was in fact more susceptible to pathogen. It is thus possible to identify genetically at least two types of cell death, namely those which feed into a pathway culminating in establishment of a disease resistant state, and those which do not.

The Isdl mutant is exceptional. In conditions permissive for wild type plant growth and in the absence of detectable microscopic lesions, the Isdl mutant is hyper-responsive to challenge by a variety of stimuli including pathogens and low doses of chemicals which trigger the onset of SAR (Dietrich et al, 1994). Mutant Isdl plants are resistant to otherwise virulent pathogens in conditions where no spontaneous cell death lesions form. Following initiation of cell death in a local spot on a leaf, lesions propagate throughout the leaf and kill it 2-4 days later. Propagation of locally initiated cell death is confined to the inoculated leaf. Thus, LSDl functions to negatively regulate both the initial response to pathogens and the subsequent spread of cell death. Superoxide is a necessary and sufficient trigger for this phenotype, and superoxide production precedes onset of cell death by 8-16 hours following initiation by three different triggers (Jabs et al., 1996). Therefore, the LSDl gene responds to either superoxide or to a signal derived from it to down regulate or dampen the cell death response, resulting in the typical locally bounded HR. The invention herein includes the LSDl gene, which encodes the first member of a new subclass of zinc- finger proteins in Arabidopsis.

It is therefore an object of the invention to provide a novel DNA molecule, LSDl, isolated from Arabidopsis which works to protect plant cells in response to pathogens, and DNA molecules encoding LSDl related proteins LOLl and LOL2. It is a further object of the invention to provide the protein encoded by LSDl, and transgenic plants comprising LSDl. Knowledge of the structure of the LSDl gene allows accurate creation of particular mutants (e.g., deletion and point mutations), for example, mutants having a dominant negative phenotype, analogous to the mutants of Drosophila PANNIER gene (Ramain et al., 1993), using methods known in the art. This in turn allows engineering of transgenic crop plants which do not suffer cell death, but are still resistant to infection. In addition, expression of the dominant negative LSDl protein may be refined so that it is expressed very quickly after infection.

The LSDl protein is also a useful target for herbicide development. Transgenic plants may be made in which LSDl mutant genes are expressed which are resistant to herbicidal compounds which normally result in cell death in combination with the wild-type LSDl. Mutants of the LSDl gene are tested in a Isdl background to determine if the mutant has a normal or novel function, and in a wild-type background to determine the existence of a dominant negative function.

Other objects and advantages will be more fully apparent from the following disclosure and appended claims.

SUMMARY OF THE INVENTION

The invention herein comprises the DNA molecule of the wild-type LSDl, which functions to monitor levels of a superoxide-dependent signal and negatively regulates a plant cell death pathway. The predicted LSDl protein contains three zinc-finger domains, defined by CxxCxRxxLMYxxGASxVxCxxC (SEQ ID NO.54). The invention further comprises a protein encoded by LSDl, and transgenic plants comprising LSDl, and mutations thereof.

In particular, the preferred embodiments of the invention herein include the following: an isolated DNA molecule, encoding the LSDl polypeptide sequence, selected from the group consisting of SEQ ID NOS: 13- 15; the LSDl DNA molecule having the nucleotide sequence as set forth in SEQ ID NO: 13; the DNA molecule that is cDNA; the DNA molecule which is genomic DNA; a chimeric construction comprising a promoter sequence and the LSDl DNA molecule or portions of the LSDl DNA molecule; a recombinant plant transformed with the LSDl DNA molecule; a transformed plant comprising a DNA molecule encoding a protein as set out in SEQ ID NO: 16 or SEQ ID NO:17; an isolated protein molecule comprising the protein set out in SEQ ID NO:16 or SEQ ID NO:17; a transformation vector comprising a LSDl DNA molecule as set forth herein; an isolated DNA molecule encoding the zinc finger consensus sequence shown in SEQ ID NOS: 1-3; and anything that hybridizes to the LSDl DNA molecule set forth herein under hybridization conditions as defined herein. Other objects and features of the inventions will be more fully apparent from the following disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-C show the physical delineation of the Isdl mutation. Figure 1A shows YAC clones at Isdl. The arrowheads imply the YAC clone extending in the direction given, solid vertical black bars denote YAC ends used to isolate genomic phage clones and subsequently converted into CAPS RFLP markers as described (refer to Figure 2 for their map position and to Tables 1 and 2, Examples II and III, for their definition). Figure IB shows the three BAC clones which contained the CAPS markers listed above BAC1G5. The arrowheads imply extension of the BAC clone in the direction shown. The scale in Figures 1A and IB are the same. Figure 1C shows the genomic phage clones positioned under an expansion of three of the BACs. The diamond-filled bar represents the 8A6-1.3 clone, which co-segregated with Isdl, used to isolate these phage. The Isdl deletion is noted at the bottom. Figure 2 is a genetic linkage map of the Isdl region. The vertical line at the left represents the section of Arabidopsis chromosome 4 between CH42 and B9-1.8 (telomeric toward bottom). CAPS-based RFLP markers discussed in the text intersect the chromosome, and their relative recombination frequencies in the F mapping population are placed in the center. The number of meioses identified among the total number of F 's scored is at the right. The arrowhead denotes the co-segregating marker.

Figures 3A-C show molecular fine mapping of the Isdl locus. Figures 3A and 3B show genomic DNA blots demonstrating the presence of a 0.8 kb deletion om the Isdl mutant. Genomic DNA (5 g) from wild type Ws-0 or Isdl was digested with (for each pair of lanes from left to right) EcoRI, Hindlll, a double digest of Hindlll and Xbal. or Kpnl. In Figure 3A, the blot was probed with the 0.8 kb EcoRI-Xbal. In Figure 3B, a duplicate blot was probed with the 4.5 kb Pstl-Xhol fragment. The probes are depicted in Figure 3C, and were isolated from phage clones depicted in Figure lC. Molecular weight markers are the Gibco-BRl 1 kb ladder. Figure 3C shows the restriction map in and around the Isdlgene. The extent of the deletion of this locus is shown as are the extent of the hybridization of the various restriction fragments with Isdl cDNAs. Genomic restriction fragments used in complementation experiments are underlined. The asterisk refers to an Xhol site derived from the phage lambda cloning junction.

Figure 4 shows that the Isdl mutation is an mRNA null allele. RNA blots (1 g of polyA+ RNA) from leaf tissue of 5 week old plants kept in short days (permissive for Isdl growth) 3 days after spraying with either IN A (0.3 mg/ml powder containing 25% active ingredient, or 4 mM), or wettable powder control. Spreading Isdl lesions had just started to appear at the time of leaf harvest. Probes were purified inserts from the LSDl cDNA as represented by EST 82D11T7 (top), a PR-1 cDNA (Uknes et al, 1993b), and an actin cDNA. The blot was probed successively in the order displayed.

Figure 5 shows the zinc finger domains (SEQ ID NOS:l-3) of the predicted LSDl protein and the alignment of the three zinc finger domains. The numbers at the left and right refer to amino acid residue position in the deduced LSDl protein. Vertical lines indicate conservation in pairwise comparison, and a colon indicates conservative substitution. A consensus sequence is listed below, with conservative substitutions noted in the second line of consensus where "+" is basic, plus charged; and "@" is amide, polar, uncharged, hydrophilic. Figure 6 shows how the carboxyl portion of the deduced LSDl protein is related to known DNA-binding and transcription factors. Vertical lines indicate conservation in pairwise comparison, and a colon indicates conservative substitution. Figure 6A shows homology of a slightly longer portion of the deduced LSDl protein with mammalian insulin receptor substrate proteins. The LSDl translation product (SEQ ID NO:4) is shown on the top, aligned with the mouse insulin receptor substrate (SEQ ID NO:5). In this region, all mammalian insulin receptor substrates are identical. Figure 6B shows the homology of LSDl, on each top line, with four known transcription factors. The LSDl translation product (SEQ ID NO:6) is shown on top, and below it are the related domains from a human early growth response (EGR) Zn-fmger protein (SEQ ID NO: 7, a human TGF —early induced Zn-fmger protein (SEQ ID NO: 8), a Xenopus laevis H-L-H transcription factor (SEQ ID NO:9), and the human ELK-1 protein (SEQ ID NO: 10). Figure 6C shows the homology of a LSDl transcription product (SEQ ID NO: 11) with a putative maize transcription initiator binding protein (SEQ ID NO: 12). GenBank accession numbers of each protein are listed at the right.

Figure 7 shows the consensus sequence of the zinc finger domains (SEQ ID NOS:63-65, respectively) of LSDl (A), LOLl (B) and LOL2 (C).

Figure 8 shows the homologies between the first (A), second (B) and third (C) zinc finger domains of LSDl, LOLl and LOL2

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The present invention provides a genomic DNA sequence (SEQ ID NO: 13) and a cDNA sequence (SEQ ID NOS: 14- 15) or the LSDl gene which is required for the regulation of initial plant response to pathogens, and cDNA proteins deduced (from short form, MG7-SEQ ID NO: 16; from long form, MG, SEQ ID NO: 17). In addition, the invention herein provides functional protein domain sequences involved in regulating genes controlling cell death. Gene expression can be regulated by attaching a promoter to the LSDl gene, which may be either the native promoter or any other promoter.

The invention herein includes the DNA molecule having the nucleotide sequence as set forth in SEQ ID NOS: 13, 14 and 15, encoding either of two LSDl polypeptides, which are preferably the LSDl polypeptides set forth in SEQ ID NOS: 16 and 17. This DNA molecule may be cDNA or genomic. The invention also includes as the open reading frame any chimeric construction comprising a promoter sequence and the DNA molecule of the invention, a recombinant plant transformed with the DNA molecule, and any transformation vector comprising the DNA of the invention. In addition, the DNA sequence of either the full-length SEQ ID NO: 13, or a shortened or otherwise modified version thereof, may be modified to optimize its expression in plants, with codons chosen for production of the same or a similar protein as encoded by the wild type LSDl gene. Other modifications of the LSDl gene that yield a protein having essentially the same properties as the LSDl gene are included within the invention herein.

The invention herein also includes anything that hybridizes to the LSDl DNA (SEQ ID NO: 13) of the invention as discussed above, under hybridization conditions, which are defined as: 7% Na dodecyl sulfate (SDS), 0.5 M sodium phosphate, pH 7.0, 1 mM EDTA at 50C, and wash in 2X SSC buffer, 1% SDS, at 50C (Church and Gilbert, 1984). Proc. Natl. Acad. Sci. USA 81 :1991-1995 (1984)).

The novel LSDl gene of the present invention, it its wild type form or as mutated by selected mutations and genetically engineered derivatives obtained as is known in the art, and proteins encoded thereby, are included in the invention herein, and may be transferred into any plant host using methodology known in the art for purposes of altering the extent and type of plant resistance to pathogens, and to change resistance to particular herbicides. The mutant phenotype of the null Isdl allele suggests that the wild type product is a negative regulator of cell death. In addition, Isdl reacts to both nominally virulent pathogens, and to chemicals which trigger the onset SAR, with an HR-like response. But it is important to note that Isdl expresses wild type timing of R gene driven HR (Dietrich et al, 1994)~it is the subsequent spread of cell death which distinguishes the mutant. Thus, cell autonomous signals required for R gene function are intact in an Isdl null, but the response to cell non-autonomous signals emanating from cells undergoing HR is perturbed. Collectively, these features of the mutant phenotype suggest that LSDl functions to limit both the initiation of defense responses and the subsequent extent of the HR. The fact that an Isdl null is hyper-responsive to signals initiating the defense response and HR-like cell death additionally suggests that these pathways are functionally intact in the wild type cell, but require a threshold level of signal for full activation.

LSDl appears to act as a transcription factor (or as a protein which sequesters a transcription factor). As outlined above, the oxidative burst in an infected cell generates a superoxide-dependent signal up-regulating the HR pathway. This signal overcomes the negative regulatory function of the available LSDl, and drives primary responding cells into the HR pathway. Additionally, the cells undergoing HR amplify the signal, probably via a sustained extracellular oxidative burst, to neighboring cells. The primary signal molecule may be diffusible over short ranges (Levine et al., 1994), could act as an autocrine signal, and could lead to the accumulation of a secondary signal molecule in a steep spatial gradient from the infection site. At a critical point in the signal gradient, a threshold is reached. Above that point the pro-death pathway operates, and below it the pro-death response would be attenuated by LSDl. Such a gradient is formed by SA and SA-conjugates (Enyedi et al., 1992); SA biosynthesis can be induced by hydrogen peroxide (Leon et al., 1995); and sub- effective doses of SA can amplify pathogen-derived signals (Kauss et al., 1992; Kauss and Jeblick, 1995; Mauch-Mani and Slusarenko, 1996). Thus, it could be that an SA gradient dictates LSDl activity.

Constitutive expression levels by LSDl could suffice to protect cells below the critical signal threshold for death induction. The time lag of 12-16 hours observed between superoxide production initiated in Isdl by a variety of triggers and the onset of cell death (Jabs et al., 1996), which could provide sufficient time for up-regulation of LSDl activity before irrevocable commitment to death during wild type responses, so that cell death could spread until sufficient active LSDl accumulates. Alternatively, this time lag could represent a requirement for biosynthesis of pro-death intermediates and LSDl normally could operate by interdicting this pathway. LSDl could positively regulate anti-cell death targets, potentially including genes involved in cell survival, ROI de-toxification, or in degradation of a key intermediate in the pro-death pathway. Alternatively, LSDl could act as a transcriptional repressor directly on genes in the pro-death effector pathway. This scenario differs from the first only in that the set of target genes would be different. The availability of extragenic suppressors of Isdl will aid in identifying LSDl targets (Jabs et al., 1996).

This model also explains the runaway cell death phenotype of the null Isdl mutant. In the absence of LSDl, the threshold normally required before commitment to HR is removed. Thus, minimal up-regulation of the superoxide-dependent signal drives the cell into the HR pathway. Hence the ability of Isdl to respond to virulent pathogens as if resistant derives from a lack of background inhibition of the HR pathway normally operating in the cell. Moreover, extracellular superoxide produced during the oxidative burst initiates the same series of events in cells immediately surrounding the site of initiation, and the cell death propagation indicative of the Isdl phenotype results. Because the null Isdl mutant still requires superoxide for initiation of cell death propagation, it is unlikely that superoxide directly regulates LSDl activity. This further suggests that a superoxide-dependent signal is the autocrine which propagates the response to neighboring cells.

The A. thaliana Isdl mutant phenotype is characterized by enhanced disease resistance, spontaneous formation of lesions in the absence of cell death initiators and failure to limit the extent of cell death. The wildtype LSDl protein therefore negatively regulates a cell death pathway involved in plant defense responses.

The LSDl gene encodes a protein containing a novel zinc finger protein, which is included in the invention herein and is defined by its three consensus zinc fingers: CxxCRxxLMYxxGASxRxVxCxxC (SEQ ID NO:52). These three zinc finger domains have not been observed before in the range of zinc finger proteins. As shown in Dietrich et al., 1997, the LSDl gene is a key negative regulator of hypersensitive cell death in plants. We sought other versions of this consensus zinc finger sequence in other plant proteins.

The data on homologies between the LSDl and LOLl and LOL2 zinc finger domains indicates that LSDl as well as LOLl and LOL2 are members of a novel subclass of zinc finger proteins that are involved in plant cell death pathways. LOLl and LOL2 might function in cell death phenomena leading to hypersensitive response and disease resistance as has been shown for LSDl . The homologues may also be involved in programmed cell death (PCD) pathways occurring in plants. Examples of PCD n plants include lateral root development, tracheary element differentiation, and abscission of leafs. Preliminary expression studies suggest that LOL2 is expressed in flowers and siliques. Thus a role for LOL2 in PCD pathways leading to petal senescence, anther dihiscence or PCD of nucellar cells is not unlikely. It is also possible that LOL2 is involved in the hypersensitive response and disease resistance in flowers, thus protecting seeds and ultimately the following generations from pathogen. Alternatively, LOL2 could be up- regulated during the hypersensitive response. Use of LOLl and LOLl should allow prediction of the protein's function with respect to protection from programmed cell death.

The consensus sequences defined by the LSDl, LOLl and 1OL2 zinc finger domains

(Figures 7-8) are thus far unique in the available deduced protein databases. Because zinc fmger domains of this type bind DNA and thereby regulate gene activation, it is highly likely that the consensus zinc finger domain defined here is required for proper regulation of related sets of genes. Furthermore, because zinc finger DNA binding domains of related sequence generally control related cellular processes, the new consensus defined here should also do so. Because LSDl is known to negatively regulate cell death induced by pathogens, it is highly likely that LOLl and LOL2 also control plant cell death. Thus, the utility of this portion of the invention lies in production of transgenic plants which have mutated versions of the LOLl or LOLl genes or which overexpress these proteins. Such plants will likely be more resistant to pathogen attack, if, in the first case, the LOL genes function to repress defense response (as does LSDl). Alternatively, if the LOL genes function to activate defense mechanisms, then overexpression will lead to a more effective pathogen response. Because zinc fmger proteins featuring other non-LSD 1 type DNA binding domains function to either activate or repress gene transcription, we cannot distinguish at present between these two models.

The invention also includes plant proteins, and the genes which encode them, which directly interact with LSDl protein. Gene regulation in response to pathogen attack is controlled, in part, by the repression and activation of genes. The LSDl, LOLl and LOL2 proteins encode a novel branch of the zinc-finger DNA binding protein superfamily with roles in controlling plant cell death. As such, they are expected to interact with other proteins. Paradigms of gene activation currently demonstrate that DNA binding proteins can have two classes of "partners". The first class sequesters the DNA binding protein in the cell's cytosol. These partner proteins hold the DNA binding protein out of the nucleus until the correct cellular stimulus is received. This stimulus disrupts the physical interaction, and the DNA binding protein is free to migrate into the nucleus and activate or repress transcription. The second class of protein which interacts with DNA binding protein is made up of proteins which are partners having the role of "enhancing" the gene activation or repression encoded by the DNA binding protein. These partners are termed "co- activators" or "co-repressors" and they may or may not have intrinsic DNA binding activity. We have identified several genes whose protein products interact physically with the LSDl protein using a common assay, called a "yeast two-hybrid interaction trap" to detect such interactions genetically (Fields and Sternglanz, 1994; Finley and Brent, 1996). Because the inactivation of LSDl by mutation leads to enhanced disease resistance, the LSDl partner proteins represent novel targets for engineering plants with enhanced resistance to pathogens. Thus, this invention includes all proteins which interact with the cell death regulator LSDl (SEQ ID NOS: 66-91 (includes sequential pairs of nucleic acids and corresponding amino acid sequences).

The features of the present invention will be more clearly understood by reference to the following examples, which are not to be construed as limiting the invention.

EXAMPLES Example I Care and maintenance of plants Plants were grown in a chamber at 9 hours light per day, 22°C day temperature and

20°C night temperature essentially as described (Dietrich et al., 1994).

Example II Isolation of DNA and RNA, probe preparation, cloning

Small scale genomic DNA preps were made from single leaves (~lcm long rosette leaves) (Lukowitz et al., 1996). The DNA pellet was re-suspended in 50 ml of Tris/EDTA (TE) and 1 ml was used in a 20 ml polymerase chain reaction (PCR). Large scale genomic DNA preps were done based on the protocol of (Rogers and Bendich, 1985), modified such that concentration in the 2X hexadecyltrimethylammonium bromide (CTAB)(Sigma, St. Louis, MO) buffer was increased to 3% and the precipitated DNA was resuspended in Tris/EDTA/sodium chloride (TEN) buffer and digested with 100 mg/ml, followed by two extractions with chloroform/iso-amyl alcohol and a final precipitation.

RNA was isolated by grinding fresh tissue in liquid nitrogen to a fine powder and extraction in 1 ml of Trizol reagent (Gibco-BRL, Gaithersburg, MD) per 100 mg tissue fresh weight. RNA was isolated according to the manufacturer's protocol. PolyA+ RNA was isolated using DynaBeads (Dynal, Oslo, Norway). RNA blots were formaldehyde agarose gels and contained either 15 mg total RNA or 1 mg polyA+ RNA. HyBond filters for DNA or RNA blots (Amersham, Little Chalfort, United Kingdom) were hybridized in 6xSSC, 5X Denhardt's solution, 0.1% SDS and 100 mg/ml sheared Herring sperm DNA at 65°C. Washes were in 0.2X SSC, 0.1 % SDS at the same temperature. RNA blots were stripped for re-hybridization in 5 mM TRIS/2mM EDTA, (pH8.0), 0.1X Denhardt's solution for 1 hour at 65°C. Example III Isolation of new CAPS markers and genetic mapping of Isdl

After establishing linkage to the agamous {AG) co-dominant amplified polymorphic sequences (CAPS) marker (Konieczny and Ausubel, 1993), we subcloned and end- sequenced a 1.6 kb Hindlll fragment from the RFLP cosmid marker g3883 (position 73.5 on the Arabidopsis RI map; Lister and Dean, 1993; see http://nasc.nott.ac.uk/RI_data/top_frame.html), and primers designed based on this sequence. This primer set amplified a rapid amplified polymorphic DNA (RAPD) marker (size difference in Ws-0 versus Col-0 without restriction digestion), and map data generated using this primer allowed us to place Isdl below (telomeric to) it. Probe B9-1.8, isolated as a 1.8 kb Sstl-EcoRI fragment from the JGB9 genomic phage clone (RI map position -75; gift of Dr. George Coupland, Cambridge Laboratories, Norwich U.K.) was converted into a CAPS marker. Mapping of this polymorphism placed Isdl above (centromeric to) it (Fig. 2). Recombinants were identified as homozygous for one of these CAPS markers, and heterozygous for the other using DNA from F2 individuals. F3 progeny from these recombinants were then scored as either homozygous Isdl, segregating Isdl, or homozygous wild-type for lesion spread. All CAPS markers we developed are described in Table 1 (below).

Table 1. New PCR based RFLP (CAPS markers derived during cloning of Isdl

Marker Enzyme PCR prod. Col-0 Ws-0 ch42 Clal 1.4 kb 750 bp 1.4 kb

650 g3883-1.6 none 1.4 kb 0.7 kb

(uncut) (uncut) gl3838-1.4 Hinfl 1.4 kb 450 bp 450 bp

330 330

280 280

200 160

B9-1.8 Hinfl 1.8 kb 420 bp 420 bp

260 260

240

180 180

160

140 140

1H1L-1.6 Ddel 1.6 kb 1.0 kb 700 bp 300 bp 300

(doublet?)

5F7R-1.5 NlalV 1.5 kb 1.0 kb 1.2 kb 250 bp 250 bp

200 bp 0B4-1.6 Ddel 1.6 kb 900 bp 700 bp

400 400

220

180 180

8A6-1.3 Taql 1.3 kb 800 bp 800 bp

400 250

220 150

Example IV Map refinement

YACs were defined (Schmidt et al., 1995; Schmidt et al, 1996, http://genom.e- www.stanford.edu/Arabidopsis/JIC-contigs.html), confirmed by DNA blotting to establish a contig and their ends were isolated by vectorette PCR as described (Matallana et al., 1992; Grant et al., 1995). These ends were also used to isolate genomic phage from a Ws-0 genomic library (Fig. 1). Insert fragments of 1-3 kb were cloned into PBS and end sequenced for derivation of primers identifying new CAPS. PCR conditions (DNA Engine MJ Research) for all CAPS primer pairs except 8A6-1.3 and Isdl deletion primers are: 92°C, 3'; 35 cycles of (denature 92°C, 30"; anneal 50°C, 30"; extend 72°C, 2'30"); 72°C, 3'. For 8A6-1.3 and the Isdl deletion primer pairs we used 53°C annealing. Table 2 shows the primer sequences used to identify new CAPS markers.

Table 2. Primer sequences used to identify new CAPS markers used for cloning Isdl ch42 for 5'-cag tgg ate ttt cct cag acg-3' (SEQ ID NO: 18) ch42 rev 5 '-cat ctt ctt ctg caa tct ggg-3' (SEQ ID NO: 19)

g3883-1.6 for 5'-cat cca tea aac aaa etc c-3' (SEQ ID NO:20) g3883-1.6 rev 5'-tgt ttc aga gta gcc aat tc-3' (SEQ ID NO:21)

gl3138-1.4 for 5'-cac gtt agt tag tta gaa gg-3' (SEQ ID NO:22) gl3138-1.4 rev 5'-ctg atg ttc tct aca aat gg-3' (SEQ ID NO:23)

B9-1.8 for 5'-cgt ate cgc att tct tea ctg c-3 ' (SEQ ID NO:24) B9-1.8 rev 5'-cat ctg caa cat ctt ccc cag-3' (SEQ ID NO:25)

1H1L-1.6 for 5'-ttg agt cct tct tgt ctg-3' (SEQ ID NO:26) 1H1L-1.6 rev 5 '-eta gag ctt gaa agt tga tg-3' (SEQ ID NO:27)

F7R-1.5 for 5'-gaa tgg tgt aac caa act c-3' (SEQ ID NO:28) F7R-1.5 rev 5 '-cat ace gta tga tgg aac-3' (SEQ ID NO:29)

0B4L-1.6 for 5'-gaa etc art gta tgg acc-3' (SEQ ID NO:30) 0B4L-1.6 rev 5 '-eta aga tgg gaa tgt tgg-3' (SEQ ID NO:31)

8A6-1.3 for 5'-cca aga aga gaa aac gga ga-3' (SEQ ID NO:32) 8A6-1.3 rev 5'-aac aat agg agg tgc aga gt-3' (SEQ ID NO:33)

Primers to amplify across the Isdl deletion:

Isdl far side: 5'-acc taa caa aaa gaa aag tgt gtg agg-3' (SEQ ID NO:34)

Isdl outside 5'-ata ata aac cct act age tct aac aag-3' (SEQ ID NO:35) Isdl alt. spl. 5' 5'-ctg eta ctt tea tec aaa c-3' (SEQ ID NO:36)

Example V Vector construction for complementation

The Agrobacterium vacuum infiltration procedure was used to generate transgenic plants (Bechtold et al., 1993; Grant et al., 1995). Vectors were derived from pGPTV-Hyg

(Becker et al., 1992) as follows: pSGCGF was made by restricting pGPTV-Hyg with

Hindlll and Sad and replacing this fragment with a Hindlll-Sacl fragment containing the polylinker from pIC20H (GenBank accession L08912; provided by Steve Goff, Novartis,

Research Triangle Park, N.C). Either the 7kb Xhol or 4.5 kb Pstl-Xhol genomic fragments were cloned into this, the former into the unique vector Sail site, the latter as a Sacl-Sall fragment derived from an intermediate cloning step into pBS as a Pstl-Xhol fragment. The pHyg35S vector was made by cloning a four enhancer-containing 35S promoter fragment as a Hindlll-Xbal fragment into pGPTV-Hyg (provided by Dr. Douglas C. Boyes, Univ. of

North Carolina, Chapel Hill). The EST 82D11 cDNA sequence was isolated as a Sall-Xbal fragment from pZLl (Newman et al., 1994) and cloned into Xhol-Xbal digested pHyg35S.

Example VI Cloning

The genomic Ws-0 library in 1GEM11 was a gift of Dr. Kenneth A. Feldmann (Univ. of Arizona). The cDNA library is an oligo-dT primed library prepared from polyA+ Col-0 mRNA from leaves cloned into IZAPII (Stratagene, La Jolla, CA) according to the manufacturer's instructions (gift of Dr. Douglas C. Boyes and Dr. Murray R. Grant). Example VII LSDl sequences

The sequences of the LSDl cDNA (SEQ ID NOS: 14 and 15) and the 4.5 kb LSDl Xhol-Pstl genomic fragment (SEQ ID NO:13; the longest 5'LSDl cDNA starts at base 1892 of this sequence) are deposited in GenBank as accessions U 87833 and U 87834, respectively. Endpoints of the various LSDl cDNAs isolated are shown in Table3A and examples are provided by SEQ ID NO: 14 (short form from cDNA MG7 as shown in Table 3) and SEQ ID NO: 15 (long form, from cDNA MG8). The polypeptides deduced from these are shown in Fig. 11-12, respectively. Table 3B shows the sizes of each intron deduced from comparison of the sequence shown in SEQ ID NO: 13.

Table 3. Sequence characteristics of the LSDl gene

Endpoints of independent LSDl cDNAs

cDNA 5' end point Alternate splice 3' end point

MG7(2) C l short A 1021

EST 82D1 1 A 27 short T 1031

MG4 C59 short A 1188*

MG10 C 59 short G 1225

MG5 G 67 short A 1205

MG2 (4) G 90 short A 1106

MG8 (2) G 98 long A 1082

MG16 (2) C 103 short A 1066

MG11 C 117 long G 1225

Numbers in parentheses refer to the number of isolates of the same clone. Nucleotide numbers at the 5' and 3' ends refer to nucleotide positions from SEQ ID NO: 13. An A at the 3' endpoint can be either an A in the genomic sequence or the first A of the polyA tail. The endpoint marked with an * had no polyA tail.

Intron sizes intron # size in nucleotides

1 88

2 (short splice) 68

2 (long splice) 129

3 89

4 489

5 100

6 92 7 87

Intron splice junction positions are located at bses 198-199, 260-261, 447-448, 552-553, 692-693, 764-765, and 836-837 in SEQ ID NO: 13.

Example VIII Genetic and physical mapping of Isdl

The Isdl mutation segregates as a monogenic recessive (Dietrich et al., 1994). F2 progeny of a cross between Isdl (Ws-0 background) and Col-0 (LSDl) were analyzed using the co-dominant amplified polymorphic sequences (CAPS) mapping procedure (Konieczny and Ausubel, 1993) to first establish linkage to the AG marker on chromosome 4. The closely linked gl3838 probe (3 recombinants in 1632 meioses) was used to identify YAC (yeast artificial chromosome) clones (Schmidt et al., 1995; Schmidt et al., 1996). We constructed a physical contig of these YACs, shown in figure 1A. We used labeled YAC ends CIC1H1L, yUP5F7R and EG20B4L to isolate genomic phage clones, subcloned fragments form each of these, end-sequenced the subclones, derived primer sequences and developed new CAPS markers (see Tables 1 and 2). The CAPS markers 1H1L-1.6 and 5F7R-1.5 mapped closest to Isdl (1 and 3 recombinants, respectively from 2054 meioses); see Tables 1 and 2 for new CAPS markers). We hybridized these two CAPS markers to filters containing bacterial artificial chromosome (BAC) clone arrays (Choi et al, 1995, distributed by the Arabidopsis Biological Resource Center, Ohio St. Univ.), and isolated the five BAC clones depicted in Figure 2B. Because 5F7R-1.5 and 1H1L-1.6 genetically flank Isdl (Figure IB), BAC clone 1G5 should contain the gene.

As 1 G5 was the only BAC clone to physically span the relevant genetic region, we connected BACs 6H3 and 8A6 by walking in a genomic phage library. We defined a 5kb Hindlll fragment from BAC 8A6 which hybridized only to itself and BAC 1G5. When used as a probe on filters containing restriction digests of the relevant BAC clones, this fragment hybridized to a 1.3 kb EcoRI fragment which also was present only on BACs 8A6 and 1G5. This 8A6-1.3 clone, (small box in Figure 1C) was used to isolate three phage clones, two of which are depicted in Figure 1C. Labeled inserts from each detected BAC clones 1G5, 6H3 and 8A6, thus providing multiple redundancy of genomic cloned DNA encompassing Isdl. We also converted 8A6-1.3 into a CAPs marker, and found that it co- segregated with Isdl in 2054 meioses. This map resolution of approximately 0.05 map units, suggested that Isdl was within 5-15 kb (at 100-300 kb per map unit; Schmidt et al., 1995; Schmidt et al., 1996) in either direction of 8A6-1.3.

We probed genomic Arabidopsis DNA blots of digested wild type Ws-0 and Isdl to confirm co-linearity of the cloned and genomic DNA immediately surrounding 8A6-1.3. We noted that a variety of fragments detected a genomic DNA rearrangement in Isdl relative to wild type Ws-0 (data not shown). This rearrangement corresponded to a loss of restriction sites and a deletion as noted in Figures 1C and 3C. The Isdl mutant comes from an Agrobacterium mutagenized population of Arabidopsis, and it is known that the transformation procedure can generate non-T-DNA associated mutations (Feldmann, 1991). We subcloned and sequenced various wild type genomic DNA fragments at this position, and compared their sequences to several databases, including the Arabidopsis EST database (Rounsley et al., 1996, http://www.tigr.org/tdb/at/at.html). One EST clone (EST 82D11T7; GenBank accession T45220) exhibited blocks of identity to our genomic DNA sequence, suggesting the presence of introns in the latter. Because the gene encoding this EST is largely deleted in Isdl, it became a candidate LSDl gene.

Example IX Complementation of Isdl

To confirm that the genomic deletion encompasses LSDl, we constructed subclones from the genomic phage as shown in Figure 3C for complementation into the T-DNA binary vector pSGCGF. Because the typical method for generation of transgenic Arabidopsis, vacuum infiltration of Agrobacterium carrying binary T-DNA vectors, triggers the propagative cell death indicative of the Isdl phenotype, we devised an alternate complementation strategy. We transformed FI plants of Isdl x Col-0, and plated surface- sterilized seeds of the next (F2) generation onto media containing hygromycin as a selective antibiotic. We then identified hygromycin resistant transformants which were homozygous for Ws-0 alleles at 5F7R-1.5, 1H1L-1.6, and 8A6-1.3, and thus were Isdl/lsdl homozygous mutants. These individuals contained both mutant and wild type alleles for the CAPS marker which spans the Isdl deletion, because a wild type allele is present on the transgene. These transgenic plants were treated with droplets of 2,6-dichloroisonicotinic acid (IN A); 0.3 mg/ml wettable powder containing 25% active ingredient, Uknes et al., 1993a) a potent inducer of SAR and the Isdl phenotype (Dietrich et al., 1994). If the mutation were complemented, then INA treatment should not lead to spreading cell death. Table 4 shows that transgenic plants carrying either the 7kb Xhol fragment or the 4.5 kb Pstl-Xhol (Figure 3C) all survived this treatment, and are thus complemented for the Isdl mutation. Selfed F3 progeny from a complemented F2 individual carrying either the 4.5 kb Xhol-Pstl fragment or the 7 kb Xhol fragment were also analyzed. All F3 progeny which inherited the transgene were complemented (Table 4), while all of their non-transgenic sibs still exhibited the Isdl phenotype (data not shown). In no case did wild type control plants exhibit spreading cell death after INA application.

Table 4. Complementation of the Isdl mutant

# of plants complemented/# transgenics tested from: Construct Independent F2s Transgenic F3 progeny 7 kb Xhol 1/1^A 20/20^B

3/3° 21/21° kb Pstl-Xhol 2/2^A 14/14^B

35S-cDNA 1/1^Λ 19/19^B

^■x Selected for hygromycin resistance and screened for homozygous Ws-0 alleles through the

Isdl genetic interval as described, except where noted in . Individual F s were both drop tested with INA and shifted to LD conditions (Dietrich et al., 1994).

B

Selfed progeny from a complemented F individual (homozygous Ws-0 alleles through the Isdl interval) were screened by PCR at F for presence of the hygromycin resistance gene and then INA tested, c F parents were identified as hygromycin resistant and heterozygous through the Isdl interval, then selfed and re-screened as hygromycin resistant and homozygous Ws-0 through the Isdl interval at F before INA testing.

Due to low numbers of independent F2 transformants which were homozygous mutant through the Isdl interval from the original transformation, we also isolated F2 transformants carrying the 7 kb Xhol fragment which were originally identified as heterozygote at the CAPS markers flanking Isdl. Selfed progeny from these should segregate both the transgene and the Isdl mutation. Among these progeny, we identified F3 individuals which were homozygous Ws-0 through the Isdl interval and carried the transgene. As shown in Table 4, these also were all complemented for protection against INA-induced spreading cell death. We conclude that the 4.5kb Pstl-Xhol fragment carries the Isdl gene and sufficient cis control elements to ensure its expression.

All transgenic plants complemented for the INA-induced Isdl mutant phenotype were also complemented for initiation of spreading cell death after transfer to non- permissive long day conditions as well (Dietrich et al., 1994; not shown). Thus, the complementing DNA corrects the mutant phenotype induced by two independent stimuli.

Example X Identification of alternately spliced LSDl transcripts We sequenced all of the complementing 4.5 kb Pstl-Xhol genomic DNA fragment

(SEQ ID NO: 13), eight independent cDNAs (Example VII) and completed the sequence of the full 82D11T7 EST sequence. Among the cDNAs, we identified two classes expressing open reading frames of either 184 or 189 amino acids (SEQ ID NO: 16 and 17). An alternate splice which adds 61bp to the 5' region of some cDNAs also provides an alternate translation start, hence, the extra five amino acids in SEQ ID NO: 17. The sequences of both cDNA classes matched exactly the genomic sequence except at the positions of 7 introns (see Table 3). Nucleotide 1 of the longest cDNA is at position 1892 in the 4.5 kb Pstl-Xhol genomic sequence (SEQ ID NO: 13 ). Thus, 1891 nucleotides of promoter are sufficient for appropriate expression in complementation of the Isdl mutation. The cDNA 5' ends are clustered (Table 3), suggesting that the longest could be full length. We also complemented the Isdl mutation by transformation of the full insert from EST clone 82D11T7 expressed from the strong and constitutive cauliflower mosaic virus 35S promoter (see Table 3) proving that this cDNA contains the entire LSDl coding region. The 3' ends of these cDNAs are very heterogeneous, suggesting the presence of multiple polyadenylation addition signals (Table 3). No other significant open reading frames were observed in the 4.5 kb Pstl-Xhol genomic clone.

When either the EST 82D11T7 clone, or a 0.8 kb EcoRI-Xbal genomic fragment covering the Isdl deletion were used as probes on RNA blots, a rare mRNA of approximately 1.2 kb was detected in leaf tissue of wild type Ws-0 plants (Figure 3). This length is consistent with the size of the longest cDNA, supporting the conclusion that we have identified a nearly full-length transcript. Importantly, this mRNA was completely lacking in mRNA prepared from Isdl leaves, furthering the argument that it encodes LSDl . The finding that Isdl is an mRNA allele was corroborated by sequencing across the genomic deletion in the mutant (Figure 3). The 5' border of the deletion is an A at nucleotide 55 and the 3' boundary is in the fourth intron (data not shown). It is noteworthy that expression of this candidate mRNA was unaffected by application of INA (Figure 4, top). The expected high level of INA-induced PR-1 mRNA accumulation in leaves of both wild type and Isdl (Figure 4, middle) served as a control in this experiment for efficacy of INA treatment.

The Isdl phenotype can be observed in all cell types examined after initiation of lesion formation (Dietrich et al., 1994). RNA blot analysis of seedlings, stems, leaves and flowers demonstrated that the LSDl gene is expressed constitutively in each of these Arabidopsis tissues (data not shown). Thus, the requirement for LSDl activity in these tissues is consistent with the gene's expression pattern.

Example XI The LSDl mRNA encodes a novel zinc-finger domain

We searched a variety of databases with the predicted translation product of the LSDl cDNA sequence. Several striking features emerged. First, there are three zinc-finger domains, depicted in Figure 5 (SEQ ID NOS: 1-3), which share remarkable homology with one another. These are C-x-x-C, or type IV, zinc-fingers, according to the classification of Sanchez-Garia and Rabbitts (1994), and they share most homology with plant relatives of the GATA-1 transcription factor (Evans and Felsenfeld, 1989; Omichinski et al., 1993). The plant members of this sub-family described to date include the CO gene, which controls transition to flowering (Putterill et al., 1995), a set of related DNA binding proteins (Yanagisawa, 1995; De Paolis et al., 1996) and a gene whose transcription is salt stress- induced (Lippuner et al., 1996). None of these proteins shares with LSDl the consensus homology within the Zn-fingers. The second homology domain is derived from the carboxy 1 portion of LSDl, from residues 129 to 180 (Figure 6-SEQ ID NO:4). This region of LSDl exhibits homology to three broad classes of regulatory proteins. First, all mammalian insulin receptor substrates; second, a set of animal transcription factors; and third, a maize transcription initiator binding protein.

The conceptual LSDl translation product also identified two additional Arabidopsis ESTs via their predicted amino acid homology. Importantly, each has at least one C-x-x-C Zn-fmger and most of the associated consensus residues found in the LSDl internal homologies. They are ESTs 172A7T7 (GenBank R6552)(SEQ ID NO: 58 and 132J21T7 (GenBank T45809). Thus, it is probable that LSDl is the first member of a widely distributed Zn-fmger sub-family in plants, defined by the internal homology within each zinc-finger. The other amino acids in the consensus section are not known to be found in any other zinc finger proteins.

Example XII Identification of expressed target sequence tags (EST) and cDNAs containing LSDl -type zinc finger domains As discussed in the text prior to the Examples, the predicted amino acid sequence of the LSDl zinc fingers was used to search the GenBank database (NCBI). Two Arabidopsis thaliana ESTs (EST132J21T7 and EST 172A7T7) were identified, each of which contains at least two zinc finger domains and most of the associated consensus residues found in the LSDl internal homologies (Dietrich, 1997). These ESTs were ordered from Ohio State University Arabidopsis Biological Resource Stock Center and resequenced. Sequences were analyzed with the Genetics Computer Group programs (Devereaux et al.,1994). A specific probe isolated from EST172A7T7 was subsequently used for screening of cDNA and genomic libraries. The bacterial strain carrying EST132J21T7, however, was not viable. Therefore, degenerated primers were designed based on the EST132J21T7 sequence. Genomic Arabidopsis thaliana Ws-0 DNA was used in the PCR reaction and gave rise to a specific PCR product of approximately 400 bp. This fragment was subcloned via the TA Cloning Kit (Invitrogen, Carlsbad, CA) into pBluescript KS(+). Two new genes were identified as described here. Their predicted protein products are highly related to that of LSDl indicating an involvement in the control of cell death in plants

Example XIII LOLl cDNA

Poly A + RNA isolated from uninduced and P. syringae DC3000 induced Arabidopsis thaliana Col-0 leaf tissue was reverse transcribed. The resulting cDNA population was subcloned unidirectionally into the EcoRI/Xhol - sites of a lambda-Zap II vector using the cDNA-synthesis Kit (Stratagene, La Jolla, CA) according to the

6 manufacturer's directions. The titer of this MG-library was calculated as 2.5 x 10 pfu

5 Approximately 8x10 pfu of the amplified MG-library were subsequently screened with α

P dCTP labeled probes (Stratagene 'Prime it' Kit) specific for EST132J21T7 or

EST172A7T7. With the probe specific for EST132J21T7, four cDNA clones were identified and subcloned via the Stratagene excision system. One clone contained an insert of less than 100 bp in length and was not further analyzed. The three remaining clones were sequenced by standard protocol (primers: M13F, M13R, PE6, and PE7); for primer sequences refer to Table 5, below). Clones 2 and 3 contained identical open reading frames (ORFs) and were homologous to EST132J21T7 and to another identical and overlapping EST clone, EST119C9T7. The fourth clone consisted of a chimeric cDNA of approximately 1500 bp, with approximately 400 bp similarity to EST132J21T7, EST119C9T7, and clones 2 and 3. It was also not analyzed further.

Table 5. Primers and primer sequences used

Primer Primer Sequence SEQ ID NO: M13F 5'-GTAAAACGACGGCCATG-3' 37

M13R 5'-GGAAACAGCTATGACCATG-3' 38

PE6 5'-TTCATGGCAATGGTGTGACCCC-3' 39

PE7 5 ' - CTG CCG GAT TCT TGA TCG A AG A -3 ' 40

PE8 5'-AGAGGAAGGTCCGCCTCCGG-3' 41 PE9 5'-CTCTGCTCTCCTGAGACTGCTT-3' 42

PE13 5'-CATCATAATGTCTCCTTTTGAGAC-3' 43

PE15 5'-GCCATCCATTATTCATCGCCT-3' 44

PE23 5'- GAG GAG GAA GAA CTG CAG ATT CC -3' 45

PE30 5'- GTG CTC CAT GTC CAA ATC ATA C -3 ' 46

Clone 2, with an insert length of 908 bp represents a full length cDNA clone, as determined by the presence of an open reading frame flanked by untranslated sequences, and was renamed LOLl (Z,sd one Λke)(SEQ ID NO:47). We confirmed that the LOLl cDNA and EST132J21T7 are encoded by the same gene using genomic DNA (Southern) blot analysis (data not shown). The LOLl protein of 154 amino acids (SEQ ID NO:48) contains three zinc fmger domains of the LSDl -type (SEQ ID NOS:49-51). The consensus sequence of the LOLl zinc finger domains is defined by CxxCxxLLMYxxGAxSxCxxC (SEQ ID NO:53).

Example XIV LOL2 cDNA By screening the MG-cDNA-library, no clones homologous to EST172A7T7 could be obtained. Therefore, the AB-cDNA-library (derived from RNA isolated from different tissues of sterile grown plants, available at the European .Arabidopsis Stock Center,

32

Cologne, Germany) was screened with α P dCTP labeled probe specific for EST172A7T7. Six homologous cDNA clones were obtained and subcloned into the Smal site of pBluescript KS(+). Restriction analysis indicated that the inserts were encoded by the same gene. Only the longest insert was sequenced following standard protocol (primers used: M13F, M13R, PE8 and PE9: for primer sequences, refer to Table 5. We demonstrated that this insert contained an ORF of 500 bp homologous to EST172A7T7. This non-full length cDNA was designated LOLl (SEQ ID NO:54). The deduced protein (SEQ ID NO:55) consisting of two LSDl -type zinc fmger domains extending from bases 130-195 and 244- 309 of SEQ ID NO:54 (SEQ ID NOS:56-57, respectively). Comparison to EST172A7T7 shows that the EST (SEQ ID NO:58) contains a 124 bp insertion (bases 386-509 after the second zinc finger of SEQ ID NO:58), leading to a different C-terminal. Comparison of these two partial cDNA sequences with the genomic LOLl sequence (see below) demonstrates that they are alternate splice forms from the same gene encoding two related proteins. This conclusion is strengthened by the fact that the LOLl cDNA and EST172A7T7 hybridize to the same genomic DNA fragment and therefore are encoded by the same gene (data not shown). Thus, sequence analysis of genomic LOLl clones shows that the non-identical C-termini of LOL2 and EST172A7T7 are due to alternative splice sites. The genomic sequence of LOLl (SEQ ID NO:59, has a putative TATA-box sequence and polyadenylation signal (bases 922-930 and 2539-2544), and the exon borders of an alternative splice site (bases 2256-2382). The derived amino acid sequence extends from bases 1231-2462.

Example XV Isolation of genomic LOL2 sequences from an Arabidopsis thaliana Col-0 library

5

8 x 10 genomic lambda clones (lambda GEM11, European Arabidopsis Stock

32

Center) were screen with a α P dCTP labeled probe specific for EST172A7T7. Nine clones homologous to LOLl EST172A7T7 could be identified. Restriction analysis demonstrated that the nine clones belonged to five different classes. Inserts ranging from two to five kb in size were isolated and subcloned into either Sad ore BamHI sites of pBluescript KS(+). Sequence information derives from two overlapping clones, sequentially sequenced with primers M13R, PE9, PE13, PE15, PE23 and PE30 (see Table 5).

The genomic LOL2 sequence has a length of 3060 bp. Promoter and 5' untranslated regions consist of approximately 1200 bp. The translation products are encoded by three exons, which are interrupted by two introns of 182 bp and 458 bp length, respectively. The overall length of the coding sequence is 1232 bp. Due to alternative splice sites, two proteins which differ in their C-terminal regions are encoded by the LOLl gene (SEQ ID NO:59). A first protein, of 155 amino acids (SEQ ID NO:60), is identical to the LOLl cDNA and contains two zinc finger domains of the LSDl -type. The other translation product corresponds to EST172A7T7, consists of 147 amino acids, and contains two and a half zinc finger domains (SEQ ID NO:61). The consensus sequence of the two zinc finger domains of LOL2 is CxxCxxLLxYxxGxxxVxCSSC (SEQ ID NO:62).

Example XVI Obtaining interacting genes

The methodology for this Example is known to those skilled in the art and summarized in Fields and Sternglanz, 1994, and Finley and Brent, 1996. The LSDl short or LDS1 long open reading frames were cloned into the "bait vector" pEG202 of the

TM commonly available LexA yeast two-hybrid system (Matchmaker , Clonetech, Palo Alto, CA) to generate plasmids pEG202-L and pEG202-S. These encode fusion proteins of the LexA DNA binding domain and the full length LSDl protein of both long and short isoforms (SEQ ID NOS 14 and 15). Yeast strain EGY48 is transformed with this plasmid, and appropriate controls performed to ascertain the LSDl fusion protein encoded by plasmids pEG202-L and pEG202-S did not intrinsically activate expression of the yeast markers used in this system. A yeast gene expression library was constructed in plasmid pJG4-5 using RNA from Arabidopsis leaves infected with Pseudomonas syringae. This library encodes fusion proteins of expressed Arabidopsis genes and the B42 transcriptional activation domain. The library was transformed en masse into the yeast strain EGY48 carrying either plasmid pEG202-L or -S. From an equivalent of 6 million clones screened, 122 were isolated. The longest insert of a member from each of these classes was sequenced using standard DNA sequencing methods. Because the novel Arabidopsis gene so identified is produced as an active translation fusion in this system, one is immediately able to identify the deduced protein sequence. The most interesting sequences thus defined, and their deduced protein sequences, are set forth herein as SEQ ID NOS: 66-91. The first main class of LSDl -interacting proteins has no database homologues.

These proteins encode putative "sequestration" proteins for LSDl whose function is to inhibit LSDl function until the correct pathogen signal is received. Their utility lies in manipulation of the interaction with LSDl in plant cells such that LSDl is altered in its ability to regulate the response to pathogen. Alternatively, these novel LSDl -interacting proteins may encode new components of the gene regulation machinery working together with LSDl to control transcription in response to pathogen infection. These proteins are valuable because of the knowledge that LSDl is a key regulator of cell death in plants in response to pathogens. Proteins which physically interact with LSDl share in this cellular function.

The second class defines proteins having database homologies to other proteins, strongly suggesting a role in control of gene transcription (e.g., CAAT box binding proteins which are known to bind the common CAAT regulatory unit in DNA preceding nearly all genes encoding eukaryotic mRNA). This finding is completely consistent with the embodiment described above, in which the LSDl partner proteins identify other components of the gene regulatory machinery required for response to pathogens. Manipulation of the expression of, for example, CAAT box binding proteins, will result in altered response to pathogen infection.

While the invention has been described with reference to specific embodiments, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly , all such variations, modifications, and embodiments are to be regarded as being within the spirit and scope of the invention.

LITERATURE CITED

Baker, C. J. and Orlandi, E. W. (1995). Active oxygen in plant pathogenesis. Annu. Rev. Phytopathol. 33, 299-322.

Bechtold, N., Ellis, J. and Pelletier, G. (1993). In planta agrobacterium-mediated gene transfer by infiltration of Arabidopsis thaliana plants. C. R. Acad. Sci., Paris 316, 1194- 1199.

Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992). New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol. Biol. 10, 1 195- 1197.

Bent, A. (1996). Function meets structure in the study of plant disease resistance genes. Plant Cell 8, 1757-1771.

Bolwell, G. P., Butt, V. S., Davies, D. R. and Zimmerlin, A. (1995). The origin of the oxidative burst in plants. Free Rad. Res. 13, 517-532.

Brisson, L. F., Tenhaken, R. and Lamb, C. J. (1994). Function of oxidative cross-linking of cell wall structural proteins in plant disease resistance. Plant Cell 6, 1703-1712.

Chen, Z., Silva, H. and Klessig, D. (1994). Involvement of reactive oxygen species in the induction of systemic acquired resistance by salicylic acid in plants. Science 161, 1883- 1886.

Choi, S., Creelman, R. A., Mullet, J. E. and Wing, R. A. (1995). Construction and characterization of a bacterial artificial chromosome library of Arabidopsis thaliana. Plant Mol. Biol. 13, 124-128.

Church and Gilbert, (1984). Proc. Natl. Acad. Sci. USA 81 :1991-1995.

Cosio, E. G., Frey, T. and Ebel, J. (1992). Identification of a high affinity binding protein for a hepta-β-glucoside phytoalexin elicitor in soybean. Eur. J. Biochem. 104, 1115-1 123.

Crute, I. R. (1985). The genetic bases of relationships between microbial parasites and their hosts. In Mechanisms of Resistance to Plant Diseases., R. S. S. Fraser, eds. (Dordrecht: Kluwer Academic Press), pp. 80-143.

Dangl, J. L. (1995). Piece de Resistance: Novel classes of plant disease resistance genes. Cell 80, 363-366.

Dangl, J. L., Dietrich, R. A. and Richberg, M. H. (1996). Death Don't Have No Mercy: Cell Death Programs in Plant-Microbe Interactions. Plant Cell 8, 1793-1807.

De Paolis, A., Sabatini, S., De Pascalis, L., Costantino, P. and Capone, I. (1996). A rolB regulatory factor belongs to a new class of single zinc fmger plant proteins. Plant J. 70, 215- 224.

Delaney, T., Uknes, S., Vernooij, B., Friedrich, L., Weymann, K., Negrotto, D., Gaffney, T., Gut-Rella, M., Kessman, H., Ward, E. and Ryals, J. (1994). A central role of salicylic acid in plant disease resistance. Science 166, 1247-1250.

Devereaux, J., Haeberli, M.J., and Smithies, O. (1984). Nucl. Acids Res. 11, 387-395.

Dietrich, R. A., Delaney, T. P., Uknes, S. J., Ward, E. J., Ryals, J. A. and Dangl, J. L. (1994). Arabidopsis mutants simulating disease resistance response. Cell 77, 565-578.

Dietrich, R.A., Richberg, M.H., Schmidt, R.., Dean, C, and Dangl, J.L. (1997). Cell 88, 685-694.

Doke, N. (1983). Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components. Physiol. Plant Pathol. 13, 345-357.

Durner, J. and Klessig, D. F. (1995). Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses. Proc. Natl. Acad. Sci., USA P2, 11312-11316.

Enyedi, A. J., Yalpani, N., Silverman, P. and Raskin, I. (1992). Localization, conjugation and function of salicylic acid in tobacco during the hypersensitive reaction to tobacco mosaic virus. Proc. Natl. Acad. Sci., USA 89, 2480-2484.

Evans, T. and Felsenfeld, G. (1989). The erytroid-specific transcription factor EryFl : a new finger protein. Cell 58, 877-885.

Feldmann, K. A. (1991). T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum. Plant J. 7, 71-82.

Fields, S. and Sternglanz, R. (1994). The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 8, 286-293.

Finley, R.L. and Brent, R. (1996). Interaction trap cloning in yeast. In Gene Probes: A Practical Approach, eds. Oxford: Oxford Univ. Press, pp.

Flor, H. H. (1971). Current status of the gene-for-gene concept. Annu. Rev. Phytopathol. 9, 275-296.

Gaffney, T., Friedrich, L., Vemooij, B., Negrotto, D., Nye, G., Uknes, S., Ward, E. and Ryals, J. (1993). Requirement for salicylic acid for the induction of systemic acquired resistance. Science 161, 754-756.

Godiard, L., Grant, M. R., Dietrich, R. A., Kiedrowski, S. and Dangl, J. L. (1994). Perception and response in plant disease resistance. Curr. Opin. Genet, and Dev. 4, 662-671.

Goodman, R. N. and Novacky, A. J. (1994). The hypersensitive response in plants to pathogens. (St. Paul: APS Press).

Gopalan, S., Bauer, D. W., Alfano, J. R., Lonlello, A. O., He, S. Y. and Collmer, A. (1996). Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific hypersensitive cell death. Plant Cell 8, 1095-1105.

Grant, M. R., Godiard, L., Straube, E., Ashfield, T., Lewald, J., Sattler, A., Innes, R. W. and Dangl, J. L. (1995). Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance. Science 169, 843-846.

Greenberg, J. T. and Ausubel, F. M. (1993). Arabidopsis mutants compromised for the control of cellular damage during pathogenesis and aging. Plant J. 4, 327-342.

Greenberg, J. T., Guo, A., Klessig, D. F. and Ausubel, F. M. (1994). Programmed cell death in plants: A pathogen-triggered response activated coordinately with multiple defense functions. Cell 77, 551-564.

Hammond-Kosack, K. E. and Jones, J. D. G. (1996a). Inducible plant defense mechanisms and resistance gene function. Plant Cell 8, in press.

Jabs, T., Dietrich, R. A. and Dangl, J. L. (1996). Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide. Science 173, 1853-1856.

Johal, G. S., Hulbert, S. H. and Briggs, S. P. (1994). Disease lesion mimics of maize: A model for cell death in plants. Bioessays 77, 685-692.

Kauss, H. and Jeblick, W. (1995). Pretreatment of parsley suspension cultures with salicylic acid enhances spontaneous and elicited production of H2O2. Plant Physiol. 108, 1171-1178.

Kauss, H., Theisinger-Hinkel, E., Mindermann, R. and Conrath, U. (1992). Dichloroisonicotinic and salicylic acid, inducers of systemic acquired resistance, enhance fungal elicitor responses in parsley cells. Plant J. 1, 655-660.

Keen, N. T. (1990). Gene-for-gene complementarity in plant-pathogen interactions. Annu. Rev. Genet. 14, 447-463.

Kiyosawa, S. (1970). Inheritance of a particular sensitivity of the rice variety Sekiguchi Asahi, to pathogens and chemicals, and linkage relationships with blast resistance genes. Bull. Nat. Inst. Agric. Sci. (Jpn) Ser D, Physiol. Genet. 27, 61-71.

Kobayashi, D. Y., Tamaki, S. J. and Keen, N. T. (1989). Cloned avirulence genes from the tomato pathogen Pseudomonas syήngae pv. tomato confer cultivar specificity on soybean. Proc. Natl. Acad. Sci., USA 86, 157-161.

Konieczny, A. and Ausubel, F. M. (1993). A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J. 4, 403-410.

Kosslak, R. M., Chamberlin, M. A., Palmer, R. G. and Bowen, B. A. (1996). Apoptosis and necrosis in adjacent root cortical cells precede the defense response in soybean ROOT NECROSIS mutants. J. Hered. 87, 413-422. Langford, A. N. (1948). Autogenous necrosis in tomatoes immune from Cladosporium fulvum Cooke. Can. J. Res. 16, 35-64.

Leon, J., Lawton, M. A. and Raskin, I. (1995). Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco. Plant Physiol. 108, 1673-1678.

Levine, A., Pennell, R., Palmer, R. and Lamb, C. J. (1996). Calcium-mediated apoptosis in a plant hypersensitive response. Curr. Biol. 6, 427-437.

Levine, A., Tenhaken, R., Dixon, R. and Lamb, C. J. (1994). H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 583-593.

Lippuner, V., Cyert, M. S. and Gasser, C. S. (1996). Two classes of plant cDNA clones differentially complement yeast calcineurin mutants and increase salt tolerance of wild-type yeast. J. Biol. Chem. 277, 12859-12866.

Lister, C. and Dean, C. (1993). Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana. Plant J. 4, 745-750..

Low, P. S. and Merida, J. R. (1996). The oxidative burst in plant defense: Function and signal transduction. Physiol. Plant. 96, 533-542.

Lukowitz, W., Mayer, U. and Jϋrgens, G. (1996). Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product. Cell 84, 61-11.

Malamy, J., Hennig, J. and Klessig, D. F. (1992). Temperature-dependent induction of Salicylic acid and its conjugates during the resistance response to tobacco mosaic virus infection. The Plant Cell 4, 359-366.

Matallana, E., Bell, C. J., Lu, P. J. and Ecker, J. E. (1992). Genetic and physical linkage of the Arabidopsis genome: Methods for anchoring yeast artificial chromosomes. In Methods in Arabidopsis Research, C. Koncz, N.-H. Chua and J. Schell, eds. (Singapore: World Scientifc), pp. 144-169.

Mauch-Mani, B. and Slusarenko, A. J. (1996). Production of salicylic acid precursors is a major function of phenylalanine-ammonia lyase in the resistance of Arabidopsis to Peronospora parasitica. Plant Cell 9, 203-212. May, M. J., Hammond-Kosack, K. E. and Jones, J. D. G. (1996). Involvement of reactive oxygen species, glutathione metabolism and lipid peroxidation in the C/-gene-dependent defense response of tomato cotyledons induced by race-specific elicitors of Cladosporium fulvum. Plant Physiol. 770, 1367-1380.

Mittler, R., Shulaev, V. and Lam, E. (1995). Coordinated activation of programmed cell death and defense mechanisms in transgenic tobacco plants expressing a bacterial proton pump. Plant Cell 7, 29-42.

Newman, T., de Bruijn, F. J., Green, P., Keegstra, K., Kende, H., Mclntosh, L., Ohlrogge, J., Raikhel, N., Somerville, S., Thomashow, M., Retzel, E. and Somerville, C. (1994). Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol. 706, 1241-1255.

Nϋrnberger, T., Nennstiel, D., Jabs, T., Sacks, W. R., Hahlbrock, K. and Scheel, D. (1994). High-affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses. Cell 78, 449-460.

Omichinski, J. G., Clore, G. M., Schaad, O., Felsenfeld, G., Trainor, C, Appella, E., Stahl, S. J. and Gronenborn, A. M. (1993). NMR stmcture of a specific DNA complex of Zn- containing DNA binding domain of GATA-1. Science 161, 438-446.

Pryor, T. P. and Ellis, J. (1993). The genetic complexity of fungal resistance genes in plants. Adv. Plant Pathol. 70, 281-307.

Putterill, J., Robson, F., Lee, K., Simon, R. and Coupland, G. (1995). The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors. Cell 80, 847-857.

Ramain, P., Heitzler, P., Haenlin, M. and Simpson, P. (1993). pannier, a negative regulator of achaete and scute in Drosophila, encodes a zinc finger protein with homology to the vertebrate transcription factor GATA-1. Development 119, 1277-1291.

Rogers, S. O. and Bendich, A. J. (1985). Extraction of DNA from milligram quantities of fresh, herbarium, and mummified plant tissue. Plant Mol. Biol. 5, 69-76. Rounsley, S. D., Glodek, A., Sutton, G., Adams, M. D., Somerville, C. R., Venter, J. C. and Kerlavage, A. R. (1996). The construction of Arabidopsis expressed sequence tag assemblies. A new resource to facilitate gene identification. Plant Physiol. 772, 1177-1183.

Rueffler, M., Steipe, B. and Zenk, M. H. (1995). Evidence against specific binding of salicylic acid to plant catalase. FEBS Letters. 377, 175-180.

Ryals, J. L., Neuenschwander, U. H., Willits, M. C, Molina, A., Steiner, H.-Y. and Hunt, M. D. (1996). Systemic acquired resistance. Plant Cell 8, 1809-1819.

Ryerson, D. E. and Heath, M. C. (1996). Cleavage of nuclear DNA into oligonucleosomal fragments during cell death induced by fungal infection or by abiotic treatments. Plant Cell 8, 393-402.

Sanchez-Garia, I. and Rabbitts, T. H. (1994). The LIM motif: a new structural motif found in zinc-finger-like proteins. Trends Genet. 70, 315-320.

Schmidt, R., West, J., Cnops, G., Love, K., Balestrazzi, A. and Dean, C. (1996). Detailed description of four YAC contigs representing 17Mb of chromosome 4 of Arabidopsis thaliana ecotype Columbia. Plant J. 9, 755-765.

Schmidt, R., West, J., Love, K., Lenehan, Z., Lister, C, Thompson, H., Bouchez, D. and Dean, C. (1995). Physical map and organization of Arabidopsis thaliana chromosome 4. Science 170, 480-483.

Scofield, S. R., Tobias, C. M., Rathjen, J. P., Chang, J. H., Lavelle, D. T., Michelmore, R. W. and Staskawicz, B. J. (1996). Molecular basis of gene-for-gene specificity in bacterial speck disease of tomato. Science in press,

Stakman, E. C. (1915). Relation between Puccinia graminis and plants highly resistant to its attack. J. Agric. Res. 4, 193-299.

Staskawicz, B. J., Ausubel, F. M., Baker, B. J., Ellis, J. and Jones, J. D. G. (1995). Molecular genetics of plant disease resistance. Science 168, 661-667.

Tang, X., Frederick, R. D., Zhou, J., Halterman, D. A., Jia, Y. and Martin, G. B. (1996). Physical interaction of avrPto and the Pto kinase defines a recognition event involved in plant disease resistance. Science in press,

Uknes, S., Dincher, S., Friedrich, L., Negrotto, D., Williams, S., Thompson-Taylor, H., Potter, S., Ward, E. and Ryals, J. (1993a). Regulation of pathogenesis-related protein 1-a gene expression in tobacco. Plant Cell 5, 159-169.

Uknes, S., Winter, A. M., Delaney, T., Vemooij, B., Morse, A., Friedrich, L., Nye, G., Potter, S., Ward, E. and Ryals, J. (1993b). Biological induction of systemic acquired resistance in Arabidopsis. Molec. Plant-Microbe Interact. 6, 692-698.

Valent, B., Farrall, L. and Chumley, F. G. (1990). Magnaporthe grisea genes for pathogenicity and virulence identified through a series of backcrosses. Genetics 727, 87- 101.

Vemooij, B., Friedrich, L., Morse, A., Reist, R., Kolditz-Jawhar, R., Ward, E., Uknes, S., Kessmann, H. and Ryals, J. (1994). Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance, but is required in signal transduction. Plant Cell 6, 959-965.

Walbot, V., Hoisington, D. A. and Neuffer, M. G. (1983). Disease lesion mimics in maize. In Genetic Engineering of Plants, T. Kosuge and C. Meredith, eds. (New York: Plenum Publishing Co.), pp. 431-442.

Wang, C.-Y., Mayo, M. W. and Baldwin Jr., A. S. (1996a). TNF- and cancer therapy- induced apoptosis: potentiation by inhibition of NF-κB. Science 174, 784-787.

Wang, H., Li, J., Bostock, R. M. and Gilchrist, D. G. (1996b). Apoptosis: A functional paradigm for programmed plant cell death induced by a host-selective phytotoxin and invoked during development. Plant Cell 8, 375-391.

Whalen, M. C, Stall, R. E. and Staskawicz, B. J. (1988). Characterization of a gene from a tomato pathogen determining hypersensitive resistance in non-host species and genetic analysis of this resistance in bean. Proc. Natl. Acad. Sci., USA 85, 6743-6747.

Yanagisawa, S. (1995). A novel DNA-binding domain that may form a single zinc finger. Nucl. Acids Res. 13, 3403-3410. 1

SEQUENCE LISTING SEQ ID NO: 1

LVCHGCRNLLMYPRGASNVRCALCNTI MV

SEQ ID NO:2

11CGGCRTMLMYTRGASSVRCSCCQTTN V

SEQ ID NO:3

INCGHCRTTLMYPYGASSVKCAVCQFVTNV

SEQ ID NO:4

MSNGRV- PLPTNRP-NGTACPPST- STSTPPSQTQTVWENPMSVDESGKLVSNV

SEQ ID NO:5

MSPG-VAPVPSNRKGNGDYMPMSPKS S P-QQIINPIRRHPQRVDPNGYMM

SEQ ID NO:6

VPLPTNRP-NGTACPPSTSTSTPPSQTQTVWENPMSVDESGKLVSNV

SEQ ID NO:7

VPLPANNPW-TTWPSTPPSQPPAVCPPW

SEQ ID NO:8

VPLPANNPW-TTVVPSTPPSQPPAVCPPVV

SEQ ID NO:9

IPVYTNSNV-GTALPPSVSPSVSPSVT

SEQ ID NOrlO

WLP-NAAPAGAAAPPSGSRSTSPS

SEQ ID NO: 11

SNGRVPLPTNRPN-GTACPPSTSTSTPPSQTQTVWENPMSVDESGKLVSNV

SEQ ID NO: 12

SRA VPVPAADPNAG-AIVPANKSKRSPEQGQRRIRR SEQ ID NO:13

10 30 50

GATCAAATCTAGTTACGCTTAAATTTGGATATATCTAAGGTTTCTTCGTCAATATATGGA

70 90 110

GCTTACGAAAACGAAAGAGTGAGCTACGAGGAACTAAATCAATGAAGATAAGAGGAATGA

130 150 170

AGGAGAGAAGATCACCAAGGTGTAGAAATTTCTGAAGTCGTCTCCTCCAATCTCCACTAT

190 210 230

TGGTTTGTTCAGAACTTGAGAAGGCCTTAGATCCAAGCCATTAGTAACCTCTCTATGGCC

250 270 290

ATAAGTGACCTTAAGAGAGACCAACCTCGTGAAAGGATCAAGAACATCTCCAACAACACT

310 330 350

GCCGACCACGAGAGGATCTCTACGACTTAAAGACATATTTATCTTGGATCTCAAGTATCT

370 390 410

CAATAAAATGTTTTGCTTCTAACCTTATGAACCCTTACTTGCTATTCTTTATATAACGTT

430 450 470

TTGGGAATTGCAATAATTAGCTATTTAGCTTTATTCTCTCCAATGAAATCATTACCAGGG

490 510 530

TCTTTTCGTGTATAGTTATCTTCGAGAATCTACAACTCGTTCAACGTACGTATATCACTT

550 570 590

ATAATTCATGTTTTTTTTTCTTTCCTTTTTTCTAAATTTATAGTATTCTTATTCCAAAAC

610 630 650

CCACCAGTATAAAACAGAAATAATCATATTCCAAATTATACATCATCCACTTGTTTCTTG

670 690 710

CTAGCCACTAGTATGTAATTTATTCTGACTTATCATTGGAACTTCATGAACTATTTAAAA

730 750 770

TAATGTCACAAGCATATAATATGCTGCATATTTGCGTACGTCACGCATTTTGCGTCACAT

790 810 830

GTCACTCATTTAAATAGTTAAGGACACTACATTACACCGATTATGTATGATGTTAATGCA

850 870 890

TTTTAGAATAACTCCTTCAACCTAAACCATCATATAAAAGTATATATGCTCCAGATAAAT

910 930 950

TGACGCCATAATTGTTCACATATCTGGTTGGTTTGTACATACGTACTAGACTCTTTTTTT

970 990 1010

TCTTTTCTTAATGTAGTACTAAACTTAATTAATACCATCAAAAATATCAATTTAACAAAA

1030 1050 1070 CAAACCAGTAAAACTTTTAAAACAATGGAGTAAATCAAATAAAACAAGTAAATTAACAAA

1090 1110 1130

TAGACACAAGGTAACAGAAGTATAATAACGACAGAAAAATGAACAATTGGCCAAAAAATT

1150 1170 1190

CGTTTTCAAACGTGATTTCAAAATTGTCTCCAAATCTTAAATGTTGATAAAGTAATTTTT

1210 1230 1250

TTTTAAATTCATTATACCTTTCAAAAACAAGTGTATTACCTAAAAGCTCAACCGTGTATT

1270 1290 1310

CTTACACTCCAAACAAATTTAGTTCCCCAAGTTTGGAAGACAAAAATTTCTAAGAAATTT

1330 1350 1370

CTGACAAAACACATGAGAAATAAACCGATAAAGACTTCTAAAAACTATTGCAGACCAGTT

1390 1410 1430

TCATTTGCTGACCACAAAAAGTCATGAGAATACAATTAGCTCAGTGATTCTTGATATTTC

1450 1470 1490

TGGTACCTAACAAAAAGAAAAGTGTGTGAGGTTAGATGGCTATGATTTTTGCTCTCCAAT

1510 1530 1550

TTATTGTCCATTTCCCCAATTTGTAATATGAAATGCGCAAATTACTCTTCTTCCGATATG

1570 1590 1610

AATAAGCAAACGAAAACATACGTGGGACGTTATGTTGAGAACATTTGATTAAAGTTTATA

1630 1650 1670

TGCGATTTTCATTTATTTACTATGAATTTTTTGTTTGGCAGCATGTACGATTTTTCATTT

1690 1710 1730

AACACACAAATATTATAGAATTTTCATTGGTTCAAAGGGGTAGACAAAAAATAATTTAAT

1750 1770 1790

ATTATTACACCATTTGCAGAAAATTAGAAAATATATTTTTACCCATAATTAATTGATCTA

1810 1830 1850

TGGACGTATGCTTGGCATAAAAATTCATATTTAATTAGCAGAAGCCAATCGCTGCGTTTG

1870 1890 1910

TATATACGCGTTTATGACCGAGAAAAAAACCCTTACGCGTCATGTAAAAAAAAAAGAAGC

1930 1950 1970

GTAAATTACGAAAAACAGAGAGATAAATCCGGGCATTGAGATTTTGGAGATAGAGAGAGA

1990 2010 2030

GAAAAATCGAAATCTATTGTCTATCTCCTCAATTTGGATTGGATTTTCTGCATATCATCG

2050 2070 2090

CTCTAGATTTCGCGGGTTTTGGATTCGATTCCTTACCCTTCTCCAATCGGTAAGAACAAG

2110 2130 2150

CTCCAAAGTTTGTTCCTTTTTTTCAATTTTCGCCAATTCTGTAATCTCATCATTGTTCTT 2170 2190 2210

GTTTGATTTGGATGCAGAAGTTTTTGGGTTTGAATTGGATTTGGGTTTCGTTCCAAAATC

2230 2250 2270

AGCTCTTTTTGTTAATCAGGTGAGTTTTTAGGTATTTGAATCTCCAATTGCTTCCCTTGC

2290 2310 2330

AATGACTAAGTATTGTGAAATGTTTAGGGTTTCATCTGTGTGGGTCTTGTTTTGAAGCAA

2350 2370 2390

TTTGTGTGTGTTTGGATGAAAGTAGCAGATATGCAGGACCAGCTGGTGTGTCATGGTTGT

2410 2430 2450

AGGAATTTATTGATGTATCCTAGAGGAGCATCTAATGTGCGTTGTGCGTTATGTAACACT

2470 2490 2510

ATCAACATGGTTCCTCCTCCTCCTCCACCTCACGGTATCGATTTCTTTGTTGAATTTGAA

2530 2550 2570

TTGAGGATGAGGTTAATATGCTCTGCAATTGTATTATAACTTGGGTTCTGATTCTGAATA

2590 2610 2630

CAGACATGGCACACATTATATGTGGTGGTTGTAGAACAATGCTTATGTATACGCGTGGGG

2650 2670 2690

CTAGTAGCGTAAGATGCTCTTGCTGTCAAACTACGAACCTTGTGCCAGGTATATTAATAA

2710 2730 2750

TATCGTGACATCCATATCAATCCTTTTAAAGACCATGTATTATATTGCTTTATAAGGTCT

2770 2790 2810

TTTAGTCCTTTAGAATCTTCTTTCACACTTTTGTTTGATAACATTGTTCTGTGGAGATGA

2830 2850 2870

TGCTTACGTAACGTATTTCCACTTTTCCCAAAGATGTATATGAATCTGAATTCTGAAAAT

2890 2910 2930

ATCTGGGATTTGTAAAGCAGCTGAAAGTACTTAAAACAAAGCTTTTAGATGGTCCCGGTG

2950 2970 2990

GACTAGGTAACTACTTGTTAGAGCTAGTAGGGTTTATTATTGTTTTGTTTGATCTACCAT

3010 3030 3050

TAGATTCTTATCTTTAATTAGCGTCTAAGCTGTTGTCATTTAGCTGTATGATTATCATTT

3070 3090 3110

ATCCATGACTGCTTAAGAACATTGCTGATTACTTCGTTCATTAGTATTTCTTGGATTTTT

3130 3150 3170

CTAGCATTAACATTGCTTGTTTTCTGAATCTGTGCGTGTCTTTTTTGAAATCGACAGCGC

3190 3210 3230

ACTCCAATCAGGTTGCCCATGCTCCTTCCAGTCAGGTTGCGCAAATCAATTGTGGGCATT ^" 3250 3270 3290

GTCGGACGACCCTCATGTATCCTTACGGTGCATCATCCGTCAAATGCGCTGTTTGTCAAT

3310 3330 3350

TCGTAACTAACGTTAATGTGATTATTCCTATCTATTAAGCCACCTCTGCATGGTTGAGTT

3370 3390 3410

AAGTATAGAGATCTTTCTGTTGGAAATTTTCATTTCTGATTCATTTTGCATCCTTAGATG

3430 3450 3470

AGCAATGGAAGGGGTACCTCTCCCAACTAACCGGCCAAATGGAACAGCTTGTCCCCCCTC

3490 3510 3530

TACATCAACTGTGAGTTATCAAATTATGAATTTGTAATAGTTCTGTATATTCTTATGGAA

3550 3570 3590

CTGGTACTTACTCTGTTCATCGATTTTTCATTTTACCAACAGTCAACACCACCCTCTCAG

3610 3630 3650

ACCCAAACCGTTGTTGTAGAAAACCCCATGTCCGTTGATGAAAGCGGAAAGTTGGTGAGT

3670 3690 3710

ATTTCTATCACCTGTGTTCTTCTTCTTATTTACCACATTAGAGGAAGATATGACAAAGTG

3730 3750 3770

ACTGAAACACACAAATTGCAGGTGAGCAATGTTGTTGTTGGAGTGACAACTGACAAAAAG

3790 3810 3830

TAATCAAGAATGAGTGAGATCTTAAAGATCAAATCCAAATTCTTCCTCTATTCCTGCGTT

3850 3870 3890

TGGTTTGTGCATATTACATACGCGGAAAAACTGTATGTTATATATCTCTTGACTCCTTTT

3910 3930 3950

TAACCCAAGAGAAAAAGCTTATCAGAATCTCTTGTTACTGCATTATTGGGGTTTATTCAA

3970 3990 4010

AGTTGAAGACACAAGGTTTTTGCTCGAATAATTTGGCATTCTTTTGCTCCATGGAACTTG

4030 4050 4070

ACCTTCTCTTCTGTTAGTTGACTTCTAAAACTCCATCGGCCCTTGTGGCATTGTTAATGT

4090 4110 4130

ATGTATGAATATAATCTGATACACCAACCAATCATTAAGATTTGGGTTTGAAATCTGTCT

4150 4170 4190

CTTCCGTGGATGAGATATGCTACATGTCACAAGAACTGGTCTTAGCTTTGGTAGATAAGA

4210 4230 4250

CTTGTCTTAGAAGCAAGTCTTGAAATCTGGAAATCTATTTTGCAGTAATCTTGTCACAAC

4270 4290 4310

AACCATAACCTAATCAGTCAGTACCCTCCAAGAAACATTAAAGTTAGATGATCCGACAAA

4330 4350 4370 ACCTCTCAACAAAACCAACTCTTTCCATATAAATACTCTTTAACACTGGACCAAATTTNC

4390 4410 4430

ACCCTTCCTCTTGATCCTCCCTGCATCACAATGGCCAAAAAAAAAATGGTGGTTGGCNGG

4450 4470 4490

TGGGTACCACAAAGAGCTGGAAACTACTCTTGGGGCTGAGAATATTTGCATTCATGGCTA

4510 CTTTAGCTGCAG

SEQ ID NO:14

10 30 50

CTTACGCGTCATGTAAAAAAAAAAGAAGCGTAAATTACGAAAAACAGAGAGATAAATCCG

70 90 110

GGCATTGAGATTTTGGAGATAGAGAGAGAGAAAAATCGAAATCTATTGTCTATCTCCTCA

130 150 170

ATTTGGATTGGATTTTCTGCATATCATCGCTCTAGATTTCGCGGGTTTTGGATTCGATTC

190 210 230

CTTACCCTTCTCCAATCGAAGTTTTTGGCTTTGAATTGGATTTGGGTTTCGTTCCAAAAT

250 270 290

CAGCTCTTTTTGTTAATCAGATATGCAGGACCAGCTGGTGTGTCATGGTTGTAGGAATTT

310 330 350

ATTGATGTATCCTAGAGGAGCATCTAATGTGCGTTGTGCGTTATGTAACACTATCAACAT

370 390 410

GGTTCCTCCTCCTCCTCCACCTCACGACATGGCACACATTATATGTGGTGGTTGTAGAAC

430 450 470

GATGCTTATGTATACGCGTGGGGCTAGTAGCGTAAGATGTTCTTGCTGTCAAACTACGAA

490 510 530

CCTTGTGCCAGCGCACTCCAATCAGGTTGCCCATGCTCCTTCCAGTCAGGTTGCGCAGAT

550 570 590

CAATTGTGGGCATTGTCGGACGACCCTCATGTATCCTTACGGTGCATCATCCGTCAAATG

610 630 650

CGCTGTTTGTCAATTCGTAACTAACGTTAATATGAGCAATGGAAGGGTACCTCTCCCAAC

670 690 710

TAACCGGCCAAATGGAACAGCTTGTCCCCCCTCTACATCAACTTCAACACCACCCTCTCA

730 750 770

GACCCAAACCGTTGTTGTAGAAAACCCCATGTCCGTTGATGAAAGCGGAAAGTTGGTGAG

790 810 830

CAATGTTGTTGTTGGAGTGACAACTGACAAAAAGTAATCAAGAATGAGTGAGATCTTAAA 850 870 890

GATCAAATCCAAATTCTTCCTCTGTTCCTGCGTTTGGTTTGTGCATATTACATACGCGGA

910 930 950

AAAACTGTATGTTATATATCTCTTGACTCCTTTTTAACCCAAGAGAAAAAGCTTATCAGA

970

AAAAAAAAAAAAAAAAA

SEQ ID NO:15

10 30 50

GAAATCTATTGTCTATCTCCTCAATTTGGATTGGATTTTCTGCATATCATCGCTCTAGCT

70 90 110

TTCGCGGGTTTTGGATTCGATTCCTTACCCTTCTCCAATCGAAGTTTTTGGCTTTGAATT

130 150 170

GGATTTGGGTTTCGTTCCAAAATCAGCTCTTTTTGTTAATCAGGGTTTCATCTGTGTGGG

190 210 230

TCTTGTTTTGAAGCAATTTGTGTGTGTTTGGATGAAAGTAGCAGATATGCAGGACCAGCT

250 270 290

GGTGTGTCATGGTTGTAGGAATTTATTGATGTATCCTAGAGGAGCATCTAATGTGCGTTG

310 330 350

TGCGTTATGTAACACTATCAACATGGTTCCTCCTCCTCCTCCACCTCACGACATGGCACA

370 390 410

CATTATATGTGGTGGTTGTAGAACAATGCTTATGTATACGCGTGGGGCTAGTAGCGTAAG

430 450 470

ATGCTCTTGCTGTCAAACTACGAACCTTGTGCCAGCGCACTCCAATCAGGTTGCCCATGC

490 510 530

TCCTTCCAGTCAGGTTGCGCAGATCAATTGTGGGCATTGTCGGACGACCCTCATGTATCC

550 570 590

TTACGGTGCATCATCCGTCAAATGCGCTGTTTGTCAATTCGTAACTAACGTTAATATGAG

610 630 650

CAATGGAAGGGTACCTCTCCCAACTAACCGGCCAAATGGAACAGCTTGTCCCCCCTCTAC

670 690 710

ATCAACTTCAACACCACCCTCTCAGACCCAAACCGTTGTTGTAGAAAACCCCATGTCCGT

730 750 770

TGATGAAAGCGGAAAGTTGGTGAGCAATGTTGTTGTTGGAGTGACAACTGACAAAAAGTA

790 810 830

ATCAAGAATGAGTGAGATCTTAAAGATCAAATCCAAATTCTTCCTCTATTCCTGCGTTTG

850 870 890

GTTTGTGCATATTACATACGCGGAAAAACTGTATGTTATATATCTCTTGACTCCTTTTTA 910 930 950

ACCCAAGAGAAAAAGCTTATCAGAATCTCTTGTTACTGCATTATTGGGGTTTATTCAAAG

970 990

TTGAAGACACAAGGTTTTTGCTCGAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO:16

MetGlnAspGlnLeuValCysHisGlyCysArgAsn euLeuMetTyrProArgGlyAla

10 20

SerAsnValArgCysAlaLeuCysAsnThrlleAsnMetValProProProProProPro

30 40

HisAspMetAlaHisIlelleCysGlyGlyCysArgThrMet euMetTyrThrArgGly

50 60

AlaSerSerValArgCysSerCysCysGlnThrThrAsn euValProAlaHisSerAsn

70 80

GlnValAlaHisAlaProSerSerGlnValAlaGlnlleAsnCysGlyHisCysArgThr

90 100

Thr euMetTyrProTyrGlyAlaSerSerValLysCysAlaValCysGlnPheValThr

110 120

AsnValAsnMetSerAsnGlyArgValProLeuProThrAsnArgProAsnGlyThrAla

130 140

CysProProSerThrSerThrSerThrProProSerGlnThrGlnThrValValValGlu

150 160

AsnProMetSerValAspGluSerGlyLysLeuValSerAsnValValValGlyValThr

170 180

ThrAspLysLys

SEQ ID NO:17

MetLysValAlaAspMetGlnAspGlnLeuValCysHisGlyCysArgAsnLeuLeuMet

10 20

TyrProArgGlyAlaSerAsnValArgCysAlaLeuCysAsnThrlleAsnMetValPro

30 40

ProProProProProHisAspMetAlaHisIlelleCysGlyGlyCysArgThrMetLeu

50 60

MetTyrThrArgGlyAlaSerSerValArgCysSerCysCysGlnThrThrAsnLeuVal 70 80

ProAlaHisSerAsnGlnValAlaHisAlaProSerSerGlnValAlaGlnlleAsnCys

90 100

GlyHisCysArgThrThrLeuMetTyrProTyrGlyAlaSerSerValLysCysAlaVal

110 120

CysGlnPheValThrAsnValAsnMetSerAsnGlyArgValProLeuProThrAsnArg

130 140

ProAsnGlyThrAlaCysProProSerThrSerThrSerThrProProSerGlnThrGln

150 160

ThrValValValGluAsnProMetSerValAspGluSerGlyLysLeuValSerAsnVal

170 180

ValValGlyValThrThrAspLysLys

SEQ ID NO: 18

5'-CAG TGG ATC TTT CCT CAG ACG-3'

SEQ ID NO: 19

5'-CAT CTT CTT CTG CAA TCT GGG-3'

SEQ ID NO:20

5 '-CAT CCA TCA AAC AAA CTC C-3'

SEQ ID NO:21

5'-TGT TTC AGA GTA GCC AAT TC-3'

SEQ ID NO:22

5'-CAC GTT AGT TAG TTA GAA GG-3'

SEQ ID NO:23

5' -CTG ATG TTC TCT AC A AAT GG-3'

SEQ ID NO:24 10

5'-CGT ATC CGC ATT TCT TCA CTG C-3'

SEQ ID NO:25

5'-CAT CTG CAA CAT CTT CCC CAG-3'

SEQ ID NO:26

5'-TTG AGT CCT TCT TGT CTG-3'

SEQ ID NO:27

5'-CTA GAG CTT GAA AGT TGA TG-3'

SEQ ID NO:28

5'-GAA TGG TGT AAC CAA ACT C-3'

SEQ ID NO:29

5 '-CAT ACC GTA TGA TGG AAC-3'

SEQ ID NO:30

5' -GAA CTC ATT GTA TGG ACC-3'

SEQ ID NO:31

5'-CTA AGA TGG GAA TGT TGG-3'

SEQ ID NO:32

5'-CCA AGA AGA GAA AAC GGA GA-3'

SEQ ID NO:33

5'-AAC AAT AGG AGG TGC AGA GT-3'

SEQ ID NO:34

5'-ACC TAA CAAAAA GAAAAG TGT GTGAGG-3'

SEQ ID NO:35

5'-ATA ATA AAC CCT ACT AGC TCT AAC AAG-3'

SEQ ID NO:36

5'-CTG CTA CTT TCA TCC AAA C-3' 11

SEQ ID NO:37

5'- GTA AAA CGA CGG CCA TG -3'

SEQ ID NO:38

5'- GGA AAC AGC TAT GAC CAT G -3'

SEQ ID NO:39

5'- TTC ATG GCAATG GTGTGA CCC C -3'

SEQ ID NO:40

5'- CTG CCG GAT TCT TGA TCG AAG A -3'

SEQ ID NO:41

5'- AGA GGA AGG TCC GCC TCC GG -3'

SEQ ID NO:42

5'- CTC TGC TCT CCT GAG ACT GCT T -3'

SEQ ID NO:43

5'- CAT CAT AAT GTC TCC TTT TGA GAC -3'

SEQ ID NO:44

5'- GCC ATC CAT TAT TCA TCG CCT -3'

SEQ ID NO:45

5'- GAG GAG GAA GAA CTG CAG ATT CC -3'

SEQ ID NO:46

5'- GTG CTC CAT GTC CAA ATC ATA C -3'

SEQ ID NO:47

10 30 50

AATATATCGAAACGAGATTCCACAATTAGTCTCTAGTCAAAGAGCTTCATGGCAATGGTG

70 90 110 2

TGACCCCAAATATAGATTTGATGAAAGTGAGGAAATAGGAGAAGAAATGAAGAACACAGG

130 150 170

ATGTGTCTTCTTCTTCTAAGTCACTAACAAAATCAACAAAGAGGAGAAGCCATTATTATA

190 210 230

TAATAGAGAGATTGAGAGAAGAGATTTATCCAAAAAAATATTGCAATTCTTCTTGGAGTG

250 270 290

AATAATGCCAGTCCCTCTTGCACCATATCCAACACCTCCGGCACCGGCACAGGCTCCGTC

310 330 350

GTACAACACTCCTCCGGCAAATGGAAGTACAAGTGGGCAGAGCCAGTTAGTGTGTTCAGG

370 390 410

TTGCAGAAACCTTCTGATGTATCCCGTCGGAGCAACCTCCGTCTGCTGCGCCGTCTGTAA

430 450 470

CGCCGTCACGGCCGTTCCTCCGCCGGGAACGGAGATGGCACAGTTAGTATGTGGAGGATG

490 510 530

CCATACACTCTTAATGTACATTCGTGGAGCTACAAGTGTTCAATGTTCTTGTTGTCACAC

550 570 590

TGTTAATCTCGCCCTCGAAGCGAACCAAGTAGCGCATGTGAATTGCGGAAACTGCATGAT

610 630 650

GCTACTAATGTATCAATATGGAGCAAGATCAGTGAAATGTGCCGTTTGTAACTTTGTCAC

670 690 710

ATCTGTTGGGGGTTCAACGAGCACGACTGATTCGAAGTTTAACAATTAAAACTTGGATCT

730 750 770

ATCTACCTATCAATATCTATTGAGTTATGAGCAATATAGAGGAAGCATCAAATCTTTTTC

790 810 830

ACTCTCTCTTCGATCAAGAATCCGGCAGTTATGAGTTTGAAACCATTTTCGGAAGTAAAT

850 870 890

GAAATATGTAATTCGTCGAAATTTCTGACTTTGGTCTCTTTGTCCGTTTGTATAGAGCTA

910 AAAAAAAAAA

SEQ ID NO: 48

MetProValProLeuAlaProTyrProThrProProAlaProAlaGlnAlaProSerTyr

10 20

AsnThrProProAlaAsnGlySerThrSerGlyGlnSerGlnLeuValCysSerGlyCys 3

30 40

ArgAsnLeuLeuMetTyrProValGlyAlaThrSerValCysCysAlaValCysAsnAla

50 60

ValThrAlaValProProProGlyThrGluMetAlaGlnLeuValCysGlyGlyCysHis

70 80

ThrLeuLeuMetTyrlleArgGlyAlaThrSerValGlnCysSerCysCysHisThrVal

90 100

AsnLe iAlaLeuGluAlaAsnGlnValAlaHiεsValAsnCysGlyAsnCysMetMetLeu

110 120

LeuMetTyrGlnTyrGlyAlaArgSerValLysCysAlaValCysAsnPheValThrSer

130 140

ValGlyGlySerThrSerThrThrAspSerLysPheAsnAsn

150

SEQ ID NO: 49

CysSerGlyCysArgAsnLeuLeuMetTyrProValGlyAlaThrSerValCysCysAlaValCys

SEQ ID NO: 50

CysGlyGlyCysHisThrLeuLeuMetTyrlleArgGlyAlaThrSerValGlnCysSerCysCys

SEQ ID NO: 51

CysGlyAsnCysMetMetLeuLeuMetTyrGlnTyrGlyAlaArgSerValLysCysAlaValCys

SEQ ID NO: 52

CysXxxXxxCysArgXxxXxxLeuMetTyrXxxXxxGlyAlaSerXxxValXxxCysXxxXxxCys

SEQ ID NO: 53

CysXxxXxxCysXxxXxxLeuLeuMetTyrXxxXxxGlyAlaXxxSerValXxxCysXxxXxxCys

SEQ ID NO:54

10 30 50

GAGGAGGAAGAGGAAGGTCCGCCTCCGGGATGGGAATCTGCAGTTCTTCCTCCTCCAATC

70 90 110

GTCACCATCACCGCCGCCGTAAACCCCAATCCCACCACCGTAGAAATTCCCGAAAAGGCC 4

130 150 170

CAAATGGTATGTGGATCTTGCAGGCGTTTGCTTTCTTATCTAAGAGGATCCAAACATGTT

190 210 230

AAGTGCTCCTCTTGTCAGACTGTTAATCTCGTTCTTGAAGCTAACCAGGTTGGTCAGGTG

250 270 290

AATTGCAACAATTGCAAACTGCTACTGATGTATCCTTATGGAGCTCCAGCTGTTAGATGT

310 330 350

TCCTCCTGCAATTCTGTCACAGATATCAGTGAAAACAACAAACGACCTCCATGGTCTGAG

370 390 410

CAGCAAGGACCACTCAAAAGTTTAAGCAGTCTCAGGAGAGCAGAGAATTAAACTTGAACC

430 450 470

GATTTTTGTCAATTTTGAACCGGTTTGACGACTAAAAACCTTGTAATAATGTCGAAGGAT

490 AGATGAAATAAAATCACACC

SEQ ID NO:55

GluGluGluGluGluGlyProProProGlyTrpGluSerAlaValLeuProProProIle

10 20

ValThrlleThrAlaAlaValAsnProAsnProThrThrValGluIleProGluLysAla

30 40

GlnMetValCysGlySerCysArgArgLeuLeuSerTyrLeuArgGlySerLysHisVal

50 60

LysCysSerSerCysGlnThrValAsnLeuValLeuGluAlaAsnGlnValGlyGlnVal

70 80

AsnCysAsnAsnCysLysLeuLeuLeuMetTyrProTyrGlyAlaProAlaValArgCys

90 100

SerSerCysAsnSerValThrAspIleSerGluAsnAsnLysArgProProTrpSerGlu

110 120

GlnGlnGlyProLeuLysSerLeuSerSerLeuArgArgAlaGluAsn

55 SEQ ID NO:56

CGSCRRLLSYLRGSKHVKCSSC 15

SEQ ID NO:57

CNNCKLLLMYPYGAPAVRCSSC

SEQ ID NO: 58

10 30 50

GGAAGAGATACAACAACAAACGCAGAAGGAAGAACAAAAGCACCGTGAAGAAGAAGAGGA

70 90 110

GGAAGAGGAAGGTCCGCCTCCGGGATGGGAATCTGCAGTTCTTCCTCCTCCAATCGTCAC

130 150 170

CATCACCGCCGCCGTAAACCCCAATCCCACCACCGTAGAAATTCCCGAAAAGGCCCAAAT

190 210 230

GGTATGTGGATCTTGCAGGCGTTTGCTTTCTTATCTAAGAGGATCCAAACATGTTAAGTG

250 270 290

CTCCTCTTGTCAGACTGTTAATCTCGTTCTTGAAGCTAACCAGGTTGGTCAGGTGAATTG

310 330 350

CAACAATTGCAAACTGCTACTGATGTATCCTTATGGAGCTCCAGCTGTTAGATGTTCCTC

370 390 410

CTGCAATTCTGTCACAGATATCAGTGTATGTATTCACAGATGGTTTTGTGCTCCATGTCC

430 450 470

AAATCATACTTGGAAGAGTTGATACATTTTGAGATCCGAGTAAGTAATCATCTGATGAAT

490 510 530

CATTTATAATAAACTGTGTTATATTTCAGGAAAACAACAAACGACCTCCATGGTCTGAGC

550 570 590

AGCAAGGACCACTCAAAAGTTTAAGCAGTCTCAGGAGAGCAGAGAATTAAACTTGAACCG

610 630 650

ATTTTTGTCAATTTTGAACCGGTTTGACGACTAAAAACCTTGTAATAATGTCGAAGGATA

670 690

GATGAAATAAAATCACCATTAATAATCTAAAAAAAAAAAAAAAA

SEQ ID NO:59

10 30 50

CTCTATCCTTACTTCAACGGAGCTTTACCAGACCCAAACTCTCTTAGGCCGCACCGAGAG 70 90 110

TTGTTTGTACGTGTGCTTAACGCAGATTACATATGACGCTTCTAACCCACAATTAATTTG

130 150 170

GTTCACTCTTTGCCGCAAACCAAATAGCTCAAAAAAGATTTTAATCCCAATTTCATATCC

190 210 230

TAAATCTGCATCATGGTCGGATAGTGTAGTGGCTGTTGGTCCTAATATCTACGCTATTGG

250 270 290

GGGATTCAGTAATAATAGAACTAAACCTTCGTCTAGCGTCATGGTCATGGATTGTCGTAC

310 330 350

TCACACATGGTGTGAGGCCCCTAGCATGCAGGTTTCCCGTGTGTTCCAATCTACTTGCGT

370 390 410

CCTTGATGGGAAAATATATGTAACAGGAGGCCGCGGAACTCTCGATTCAACGAAATGGAT

430 450 470

GGAGGTTTTTGATACGAAAACCCAAACTTGGGAGTTTTTGCAATTCCCGAGTGAGGAGAA

490 510 530

GATATGCACAGGCTATAAGTGTGAGAGCATAGTGTATGAAGGAACTGTCTATGTAAGGTC

550 570 590

GTATTTTCATAATGTGACTTACAAGCTGCATAAAGGTAGATGGATTCAGCGGCAGACTTT

610 630 650

AGGCGATGAATAATGGATGGCCGTTGCTCATCATTTTTTTGTGTGATAAAGAACGTGTTC

670 690 710

TACTTGTTGCAATAGAAGTGGTAACGGTATGATCGATTGGTATGACTCGGAAAAAGGATC

730 750 770

ATGGACAACTATGAAGGGGTTGGAAAGATTGCCTAAAGTTTATGGTAATGTTAAATTGGC

790 810 830

ATATTATGGTGGAAAAATGGTGGTGGTGTACGTGGAGTGCTAAGGAGTGGGGTAACGTGA

850 870 890

GAAAAATTTGGTGTGCGGAAATTACGATTGAAAAACGCAAGGATGGAGAGATTTGGGGGA

910 930 950

TACTAGAATGGTTTGACGATGTATATAAAGCCAAGGATGAGCTAGAATATTTAGCTGTAG

970 990 1010

TGCATGCTGTTGTTACTACCATCTGATTGATAAGAGAGTCATGTGAACATTGTTCATTGA

1030 1050 1070

TTCACCGATGCAATAACGAATTTATCTACTATCATTTGTTTTGATTTTCTTTCTAAATCT

1090 1110 1130

TTTTTGTTTGTTCTTGTATTGAATTTTACCTTACATTTATTAAGAAAGTCAACTATTTGT 1150 1170 1190

CAACGTTACTGGAAAGTTAAAAAGGTAAAAGTAATAATAATCTGAGAGTTAACTTTGGAC

1210 1230 1250

ATCTTCGCCGGAGCCGAGACGGAAGGCGTGATGGAAGAGATACAACAACAAACGCAGAAG

1270 1290 1310

GAAGAACAAAAGCACCGTGAAGAAGAAGAGGAGGAAGAGGAAGGTCCGCCTCCGGGATGG

1330 1350 1370

GAATCTGCAGTTCTTCCTCCTCCAATCGTCACCATCACCGCCGCCGTAAACCCCAATCCC

1390 1410 1430

ACCACCGTAGAAATTCCCGGTATTCTTGTAGTCTTGTCTATTTTAGGGTTTATCGATTTG

1450 1470 1490

CTTCCATTTCTTGCTACAGTCTGATCAAATTAGAGATTTTTAGTGGAGTTTGTAGACTTT

1510 1530 1550

TAGAGATAACCCATTTTCGATTCCGAGAATTGATTAGTGTTTTTTTTCTGCAAATCTTCT

1570 1590 1610

TTGTTTTTGGGGTTGTTGCAGAAAAGGCCCAAATGGTATGTGGATCTTGCAGGCGTTTGC

1630 1650 1670

TTTCTTATCTAAGAGGATCCAAACATGTTAAGTGCTCCTCTTGTCAGACTGTTAATCTCG

1690 1710 1730

TTCTTGAAGGTTCGTTCTTCCATGGCTTTTTTATCTCTTATTCATTACTTGAAAAGCTTT

1750 1770 1790

TGTTGATAATCTCAGTCACTTGAAACTCTTAATGGAACAATCTTGGAATGCTCTCTCAGT

1810 1830 1850

CTAGTTTTACTTAGCATGTGTGAATGATATATCTATGTTCTTTTGAGAATCTCAAAATGT

1870 1890 1910

AAGCTTCCTGAGACCAAATGAGTTTAGTTCTTAACTGACACAAGAATGATCTTTGGTTAG

1930 1950 1970

GATTCTTCTCTTAAGCTTTTGTGAGCCTTTTGGTCTCTACTCCATCATAATGTCTCCTTT

1990 2010 2030

GTAGACCATTTATGTGGTCTTTATCCTTTACTCTTACTACTCTTGGGGAAATTGTGTGAT

2050 2070 2090

CTTAAGACCAAGATTGTTCTTCTTAGCTTGTGAATCACTTGGCCTCATTATTGATGAAAT

2110 2130 2150

AGCCTTCTTCTCTTATCGGTTCTGGACTTGTCGTTCTTTGTTTGCAGCTAACCAGGTTGG

2170 2190 2210

TCAGGTGAATTGCAACAATTGCAAACTGCTACTGATGTATCCTTATGGAGCTCCAGCTGT

2230 2250 2270 TAGATGTTCCTCCTGCAATTCTGTCACAGATATCAGTGTATGTATTCACAGATGGTTTTG

2290 2310 2330

TGCTCCATGTCCAAATCATACTTGGAAGAGTTGATACATTTTGAGATCCGAGTAAGTAAT

2350 2370 2390

CATCTGATGAATCATTTATAATAAACTGTGTTATATTTCAGGAAAACAACAAACGACCTC

2410 2430 2450

CATGGTCTGAGCAGCAAGGACCACTCAAAAGTTTAAGCAGTCTCAGGAGAGCAGAGAATT

2470 2490 2510

AAACTTGAACCGATTTTTGTCAATTTTGAACCGGTTTGACGACTAAAAACCTTGTAATAA

2530 2550 2570

TGTCGAAGGATAGATGAAATAAAATCACCATTAATAATCTCATTGAATTCCCATTCTTTC

2590 2610 2630

AGATATTACTTGCTCATCATCCTTTACTGTTTTAAGCTTTAGTGGTTAAAAAGAATGTGT

2650 2670 2690

ATATATCCATACAAAAGTTGATATATGTACTGGACCAATATAAACAAACACAGCTCACAG

2710 2730 2750

TCTCACACAATACATAAAAACAAAATTCATATTTCACAGGTGAGAAAAACTAACTAGTAG

2770 2790 2810

TCTACTTGGCCGAATTTGTCAATGAATTTCAATAATTAGGTCGTATAAATAGCAAACAAA

2830 2850 2870

ACATGGACTCTTACCCAACCAAATATGCATAAATAATTTACATTACAGTTTCATATAAAA

2890 2910 2930

TACAAACTAATGGTGGGTCCTCGAGAGAGCTAACAAGAGCTGTGTGTGGGTGAAGAACCA

2950 2970 2990

ACTTGTCAACGAAACCAATTTAATGGAAATCAACCCTAAATTTAATGAAACCTTGGACGA

3010 3030 3050

AACTTACATTTTGTTAACCAGTTTATCCTTTTAAATCAAACCTGCATAGAATTTTGATTT

SEQ ID NO:60

MetGluGluIleGlnGlnGlnThrGlnLysGluGluGlnLysHisArgGluGluGluGlu

10 20

GluGluGluGluGlyProProProGlyTrpGluSerAlaValLeuProProProIleVal

30 40

ThrlleThrAlaAlaValAsnProAsnProThrThrValGluIleProGluLysAlaGln

50 60

MetValCysGlySerCysArgArgLeuLeuSerTyrLeuArgGlySerLysHisValLys

70 80 19

CysSerSerCysGlnThrValAsnLeuValLeuGluAlaAsnGlnValGlyGlnValAsn

90 100

CysAsnAsnCysLysLeuLeuLeuMetTyrProTyrGlyAlaProAlaValArgCysSer

110 120

SerCysAsnSerValThrAspIleSerGluAsnAsnLysArgProProTrpSerGluGln

130 140

GlnGlyProLeuLysSerLeuSerSerLeuArgArgAlaGluAsn

150

SEQ ID NO:61

MetGluGluIleGlnGlnGlnThrGlnLysGluGluGlnLysHisArgGluGluGluGlu

10 20

GluGluGluGluGlyProProProGlyTrpGluSerAlaValLeuProProProIleVal

30 40

ThrlleThrAlaAlaValAsnProAsnProThrThrValGluIleProGluLysAlaGln

50 60

MetValCysGlySerCysArgArgLeuLeuSerTyrLeuArgGlySerLysHisValLys

70 80

CysSerSerCysGlnThrValAsnLeuValLeuGluAlaAsnGlnValGlyGlnValAsn

90 100

CysAsnAsnCysLysLeuLeuLeuMetTyrProTyrGlyAlaProAlaValArgCysSer

110 120

SerCysAsnSerValThrAspIleSerValCysIleHisArgTrpPheCysAlaProCys

130 140

ProAsnHisThrTrpLysSer

SEQ ID NO:62

CysXxxXxxCysXxxXxxLeuLeuXxxTyrXxxXxxGlyXxxXxxXxxValXxxCysSerSerCys

SEQ ID NO: 63

LeuValCysHisGlyCysArgAsnLeuLeuMetTyrProArgGlyAlaSerAsnValArgCysAlaLeuCysA snThrlleAsnMetVal

IlelleCysGlyGlyCysArgThrMetLeuMetTyrThArgGlyAlaSerSerValArgCysSerCysCysG InThrThrAsnLeuVal 20

IleAsnCysGlyHisCysArgThrThrLeuMetTyrProTyrGlyAlaSerSerValLysCysAlaValCysG InPheValThrAsnVal

SEQ ID NO: 64

LeuValCysSerGlyCysArgAsnLeuLeuMetTyrProValGlyAlaThrSerValCysCysAlaValCysA snAlaValThrAlaVal

LeuValCysGlyGlyCysHisThrLeuLeuMetTyrlleArgGlyAlaThrSerValGlnCysSerCysCysH isThrVa1 snLeuAla

ValAsnCysGlyAsnCysMetMetLeuLeuMetTyrGlnTyrGlyAlaArgSerValLysCysAlaValCysA snPheValThrSerVal

SEQ ID NO: 65

MetValCysGlySerCysArgArgLeuLeuSerTyrLeuArgGlySerLysHisValLysCysSerSerCysG InThrValAsnLeuVal

ValAsnCysAsnAsnCysLysLeuLeuLeuMetTyrProTyrGlyAlaProAlaValArgCysSerSerCysA snSerValThrAspIle

SEQ ID NO: 66

Nucleic acid sequence of C

10 30 50

AGCAACAACAACAACAACCAGCAACCACCACCAACCTCCGTCTATCCACCTGGCTCCGCC

70 90 110

GTCACAACCGTAATCCCTCCTCCACCATCTGGATCTGCATCAATAGTCACCGGAGGAGGA

130 150 170

GCGACATACCACCACCTCCTCCAGCAACAACAGCAACAGCTTCAAATGTTCTGGACATAC

190 210 230

CAGAGACAAGAGATCGAACAGGTAAACGATTTCAAAAACCATCAGCTCCCTCTAGCTCGT

250 270 290

ATCAAAAAAATCATGAAAGCTGATGAAGATGTGCGTATGATCTCCGCCGAAGCACCGATT

310 330 350

CTCTTCGCGAAAGCTTGTGAGCTTTTCATTCTCGAACTTACGATTAGATCTTGGCTTCAC

370 390 410

GCTGAAGAGAACAAACGTCGTACGCTTCAGAAAAACGATATCGCTGCTGCGATTACTAGA

430 450 470

ACCGATATCTTCGATTTCCTTGTTGATATTGTTCCTAGGGAAGAGATCAAGGAAGAGGAA

490 510 530

GATGCAGCATCGGCTCTTGGTGGAGGAGGTATGGTTGCTCCCGCCGCGAGCGGTGTTCCT 1

550 570 590

TATTATTATCCACCGATGGGACAACCGGCGGTTCCTGGAGGGATGATGATTGGAAGACCG

610 630 650

GCGATGGATCCTAGCGGTGTTTATGCTCAGCCTCCTTCTCAGGCATGGCAAAGCGTTTGG

670 690 710

CAGAATTCAGCTGGTGGTGGTGATGATGTGTCTTATGGAAGTGGAGGAAGTAGCGGCCAT

730 750 770

GGTAATCTCGATAGCCAAGGTTGAGCTATGGAACCAGAAGCTTAGAGATTTAATCATCAT

790 810 830

TTCGACCCTGCAAGTGTCTGATTCTTATATGTCTATGATTCGAATGACTTA

SEQ ID NO: 67

Amino acid sequence of C

SerAsnAsnAsnAsnAsnGlnGlnProProProThrSerValTyrProProGlySerAla

10 20

ValThrThrVal lleProProProProSerGlySerAlaSerlleValThrGlyGlyGly

30 40

AlaThrTyrHisHisLeuLeuGlnGlnGlnGlnGlnGlnLeuGlnMetPheTrpThrTyr

50 60

GlnArgGlnGluIleGluGlnValAsnAspPheLysAsnHisGlnLeuProLeuAlaArg

70 80

IleLysLysIleMetLysAlaAspGluAspValArgMetlleSerAlaGluAlaProIle

90 100

LeuPheAlaLysAlaCysGluLeuPhelleLeuGluLeuThrlleArgSerTrpLeuHis

110 120

AlaGluGluAsnLysArgArgThrLeuGlnLysAsnAspI leAlaAlaAlalleThrArg

130 140

ThrAspIlePheAspPheLeuValAspIleValProArgGluGluIleLysGluGluGlu

150 160

AspAlaAlaSerAlaLeuGlyGlyGlyGlyMetValAlaProAlaAlaSerGlyValPro

170 180

TyrTyrTyrProProMetGlyGlnProAlaValProGlyGlyMetMetlleGlyArgPro

190 200

AlaMetAspProSerGlyValTyrAlaGlnProProSerGlnAlaTrpGlnSerValTrp

210 220 2

GlnAsnSerAlaGlyGlyGlyAspAspValSerTyrGlySerGlyGlySerSerGlyHis

230 240

GlyAsnLeuAspSerGlnGly

SEQ ID NO: 68

Nucleic acid sequence of CC

10 30 50

AGTATGGATGAGCTTTCAGAAGCTTCTCAGATACTCACATGTTGCTCTGACATGGTGTAC

70 90 110

TGCACGGTTTGCGCATGTATGCAGACACAACACAAGATGGAAATGGACAAGAGGGACGGT

130 150 170

AAGTTCGGGCCACAGCCAATGGCAGTGCCTCCGGCTCAGCAAATGTCACGGTTTGATCAA

190 210 230

GCCACCCCACCCGCAGTCGGTTATCCTCCACAACAAGGTTATCCACCTTCTGGTTATCCT

250 270 290

CAACACCCTCCACAAGGTTATCCACCTTCTGGCTATCCTCAAAACCCTCCTCCCTCAGCT

310 330 350

TATTCTCAATACCCTCCTGGGGCTTATCCTCCTCCTCCCGCTTACCCAAAGTGATCACTC

370 390 410

TTTGCCTGTTTTCTCTCCCGATTGGAAAATTTTATTTCATCTTTTTTTAATGCTGTCTTG

430 450 470

TTACGGGTCAAGAATTGAACGTTCGCTGATTGTTTTGAGGTCGTTGTTTGTATGAGATTT

490 510 530

TGACCTCGCATGTTGTTGTTGTTTTCTGAAACGTCCCTCTTGGACTAAGAGATTTCATGA

550 CTTAAAAAAAAAAAAAAAAAA

SEQ ID NO: 69

Amino acid sequence of CC

SerMetAspGluLeuSerGluAlaSerGlnlleLeuThrCysCysSerAspMetValTyr

10 20

CysThrValCysAlaCysMetGlnThrGlnHisLysMetGluMetAspLysArgAspGly 3

30 40

LysPheGlyProGlnProMetAlaValProProAlaGlnGlnMetSerArgPheAspGln

50 60

AlaThrProProAlaValGlyTyrProProGlnGlnGlyTyrProProSerGlyTyrPro

70 80

GlnHisProProGlnGlyTyrProProSerGlyTyrProGlnAsnProProProSerAla

90 100

TyrSerGlnTyrProProGlyAlaTyrProProProProAlaTyrProLys

110

SEQ ID NO: 70

Nucleic acid sequence of FF

10 30 50

AGGTTTCCGACGTTGATGACCCAATTTCCGTCGTCGACGAAGACGATTCCGGCATCGTAT

70 90 110

TTGCTTCCGTTACAATGGCCTCAGCCGCAGAACGAGGAGATTCTTCTCGCCATGGAAGAA

130 150 170

GCTGAGTTCGAAGAAAAGTGCAACGAGATCAGAAAGATGAGTCCTGCTTTACCGGTAATT

190 210 230

GGAAAACCAGTCGTCAACAACGAACAAGAAGAGGATGATAATGAATCAGAGGATGATGAT

250 270 290

GCAGATAATGCAGAGGAATCAGATGGTGAAGAGTTTGAGCAAGAAACCGGATAAATAATC

310 330 350

TTGAGGCCGAAAATACACAAGGGTTATTGATGGCATTGGCTTGAAACTTGAGGACCCTTA

370 390 410

TCTAAATCTTCTTGTGATAAAACGACTGTGATTCTGACTTTGTAAACCANGTTTTTTTCT

430 450 470

TTTCTTAGGAACGACTGAAATGTTCACTTTTGGCCCTAAGGTTAGTCAGTGGATTATTCG

490 510 530

TAGTTAATTGTCTCAATCTCATGGTGTTAATTGTGTTAGTGTATTGACATTGAATTTTAT

550 570

GGTTTATAGATTGTAGTGATTTGATGAAAAAAAAAAAAAAAAAAAAA 24

SEQ ID NO: 71

Amino acid sequence of FF

ArgPheProThrLeuMetThrGlnPheProSerSerThrLysThrlleProAlaSerTyr

10 20

LeuLeuProLeuGlnTrpProGlnProGlnAsnGluGluIleLeuLeuAlaMetGluGlu

30 40

AlaGluPheGluGluLysCysAsnGluIleArgLysMetSerProAlaLeuProVallle

50 60

GlyLysProValValAsnAsnGluGlnGluGluAspAspAsnGluSerGluAspAspAsp

70 80

AlaAspAsnAlaGluGluSerAspGlyGluGluPheGluGlnGluThrGly

90

SEQ ID NO: 72

Nucleic acid sequence of GG

10 30 50

AGGGAAACAATGAGCCAGTACAATCAACCTCCCGTTGGTGTTCCTCCTCCTCAAGGTTAT

70 90 110

CCACCGGAGGGATATCCAAAAGATGCTTATCCACCACAAGGATATCCTCCTCAGGGATAT

130 150 170

CCTCAGCAAGGCTATCCACCTCAGGGATATCCTCAACAAGGTTATCCTCAGCAAGGATAT

190 210 230

CCTCCACCGTACGCGCCTCAATATCCTCCACCACCGCAGCATCAGCAACAACAGAGCAGT

250 270 290

CCTGGCTTTCTAGAAGGATGTCTTGCTGCTCTGTGTTGTTGCTGTCTCTTGGATGCTTGC

310 330 350

TTCTGATTGGAGTCTCTCTCTCTCTGCATAAAGCTTCGGGATTTATTTGTAAGAGGGTTT

370 390 410

TGGTTAAACAAAAACCTTAATTGATTTGTGGGGCATTAAAAATGAATCTCTCGATGATTC

430 450 470

TCTTTCGTTTTATGTGTAATGTTCTTCGGTTCATAACATTTTAACTATTGTCTATCGACG

490 510 530

TTCTGCCTTAGTTTGTATTTGATTATGGGAATGTAAATTGGTTGGGAGACACTATTCTAT 5

550 570

GCCATAGTTTATTGCTTGGATCTTCAAAAAAAAAAAAAAAAAA

SEQ ID NO: 73

Amino acid sequence of GG

ArgGluThrMetSerGlnTyrAsnGlnProProValGlyValProProProGlnGlyTyr

10 20

ProProGluGlyTyrProLysAspAlaTyrProProGlnGlyTyrProProGlnGlyTyr

30 40

ProGlnGlnGlyTyrProProGlnGlyTyrProGlnGlnGlyTyrProGlnGlnGlyTyr

50 60

ProProProTyrAlaProGlnTyrProProPrσProGlnHisGlnGlnGlnGlnSerSer

70 80

ProGlyPheLeuGluGlyCysLeuAlaAlaLeuCysCysCysCysLeuLeuAspAlaCys

90 100

Phe

SEQ IDNO: 74

Nucleic acid sequence of HH

10 30 50

AGTGATGTTCTTCCTAAGTCCGTTGACTGGAGAAACGAAGGCGCAGTGACTGAAGTCAAA

70 90 110

GATCAAGGCCTTTGCAGGAGTTGTTGGGCTTTCTCCACTGTGGGAGCAGTGGAAGGCTTA

130 150 170

AACAAGATTGTGACTGGAGAGCTAGTAACTTTGTCTGAGCAAGATTTGATCAATTGTAAC

190 210 230

AAAGAAAACAATGGTTGCGGAGGAGGCAAAGTCGAGACAGCCTATGAGTTCATCATGAAC

250 270 290

AATGGTGGTCTTGGTACCGACAACGATTATCCTTACAAAGCTCTCAATGGAGTCTGCGAA

310 330 350

GGCCGCCTCAAGGAAGACAACAAGAATGTTATGATTGATGGGTATGAGAATTTGCCTGCA

370 390 410

AACGATGAAGCCGCTCTCATGAAAGCGGTTGCTCACCAGCCTGTGACTGCCGTTGTCGAT 6

430 450 470

TCCAGCAGCCGAGAGTTTCAGCTTTATGAATCGGGAGTGTTTGACGGAACTTGCGGAACA

490 510 530

AACCTAAACCATGGTGTTGTTGTGGTCGGGTATGGAACCGAGAATGGTCGTGACTACTGG

550 570 590

ATTGTGAAAAACTCGAGGGGCGACACATGGGGGGAGGCTGGCTACATGAAGATGGCTCGC

610 630 650

AACATTGCCAATCCAAGAGGCATATGTGGCATCGCAATGCGAGCTTCATACCCTCTCAAG

670 690 710

AACTCGTTTTCTACGGATAAAGTTTCGGTTGCCTAATAATATGAACTAAATGTATGCCAT

730 750 770

GGAACGGATCGGTTAAGCCATTATCGTTATTCGACTTTGAAGGAAACTAAAAAATAATGT

790 810 830

GGTCGATTGGTTTGGTTTTGTTATATATTATGCATTTGTATGGGGGTCAGTCAATGTTTG

850 870 890

AACTTTGTATAATATTTCTTTGGGTCTAGTGATAAATATTTTCCCTTTTGCGAAAAAAAA

910 AAAAAAAAAA

SEQ ID NO: 75

Amino acid sequence of HH

SerAspValLeuProLysSerValAspTrpArgAsnGluGlyAlaValThrGluValLys

10 20

AspGlnGlyLeuCysArgSerCysTrpAlaPheSerThrValGlyAlaValGluGlyLeu

30 40

AsnLysIleValThrGlyGluLeuValThrLeuSerGluGlnAspLeuIleAsnCysAsn

50 60

LysGluAsnAsnGlyCysGlyGlyGlyLysValGluThrAlaTyrGluPhelleMetAsn

70 80

AsnGlyGlyLeuGlyThrAspAsnAspTyrProTyrLysAlaLeuAsnGlyValCysGlu

90 100

GlyArgLeuLysGluAspAsnLysAsnValMetlleAspGlyTyrGluAsnLeuProAla

110 120

AsnAspGluAlaAlaLeuMetLysAlaValAlaHisGlnProValThrAlaValValAsp

130 140 7

SerSerSerArgGluPheGlnLeuTyrGluSerGlyValPheAspGlyThrCysGlyThr

150 160

AsnLeuAsnHisGlyValValValValGlyTyrGlyThrGluAsnGlyArgAspTyrTrp

170 180

IleValLysAsnSerArgGlyAspThrTrpGlyGluAlaGlyTyrMetLysMetAlaArg

190 200

AsnlleAlaAsnProArgGlylleCysGlylleAlaMetArgAlaSerTyrProLeuLys

210 220

AsnSerPheSerThrAspLysValSerValAla

230

SEQ ID NO: 76

Nucleic acid sequence of I

10 30 50

AGCGAAATGCCAGTTTCAGCTCCATCTCCGCCTCGTCTTCATTCTCCGTTCATTCACTGT

70 90 110

CCCATCAATTTCACTCCTTCTTCTTTCTCGGCGAGGAATCTCCGGTCGCCGTCAACATCT

130 150 170

TATCCCCGAATCAAAGCTGAACTCGATCCCAACACGGTAGTCGCGATATCTGTAGGCGTA

190 210 230

GCAAGCGTCGCATTAGGAATCGGAATCCCTGTGTTCTACGAGACTCAAATCGACAATGCG

250 270 290

GCTAAGCGAGAGAATACTCAACCTTGTTTTCCCTGTAATGGCACCGGAGCTCAGAAATGC

310 330 350

AGATTGTGTGTGGGAAGTGGTAATGTGACCGTAGAGCTTGGTGGAGGAGAGAAAGAAGTC

370 390 410

TCAAACTGTATCAACTGTGATGGTGCTGGTTCCTTAACTTGCACTACTTGTCAAGGCTCT

430 450 470

GGTGTTCAACCTCGATACCTTGATCGAAGGGAGTTCAAGGACGATGACTAAATACCTTGC

490 510 530

TCTAAGGAACATTTCTTTTCTTCTCCCTTCTCACATTTCTTCATTGTACAATGCTGTTTT

550 570 590

GTTCACCAAACATGTTGAGAGAACATCATGACATGGATATTGTAATTGTGAAAGAAAACC

610 630 650 28

ACCAGAGTTCAATCAAATGTTTCTTCTTGTACTTAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 77

Amino acid sequence of I

SerGluMetProValSerAlaProSerProProArgLeuHisSerProPhelleHisCys

10 20

ProIleAsnPheThrProSerSerPheSerAlaArgAsnLeuArgSerProSerThrSer

30 40

TyrProArglleLysAlaGluLeuAspProAsnThrValValAlalleSerValGlyVal

50 60

AlaSerValAlaLeuGlylleGlylleProValPheTyrGluThrGlnlleAspAsnAla

70 80

AlaLysArgGluAsnThrGlnProCysPheProCysAsnGlyThrGlyAlaGlnLysCys

90 100

ArgLeuCysValGlySerGlyAsnValThrValGluLeuGlyGlyGlyGluLysGluVal

110 120

SerAsnCysIleAsnCysAspGlyAlaGlySerLeuThrCysThrThrCysGlnGlySer

130 140

GlyValGlnProArgTyrLeuAspArgArgGluPheLysAspAspAsp

150

SEQ IDNO: 78

Nucleic acid sequence of II

10 30 50

AGAGAAAACATGGGAGGTGACAATGATAATGACAAAGACAAAGGGTTTCATGGGTATCCT

70 90 110

CCCGCTGGATACCCACCCCCTGGGGCTTATCCACCCGCTGGATACCCACAACAAGGTTAC

130 150 170

CCTCCACCACCCGGTGCTTACCCGCCTGCAGGTTATCCTCCGGGTGCCTACCCACCTGCT

190 210 230

CCTGGTGGTTATCCTCCCGCCCCTGGTTATGGTGGTTATCCTCCAGCTCCTGGTTATGGA

250 270 290 9

GGTTATCCTCCTGCACCTGGTCATGGTGGTTACCCTCCTGCTGGCTATCCTGCTCATCAC

310 330 350

TCAGGACACGCAGGAGGAATTGGGGGTATGATTGCAGGTGCTGCAGCTGCCTATGGAGCT

370 390 410

CACCACGTATCTCATAGCTCTCACTGTCCTTACGGACATGCTGCATATGGTCACGGTTTT

430 450 470

GGCCATGGTCATGGCTATGGCTATGGTCATGGTCATGGTAAGTTCAAGCATGGGAAGCAC

490 510 530

GGGAAGTTCAAGCATGGGAAGCATGGAATGTTTGGAGGAGGCAAGTTCAAGAAGTGGAAG

550 570 590

TGATCTAGCTATTACCTTGTGTGAATTTGTCTGGACTGACCAATGTTTCAAATAAGCCCT

610 630 650

AAACATTATATAAGTTGACTTTCGTCGGTTAGATTGCTGGTTCGAGTTGGAATAATTGAA

670 690 710

ACTTAATTAGTATCAAATCTTATTGTGTACTTTAAAGCTATCGTTGGCTTTATAATGACA

730 750 770

GATTCTGGTTTCGGTGTGTTGTTTTAAGATTTTTGTATATACTGTTTTTTACATTGCTTA

790 810

AGCTTATAGAAGTCATGATTATGATTAAAAAAAAAAAAAAAAAA

SEQ ID NO: 79

Amino acid sequence of II

ArgGluAsnMetGlyGlyAspAsnAspAsnAspLysAspLysGlyPheHisGlyTyrP.ro

10 20

ProAlaGlyTyrProProProGlyAlaTyrProProAlaGlyTyrProGlnGlnGlyTyr

30 40

ProProProProGlyAlaTyrProProAlaGlyTyrProProGlyAlaTyrProProAla

50 60

ProGlyGlyTyrProProAlaProGlyTyrGlyGlyTyrProProAlaProGlyTyrGly

70 80

GlyTyrProProAlaProGlyHisGlyGlyTyrProProAlaGlyTyrProAlaHisHis

90 100

SerGlyHisAlaGlyGlylleGlyGlyMetlleAlaGlyAlaAlaAlaAlaTyrGlyAla

110 120

HisHisValSerHisSerSerHisCysProTyrGlyHisAlaAlaTyrGlyHisGlyPhe 0

130 140

GlyHisGlyHisGlyTyrGlyTyrGlyHisGlyHisGlyLysPheLysHisGlyLysHis

150 160

GlyLysPheLysHisGlyLysHisGlyMetPheGlyGlyGlyLysPheLysLysTrpLys

170 180

SEQ ID NO: 80

Nucleic acid sequence of K

10 30 50

AGTGTCACTACTCCATCCGAGGAGGATTCAAACAACGGTTTACCGGTTCAGCAACCCGGT

70 90 110

ACACCGAACCAGCGAACCAGAGTTCCCGTGAGTCAATTCGCGCCGCCGAATTATCAGCAA

130 150 170

GCTAATGTTAACCTATCTGTTGGGAGGCCATGGAGCACTGGTTTGTTTGATTGTCAAGCA

190 210 230

GACCAAGCCAATGCCGTTTTGACCACAATTGTACCTTGTGTAACATTTGGACAAATAGCA

250 270 290

GAAGTGATGGATGAAGGAGAGATGACTTGTCCTCTTGGAACTTTCATGTACTTATTGATG

310 330 350

ATGCCGGCTTTATGCTCTCACTGGGTGATGGGATCAAAGTATAGAGAAAAAATGAGGAGA

370 390 410

AAATTTAATCTTGTGGAAGCTCCATATTCAGATTGTGCCAGTCATGTCCTATGCCCTTGT

430 450 470

TGCTCTCTTTGTCAAGAATACAGAGAGCTCAAGATTAGGAATCTTGATCCTTCTCTAGGT

490 510 530

TGGAATGGGATACTTGCTCAAGGACAAGGACAATATGAGAGAGAAGCACCAAGTTTTGCT

550 570 590

CCTACAAATCAATATATGTCTAAGTAAACATTTGATTTTAGTTGACTTCCATATTTATTA

610 630 650

AAACATTATTTGTGGACCATTGTACAATGAAAGTGTGCTATATTAAAATTTGCAATGCAA

670 690

GTGTGAGATTGATAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 81 1

Amino acid sequence of K

SerValThrThrProSerGluGluAspSerAsnAsnGlyLeuProValGlnGlnProGly

10 20

ThrProAsnGlnArgThrArgValProValSerGlnPheAlaProProAsnTyrGlnGln

30 40

AlaAsnValAsnLeuSerValGlyArgProTrpSerThrGlyLeuPheAspCysGlnAla

50 60

AspGlnAlaAsnAlaValLeuThrThrlleValProCysValThrPheGlyGlnlleAla

70 80

GluValMetAspGluGlyGluMetThrCysProLeuGlyThrPheMetTyrLeuLeuMet

90 100

MetProAlaLeuCysSerHisTrpValMetGlySerLysTyrArgGluLysMetArgArg

110 120

LysPheAsnLeuValGluAlaProTyrSerAspCysAlaSerHisValLeuCysProCys

130 140

CysSerLeuCysGlnGluTyrArgGluLeuLysIleArgAsnLeuAspProSerLeuGly

150 160

TrpAsnGlylleLeuAlaGlnGlyGlnGlyGlnTyrGluArgGluAlaProSerPheAla

170 180

ProThrAsnGlnTyrMetSerLys

SEQ ID NO: 82

Nucleic acid sequence of M

10 30 50

AGAAAATACGAAAAGGTCTCCCTCCCAGCACCTTACGTGGCTGGACACTCGAGCCATCAC

70 90 110

GAAGACGACGGTCAATACTATCCCGGCAAATACGAAAAAGCCTCCCTCCCAGCACCTTAC

130 150 170

GTGGCCGGATATCCGAGCCATCATGAAGACGATGGTCAATACTATCCTGGCAAATACGAA

190 210 230

AAGGTCTCCCTCCCAGCACCTTACGTGGTCGGACACCCGAGCCACTCCGAAGATGATGGC

250 270 290

CAATACTATCCCGGCAAATACGAAAAGGCCTCCGTCCCATCAGCTTACGTGGCCGAACAC

310 330 350

TCGAGCCACTCCGAAGATGATGGCCAATACTATCCTGGCAAATACGAAAAGCCCGAACAC 2

370 390 410

CATTACTGAAAACTCTCACACAACAATGATTCTCATCCTTCCGTAGTCTTTTAATTCGAC

430 450 470

TTTTAACAATAAAAACGTGATCTTAATTTTTCATCAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 83

Amino acid sequence of M

ArgLysTyrGluLysValSerLeuProAlaProTyrValAlaGlyHisSerSerHisHis

10 20

GluAspAspGlyGlnTyrTyrProGlyLysTyrGluLysAlaSerLeuProAlaProTyr

30 40

ValAlaGlyTyrProSerHisHisGluAspAspGlyGlnTyrTyrProGlyLysTyrGlu

50 60

LysValSerLeuProAlaProTyrValValGlyHisProSerHisSerGluAspAspGly

70 80

GlnTyrTyrProGlyLysTyrGluLysAlaSerValProSerAlaTyrValAlaGluHis

90 100

SerSerHisSerGluAspAspGlyGlnTyrTyrProGlyLysTyrGluLysProGluHis

110 120

HisTyr

SEQ ID NO: 84

Nucleic acid sequence of 00

10 30 50

AGCCGATCTCAGATTCTTCCATCTTCCAGGAGGAATTTCAGTGTGGCGACCACACAGCTT

70 90 110

GGCATTCCAACAGACGATCTAGTCGGCAATCACACCGCCAAATGGATGCAGGATAGAAGC

130 150 170

AAGAAATCACCTATGGAACTGATTAGTGAGGTTCCACCTATCAAAGTTGATGGAAGGATT

190 210 230

GTTGCTTGTGAAGGAGACACCAATCCGGCCCTAGGTCATCCAATCGAGTTCATATGCCTC 3

250 270 290

GACCTAAATGAGCCTGCGATCTGCAAGTACTGCGGCCTTCGTTATGTTCAAGATCATCAC

310 330 350

CATTGAGGCAAATTCTGAAAGTGAACTGCTGGTCTCTCTCCCCTTTTTATTGCATTTTTA

370 390 410

AGTTTGTGTATTGTTTTTTTCTGGTGTGCCTACTACATCTTCAGCTATATTATCTAATAA

430 450 470

AGGATTCGATCAAAGTCGGGTAAGTTTGATTTTTGTTTGATCTCACTTCAGCACTTGTCA

490 510 530

TGTTGTAACATTCAATCTCTGATATCACTGTCTTTTACATGCCAAAAAAAAAAAAAAAAA

550 AAAAAAAAAAAAAAAA

SEQ ID NO: 85

Amino acid sequence of OO

SerArgSerGlnlleLeuProSerSerArgArgAsnPheSerValAlaThrThrGlnLeu

10 20

GlylleProThrAspAspLeuValGlyAsnHisThrAlaLysTrpMetGlnAspArgSer

30 40

LysLysSerProMetGluLeuIleSerGluValProProlleLysValAspGlyArglle

50 60

ValAlaCysGluGlyAspThrAsnProAlaLeuGlyHisProIleGluPhelleCysLeu

70 80

AspLeuAsnGluProAlalleCysLysTyrCysGlyLeuArgTyrValGlnAspHisHis

90 100

HisEndGlyLysPhe

SEQ ID NO: 86

Nucleic acid sequence of P

10 30 50

AGAACAGCTCGAGTTCCTTATGGGCCTAGACTCTCTGGTGGTGGTTACAACCGATCTGGA

70 90 110

AACAGGGTTCCGCGTAACAAACCAAGCTTCCCCAATAGCACCGAGTCCAATGGTGAGGCT 4

130 150 170

AATCAATTCAATGGCCCAAGAATAATGAACCCCCATGCTGCTGAGTTCATACCGAGTCAA

190 210 230

CCTTGGGTTTCTAATGGGTATCCAGTGTCACCAAATGGCTATTTAGCATCCCCAAATGGT

250 270 290

GCAGAAATAACACAGAATGGGTACCCTTTGTCACCAGTAGCAGGTGGATATCCGTGTAAC

310 330 350

ATGTCCGTTACACAGCCTCAGGATGGACTTGTTTCAGAGGAATTACCTGGTGCTGGAAGC

370 390 410

TCTGAGGAGAAGAGCGGAAGCGAAGAAGAAAGCAACAACGACAAAAATGCTGGAGAGGAT

430 450 470

GACGAAGCCGTTGGACAAGAAACTACAGATACACCTGAAAATGGACATTCGACAGTAGGT

490 510 530

GAAGTGGAAACCACATCACATGAGACTTGTGATGAGAAAAATGGAGAACGACAAGGAGGC

550 570 590

AAGTGCTGGGGAGATTACAGCGATAATGAAATCGAGCAAATTGAAGTTACAAGTTGAAGA

610 630 650

CGCAACTGTCTGTTACTGAAGTATTAACATTGAGGCTAAAGGAATGCGGAGACATTTTGG

670 690 710

CTCCATTGATGAGGTTAAAGGTAAACAATCATCATAGTCGAGAAAAGCATTTTTACATGT

730 750 770

GAATGTTTTGTGTTGTAGCGCAGGACCAAGGCTCGTCACTCCTGCTTTAACAACTTTTCT

790 810 830

CCTGCTTTCAGTTTTTGGTTTCATAGCTGAAAACTAGATATATTCAACTCCTTAATAAAA

850 870

GATTTGTCCCTTTGTTTAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 87

Amino acid sequence of P

ArgThrAlaArgValProTyrGlyProArgLeuSerGlyGlyGlyTyrAsnArgSerGly

10 20

AsnArgValProArgAsnLysProSerPheProAsnSerThrGluSerAsnGlyGluAla

30 40

AsnGlnPheAsnGlyProArglleMetAsnProHisAlaAlaGluPhelleProSerGln

50 60

ProTrpValSerAsnGlyTyrProValSerProAsnGlyTyrLeuAlaSerProAsnGly 5

70 80

AlaGluIleThrGlnAsnGlyTyrProLeuSerProValAlaGlyGlyTyrProCysAsn

90 100

MetSerValThrGlnProGlnAspGlyLeuValSerGluGluLeuProGlyAlaGlySer

110 120

SerGluGluLysSerGlySerGluGluGluSerAsnAsnAspLysAsnAlaGlyGluAsp

130 140

AspGluAlaValGlyGlnGluThrThrAspThrProGluAsnGlyHisSerThrValGly

150 160

GluValGluThrThrSerHisGluThrCysAspGluLysAsnGlyGluArgGlnGlyGly

170 180

LysCysTrpGlyAspTyrSerAspAsnGluIleGluGlnlleGluValThrSer

190

SEQ ID NO: 88

Nucleic acid sequence of T

10 30 50

AGAGACCATCCAGCTTACCATCAGATCCACCAGCAACAACAACAACAGCTCACTCAACAG

70 90 110

CTTCAATCTTTCTGGGAGACTCAATTCAAAGAGATTGAGAAAACCACTGATTTCAAGAAC

130 150 170

CATAGCCTTCCATTGGCAAGAATCAAGAAAATCATGAAAGCTGATGAAGATGTGCGTATG

190 210 230

ATCTCGGCCGAGGCGCCTGTTGTGTTCGCCAGGGCCTGCGAGATGTTTATTCTGGAGCTT

250 270 290

ACGTTAAGGTCTTGGAACCATACTGAGGAGAACAAGAGAAGGACGTTGCAGAAGAATGAT

310 330 350

ATCGCGGCTGCGGTGACTAGAACTGATATTTTTGATTTTCTTGTGGATATTGTTCCTCGG

370 390 410

GAGGATCTTCGTGATGAAGTCTTGGGTGGTGTTGGTGCTGAAGCTGCTACAGCTGCGGGT

430 450 470

TATCCGTATGGATACTTGCCTCCTGGAACAGCTCCAATTGGGAACCCGGGAATGGTTATG

490 510 530

GGTAACCCGGGCGCGTATCCGCCGAACCCGTATATGGGTCAGCCAATGTGGCAACAACCA

550 570 590

GGACCTGAGCAGCAGGATCCTGACAATTAGCTTGGCCTAATAAACTAGCCGTCTAATTCG 6

610 630 650

AAGCTCTCCCCGGTGGATCTACTCAAGAAGAAGAATGTTAATAGAAAACTATTGCGACAT

670 690 710

AAAAAGTTTGGTGTAGTAGAATAATTTCTGTTTTATGATCCATGGATTTATCTATTGTTA

730 750 770

TTCAGTTTGGTTTATCTTGTCATCAAACTGTTTTCGGTCAATGTAACAAATTCATAAACT

790 810 830

GAGAATTGAACTTACAAAAGGCTAGATTACTACTTATAAAGTTCAAAGCTAAAAAAAAAA

AAAAAAAA

SEQ ID NO: 89

Amino acid sequence of T

ArgAspHisProAlaTyrHisGlnlleHisGlnGlnGlnGlnGlnGlnLeuThrGlnGln

10 20

LeuGlnSerPheTrpGluThrGlnPheLysGluIleGluLysThrThrAspPheLysAsn

30 40

HisSerLeuProLeuAlaArglleLysLysIleMetLysAlaAspGluAspValArgMet

50 60

IleSerAlaGluAlaProValValPheAlaArgAlaCysGluMetPhelleLeuGluLeu

70 80

ThrLeuArgSerTrpAsnHisThrGluGluAsnLysArgArgThrLeuGlnLysAsnAsp

90 100

IleAlaAlaAlaValThrArgThrAspIlePheAspPheLeuValAspIleValProArg

110 120

GluAspLeuArgAspGluValLeuGlyGlyValGlyAlaGluAlaAlaThrAlaAlaGly

130 140

TyrProTyrGlyTyrLeuProProGlyThrAlaProIleGlyAsnProGlyMetValMet

150 160

GlyAsnProGlyAlaTyrProProAsnProTyrMetGlyGlnProMetTrpGlnGlnPro

170 180

GlyProGluGlnGlnAspProAspAsn 37

SEQ ID NO: 90

Nucleic acid sequence of X

10 30 50

AGATTCGCTATTCCTGGCAAAGAAAGACAAGATTCTGTTTACAGTGGACTTCAGGAAATC

70 90 110

GATGTGAACTCTGAGCTTGTTTGTATCCACGACTCTGCCCGACCATTGGTGAATACTGAA

130 150 170

GATGTCGAGAAGGTCCTTAAAGATGGTTCCGCGGTTGGAGCAGCTGTACTTGGTGTTCCT

190 210 230

GCTAAAGCTACAATCAAAGAGGTCAATTCTGATTCGCTTGTGGTGAAAACTCTCGACAGA

250 270 290

AAAACCCTATGGGAAATGCAGACACCACAGGTGATCAAACCAGAGCTATTGAAAAAGGGT

310 330 350

TTCGAGCTTGTAAAAAGTGAAGGTCTAGAGGTAACAGATGACGTTTCGATTGTTGAATAC

370 390 410

CTCAAGCATCCAGTTTATGTCTCTCAAGGATCTTATACAAACATCAAGGTTACAACACCT

430 450 470

GATGATTTACTGCTTGCTGAGAGAATCTTGAGCGAGGACTCATGAGATATTATATCATTT

490 510 530

ACTTAGTAAGAAGACGTGTCAAGGGTATGCATGAAAAATGTTTTATTGAAATCTTTGCAT

550 570 590

CCTAGTTTGGTGGTTTATAAAATGTGCAAGATAATTGTTTCACTGAAAACTACTTGCTGT

610 630 650

GAATATGGATTCGAACAGAGCCAATTCGAAGTAGAATTTGCATATTGTAAAAAAAAAAAA

670 AAAAAAAAAA

SEQ ID NO: 91

Amino acid sequence of X

ArgPheAlalleProGlyLysGluArgGlnAspSerValTyrSerGlyLeuGlnGluIle

10 20

AspValAsnSerGluLeuValCysIleHisAspSerAlaArgProLeuValAsnThrGlu

30 40

AspValGluLysValLeuLysAspGlySerAlaValGlyAlaAlaValLeuGlyValPro

50 60 AlaLysAlaThrlleLysGluValAsnSerAspSerLeuValValLysThrLeuAspArg

70 80

LysThrLeuTrpGluMetGlnThrProGlnVallleLysProGluLeuLeuLysLysGly

90 100

PheGluLeuValLysSerGluGlyLeuGluValThrAspAspValSerlleValGluTyr

110 120

LeuLysHisProValTyrValSerGlnGlySerTyrThrAsnlleLysValThrThrPro

130 140

AspAspLeuLeuLeuAlaGluArglleLeuSerGluAspSer

150

Claims

THE CLAIMSWhat is Claimed Is:

1. An isolated DNA sequence that encodes a LSDl polypeptide.

2. The isolated DNA sequence of claim 1, wherein the sequence is selected from the group consisting of SEQ ID NO13, SEQ ID NO 14 and SEQ ID NO 15.

3. The isolated DNA sequence of claim 1, wherein the sequence comprises SEQ ID NO 13.

4. The isolated DNA sequence of claim 1 , wherein the sequence comprises SEQ ID NO 14.

5. The isolated DNA sequence of claim 1, wherein the sequence comprises SEQ ID NO 15.

6. The isolated DNA sequence of claim 1 , wherein the DNA is cDNA.

7. The isolated DNA sequence of claim 1 , wherein the DNA is genomic.

8. The isolated DNA sequence of claim 1 , wherein the polypeptide comprises SEQ ID NO 16.

9. The isolated DNA sequence of claim 1 , wherein the polypeptide comprises SEQ ID NO 17.

10. A protein encoded by the isolated DNA sequence of claim 1.

11. A chimeric construction comprising a promoter sequence and a DNA sequence according to claim 1.

12. A transformation vector comprising the isolated DNA sequence of claim 1.

13. A mutated DNA sequence derived from the DNA sequence of claim 1.

14. A transgenic plant expressing LSDl mutant genes that affect resistance to herbicidal compounds that normally result in plant cell death.

15. A transgenic plant expressing LSDl mutant genes which affect resistance to plant pathogens that normally result in plant cell death.

16. A messenger RNA encoding LSD 1.

17. An isolated DNA sequence that encodes the zinc finger consensus selected from the group consisting of SEQ ID NOS 1-3.

18. A protein containing a zinc fmger protein selected from the group consisting of CxxCxRxxLMYxxGASxVxCxxC, CxxCRxxLMYxxGASxRxVxCxxC, CxxCxxLLMYxxGAxSxCxxC, CxxCxxLLxYxxGxxxVxCSSC,

CSGCRNLLMYPVGATSVCCAVC, CGGCHTLIMYIRGATSVQCSCC, CGNCMMLLMYQYGARSVKCAVC, CGSCRRLLSYLRGSKHVKCSSC, and CNNCKLLLMYPYGAPAVRCSSC, wherein x is any substituted amino acid.

19. A gene encoding a zinc fmger protein according to claim 18.

20. An isolated DNA sequence encoding a protein according to claim 18.

21. A recombinant plant transformed with the DNA sequence as claimed in claim 1.

22. A recombinant plant transformed with the DNA sequence as claimed in claim 20.

23. An isolated DNA molecule that hybridizes under hybridization conditions to a DNA sequence as claimed in claim 1.

24. An isolated DNA molecule that hybridizes under hybridization conditions to a DNA sequence as claimed in claim 20.

25. An isolated DNA sequence that encodes a LSDl homologue.

26. The isolated DNA sequence of claim 25, wherein the homologue is selected from the group consisting of LOLl and LOL2.

27. The isolated DNA sequence of claim 25, wherein the homologue is selected from the group consisting of SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:60 and SEQ ID NO:62.

28. The isolated DNA sequence of claim 25, wherein the sequence is selected from the group consisting of SEQ ID NO:47, SEQ ID NO:54, and SEQ ID NO:59.

29. The isolated DNA sequence of claim 25, wherein the sequence comprises SEQ ID NO 47.

30. The isolated DNA sequence of claim 25, wherein the sequence comprises SEQ ID NO 54.

31. The isolated DNA sequence of claim 25, wherein the sequence comprises SEQ ID NO 59.

32. The isolated DNA sequence of claim 25, wherein the DNA is cDNA.

33. The isolated DNA sequence of claim 25, wherein the DNA is genomic.

34. A recombinant plant transformed with the DNA sequence as claimed in claim 25.

35. An isolated DNA molecule that hybridizes under hybridization conditions to a DNA sequence as claimed in claim 25.

36. A protein encoded by the isolated DNA sequence of claim 25.

37. A chimeric construction comprising a promoter sequence and a DNA sequence according to claim 25.

38. A transformation vector comprising the isolated DNA sequence of claim 25.

39. A mutated DNA sequence derived from the DNA sequence of claim 25.

40. A transgenic plant expressing LOLl mutant genes that affect resistance to herbicidal compounds that normally result in plant cell death.

41. A transgenic plant expressing LOLl mutant genes which affect resistance to plant pathogens that normally result in plant cell death.

42. A messenger RNA encoding LOLl .

43. A transgenic plant expressing LOLl mutant genes that affect resistance to herbicidal compounds that normally result in plant cell death.

44. A transgenic plant expressing LOLl mutant genes which affect resistance to plant pathogens that normally result in plant cell death.

45. A messenger RNA encoding LOL2.

46. A nucleic acid that interacts with LSDl, selected from the group consisting of the nucleic acid sequences set forth in SEQ ID NOS:66-91.

47. A protein encoded by a nucleic acid according to claim 46.