# Patent

## Title
DNA molecules and polypeptides of Pseudomonas syringae Hrp pathogenicity island and their uses

## Abstract

One aspect of the present invention relates to isolated nucleic acid molecules (i) encoding proteins or polypeptides of Pseudomonas CEL and EEL genomic regions, (ii) nucleic acid molecules which hybridize thereto under stringent conditions, or (iii) nucleic acid molecules that include a nucleotide sequence which is complementary to the nucleic acid molecules of (i) and (ii). Expression vectors, host cells, and transgenic plants which include the DNA molecules of the present invention are also disclosed. Another aspect relates to the isolated proteins or polypeptides and compositions containing the same. The nucleic acid molecules and proteins of the present invention can be used to imparting disease resistance to a plant, making a plant hypersusceptible to colonization by nonpathogenic bacteria, causing eukaryotic cell death, and treating cancerous conditions.

## Background
This application claims benefit of U.S. Provisional Patent Application Ser. Nos. 60/194,160, filed Apr. 3, 2000, 60/224,604, filed Aug. 11, 2000, and 60/249,548, filed Nov. 17, 2000, which are hereby incorporated by reference in their entirety.


This work was supported by National Science Foundation Grant No. MCB-9631530 and National Research Initiative Competitive Grants Program, U.S. Department of Agriculture, Grant No. 98-35303-4488. The U.S. Government may have certain rights in this invention.


FIELD OF THE INVENTION
The present invention relates to isolated DNA molecules corresponding to the open reading frames in the conserved effector loci and exchangeable effector loci of the Pseudomonas syringae, the isolated proteins encoded thereby, and their various uses.
BACKGROUND OF THE INVENTION
The plant pathogenic bacterium Pseudomonas syringae is noted for its diverse and host-specific interactions with plants (Hirano and Upper, 1990). A specific strain may be assigned to one of at least 40 pathovars based on its host range among different plant species and then further assigned to a race based on differential interactions among cultivars of the host. In host plants the bacteria typically grow to high population levels in leaf intercellular spaces and then produce necrotic lesions. In nonhost plants or in host plants with race-specific resistance, the bacteria elicit the hypersensitive response (HR), a rapid, defense-associated programmed death of plant cells in contact with the pathogen (Alfano and Collmer, 1997). The ability to produce either of these reactions in plants appears to be directed by hrp (HR and pathogenicity) and hrc (HR and conserved) genes that encode a type III protein secretion pathway and by avr (avirulence) and hop (Hrp-dependent outer protein) genes that encode effector proteins injected into plant cells by the pathway (Alfano and Collmer, 1997). These effectors may also betray the parasite to the HR-triggering R-gene surveillance system of potential hosts (hence the avr designation), and plant breeding for resistance based on such gene-for-gene (avr-R) interactions may produce complex combinations of races and differential cultivars (Keen, 1990). hrp/hrc genes are probably universal among necrosis-causing gram-negative plant pathogens, and they have been sequenced in P. syringae pv. syringae (Psy) 61, Erwinia amylovora Ea321, Xanthomonas campestris pv. vesicatoria (Xcv) 85-10, and Ralstonia solanacearum GMI1000 (Alfano and Collmer, 1997). Based on their distinct gene arrangements and regulatory components, the hrp/hrc gene clusters of these four bacteria can be divided into two groups: I (Pseudomonas and Erwinia) and II (Xanthomonas and Ralstonia). The discrepancy between the distribution of these groups and the phylogeny of the bacteria provides some evidence that hrp/hrc gene clusters have been horizontally acquired and, therefore, may represent pathogenicity islands (Pais) (Alfano and Collmer, 1997).
Pais have been defined as gene clusters that (i) include many virulence genes, (ii) are selectively present in pathogenic strains, (iii) have different G+C content compared to host bacteria DNA, (iv) occupy large chromosomal regions, (v) are often flanked by direct repeats, (vi) are bordered by tRNA genes and/or cryptic mobile genetic elements, and (vii) are unstable (Hacker et al., 1997). Some Pais have inserted into different genomic locations in the same species (Wieler et al., 1997). Others reveal a mosaic structure indicative of multiple horizontal acquisitions (Hensel et al., 1999). Genes encoding type III secretion systems are present in Pais in animal pathogenic Salmonella spp. and Pseudomonas aeruginosa and on large plasmids in Yersinia and Shigella spp. Genes encoding effectors secreted by the pathway in these organisms are commonly linked to the pathway genes (Hueck, 1998), although a noteworthy exception is sopE, which is carried by a temperate phage without apparent linkage to SPI1 in certain isolates of S. typhimurium (Mirold et al., 1999). Three avr/hop genes have already been shown to be linked to the hrp/hrc cluster in P. syringae: avrE and several other Hrp-regulated transcriptional units are linked to the hrpR border of the hrp cluster in P. syringae pv tomato (Pto) DC3000 (Lorang and Keen, 1995); avrPphE is adjacent to hrpY (hrpK) in Pseudomonas phaseolicola (Pph) 1302A (Mansfield et al., 1994); and hopPsyA (hrmA) is adjacent to hrpK in Psy 61 (Heu and Hutcheson, 1993). Other Pseudomonas avr genes are located elsewhere in the genome or on plasmids (Leach and White, 1996), including a plasmid-borne group of avr genes described as a Pai in Pph 1449B (Jackson et al., 1999).
Because Avr, Hop, Hrp, and Hrc proteins represent promising therapeutic treatments in both plants and animals, it would be desirable to identify other proteins encoded by the Pai's in pathogenic bacteria and identify uses for those proteins.
The present invention overcomes these deficiencies in the art.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to isolated nucleic acid molecules (i) encoding proteins or polypeptides of Pseudomonas Conserved Effector Loci (“CEL”) and Exchangeable Effector Loci (“EEL”) genomic regions, (ii) nucleic acid molecules which hybridize thereto under stringent conditions, or (iii) nucleic acid molecules that include a nucleotide sequence which is complementary to the nucleic acid molecules of (i) and (ii). Expression vectors, host cells, and transgenic plants which include the DNA molecules of the present invention are also disclosed. Methods of making such host cells and transgenic plant are disclosed.
A further aspect of the present invention relates to isolated proteins or polypeptides encoded by the nucleic acid molecules of the present invention. Compositions which contain the proteins are also disclosed.
Yet another aspect of the present invention relates to methods of imparting disease resistance to a plant. According to one approach, this method is carried out by transforming a plant cell with a heterologous DNA molecule of the present invention and regenerating a transgenic plant from the transformed plant cell, wherein the transgenic plant expresses the heterologous DNA molecule under conditions effective to impart disease resistance. According to another approach, this method is carried out by treating a plant with a protein or polypeptide of the present invention under conditions effective to impart disease resistance to the treated plant.
A still further aspect of the present invention relates to a method of making a plant hypersusceptible to colonization by nonpathogenic bacteria. According to one approach, this method is carried out by transforming a plant cell with a heterologous DNA molecule of the present invention and regenerating a transgenic plant from the transformed plant cell, wherein the transgenic plant expresses the heterologous DNA molecule under conditions effective to render the transgenic plant hypersusceptible to colonization by nonpathogenic bacteria. According to an alternative approach, this method is carried out by treating a plant with a protein or polypeptide of the present invention under conditions effective to render the treated plant susceptible to colonization by nonpathogenic bacteria.
Another aspect of the present invention relates to a method of causing eukaryotic cell death by introducing into a eukaryotic cell a cytotoxic Pseudomonas protein, where the introducing is performed under conditions effective to cause cell death.
A further aspect of the present invention relates to a method of treating a cancerous condition by introducing a cytotoxic Pseudomonas protein into cancer cells of a patient under conditions effective to cause death of cancer cells, thereby treating the cancerous condition.
The benefits of the present invention result from three factors. First, there is substantial and growing evidence that phytopathogen effector proteins have evolved to elicit exquisite changes in eukaryote metabolism at extremely low levels, and at least some of these activities are potentially relevant to mammals and other organisms in addition to plants. For example, ORF5 in the Psy B728a EEL is similar to Xanthomonas campestris pv. vesicatoria AvrBsT, a phytopathogen protein that appears to have the same active site as its animal pathogen homolog YopJ, which inhibits mammalian MAPKK defense signaling (Orth et al., 2000). Second, the P. syringae CEL and EEL regions are enriched in effector protein genes, which makes these regions fertile targets for effector gene bioprospecting. Third, rapidly developing technologies for delivering genes and proteins into plant and animal cells improve the efficacy of protein-based therapies.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram illustrating the conserved arrangement of hrp/hrc genes within the Hrp Pais of Psy 61, Psy B728a, and Pto DC3000. Regions sequenced in B728a and DC3000 are indicated by lines beneath the strain 61 sequence. Known regulatory genes are shaded. Arrows indicate the direction of transcription, with small boxes denoting the presence of a Hrp box. The triangle denotes the 3.6-kb insert with phage genes in the B728a hrp/hrc region.
FIGS. 2A-C show the EEL of Pto DC3000, Psy B728a, and Psy 61, the tgt-queA-tRNALeu locus in P. aeruginosa (Pa), and EEL border sequences. FIG. 2A is a diagram of the EELs of three P. syringae strains shown aligned by their hrpK sequences and are compared with the tgt-queA-tRNALeu locus in Pa PA01. Arrows indicate the direction of transcription, with small boxes denoting the presence of a Hrp box. Shaded regions are conserved, striped regions denote mobile genetic elements, and open boxes denote genes that are completely dissimilar from each other. FIG. 2B is an alignment of the sequences of the DC3000 (DC) (SEQ. ID. No. 85), B728a (B7) (SEQ. ID. No. 86), and 61 (SEQ. ID. No. 87) EELs at the border with tRNALeu, with conserved nucleotides shown in upper case. FIG. 2C is an alignment of the sequences of the DC3000 (DC) (SEQ. ID. No. 88), B728a (B7) (SEQ. ID. No. 89), and 61 (SEQ. ID. No. 90) EELs at the border with hrpK, with conserved nucleotides shown in upper case.
FIG. 3 is a diagram illustrating the Hrp Pai CEL of P. syringae. The Pto DC3000 CEL is shown with the corresponding fragments of Psy B728a that were sequenced aligned below. The nucleotide identity of the sequenced fragments in coding regions ranged from 72% to 83%. Arrows indicate the direction of transcription, with small boxes denoting the presence of a Hrp box.
FIGS. 4A-E illustrate the plant interaction phenotypes of Pto mutants carrying deletions of the EEL (CUCPB5110) and CEL (CUCPB5115). FIG. 14A is a graph illustrating growth in tomato of DC3000 and CUCPB5110 (mean and SD). FIG. 14B is a graph illustrating growth in tomato of DC3000, CUCPB5115, and CUCPB5115(pCPP3016) (mean and SD). FIG. 14C is an image showing HR collapse in tobacco leaf tissue 24 h after infiltration with 107 cfu/ml of DC3000 and CUCPB5115. FIG. 14D is an image showing the absence of disease symptoms in tomato leaf 4 days after inoculation with 104 cfu/ml of CUCPB5115. FIG. 14E is an image showing disease symptoms typical of wild-type in tomato leaf 4 days after inoculation with 104 cfu/ml of CUCPB5115(pCPP3016).
FIG. 5 is an image of the immunoblot analysis showing AvrPto secretion by Pto DC3000 derivatives with deletions affecting the three major regions of the Hrp Pai. Bacteria were grown in Hrp-inducing minimal medium at pH 5.5 and 22° C. to an OD600 of 0.35 and then separated into cell-bound (C) and supernatant (S) fractions by centrifugation. Proteins were then resolved by SDS-PAGE, blotted, and immunostained with antibodies against AvrPto and β-lactamase as described (Manceau and Harvais, 1997), except that supernatant fractions were concentrated 3-fold relative to cell-bound fractions before loading. Pto DC3000, CUCPB5115 (CEL deletion), CUCPB5114 (hrp/hrc deletion), and CUCPB5110 (EEL deletion) all carried pCPP2318, which expresses β-lactamase without a signal peptide as a cytoplasmic marker.
FIGS. 6A-B illustrate, enlarged as compared to FIG. 1, the organization of the shcA and hopPsyA operon in the EEL of the Hrp Pai of Psy 61. In FIG. 6A, the shcA and hopPsyA are depicted as white boxes. At the border of the Hrp Pai are the tRNALeu and queA genes depicted as gray boxes. A 5′ truncated hrpK gene is represented as a hatched box. The arrows indicate the predicted direction of transcription and the black box denotes the presence of a putative HrpL-dependent promoter upstream of shcA. FIG. 6B illustrates schematically the construction of the deletion mutation in the shcA ORF marker-exchanged into Psy 61. Black bars depict regions that were amplified along with added restriction enzyme sites and each are aligned with the corresponding DNA region represented in FIG. 6A. The striped box depicts the nptII cassette that lacks transcriptional and translational terminators used in making the functionally nonpolar shcA Psy 61 mutant. EcoRI, E; EcoRV, V; XbaI, X; and XhoI, Xh.
FIG. 7 is an image of an immunoblot showing that shcA encodes a protein product. pLV9 is a derivative of pFLAG-CTC in which the shcA ORF is cloned and fused to the FLAG epitope and translation is directed by a vector ribosome binding site (RBS). pLV26 contains an amplified product containing the shcA coding region and its native RBS site. Cultures of E. coli DH5α carrying either pFLAG-CTC (Control), pLV9, or pLV26 were grown to an OD600 of 0.8 and then 100 μl aliquots were taken, centrifuged, resuspended in SDS-PAGE buffer, and then subjected to SDS-PAGE and immunoblot analysis with anti-FLAG antibodies and secondary antibodies conjugated with alkaline phosphatase.
FIG. 8 is an image of an immunoblot showing that Psy 61 shcA mutant UNLV102 does not secrete HopPsyA and shcA provided in trans complements this defect. Psy 61 cultures were grown at 22° C. in hrp-derepressing medium and separated into cell-bound (C) and supernatant fractions (S). The cell-bound fractions were concentrated 13.4-fold and the supernatant fractions were concentrated 100-fold relative to the initial culture volumes. The samples were subjected to SDS-PAGE and immunoblot analysis, and HopPsyA and β-lactamase (Bla) were detected with either anti-HopPsyA or anti-β-lactamase antibodies followed by secondary antibodies conjugated to alkaline phosphatase as described in the experimental procedures. The image of the immunoblot was captured using the Bio-Rad Gel Doc 2000 UV fluorescent gel documentation system with the accompanying Quantity 1 software.
FIG. 9 is an image of an immunoblot showing that shcA is required for the type III secretion of HopPsyA, but not secretion of HrpZ. P. fluorescens 55 cultures were grown in hrp-derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. The cell-bound fractions were concentrated 13.4-fold and the supernatant fractions were concentrated 100-fold relative to the initial culture volumes. The samples were subjected to SDS-PAGE and immunoblot analysis, and HopPsyA and HrpZ were detected with either anti-HopPsyA or anti-HrpZ antibodies followed by secondary antibodies conjugated to alkaline phosphatase as described in experimental procedures. The image of the immunoblot was captured using the Bio-Rad Gel Doc 2000 UV fluorescent gel documentation system with the accompanying Quantity 1 software.
FIG. 10 is a series of four images of tobacco leaves showing that P. fluorescens 55 carrying a pHIR11 derivative with a functionally nonpolar shcA mutation is impaired in its ability to translocate HopPsyA into plant cells. P. fluorescens 55 cultures were grown overnight in King's B and suspended in 5 mM MES pH 5.6 to an OD600 of 1.0, and infiltrated into tobacco leaf panels. Because the pHIR11-induced HR is due to the translocation of HopPsyA inside plant cells, a reduced HR indicates that HopPsyA is not delivered well enough to induce a typical HR. The leaf panels were photographed with incident light 24 hours later.
FIG. 11 is an image of an immunoblot showing that ShcA binds to HopPsyA. Soluble protein samples from sonicated cultures (Sonicate) of Psy 61 shcA mutant UNLV102 carrying pLN1 (HopPsyA) or pLN2 (ShcA-FLAG, HopPsyA) were mixed with anti-FLAG M2 affinity gel (Gel). The gel was washed (Wash) with TBS buffer, mixed with SDS-PAGE buffer, and subjected to SDS-PAGE and immunoblot analysis along with the sonicate and wash samples. HopPsyA and ShcA-FLAG were detected with anti-HopPsyA or anti-FLAG antibodies followed by secondary antibodies conjugated to alkaline phosphatase as described in experimental procedures.
FIG. 12 is a diagram illustrating the spindle checkpoint in S. cerevisiae. The spindle checkpoint is activated by a signal emitted from the kinetochores when there are abnormalities with the microtubules. This signal is somehow received by the spindle checkpoint components, which respond in a variety of ways. Mad2 is thought to bind to Cdc20 at the APC inhibiting its ubiquitin ligase activity. In the absence of Mad2 (and presumably damage to the spindle), the APC is active and it marks Pds1 and other inhibitors of anaphase for degradation via the ubiquitin proteolysis pathway; anaphase ensues.
FIGS. 13A-B illustrate the effects of transgenically expressed HopPsyA on Nicotiana tabacum cv. Xanthi, Nicotiana benthamiana, and Arabidopsis thaliana. FIG. 13A shows N. tabacum cv. Xanthi and N. benthamiana leaves infiltrated with Agrobacterium tumefaciens GV3101 with or without pTA7002::hopPsyA. FIG. 13B illustrates Arabidopsis thaliana Col-1 infiltrated with A. tumefaciens +/− pTA7002::hopPsyA. For all plants shown in FIGS. 13A-B, 48 h after Agrobacterium infiltration, plants were sprayed with the glucocorticoid dexamethasone (DEX). Images were collected 24 h after DEX treatment. A.t.=Agrobacterium tumefaciens; pA=pTA7002::hopPsyA.
FIG. 14 is an image of an SDS-PAGE which shows the distribution of HopPsyA and β-lactamase in cultures of Psy 61 (pCPP2318) or a hrp mutant, Psy 61-4 2089 (pCPP2318). Bacterial cultures were grown at 22° C. in hrp-depressing medium and separated into cell-bound (C) and supernatant fractions (S). The cell-bound fractions were concentrated 13.4 fold, and the supernatant fractions were concentrated 100 fold relative to initial culture volumes. The samples were subjected to SDS-PAGE and immunoblot analysis and HopPsyA and β-lactamase were detected with either anti-HopPsyA or anti-β-lactamase antibodies followed by secondary antibodies conjugated to alkaline phosphatase. Pss wild-type=Pseudomonas syringae pv. syringae 61 (pCPP2318); Pss hrcC=Pseudomonas syringae pv. syringae 61-2089 (pCPP2318).
FIG. 15 is a graph illustrating the ability of wild-type Pseudomonas syringae pv. syringae and a hopPsyA mutant to multiply in bean leaves. Values represent the average plate counts from crushed plant leaves of two independent inoculations. Wild-type (●), Pseudomonas syringae pv. syringae 61; hopPsyA mutant (◯), Pseudomonas syringae pv. syringae 61-2070.
FIGS. 16A-B illustrate the interaction of HopPsyA and Mad2 in a yeast two-hybrid assay. FIG. 16A illustrates cultures of yeast EGY48 strains containing either pLV24 (pEG202::'hopPsyA) and pJG4-5 (fish-vector), pLV24 and pLV116 (pJG4-5::mad2), or pEG202 (bait vector) and pLV116 on medium containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal) to check for β-galactosidase activity with either glucose (Glc) or galactose (Gal). β-galactosidase activity was indicated only in the presence of both HopPsyA and Mad2. FIG. 16B illustrates cultures of the same yeast strains on minimal medium leucine dropout plates with either Glc or Gal sugars. 1=EGY48 (pLV24, pJG4-5); 2=EGY48 (pLV24, pLV116); 3=EGY48 (pEG202, pLV116).



DETAILED DESCRIPTION OF THE INVENTION
A DNA molecule which contains the CEL of Pseudomonas syringae pv. tomato DC3000 has a nucleotide sequence (SEQ. ID. No. 1) as follows:









ggtaccgggc tctgtgacgc agagcgtcac gcaaggcatt ccactggagc gtgaggaacg
60



ataatcctga cgacaactat cgtgcgacgc tccgcgtcgg catgccgttc tggacgctct
120


gcgtcctgtc ttgagaggtg cgccaagcgc aaagcacggt aagtatcagg gaggggtgta
180


taggagggtt gcaaggcggg aggtgttcat atcaaggcag tgttcatgaa cccgtcttgc
240


ctgggctcat gaacacgttc ggcttacgcg gtcagtgcat ttcctcgctc aaatggtcca
300


gccctgccag catcaactca tgccggtgga tgtcgtccag gctggcgtag gaacccggtt
360


tttcgttgac cgcgtgccac accacaaagt cgcgtcgtac gtccagaaac aggaagtagt
420


gattgaaacg ctctgactcc ataaaacgtc gttgcagtgc atcacgcagt tgatcgggac
480


gcaacgcgcg gccttctatg tgcaaggcga tcccccaatc atggtgttcg cgccgactga
540


caaacgcgac gccattggcc actggccata ctgctgggct ctgggcggca acctgagcgt
600


aaaatgccga cttttccgtt acctcaatca tttctaatcc tttaactgca cgacagtgta
660


atcccgctca tggtcccggt cgtccagacc ttcgcgcatg tcgggcggcc accaaatgac
720


cagctcgcgg ttgttggagt ccgggcgttt gcaagcgttc cccgcacagc cgtgggtggc
780


acaccctgtc agcgtagcaa acagcaagag caagagcgtt aggctacgaa tcatcatggt
840


ttcgctcccc ggagcagtga cggcctgctt tctttggcca ttttagatat ctgcggctgg
900


cgcacagcga tgtacacctc actttcttca cccggctgca gccatgcatg aggccaggcc
960


gcaacgccga tgacccagcg accgccgcat cggctttcgt cgatacgtac cggcttgtcc
1020


gtgttgttac gcgcaaccac cacagcaaca ccccagtctt ttttgacgaa ccactgcgag
1080


cgctgcccat caagcgtcag accttcgccc ggatcacaca gacttcgtgt ttcaaagggc
1140


agggtctggc cagcgcgcag gccttccggg gcggggccgt cgatcatttg ggtaaagact
1200


ttctggatgt cgccccgcgt tggcagtcgg cctccgtcac gtcgttcctt gattttcttc
1260


atctggtcat cgacgtcatg ggggttgccg ttctgtacat agcgtgctgg attgacctga
1320


tcgccgatca gtcgaggggt cagaatgaac agccgctcgc gctgactcag ttcgcgactg
1380


cgggactgga acagcagctt gccgatatag ggaatgtcgc ccaacagcgg gatcttgtga
1440


atcctgtcat tggcttccag accgtggaag ccgccgatga ccagcgagcc gtgctcggca
1500


atcaccgcct gggtgctgac attgcctcgg cgcacactgg gttgggtgtc attgatcgtc
1560


gacacatcga tctggccatc ctcgatgtcc acgatcattt ggacctgagg cttgccatcg
1620


ttgtccagcg aacgcggaat cacttgaagg ctggtgcccg ccgtgatggg cagaatgtca
1680


gcggcccgct cggaagtggg cgtcaggtat tcggtgcgac tgaggtcgat cactgcaggc
1740


tgattctcca gggtcaggat cgacgggttg gcgatgactg acgcagaacc attgccttca
1800


agcgcatgca attcggcaga aaacttgctg gcgttctgca agaacaacgt tgaactggtg
1860


ccgccatcaa acaggttggc acccacctcc gacgctgccg ggcattgaaa ttccagccga
1920


ctggacagtt cagccagttc attggggtcg atgtcgagaa tgaccgcatc gatttcgatc
1980


aggttgcgcg gaacgtccag ctccttgacc agtttctggt acatggcctt gcgctctggc
2040


aggtcgtaaa tcaatacgga gttgttacgc acatcagcgc ttacgcggat attgccttgc
2100


ctgaggcatg acccttggca gtttttttgc tgttgaagtt caatacgcgg tgcaatgccc
2160


ctgttgcagt gctcccgtat cgataccatt ggagcccagg ttgtaaggca ggccggggcc
2220


gcgacacctg tgctgttggc aacactgctg ccctgccccg ccaacaagtt cacgctgtca
2280


atgctttcgc cacgcgaacg gctttccagc agctcttgaa gaatactggc gacaccggcc
2340


accactaact gctggtcacg gtagcgaata gtccgatcag ccgcgttggc gtatttgagt
2400


ggcagcacga caacatcttg cttgtcggcc ttctcgtcgg gcttttcgac tttcttgctg
2460


tagtcgcgca caaactccac gtatttggcc ggaccacgaa ccagaaccac gccttcgtca
2520


ggcagcgagc cccagcccaa acgcttgtca acaagaccga catcggtcag cgccgtttgc
2580


aggtcgtcca ccgcatccgg cgagacttcg atgcgccccg aggtgtgctc gctggaaggg
2640


ctgacataca gcgtgtcgtt atagacgaac cactggaagt ggtattcctg actcagccgc
2700


tcaagaaact cttcagggtt ctgagcacga atacgtccat cgaggtttcc ctggacaggc
2760


gacatgtcga gcgacatacc gaactccctg gcaaagtcag ccagggcagt agacaactcg
2820


gtctgccggg catcataggc gtaggcggtg tgtttccagg cttctggggt gaccgcccac
2880


gtggcaggga tcaccccgat caacaataaa ggcaaccaca ttaaggcctt gcgcatttca
2940


cactcccggt tgccggtgat tgaggatcga acgcccggac aaagtgggcg tcgtgttacg
3000


aatagtggtt tgcatcaggc tgagcatgcc cgcgcgctga ttggccaggc tttccagacg
3060


atcgagcagg tcaccgaggc tgcaggggtt tgccatccag ctgaccagca ctacgcagcg
3120


ggtctgcgga tcgatggcca gcgcgccgtc gcaggcacac gccaggcttg cgccgccctc
3180


gccaagcaag gcttcgagcc gttgcgggtc accggcgtcg tacgggtcga gcagttcgat
3240


actgcaacgc accccgtcgc cgacgaccgc cagccgagca ttggcgtcat cgatccagca
3300


gtccagcggc atcgctggac gctgggcaga ccactggcca acgatctcgg tgaattcact
3360


gaattccatc gatgactgct ttattgatac cgtgcttggc acgcaggcat tcattgacgg
3420


caataccggc gacatcgacc tgctgctggg acatcgtgaa tgcctgcagg tcttcgacgg
3480


tgccactctc ggaggcttcc atcgctgcct ggtccatgtt ggtgtgagca cggctcaccg
3540


aattgtcgag atggcgttgc aagctgttga aactgatcat gtcctggtgc tccagcagaa
3600


gggttcaaac cttgagtgga gcaaacccgc cgagcggttc catcatgcga tcaagtgagt
3660


gcagagagtg tgtatcaggc agcaggctcg acacccagca gccccttgcg caggtctgcc
3720


caagcgatat cgaacgcgcc attggcatcg ctcagacgca agctgtccga ggcgatcgtt
3780


gcatcgcgct tgagttgcca gtgctcggaa aaacggctgt ctgccagcca ctcagccacg
3840


gggtcggcta tttgggggtg aacactgagc gtcgcgaccg cttcattgag ctggctggcg
3900


gccaggtttc tggccagcgc ccgcgcacgt tcggccagcg tggtgtcgtc taacaagtgc
3960


cgcagggatt cactcaacag ttcttctacg gcggtcattg cctgctcctg caacgcctcg
4020


cgctgcacct gaagctcgcc gagaaacgcg ttggcgtttt cccagaactg cgccagcgcc
4080


tgctgctgaa ggtgctcggc tttctcttgc tcaagggcca gtatctgcgt ggcctgctgc
4140


cgcgcgtctg ccaggatgtc gcgcgccagc aggctgtcgg cgatgtcttc gcggcgcaag
4200


atcggttcgc gcagcagcgt agcggccgtc agagcaatac tgcgtttggc gagcatgggc
4260


gtattcctga tgcagagaag ctggttcgga ttcaggcagc cgtgacgcgc cacatgatgg
4320


cctgccataa cgcctgaagt ttgttttcgg gtgccttgcc gggggtgtcg ggcacttcat
4380


tgggcgggca ctccagacac agtcgcgacc agtattgcgg cccaagccag gcgcccagca
4440


gaagacgcgc gtcctcgtgt tcaaactcca gccagacacc ggggcgcagc gctttggtca
4500


acccccagca ccattgaccg tcaggtccgt cgctttcgtt acgggagaag cagatgcact
4560


gcgccaggct tagcgcctgc tcacgctgcg agggcgtcag cgccaaccag cgcagcaccg
4620


gttccgcggg cgctggcggc tgagccgggt caatgcccag actctgcaga aacacgccat
4680


gacggctggc catgagcgca tcgcagtcac tgaccgataa cccacgagcg ttggcgaatc
4740


ggtcatgcca ctccgaatgt gcccactgcc aggggttgca ccaccagtga atccagtgat
4800


cctcggcaga aaggctcatc atgcacgtgc cggcagcgtt gaacgaccgc gactgccaaa
4860


cccgatccgt cgcaacagac tggcgcgcca gtcactgcgc accagcagtg caccgatcag
4920


caacaccaac gcaagaccga caggtgccac ccagagcatc aggttccaga acggcaagtt
4980


cgtgctgtcc agcttgaagg gcccgaagct cacccattgc gtggtctctt ggaactctgc
5040


agcaggcaca aacacgatgg aaaacttttt cgaatcgaca gattgcgtgg acataccggg
5100


aatactgctg gcgaccatct gttgaatacg tccgcgcaca ctgtcgggat caagtgcagc
5160


agagtgcttg atgaacaccg cagcagaagc cggttgaaca ggttcgcccg gcgcgatgcg
5220


ctcgggcagc accacatgca ccctggccac aatgactccg tcgatctgcg acagcgtggc
5280


ttcaagttcc tgggacaagg cgtagatgta acgggcacgc tcttcaagcg gcgtcgaaat
5340


caccccttcc ttcttgaaaa tctcccccag cgtggtgcgc gagcgccgag gcagacccgc
5400


agcgtcgagc acgcgcacgg cgcggttcat ttcgctggtg gcgacagtca cgacaacgcc
5460


ggttttctcc agacgtttac gcgcatcgat atgctgatcg gcgaggcgcg ctacgacctc
5520


attggaatcc tgctcggaca agccagtgaa caaatcagtc tcatcactgc agccgccgag
5580


cagcagcatg cacaacagca gcagccctgc gctcagaaaa ttcacggaaa cctctactgc
5640


aggttggtca acttgtcgag cgcctgagcg ctcttgctca cgaccttggt cgtcaacgcc
5700


atttgcaacg agcactgcga caacgcccga ctcatctgca cgatgtctcc aggatcttcg
5760


gtgttcgaca ctttcttcat ctggcgtaat gcttgctgtg aaagcttctc ggtactgccc
5820


agccgctcgg acagcgcact ggctatccgg tcggacaggt gcgacgctgc tggcccgctg
5880


tcagggcgca tcgccgcatt gaataggtcg acatccgcct gaacgggttc ggagccgagc
5940


ccctgatgag cattctgccc aagctccggc gatacacttt tcaaattgct gagttgggaa
6000


atggtcacac tggttctccg tcaggcggct gtcagtcagg ccacagcctg gttagtctgg
6060


ttattggtgc cttgcaacag cgcattgatc agctgagctg ccacttgcgc agcgctcgat
6120


tgcaggtcgg cgccggtgtt gccagcatcc tgaagcgtcg cttccagccc gcgttgacgc
6180


aagccgctca gcagttgacc caggtcctga ttggacacgt tgcccgtcgg gttagccact
6240


ggcgtgccac ctgtcggctg cgtggaattg tcgaccggtg taccaagacc accacccgac
6300


gaaaccgact gcaaaccacg gtcgatgagt tgaccgatca gttgacctac gtcgacgctg
6360


gcattgccat tggccgcggg acctgtgttg gcatcgattg caggattacc cagggagctg
6420


tcactcacgg gcgaacccag accgccgcca ctggtaacgc cactggcatc accttgttgc
6480


tggccgagct gttgaccaat gacgtcgaga gccgaacgaa actgagcggt ttcctgtgca
6540


tccaggccat tgtcttcctt cagctcgttc atccacgagc cgccgtcccg agtagggaac
6600


tgggccttgt tgtcgtccat gaactgggca actttttcca gggtcggcat gtcatcactg
6660


gaaaaggttg ttccgccttc accactcggt gtcagcagat cgtccagcac ggctttgccg
6720


aggccgttca ggacctggct catcagatcg gattgcccgg cacccgcgtc gctgctcaga
6780


ccgccaccga cacccgaacc agaacccgcc ccgccaatgc caccgccacc gccacccgcg
6840


ccgatgccgg cagaggcacc gaaattgtcg ccgagctttt cgtggatcag cttgtcgagc
6900


gatgcagtga tgtcatcgat gctgttagcc gacttgccat ccgcagccat ggccttggcg
6960


agcattttgc cgagcggtga ggtttcatcg agctgcccac tttgggtcag cgcctgaacc
7020


agctgatcga tcacagcctt gagctctttg ctggaagtgc tggtgttggc gctcacatcg
7080


ctgttgagcg acacggggaa caatgatgca gaggtttgca acgaactgat gctgttaagt
7140


gcttgcataa aacgcccatc ccaaggtagc ggccccctct gatgaggggg caatcagaaa
7200


taattagtaa ctgatacctt tagcgttcgt cgctgtggca ctgatcttct tgttggtaga
7260


gtcttctttg ccggcctgga tggcgttgag cacgtccatg gtctgcttct tcattgtttc
7320


ctgggcctgc atcgcgatca gcttcgcgcc gttggcgtcg gactctttac tggccttggc
7380


ttgtgcatca accgacaggc tgtcgccggt gcccaaaaga atgtttttct gaagagtggc
7440


gttggaagca accgtgttga caccctgcaa tgcgccgccg acaccgccaa cggcgctgtt
7500


accaaggttg gtgagtttgg aggttaatcc tgcaaatgcg accatgattt gatgcccctt
7560


aagatttacc agcgtgattg cttggtactc actaggtggc agcagcctgc gatacggttc
7620


cagcgtcttt gcaaaaaatc agatctgcaa ttctttgatg cgtcgataga gcgtacgggc
7680


gtggcagtcc agttccaggc ttaccgaatc caaacaattg tcgtggcgct tgagcgactc
7740


ctgaatcagg gctttttcat caactcgcaa ttgcgatttg agcccacagg ccaagtgctc
7800


ttcgccctgc ggctcggcgc ccagcaaggg gaaacccagc acatggcgtt tggctgcagc
7860


cttgagctca cggatattgc cgggccagtc gtggcccagc agcactttgt gcagcagtgg
7920


gcaaacatcg ggaacgggaa caccgagctc cctcgcggcg gcggccgtaa aacgtgtgaa
7980


caggggaact atgcgatcag actggttacg tagcggagga agcttgagtg tcaggacgtt
8040


caggcgaaaa tacagatcgc gacgaaactg cccccgctcg acggcgtcgt ccagcgagca
8100


ttgggcggag gcgatcacgc agatatccag gttgatcgtc gacgtcgaac ccagccgttc
8160


aagcgctcgg gtttccagca ccctcagcaa tttggcttgc agggccagcg gcatgctatc
8220


gatctcatcc aggtacagcg tgccgccctg cgccgcttcg acataaccga ctctggagcg
8280


atcagcgccg gtgtaggcac cgctgaccac gccgaataac tcgctctcgg cgagggactc
8340


cggaatggcc gcgcaattca tcgccaccag gcgccctttg cgggctgaca tctcatgaat
8400


ccgtcgggca atcgtgtctt tgcccgtgcc ggtctcaccc gatagcagca cgtcgatacc
8460


cagttgcgaa atactttcgg caactatccc cagattcgga acccgctcct cgtccagatc
8520


atcctcaaac ctttcatcaa gactcatccc atgaccccca ggacatcaac gttggataac
8580


cacacctgcg tcacagaccc cggacctcgc agagtatcgg cgctgcaact cccagttcct
8640


tcatgcggtg atacagggtg cgtcttggca actccaactc ctgaagcacc gcgtcgaaat
8700


tgtgcctgtg ccgcttcaag gcatcctgga tgagcatttt ctcgatgatg cgcatttgcg
8760


tgcgcagccc cgtggcaggg tcaagcgctt ccacagggtc ggcgcccagc aaggggaagc
8820


cgagtacgaa gcgcttggct gcagacttca attcgcggat gttgcccggc cagtcgtggc
8880


tgagcagcag ctgcacacgc ccgctgtcca gcgcaggagc gggacgtccg aactcggcag
8940


cgataccctg ggtgaactgg tcgaacaatg gcaggatctg ttcacgacgt ttgcgcaagg
9000


ctggcaagtg aagcgtcagc acgttgagcc gaaaaaacag gtcgcgacgg aaaagtcctt
9060


gttccaccag ttcatccagt ggccgctggg ccgaggcaat gatccgcaga tccaccggga
9120


tgaattcggt cgagcccaga cgctcgatac ctcgactctc caacacacgc agcagtttgg
9180


cctgcaggct caacggcatg ctgtcgattt catccaggta caaggtgcca ccactggagg
9240


cctctatgta gccctcgcga gcccggcata cgccggtgaa tgcaccgttg accacaccga
9300


ataactggct ctctgccagc gactcgggaa tggcggcgca gttcatgccc acaaagggtc
9360


ccgacctgct ggacaactcg tgaatgcggt tggccagtgt gtccttgccg gtgccggttt
9420


ccccgcacaa cagcaagtcc atatccagaa acgcgctatt cattgcaatt tgatgacccg
9480


ctgataatgc agttacgccc caacactctc ggacgtcctt atcgatgcct gtactcatcg
9540


ttgcactctc atggtgggtg gcaagcggag tattaatacc acgtcttaca aggcagaaat
9600


atattaattt agttccccgg gaaatgagaa aaagatcaca aagttgagaa ttactatcat
9660


attaatatca ccataccaag acgaccctac cgatagactc aggctcttga gatgattgct
9720


ttaatctatc gttactccaa tgcgaacaag cgcttacagc gtccatgcgc tggctcgccc
9780


cgcaagccat agggcctctc cacacctcaa agcagctgtg atccgggaca agagcaggca
9840


cctttgagca gcaagcgccc caaaatcgcg caatgaaacg caactaactt ctcgtcacta
9900


ctcgagagaa acatataaga cttttccaaa acaactaaag gggtcacaag taaggaagca
9960


gaagaaaacc gaacacacaa aacaagaaaa ccaaacggtt tttagcggcg agcttaaaga
10020


agcgaacaac aataacacga gaaaacaaaa aacagcctga cactaactat ttgcacttta
10080


gaacagtcga taccaaccag cttagttccg ccccacgagc agtcggattt ccgaacaaca
10140


cagaggcttg gatactggca aagcggtcat agccccggtt tttcggcacc actcagtact
10200


ggcatttagt catcatcgca ttcggcaatc cgaacaaaag cccacctgct tagactattt
10260


ccaggcacag ccatctaagg aatcgcggaa aggattcagc gtagcttaat accggaaccg
10320


caggtttagg ttctgtgaac caggcggtta atacgatcga tgatcgcgtg ccatcaccta
10380


gaatgtttct aaatgtgtgt aatctttcac ttacattcgg ctaaaaaagt tcatcaaaat
10440


aatcatatgt agcgctctac atcatatggc taagcgccat ctttagggtc caaaaaacgg
10500


gtaacgctca ataaaagaag ttgtattgag gcagatcaat attgtccgac aacgagaaaa
10560


agcaccaaaa aagtgcgctt ttcaggggtt ttcaatagaa caatcgagta aaaccggggt
10620


tattggcgtg gatcactggc aaaaaccacg acgcgcggcc ccgtaggcag ctcgcgcgga
10680


ccgctgcgat actcgtcgtc atcacgcttg cgaggcgacg aacggtcatc cctgatgcgg
10740


ggcaactgta tccggtttgt aagcggatca ggttccacaa caggtgcgga ttgggcgatc
10800


tctaccgccg gcgctgattc agctgcagga gctggctgta acgcctcagg cgcagtgggc
10860


tgctgagcca ccggcaacgg ctgagccgtt ttgggcgaag gcaggttctc ggctaactgg
10920


gccgactgca cgggcttggg cagcggcgga cgctctgcaa cgcgcactgg acgctcagcc
10980


acaggcgcgg gcgcgggcag acgctcagcc gcccgtttca caatggctga aggggtgacc
11040


agcgggatgc tggcagtcac cggggactca ccggtaatgc gcgcgatgct ggtcgtgagc
11100


acgcgattct gggttttagg tatcagcaga cgtcccggtc catcgaaggt ctttttgcgc
11160


aggaatgccg agttcagccg caacaactgg ccctcatcca cacccgccgt ggccgcgagc
11220


tgggtcaggt ctacggcatg gttaagctcg actacgtcaa aatacggcgt gttggcgacc
11280


ggggtcagtt tcacaccgta ggcattgggg ttgcgcacaa ccattgagag cgccaacagt
11340


ctgggcacgt aatcctgggt ttccttgggt aaattcagat tccagtagtc cacaggcaga
11400


ccacgccgtc ggttggcctc aatcgcccga ccgacggtgc cctcccccgc gttataggcg
11460


gccagcgcca gcagccagtc attattgaac tgatcatgca agcgggtcag gtaatccatc
11520


gccgccttgc tggaggccac cacgtcacgg cgagcgtcgt aggtcgcgct ttgatgcaga
11580


ttgaagctgc gccccgtgga tggaatgaat tgccacaaac ctgccgcagc ggccggagag
11640


ttggccatgg ggttataaga gctttcgatc atcggcagca gtgccagctc cagcggcatg
11700


ttgcgctcgt ccaggcgctc gacaataaaa tgcagataag ggctggcccg gacactggct
11760


cccgtgataa atccgcgatt gctcagcaac cagtcgcgct ggcgagcgat acgctcattc
11820


atgccttggc catcgaccag cctgcagcgc tgggcaaccc gctgccacac gtcctcgccg
11880


ttataaacag gcagatcgga gattttgtct gcagcccgcg aaccttcctt atcatctccc
11940


ccccaataga ccagccccga caccagccgc ggcggacggt cctgacgcgg cggcgaatag
12000


tccacagact ggcagcccac acacaaggcg cccatagcga ggactgcgat ttgaacagcg
12060


cgagccagca agcgtgggct cgatacgggg aaggcgacgg cgggcatggg cgggaatgtc
12120


ctgagcgtgt ccaccctacg tggcacgctc gccgttacgg ttcccttttg aaaccgagat
12180


cggcgcacac aacgcattgc tgaatccttt cagccgtaag tttttccgat ggaacccgct
12240


ggcattgcat gccactcatc ctgtgaagga attttcacgt ttggtatcag gcggctatca
12300


gcgataaaat ggacagagag attcaccgtg cagtcaccat cgatccaccg gaacaccgga
12360


agcatcattc agccaaccgt cacccctgac gcacgtgctg caactgacct gcaggaaaga
12420


gccgaacaac ccaggcaacg ctcttcgcac tcgttgagca gtgtcggcaa gcgggcgctg
12480


aaaagcgtcg gtaaattgtt ccagaaatcc aaagcgccgc agcagaaagc tgccacgccg
12540


cccaccgcga aaaacgtcaa gacgcccccg cctgcttcaa atgtggctac gcccagaaac
12600


aaagcccgcg aatccggttt ttccaacagc agcccgcaaa atacccatag ggcacccaag
12660


tggattctgc gtaaccaccc caaccaggcg agcagctcgg gcgcgcagac gcatgaaata
12720


cacccggagg cagccccccg taaaaacctg cgcgtaaggt ttgatctgcc gcaagaccgc
12780


cttgagcgca gcccgtcgta cctcgattca gacaacccga tgaccgatga agaagcggtc
12840


gcaaatgcca ctcgccaatt ccggtcacct gacagtcacc tgcagggctc tgacggtacg
12900


cgcatttcaa tgctggccac agatcctgat cagcccagca gctccggcag caaaatcggt
12960


gattcggacg gaccgattcc gccgcgcgag cccatgctgt ggcgcagcaa cggaggccgt
13020


ttcgagctga aagacgaaaa actggttcgc aactcagagc cacaaggcag cattcagctg
13080


gatgccaagg gaaagcctga cttctccacg ttcaatacgc ccggcctggc tccattgctc
13140


gattccattc ttgccacacc caagcaaacc tacctggccc accaaagcaa agacggcgtg
13200


cacgggcacc agttgctaca ggccaacggg cactttctgc acctggcgca agacgacagc
13260


tcgctggccg tgatccgtag cagcaacgaa gcactcctta tagaaggaaa gaaaccaccg
13320


gccgtgaaaa tggagcgtga agacggcaac attcacatcg acaccgccag cggccgcaaa
13380


acccaagagc tcccaggcaa ggcacacatc gctcacatta ccaatgtgct tctcagtcac
13440


gacggcgagc gtatgcgtgt gcatgaggac cgtctctatc agttcgaccc gataagcact
13500


cgctggaaaa taccggaagg cctggaggat accgctttca acagcctgtc cactggcggc
13560


aacggctcgg tttatgcaaa aagtgacgat gccgtggtcg acttgtcgag cccgttcatg
13620


ccgcacgtgg aagtcgaaga cctgcagtca ttttcagtcg cgccggacaa cagagcagcg
13680


ttgctcagcg gcaaaacgac ccaggcgatc ctactgactg acatgagccc ggtgattggc
13740


gggctgacgc cgaaaaaaac caaaggcctt gagctcgacg gcggcaaggc gcaggcggcg
13800


gcggtcggtt tgagtggcga caagctgttt atcgctgaca ctcagggcag actttacagt
13860


gcggaccgta gcgcattcga gggcgatgac ccgaaattga agctgatgcc cgagcaggca
13920


aactttcagc tggaaggcgt gcccctcgga ggccacaacc gcgtcaccgg attcatcaac
13980


ggggacgacg gcggtgttca cgcgctgatc aaaaaccgtc agggcgagac tcactcccac
14040


gctttagacg agcaaagctc aaaactgcaa agcggctgga acctgaccaa tgcgctggta
14100


ctgaacaaca atcgcggcct gaccatgccc ccgccaccca ccgccgctga ccggctcaac
14160


ctcgatcgtg cgggcctggt tggcctgagt gaaggacgca ttcaacgctg ggacgcaacg
14220


ccagaatgct ggaaagacgc aggcataaaa gatatcgatc gcctgcaacg cggcgccgac
14280


agcaatgctt atgtactcaa gggcggcaag ctgcacgcac tcaagattgc ggccgaacac
14340


cccaacatgg cttttgaccg caacacagca ctggcccaga ccgcacgctc gacaaaagtc
14400


gaaatgggca aagagatcga aggcctcgac gaccgagtga tcaaagcctt tgcaatggtc
14460


agcaacaaac gcttcgtcgc cctcgatgac cagaacaagc tgaccgccca cagtaaggat
14520


cacaaacccg tcacactcga cattcccggg ctggaaggcg atatcaagag cctgtcgctg
14580


gacgaaaaac acaacctgca cgccctcacc agtaccggcg ggctttactg cctgcccaag
14640


gaagcctggc aatcgacaaa gctgggggac cagttgcgag cccgctggac gccggttgcg
14700


ctgcccggag ggcagccggt aaaggcactt ttcaccaacg acgacaacgt gctcagcgcc
14760


cagatcgaag acgccgaggg caagggtctt atgcagctca aggcaggcca atggcaaagg
14820


ttcgaacagc gcccggtaga agaaaacggt ttgaatgatg tgcactcgcg catcacaggt
14880


tcaaacaaga cctggcgaat tccaaaaacc gggctgacgc tcagaatgga cgtcaataca
14940


ttcgggcgca gcggtgtgga gaaatccaaa aaagccagca ccagcgagtt catccgcgcc
15000


aacatctaca aaaacaccgc agaaacgccc cgctggatga agaacgtagg tgaccatatt
15060


cagcatcgct accagggtcg cctgggtctg aaagaggttt atgaaaccga gtcgatgctg
15120


ttcaagcaac tggagctgat ccatgagtcc gggggaaggc ctccggcacg gggtcaagac
15180


ctgaaagcgc gcatcaccgc actggaagca aaactggggc ctcaaggcgc tacgctggtc
15240


aaggaactgg aaaccctgcg cgacgagctg gaaaatcaca gctacaccgc gctgatgtcg
15300


atcggtcaga gctatggcaa ggcgaaaaac cttaaacagc aggacggcat tctcaaccag
15360


catggcgagc tggccaagcc gtcggtgcgc atgcagtttg gcaagaagct tgctgatctg
15420


ggcacaaagc tcaacttcaa aagctctgga catgacttgg tcaaggagct gcaggatgcc
15480


ttgactcaag tggctccgtc tgctgaaaac cccaccaaaa agttgctcgg cacgctgaag
15540


catcaagggc tgaaactcag ccaccagaaa gccgacatac ctttgggaca gcgccgcgat
15600


gccagcgagg atcatggcct gagcaaagcg cgcctggcgc tggatctggt cacactgaaa
15660


agccttggcg cgctgctcga ccaggtcgaa cagctaccgc cgcaaagcga catagagccg
15720


ttacaaaaaa agctggcgac gctgcgtgat gtgacttacg gcgaaaaccc ggtcaaggtg
15780


gtcacagaca tgggctttac cgataacaaa gcgctggaaa gcggttacga atcggtcaag
15840


acattcctca agtcgttcaa aaaagcggac catgccgtca gcgtcaatat gcgcgcagcc
15900


acaggcagca aggaccaggc cgagctggcc ggaaaattca aaagcatgct caagcaactg
15960


gagcatggcg acgacgaagt cgggctgcag cgcagctacg gagtgaacct caccaccccg
16020


ttcatcattc ttgccgacaa ggctacaggg ctctggccaa cggcaggtgc caccggtaac
16080


cgtaactaca tactcaatgc cgagcgttgc gagggcggcg ttacgctgta cctcattagc
16140


gaaggtgcgg gaaacgtgag cggcggtttc ggtgccggca aagactactg gccgggcttt
16200


tttgacgcaa ataatcctgc acgcagtgtt gatgtcggca acaaccgcac actgaccccc
16260


aactttcgcc tgggcgtgga cgtgaccgcc accgtcgccg ccagccagcg cgccggggtg
16320


gtcttcaatg ttccggatga agacatcgac gcattcgtcg acgacctgtt tgaaggtcag
16380


ttgaatccat tgcaggtgct gaaaaaagca gtggaccatg agagctacga ggctcggcga
16440


ttcaacttcg acctcacggc aggtggaact gccgatatac gcgccggaat aaacctgacc
16500


gaagaccgag acccgaatgc cgaccccaac agcgattcgt tttctgcggt agtgcgcggc
16560


ggattcgctg cgaacatcac cgttaacctg atgacctaca ccgattattc gttgacccag
16620


aaaaacgaca agaccgaact gaaggaaggc ggtaaaaacc gcccgcgctt tttgaataac
16680


gtgacggccg gcgggcagct tcgcgctcag atcggcggca gccacacggc ccccacaggc
16740


acacccgcct ccgccccagg ccccactccc gcatcacaaa cagccgccaa caacttgggc
16800


ggagcgctca atttcagtgt ggaaaacagg acggtcaaac ggatcaagtt tcgttacaac
16860


gtcgccaagc cgataacgac tgaaggtctg agcaaattgt cgaagggcct tggggaagcg
16920


ttcctggaca acacgaccaa agcaaaactg gcggagctgg ccgaccctct gaatgcacgc
16980


tacacaggca agaaaccgga tgaggttatt caggcgcaac tcgacgggct tgaagaactg
17040


tttgccgaca taccaccgcc caaagacaac gacaagcagt acaaggcatt gcgcgacttg
17100


aaacgcgcgg cggtcgagca tcgggcatca gccaacaagc acagcgtgat ggacaacgca
17160


cgctttgaaa ccagcaaaac caacctctcc ggcctgtcca gtgaaagcat acttaccaaa
17220


ataatgagtt ccgtgcgcga cgcgagcgcc ccgggcaatg cgacaagagt tgccgaattc
17280


atgcgccagg acccgaaact tcgcgccatg ctcaaggaga tggagggcag tatcgggacg
17340


ctggcacgcg tacggctgga accgaaggac tcactggtcg acaagatcga tgaaggcagc
17400


ctcaacggca ccatgactca aagcgacctc tccagcatgc tggaggatcg caacgagatg
17460


cgcatcaagc gtctggtggt attccacacc gcgacccagg ctgaaaactt cacctcacca
17520


acaccgttgg tcagctataa cagtggagcg aatgtgagcg tcactaaaac actggggcgc
17580


atcaacttcg tttatggcgc agaccaggac aagccgattg gttacacctt cgacggcgaa
17640


ttgtcacgac catcggcatc gctcaaggaa gcggctggcg acttgaagaa agaggggttc
17700


gaactgaaga gctaataacg aaaacagtaa aaaaagcgcc gcattgaagt ggcgcttttt
17760


tattcaagcc tgtaaaaaag cacgcgcttc acgtgcctgg gaaatgaacc cgcgcgtcac
17820


gtcacaaaac gctggctcat cgagtgaggc cagttcacgc tgcgcgcata gacggacatc
17880


tccctgatcg accgcaaacc agcagccatg caagcgcgct acgtcgaagt tcagactcaa
17940


cagacgcagc aaatcggggg ctcgttccgg gcagcggcca atgcggcaat gaaagatgac
18000


catctcactg tgctcgggca attcaatgat cgccgcttcg ttgttctgac cgtcataaag
18060


agcgcatacg ccgttctgca aggtcagtga cgtgccgagc tgggcgccca gagaattgat
18120


gaagcgggcg aaatcgggtt gcgaagtttt catcgtcata gtcctttaag gttaaaacag
18180


catgaagcat gccggacagc aggcgcctgc agcctgtgtc cggcgccggg attaacgcgg
18240


gtcaagcaag ccctcttcaa gtgccctCaa tgcgtcatcg tcttttgtcg gctgcttaag
18300


cgcctcgcgt gctgacgcga ctgcgttcaa cacaccttca tccacgaccc gaaccgtatc
18360


cacggccatc tgggtaggca actgcaatgc gcctcgtccc atgtgatagg cgttttccgc
18420


gactcgtggg ataccgctca acgtgctctt ctggaacgta tgtggcagag actccctgtt
18480


cggatgacgg atgttattca aagcgtctcg gtacggtcca gcataggtgt tgcaccgccc
18540


atgcctgccg ctttcaacgc cttggcttct gcggtaaccg actggttggt gtacaacgtg
18600


gacagatagg acaccgaacc cgtcgctgcc agggccatgt tgcgcaaaat agcccccgca
18660


ctgagcgtgc cacttgcgcc ttcagcctga gcggtcacag gcggcagtgc cgaggtcagt
18720


gcagaactct gaatacccga aagagccttg ctgtagaacg tggtgcgtac cgacggctcg
18780


cgcaggtcca tacctttgag caggtccttt ttcagatcgc tctcggcgcg gtccggggta
18840


aataccggaa ttttgcgccc ttgcgggtcg acataattcg acttcaattg cagcagcgtt
18900


tgcgaactgg cagacaccgc cccgccaaaa ccggatgcca gagctcttgc actcagcgtc
18960


tgcccattga tctggtgaac atcgttgagc atctggcgca cagcctgaga accaccgaag
19020


gcactgtaag ccatcagctc acctaccgga tgggtggacg aaccctgaac cttcttctgg
19080


ttcagcagcg cgcgttcact tttcacgaac gccttgtcct gagcgacttc ctcgggcgtt
19140


tttttgacca gctcaccgtg ttcgcttttc agctcgaagg ggtcaggaat aaccgtattg
19200


gtatccacag ccttcattgg caccatgttc aggcgttcgt tgaggccagt cttctgcaag
19260


gcggcctgaa acatcggctt gaccacgctg ttgaccgtct cgtgagcaat gcccgccacc
19320


atcccgatta tcgaagcctt gagcatgttg gcgtcgctgc tggtctcggg aatcgtgtct
19380


cgcagcttgt cgctggtgga caaacgcaca taacccaagt gtgtcattga agacaagaac
19440


tgcggaaccg cagccgcgac aatcggccct gcacctttcc agccacccac cgtgttacgg
19500


gcagtgacga gatcgctgac gacgttgtcc agttgcgtat gtgcggcgac cgaagcaagg
19560


cgcttggcct ccggcgactt gacgaaatcg gcgtgcaaac ctaccagggt ggttttggcg
19620


tcgaccagcg cctgcctgtc agcgtgcaga gactccttgt tgccctgttc ggcatcttgc
19680


agagtgagat ccagcgcact gatgtgctca tccagcgacg cgatgctgtt gctcaggcct
19740


tcgccgattg ccttgcttgc acgaccggcg tattcgccaa gggcagtctg actgacggca
19800


agcgtcgcct tgtccgcttt tgcatgctgg cctaccgttg cgggcgaagc gtcatgcatc
19860


agttgaaagt gctccagttg atcagcgacc gactgagcaa aacccttgat cagttgcccg
19920


acctcggctt tatccggtat ctgacccggc tgggcgaatt tttccagccg ctgctgcaag
19980


tccgagccct gaaactgctt cagttgatag cgctcaggag acaatttctc ggccatgact
20040


tcaaaaggca aaggctcggc ctgcagcaga ctaccgatca acaacgcagc acgcgaactg
20100


atcatcggcg cgccgctgac cggagccgtc ccatgctcag ccttgaaggc ctgcaaaagc
20160


tgtgtgtgtc gagccgcgac attcagccgc gccgcgccgg cagacgagct ttctgtcgcg
20220


tgtgaccctg actgatcggg agtcagcggc ggattcatgc ctgcagtgac tgcatttggg
20280


tgagctgtct gggcgggaac agtatcgtgc tgctggttta cccggctgag tttgacgcca
20340


ccggccccgc cgatccgcga actgatcatt ggaatctccc aggagccgaa aggctctcgc
20400


gtttggctgc tggggcaaca ggttggtccg tcgaggagcc tgcagttgtg gcctgcccca
20460


tgaatccatg ctcgcgccac tctttggcca ggtcggaaaa cgacttcatc aacaacagca
20520


cgccttcggc agaggctcgt tcaagggcca cagagcccat cagcagcaca cgaccggtct
20580


gcgcattaaa ggaaaatgcc gggctgtggg cgcccgcgaa catgtgaaag ttgatgtcca
20640


tcaacgccag caacgcgctc tcacggccgc gcgcgggcaa cgcgcccatg tcaccgtaga
20700


tcagaacggc acggccttcg tcgcggtcct gaaactgcag ggtgaagtcc acttcgctga
20760


ttttgaaatt ggcagattca tagaaacgtt caggtgtgga aatcaggctg agtgcgcaga
20820


tttcgttgat aagggtgtgg tactggtcat tgttggtcat ttcaaggcct ctgagtgcgg
20880


tgcggacgaa taccagtctt cctgctggcg tgtgcacact gagtcgcagg cataggcatt
20940


tcagttcctt gcgttggttg ggcatataaa aaaaggaact tttaaaaaca gtgcaatgag
21000


atgccggcaa aacgggaacc ggtcgctgcg ctttgccact cacttcgagc aagctcaacc
21060


ccaaacatcc acatccctat cgaacggaca gcgatacggc cacttgctct ggtaaaccct
21120


ggagctggcg tcggtccaat tgcccactta gcgaggtaac gcagcatgag catcggcatc
21180


acaccccggc cgcaacagac caccacgcca ctcgattttt cggcgctaag cggcaagagt
21240


cctcaaccaa acacgttcgg cgagcagaac actcagcaag cgatcgaccc gagtgcactg
21300


ttgttcggca gcgacacaca gaaagacgtc aacttcggca cgcccgacag caccgtccag
21360


aatccgcagg acgccagcaa gcccaacgac agccagtcca acatcgctaa attgatcagt
21420


gcattgatca tgtcgttgct gcagatgctc accaactcca ataaaaagca ggacaccaat
21480


caggaacagc ctgatagcca ggctcctttc cagaacaacg gcgggctcgg tacaccgtcg
21540


gccgatagcg ggggcggcgg tacaccggat gcgacaggtg gcggcggcgg tgatacgcca
21600


agcgcaacag gcggtggcgg cggtgatact ccgaccgcaa caggcggtgg cggcagcggt
21660


ggcggcggca cacccactgc aacaggtggc ggcagcggtg gcacacccac tgcaacaggc
21720


ggtggcgagg gtggcgtaac accgcaaatc actccgcagt tggccaaccc taaccgtacc
21780


tcaggtactg gctcggtgtc ggacaccgca ggttctaccg agcaagccgg caagatcaat
21840


gtggtgaaag acaccatcaa ggtcggcgct ggcgaagtct ttgacggcca cggcgcaacc
21900


ttcactgccg acaaatctat gggtaacgga gaccagggcg aaaatcagaa gcccatgttc
21960


gagctggctg aaggcgctac gttgaagaat gtgaacctgg gtgagaacga ggtcgatggc
22020


atccacgtga aagccaaaaa cgctcaggaa gtcaccattg acaacgtgca tgcccagaac
22080


gtcggtgaag acctgattac ggtcaaaggc gagggaggcg cagcggtcac taatctgaac
22140


atcaagaaca gcagtgccaa aggtgcagac gacaaggttg tccagctcaa cgccaacact
22200


cacttgaaaa tcgacaactt caaggccgac gatttcggca cgatggttcg caccaacggt
22260


ggcaagcagt ttgatgacat gagcatcgag ctgaacggca tcgaagctaa ccacggcaag
22320


ttcgccctgg tgaaaagcga cagtgacgat ctgaagctgg caacgggcaa catcgccatg
22380


accgacgtca aacacgccta cgataaaacc caggcatcga cccaacacac cgagctttga
22440


atccagacaa gtagcttgaa aaaagggggt ggactcgtcg agtccacccc ctttttactg
22500


tttagctaca gctcacagat tgcttacgac cgcataggcc gaaacggtat ttcacttgga
22560


gaagccgccg tgcccccctc ttctatatca gcttcacgag ccgggcgttg acgcaggtta
22620


ttgaccgtat tgcgcaagct ggcgccggta tgggtgatcg cctccccgcc catgtctttg
22680


acggtcttcg ccagtttgac ggtctggtcg gctacgtagc ctgtggtact ggatgcagtc
22740


gatttcaccg tgtcctgtat gaacgactcg gcttttttca ccgcgggatc ggttgtcagc
22800


gcggccgtgg tccagcctgc gaaaacggct gccgaacctg ccaggttggt caactgactg
22860


accgcggcct tggtcgccgg gtcggtgata tttttcgtcg ccatctcctg caacttgcct
22920


acccctgcaa agccacccgc cagggccaga ccgttttggg tcaggctgga cgctgacacc
22980


aggcttctta ccgcacccat tgcgtcggtc gccatatcca gtggcagacc ggccatccgc
23040


ttgccagcgt tgagcgccgc acccgagtag ctggccgatt tgattgcttt ataagcctcg
23100


agccagtcgt tttcttcgct cagttgagcc ttgggctctt tatccttcaa accgagcact
23160


aatgcaccgc cacgctggtg atcacgcgac tgcacactga gcaggcggtt gccaaagcct
23220


gcgttggcag ccagaccacc cgccatcgat acaccaaggt ccacagcacc ctgcacggcg
23280


ggtctggacg ccagtgccgg agccaatacg gtacgtacgg cgttgcgcgc cgagtacgtc
23340


tgaaccgcaa cccccgtgtc cagaacctgt cgagcaaggc ttggcgagtg gcgcttcacc
23400


gaagcggcca tcgcatcgtg gagcctgtcc ggcgaggcgc tcaggtaatg cagatcaccc
23460


gtcgcgcggt ccatcatctt ggtgcccacc tggtccatgg cgcccgacag cgctccggaa
23520


atgagcgggg tcagcggttt gagcggagcc ggcagccaat cgcccttgtt gatcgcaggc
23580


tgcatgtact gaagcaacga ggccatggca aagggcgtcg cccgcaacgc gcctgatgta
23640


gtcgtcgcca atcggtcgag cttttccgcc ttggcgaagg tgtcggcgat ggttgccggg
23700


gtttcccctt cgaagtgcag gcggctggcg cgcgtctcga tcagcgcagt gatctgcgca
23760


ttgtgtacgt caactgcagc ttggccatca gccgaatcgg ccggcggcag tttatgcgca
23820


gcgaacacat gatctgtcag gtaatcggca atcgcattta tctcgcgttg ctgatcggag
23880


ctgacagatc gcacagagct ggaggcaaga gacgcgtcgg acgctgtccg aaagctatcc
23940


gtcgcagtca caggcggttg ttggacgcgt cggttgatgt gcatggaaat tccctctcgt
24000


tctacggaag tttgaacagc gcagtgctga agcgggcgtg tccggagcga ctacttgcgt
24060


gaaagcaata cagtgaactg tcgatcaaac agcgccagaa acagcgaaac gtccggtcgt
24120


ccgccggttt aaaaggatcg acgaaggctg tgtggtcccg gatcggttga cggttccact
24180


gaataatctg cgtacgccca ctaccaagga ctgcgccgaa aaatcaccgt cgtttgtgtt
24240


gcagattacg caaattgaaa ttaagcgagc tttaaggatg gcagcgtaag ttcacaacat
24300


ggcttggcgc ttagcgagta agcgccttCt tccaaaccag caaaggagtg ccgcaatgtc
24360


tggtcctttc gagaaaaaat ggcggtgttt cacccgaacc gtgacctacg ttggctggtc
24420


gctgttctgg cttctgctct gggacgtggc cgtcaccgtg gacgtcatgc tgatagaagg
24480


caaaggcatc gacttccccc tgatgcccct cacgttgctt tgctcggcac tgatcgtgct
24540


gatcagcttt cgcaactcga gtgcctataa ccgttggtgg gaagcgcgca ccttgtgggg
24600


cgcaatggtc aacacttcac gcagttttgg ccggcaggta ctgacgctga tcgatggcga
24660


acgggatgac ctcaacaacc ctgtcaaagc catactcttt caacgtcatg tggcttactt
24720


gcgtgccctg cgcgcgcacc tcaaaggcga cgtcaaaaca gcaaaactcg acgggttact
24780


gtcgcccgac gagattcagc gcgccagcca gagcaacaac ttccccaatg acatcctcaa
24840


tggctctgct gcggttatct cgcaagcctt tgccgccggc cagttcgaca gcatccgtct
24900


gacccgcctg gaatcgacca tggtcgatct gtccaactgt cagggcggca tggagcgcat
24960


cgccaacacg ccactgccct acccctacgt ttatttccca cggctgttca gcacgctgtt
25020


ctgcatcctg atgccgctga gcatggtcac caccctgggc tggttcaccc cggcgatctc
25080


cacggtggta ggctgcatgc tgctggcaat ggaccgcatc ggtacagacc tgcaagcccc
25140


gttcggcaac agtcagcacc ggatccgcat ggaagacctg tgcaacacca tcgaaaagaa
25200


cctgcaatcg atgttctctt cgccagagag gcagccgctg ctggctgacc tgaaaagccc
25260


cgtaccgtgg cgcgtggcca acgcatcaat tggcggtctg agcaggcaga aaaacaggtt
25320


aggggaaggc gcgaggctta tcgcaagtga aagtctgctc tgggcaccat ttcgctcagt
25380


tgcagacgtt gctccgtgcc acgccagtgc gtacctacgt cgcgcttgaa cacatcagca
25440


agaaaatggc tcatgttgct gaagctgtct gcctgaacca cgccaaaaag aggatcaaaa
25500


aaatgcagac atccctgact gtcctgatgc agagccatcg catggctatc actcaaaaac
25560


agaagcatct ggtctttacc gggctgcaac actgctttga gatcgcgatc aaggttttcc
25620


agagcaaccg catagtgcgc gtgctgtgct ctgcccagcc cttttccaag tgtcatgccc
25680


aacttgggaa gtgtgtccag aagcataggt gctgcgttct gcaacttgtt tgaataggcc
25740


tgctgctcga tatgctggaa gcccattacc ctgggtagca atgcatcgcc ctgatagtcc
25800


tccagtttgt gaaagaaggc ctcatccgac tgcccttttg cacggctctg acaccaattt
25860


actgatagcc ccagacaagc gtgcccgtcg ccacccgcgc ggccatagtc agcagcaaac
25920


gctctatcat cgatagtttt ttcaaataga aatttgctct ggtgaaacgg gtggacaagc
25980


tgacagccgt gctcttgggc aatctttctt ttggcttcga tgttcgcagt cgcgcctatg
26040


ctgttgtccg ccatagcctt gattctggtc ttgatgtatt gcgtggcgcc gtcacgtaat
26100


gaggcgatag agaccatcag atccggtagc agggtacgca acgaatgaag ctggggttgt
26160


acctgctcgg gactgggaag atcagcggca tcgaccgacg aaaaggaaga gcgcgcatcg
26220


aaaaagacct cttcatgccc ctccaatggg acaaaggcgc ccgccttttc gggatgaaaa
26280


cgggcgaacg catccgacga accgggggcg agtccggaca atgacgaggg cttatcgtgt
26340


tgcgtcttag cggcaacccc tgattgggcg ccagattgct ggatatacat aaaccgccct
26400


ctgtcaggtc atgaacgttc gtggggtcag atggacagcc ggtaagaacc gaggctcttt
26460


ctgggcggtt tttccggctt gctcctggcg tcgataatct tccagatagc gctgcaacga
26520


gacggccaat gtgctaattc gcgtcatgag gtgatcaagt ccggtctcat ccagatccgc
26580


cattgagtgc acactgcgca acaacagttc ccttgaatca gggttatagc caagcgcagc
26640


gccacctgtg cgagcaggct ccagattcag cgccattgcc agaatcaaaa tgacgttgtc
26700


ctgcggcatc gtcagccttt cgatctgtgt gaagatgaac aacgaagtgt cctgttctgg
26760


caaccagagc agacactcgc ttccattcgc ggtccttacg ttgtggcgtt gaccctcctg
26820


cgcatcgatg cctcgattgc gcagccactg ataaagccga tcttttgcct cgacaggccg
26880


catggaaatt ccccgctcgt ttaacgatga ttttcctctg tggttcaaga cgtgatgcgg
26940


ttccctttag ggtttgcact aatatcaatg cgattcttgt aaaaatcgac tcgtgagtgc
27000


cgccgatggc aaaggtaacg ggatgggcag cgagtttttg gtaacgttgc cgttgttgca
27060


gggttgaatt tgttgggtga cgttaaaacg aaggaatgta tgcttaaaaa atgcctgcta
27120


ctggttatat caatgtcact tggcggctgc tggagcctga tgattcatct ggacggcgag
27180


cgttgcatct atcccggcac tcgccaaggt tgggcgtggg gaacccataa cggagggcag
27240


agttggccca tacttataga cgtgccgttt tccctcgcgt tggacacact gctgctgccc
27300


tacgacctca ccgcttttct gcccgaaaat cttggcggtg atgaccgcaa atgtcagttc
27360


agtggaggat tgaacgtgct cggttgatcc atatttttac tgcgacagaa gagtgcggcc
27420


ccgacgcttt tggagagcac accagggatt caaacccgcc ttaaaagctt tatatgcgtg
27480


gcatgcacct cgtcaactgc ctgaaagccg caacgtaagt aaaattttgc tccgctcgga
27540


gtatcagtga acaggcgcac ggcgaaaaat tcctgcgccg catgctccac aagtcgattc
27600


accagagtct ttccaaggcc ttgacctctt gatgcgcttg cgacgtataa ccgtcgtagc
27660


ctgcccatat caccccgggc atgcggatca cgcgaaaggc ctccgatacc tgccagagcg
27720


ccgtccagaa gtacgaccat gaggcattca cccttggcct cgaatcgatt ctttccggac
27780


ctccactcct cgatcaagcg ggtaagaaac ctgaagccct ctgctactgc ctcttgctcc
27840


aggatcagaa cctgacaagg caattcagta atgatctgga cttctacctg tttcatctaa
27900


tgacctcatc cacagtggtc ctgcgctggc gaaaacacga gcaggtctgg acagaatgca
27960


tatgcaacag caaaggctgc aaccagtgca caccaccaga accgggttcg acagttaagc
28020


tgatatcatt caagcacctg caagccgagt agaagcacat gaaccgtcgc aagaaaatac
28080


agcaactgtt aaaggctcat gccaagaaag ccagcgctaa actggcaccg gcaaacaaat
28140


ccagctacgt gagcaaggct gatcggttga agctggcggc agagtccggt aacgacccga
28200


tcagttccgt cgaggactga acagcgacgt ttacgcgcca ccggtatggt caggctgttc
28260


attccgatgg agcgtattgc aaggagcctg ttcaacagct cacttacttc gcaaacgagt
28320


actcaccgcc ctgctccagc gcctggcgat acgcaggtct ttcctggcat cgttgtaccc
28380


aggctgcaag gttaggatgc ggctgcagca ttccctgcat tttggcgaat tcgccaatga
28440


agctcatctg aatatccgcg ccactcaatt cgtcgcccag cagataaggc gtcagcccca
28500


gagcttcatt cagatagccc agatagttgg ccagttcaga gtgaatgcgc ggatgcaaag
28560


gcgcgcccgc gtcacccagg cgaccgacgt acaggttgag catcagcggc agaatggccg
28620


aaccttcggc gaagtgcagc cattgtacgt actcatcgta ggtggcgctg gcaggatccg
28680


gttgcaggcg gccgtcgcca tgacggcgga tcaggtaatc gacgatggcg ccagactcga
28740


taaccacatg gggaccgtct tcgatcaccg gggatttgcc cagcggatga atggccttca
28800


gctcaggcgg cgcgaggttg gttttcgggt cgcgctggta gcgttttatc tcgtacggca
28860


ggccaagttc ttcgagtaac cacagaatgc gctgcgaacg tgagttgttc aggtggtgga
28920


caataatcat gtgggtctcc gctgggtgag agtgggatgt ctagaaaaag actgctgggc
28980


cgccgtagag tgccgtgaat cgaatgtcct ctggcgacct cagacgcgtc tgtcggcgca
29040


gagcgctgcc gactcaccgc gaagctgacg ctccactgcc gctttatcga ttaccgacca
29100


aacgccgatt atcttgccat cgctgaatgt gtagaacaca ttttcggaaa aggtgatgcg
29160


ccgtccctgt gtgtcctgcc ccagaaatcg accctgtggc gagcagttga agaccagccg
29220


ggcagcgacc tgtggtgctt caacgaccag caaatcgatc ttgaaacgca agtcggggat
29280


aatcctgacg tcgttttcca gcattgtttt gtagccggaa aggctgatca gctcaccgtt
29340


gtaatgcaca ttgtcatcga cgaagttgcc caactggtgc caactacggt cattcagaca
29400


ggcgatgtaa gcccgatagt gatcggtcag gttcatggcg cgccctcctt caggtgctca
29460


aagcagtcac tgtcaatcat ccagataacc cgcacagttt taacagagtc atagggaact
29520


cgtgcggccg acatcgccct aagcctcaca tctatgtact ggcgcgacgc tggtttcaag
29580


cgaaggactt cagattcatg tcttcaagta gcactacagc agcggctgac acgcaaggtc
29640


ggcaaaacgc ctcgcctaac cgactgattt tcatctccgt acttgtggca accatgggcg
29700


cgctcgcgtt tggttatgac accggtatta tcgncggcgc attgcccttc atgacgctgc
29760


cggccgatca gggcgggctg ggtttgaatg cctacagcga agggatgatc acggcttcgc
29820


tgatcgtcgg tgcagccttc ggctcactgg ccagtggcta tatttccgac cgtttcggac
29880


gacgcctgac cctgcgcctc ctgtcggtgc tgttcatcgc gggtgcgctg ggtacggcca
29940


ttgcgccgtc cattccgttc atggtcgccg cgcgcttcct gctgggtatc gcggtgggtg
30000


gcggctcggc gacggtgccg gtgttcattg ccgaaatcgc cggcccctcg cgtcgtgcgc
30060


ggctggtcag ccgcaacgaa ctgatgatcg tcagcggcca gttgctcgcc tatgtgctca
30120


gcgcggtcat ggccgcgctg ctgcacacgc cgggcatctg gcgctatatg ctggcgatcg
30180


cgatggtgcc gggggtgttg ctgctgatcg gcaccttctt cgtacctcct tcgccgngct
30240


ggctggcgtc caaaggccgt tttgacgaag ctcaggatgt gctggagcaa ctgcgcagca
30300


acaaggacga tgcgcancgt gaagtggacg aaatgaaagc tcatgacgag caggcgcgca
30360


atcgt
30365






Several undefined nucleotides exist in SEQ. ID. No. 1, however these appear to be present in intergenic regions. The CEL of Pseudomonas syringae pv. tomato DC3000 contains a number of open reading frames (ORFs). Two of the products encoded by the CEL are HrpW and AvrE, both of which are known. An additional 10 products are produced by ORF1-10, respectively, as shown in FIG. 3. The nucleotide sequences for a number of these ORFs and their encoded protein or polypeptide products are provided below.

The DNA molecule of ORF3 from the Pseudomonas syringae pv. tomato DC3000 CEL has a nucleotide sequence (SEQ. ID. No. 2) as follows:









atgatcagtt cgcggatcgg cggggccggt ggcgtcaaac tcagccgggt aaaccagcag
60



cacgatactg ttcccgccca gacagctcac ccaaatgcag tcactgcagg catgaatccg
120


ccgctgactc ccgatcagtc agggtcacac gcgacagaaa gctcgtctgc cggcgcggcg
180


cggctgaatg tcgcggctcg acacacacag cttttgcagg ccttcaaggc tgagcatggg
240


acggctccgg tcagcggcgc gccgatgatc agttcgcgtg ctgcgttgtt gatcggtagt
300


ctgctgcagg ccgagccttt gccttttgaa gtcatggccg agaaattgtc tcctgagcgc
360


tatcaactga agcagtttca gggctcggac ttgcagcagc ggctggaaaa attcgcccag
420


ccgggtcaga taccggataa agccgaggtc gggcaactga tcaagggttt tgctcagtcg
480


gtcgctgatc aactggagca ctttcaactg atgcatgacg cttcgcccgc aacggtaggc
540


cagcatgcaa aagcggacaa ggcgacgctt gccgtcagtc agactgccct tggcgaatac
600


gccggtcgtg caagcaaggc aatcggcgaa ggcctgagca acagcatcgc gtcgctggat
660


gagcacatca gtgcgctgga tctcactctg caagatgccg aacagggcaa caaggagtct
720


ctgcacgctg acaggcaggc gctggtcgac gccaaaacca ccctggtagg tttgcacgcc
780


gatttcgtca agtcgccgga ggccaagcgc cttgcttcgg tcgccgcaca tacgcaactg
840


gacaacgtcg tcagcgatct cgtcactgcc cgtaacacgg tgggtggctg gaaaggtgca
900


gggccgattg tcgcggctgc ggttccgcag ttcttgtctt caatgacaca cttgggttat
960


gtgcgtttgt ccaccagcga caagctgcga gacacgattc ccgagaccag cagcgacgcc
1020


aacatgctca aggcttcgat aatcgggatg gtggcgggca ttgctcacga gacggtcaac
1080


agcgtggtca agccgatgtt tcaggccgcc ttgcagaaga ctggcctcaa cgaacgcctg
1140


aacatggtgc caatgaaggc tgtggatacc aatacggtta ttcctgaccc cttcgagctg
1200


aaaagcgaac acggtgagct ggtcaaaaaa acgcccgagg aagtcgctca ggacaaggcg
1260


ttcgtgaaaa gtgaacgcgc gctgctgaac cagaagaagg ttcagggttc gtccacccat
1320


ccggtaggtg agctgatggc ttacagtgcc ttcggtggtt ctcaggctgt gcgccagatg
1380


ctcaacgatg ttcaccagat caatgggcag acgctgagtg caagagctct ggcatccggt
1440


tttggcgggg cggtgtctgc cagttcgcaa acgctgctgc aattgaagtc gaattatgtc
1500


gacccgcaag ggcgcaaaat tccggtattt accccggacc gcgccgagag cgatctgaaa
1560


aaggacctgc tcaaaggtat ggacctgcgc gagccgtcgg tacgcaccac gttctacagc
1620


aaggctcttt cgggtattca gagttctgca ctgacctcgg cactgccgcc tgtgaccgct
1680


caggctgaag gcgcaagtgg cacgctcagt gcgggggcta ttttgcgcaa catggccctg
1740


gcagcgacgg gttcggtgtc ctatctgtcc acgttgtaca ccaaccagtc ggttaccgca
1800


gaagccaagg cgttgaaagc ggcaggcatg ggcggtgcaa cacctatgct ggaccgtacc
1860


gagacgcttt ga
1872






The protein or polypeptide encoded by Pto DC3000 CEL ORF3 has an amino acid sequence (SEQ. ID. No. 3) as follows:









Met Ile Ser Ser Arg Ile Gly Gly Ala Gly Gly Val Lys Leu Ser Arg



  1               5                  10                  15


Val Asn Gln Gln His Asp Thr Val Pro Ala Gln Thr Ala His Pro Asn


             20                  25                  30


Ala Val Thr Ala Gly Met Asn Pro Pro Leu Thr Pro Asp Gln Ser Gly


         35                  40                  45


Ser His Ala Thr Glu Ser Ser Ser Ala Gly Ala Ala Arg Leu Asn Val


     50                  55                  60


Ala Ala Arg His Thr Gln Leu Leu Gln Ala Phe Lys Ala Glu His Gly


 65                  70                  75                  80


Thr Ala Pro Val Ser Gly Ala Pro Met Ile Ser Ser Arg Ala Ala Leu


                 85                  90                  95


Leu Ile Gly Ser Leu Leu Gln Ala Glu Pro Leu Pro Phe Glu Val Met


            100                 105                 110


Ala Glu Lys Leu Ser Pro Glu Arg Tyr Gln Leu Lys Gln Phe Gln Gly


        115                 120                 125


Ser Asp Leu Gln Gln Arg Leu Glu Lys Phe Ala Gln Pro Gly Gln Ile


    130                 135                 140


Pro Asp Lys Ala Glu Val Gly Gln Leu Ile Lys Gly Phe Ala Gln Ser


145                 150                 155                 160


Val Ala Asp Gln Leu Glu His Phe Gln Leu Met His Asp Ala Ser Pro


                165                 170                 175


Ala Thr Val Gly Gln His Ala Lys Ala Asp Lys Ala Thr Leu Ala Val


            180                 185                 190


Ser Gln Thr Ala Leu Gly Glu Tyr Ala Gly Arg Ala Ser Lys Ala Ile


        195                 200                 205


Gly Glu Gly Leu Ser Asn Ser Ile Ala Ser Leu Asp Glu His Ile Ser


    210                 215                 220


Ala Leu Asp Leu Thr Leu Gln Asp Ala Glu Gln Gly Asn Lys Glu Ser


225                 230                 235                 240


Leu His Ala Asp Arg Gln Ala Leu Val Asp Ala Lys Thr Thr Leu Val


                245                 250                 255


Gly Leu His Ala Asp Phe Val Lys Ser Pro Glu Ala Lys Arg Leu Ala


            260                 265                 270


Ser Val Ala Ala His Thr Gln Leu Asp Asn Val Val Ser Asp Leu Val


        275                 280                 285


Thr Ala Arg Asn Thr Val Gly Gly Trp Lys Gly Ala Gly Pro Ile Val


    290                 295                 300


Ala Ala Ala Val Pro Gln Phe Leu Ser Ser Met Thr His Leu Gly Tyr


305                 310                 315                 320


Val Arg Leu Ser Thr Ser Asp Lys Leu Arg Asp Thr Ile Pro Glu Thr


                325                 330                 335


Ser Ser Asp Ala Asn Met Leu Lys Ala Ser Ile Ile Gly Met Val Ala


            340                 345                 350


Gly Ile Ala His Glu Thr Val Asn Ser Val Val Lys Pro Met Phe Gln


        355                 360                 365


Ala Ala Leu Gln Lys Thr Gly Leu Asn Glu Arg Leu Asn Met Val Pro


    370                 375                 380


Met Lys Ala Val Asp Thr Asn Thr Val Ile Pro Asp Pro Phe Glu Leu


385                 390                 395                 400


Lys Ser Glu His Gly Glu Leu Val Lys Lys Thr Pro Glu Glu Val Ala


                405                 410                 415


Gln Asp Lys Ala Phe Val Lys Ser Glu Arg Ala Leu Leu Asn Gln Lys


            420                 425                 430


Lys Val Gln Gly Ser Ser Thr His Pro Val Gly Glu Leu Met Ala Tyr


        435                 440                 445


Ser Ala Phe Gly Gly Ser Gln Ala Val Arg Gln Met Leu Asn Asp Val


    450                 455                 460


His Gln Ile Asn Gly Gln Thr Leu Ser Ala Arg Ala Leu Ala Ser Gly


465                 470                 475                 480


Phe Gly Gly Ala Val Ser Ala Ser Ser Gln Thr Leu Leu Gln Leu Lys


                485                 490                 495


Ser Asn Tyr Val Asp Pro Gln Gly Arg Lys Ile Pro Val Phe Thr Pro


            500                 505                 510


Asp Arg Ala Glu Ser Asp Leu Lys Lys Asp Leu Leu Lys Gly Met Asp


        515                 520                 525


Leu Arg Glu Pro Ser Val Arg Thr Thr Phe Tyr Ser Lys Ala Leu Ser


    530                 535                 540


Gly Ile Gln Ser Ser Ala Leu Thr Ser Ala Leu Pro Pro Val Thr Ala


545                 550                 555                 560


Gln Ala Glu Gly Ala Ser Gly Thr Leu Ser Ala Gly Ala Ile Leu Arg


                565                 570                 575


Asn Met Ala Leu Ala Ala Thr Gly Ser Val Ser Tyr Leu Ser Thr Leu


            580                 585                 590


Tyr Thr Asn Gln Ser Val Thr Ala Glu Ala Lys Ala Leu Lys Ala Ala


        595                 600                 605


Gly Met Gly Gly Ala Thr Pro Met Leu Asp Arg Thr Glu Thr Leu


    610                 615                 620






The DNA molecule of ORF4 from the Pseudomonas syringae pv. tomato DC3000 CEL has a nucleotide sequence (SEQ. ID. No. 4) as follows:









atgaccaaca atgaccagta ccacaccctt atcaacgaaa tctgcgcact cagcctgatt
60



tccacacctg aacgtttcta tgaatctgcc aatttcaaaa tcagcgaagt ggacttcacc
120


ctgcagtttc aggaccgcga cgaaggccgt gccgttctga tctacggtga catgggcgcg
180


ttgcccgcgc gcggccgtga gagcgcgttg ctggcgttga tggacatcaa ctttcacatg
240


ttcgcgggcg cccacagccc ggcattttcc tttaatgcgc agaccggtcg tgtgctgctg
300


atgggctctg tggcccttga acgagcctct gccgaaggcg tgctgttgtt gatgaagtcg
360


ttttccgacc tggccaaaga gtggcgcgag catggattca tggggcaggc cacaactgca
420


ggctcctcga cggaccaacc tgttgcccca gcagccaaac gcgagagcct ttcggctcct
480


gggagattcc aatga
495






The protein or polypeptide encoded by Pto DC3000 CEL ORF4 has an amino acid sequence (SEQ. ID. No. 5) as follows:









Met Thr Asn Asn Asp Gln Tyr His Thr Leu Ile Asn Glu Ile Cys Ala



  1               5                  10                  15


Leu Ser Leu Ile Ser Thr Pro Glu Arg Phe Tyr Glu Ser Ala Asn Phe


             20                  25                  30


Lys Ile Ser Glu Val Asp Phe Thr Leu Gln Phe Gln Asp Arg Asp Glu


         35                  40                  45


Gly Arg Ala Val Leu Ile Tyr Gly Asp Met Gly Ala Leu Pro Ala Arg


     50                  55                  60


Gly Arg Glu Ser Ala Leu Leu Ala Leu Met Asp Ile Asn Phe His Met


 65                  70                  75                  80


Phe Ala Gly Ala His Ser Pro Ala Phe Ser Phe Asn Ala Gln Thr Gly


                 85                  90                  95


Arg Val Leu Leu Met Gly Ser Val Ala Leu Glu Arg Ala Ser Ala Glu


            100                 105                 110


Gly Val Leu Leu Leu Met Lys Ser Phe Ser Asp Leu Ala Lys Glu Trp


        115                 120                 125


Arg Glu His Gly Phe Met Gly Gln Ala Thr Thr Ala Gly Ser Ser Thr


    130                 135                 140


Asp Gln Pro Val Ala Pro Ala Ala Lys Arg Glu Ser Leu Ser Ala Pro


145                 150                 155                 160


Gly Arg Phe Gln






The DNA molecule of ORF5 from the Pseudomonas syringae pv. tomato DC3000 CEL has a nucleotide sequence (SEQ. ID. No. 6) as follows:









atgcacatca accgacgcgt ccaacaaccg cctgtgactg cgacggatag ctttcggaca
60



gcgtccgacg cgtctcttgc ctccagctct gtgcgatctg tcagctccga tcagcaacgc
120


gagataaatg cgattgccga ttacctgaca gatcatgtgt tcgctgcgca taaactgccg
180


ccggccgatt cggctgatgg ccaagctgca gttgacgtac acaatgcgca gatcactgcg
240


ctgatcgaga cgcgcgccag ccgcctgcac ttcgaagggg aaaccccggc aaccatcgcc
300


gacaccttcg ccaaggcgga aaagctcgac cgattggcga cgactacatc aggcgcgttg
360


cgggcgacgc cctttgccat ggcctcgttg cttcagtaca tgcagcctgc gatcaacaag
420


ggcgattggc tgccggctcc gctcaaaccg ctgaccccgc tcatttccgg agcgctgtcg
480


ggcgccatgg accaggtggg caccaagatg atggaccgcg cgacgggtga tctgcattac
540


ctgagcgcct cgccggacag gctccacgat gcgatggccg cttcggtgaa gcgccactcg
600


ccaagccttg ctcgacaggt tctggacacg ggggttgcgg ttcagacgta ctcggcgcgc
660


aacgccgtac gtaccgtatt ggctccggca ctggcgtcca gacccgccgt gcagggtgct
720


gtggaccttg gtgtatcgat ggcgggtggt ctggctgcca acgcaggctt tggcaaccgc
780


ctgctcagtg tgcagtcgcg tgatcaccag cgtggcggtg cattagtgct cggtttgaag
840


gataaagagc ccaaggctca actgagcgaa gaaaacgact ggctcgaggc ttataaagca
900


atcaaatcgg ccagctactc gggtgcggcg ctcaacgctg gcaagcggat ggccggtctg
960


ccactggata tggcgaccga cgcaatgggt gcggtaagaa gcctggtgtc agcgtccagc
1020


ctgacccaaa acggtctggc cctggcgggt ggctttgcag gggtaggcaa gttgcaggag
1080


atggcgacga aaaatatcac cgacccggcg accaaggccg cggtcagtca gttgaccaac
1140


ctggcaggtt cggcagccgt tttcgcaggc tggaccacgg ccgcgctgac aaccgatccc
1200


gcggtgaaaa aagccgagtc gttcatacag gacacggtga aatcgactgc atccagtacc
1260


acaggctacg tagccgacca gaccgtcaaa ctggcgaaga ccgtcaaaga catgggcggg
1320


gaggcgatca cccataccgg cgccagcttg cgcaatacgg tcaataacct gcgtcaacgc
1380


ccggctcgtg aagctgatat agaagagggg ggcacggcgg cttctccaag tgaaataccg
1440


tttcggccta tgcggtcgta a
1461






The protein or polypeptide encoded by Pto DC3000 CEL ORF5, now known as HopPtoA, has an amino acid sequence (SEQ. ID. No. 7) as follows:









Met His Ile Asn Arg Arg Val Gln Gln Pro Pro Val Thr Ala Thr Asp



  1               5                  10                  15


Ser Phe Arg Thr Ala Ser Asp Ala Ser Leu Ala Ser Ser Ser Val Arg


             20                  25                  30


Ser Val Ser Ser Asp Gln Gln Arg Glu Ile Asn Ala Ile Ala Asp Tyr


         35                  40                  45


Leu Thr Asp His Val Phe Ala Ala His Lys Leu Pro Pro Ala Asp Ser


     50                  55                  60


Ala Asp Gly Gln Ala Ala Val Asp Val His Asn Ala Gln Ile Thr Ala


 65                  70                  75                  80


Leu Ile Glu Thr Arg Ala Ser Arg Leu His Phe Glu Gly Glu Thr Pro


                 85                  90                  95


Ala Thr Ile Ala Asp Thr Phe Ala Lys Ala Glu Lys Leu Asp Arg Leu


            100                 105                 110


Ala Thr Thr Thr Ser Gly Ala Leu Arg Ala Thr Pro Phe Ala Met Ala


        115                 120                 125


Ser Leu Leu Gln Tyr Met Gln Pro Ala Ile Asn Lys Gly Asp Trp Leu


    130                 135                 140


Pro Ala Pro Leu Lys Pro Leu Thr Pro Leu Ile Ser Gly Ala Leu Ser


145                 150                 155                 160


Gly Ala Met Asp Gln Val Gly Thr Lys Met Met Asp Arg Ala Thr Gly


                165                 170                 175


Asp Leu His Tyr Leu Ser Ala Ser Pro Asp Arg Leu His Asp Ala Met


            180                 185                 190


Ala Ala Ser Val Lys Arg His Ser Pro Ser Leu Ala Arg Gln Val Leu


        195                 200                 205


Asp Thr Gly Val Ala Val Gln Thr Tyr Ser Ala Arg Asn Ala Val Arg


    210                 215                 220


Thr Val Leu Ala Pro Ala Leu Ala Ser Arg Pro Ala Val Gln Gly Ala


225                 230                 235                 240


Val Asp Leu Gly Val Ser Met Ala Gly Gly Leu Ala Ala Asn Ala Gly


                245                 250                 255


Phe Gly Asn Arg Leu Leu Ser Val Gln Ser Arg Asp His Gln Arg Gly


            260                 265                 270


Gly Ala Leu Val Leu Gly Leu Lys Asp Lys Glu Pro Lys Ala Gln Leu


        275                 280                 285


Ser Glu Glu Asn Asp Trp Leu Glu Ala Tyr Lys Ala Ile Lys Ser Ala


    290                 295                 300


Ser Tyr Ser Gly Ala Ala Leu Asn Ala Gly Lys Arg Met Ala Gly Leu


305                 310                 315                 320


Pro Leu Asp Met Ala Thr Asp Ala Met Gly Ala Val Arg Ser Leu Val


                325                 330                 335


Ser Ala Ser Ser Leu Thr Gln Asn Gly Leu Ala Leu Ala Gly Gly Phe


            340                 345                 350


Ala Gly Val Gly Lys Leu Gln Glu Met Ala Thr Lys Asn Ile Thr Asp


        355                 360                 365


Pro Ala Thr Lys Ala Ala Val Ser Gln Leu Thr Asn Leu Ala Gly Ser


    370                 375                 380


Ala Ala Val Phe Ala Gly Trp Thr Thr Ala Ala Leu Thr Thr Asp Pro


385                 390                 395                 400


Ala Val Lys Lys Ala Glu Ser Phe Ile Gln Asp Thr Val Lys Ser Thr


                405                 410                 415


Ala Ser Ser Thr Thr Gly Tyr Val Ala Asp Gln Thr Val Lys Leu Ala


            420                 425                 430


Lys Thr Val Lys Asp Met Gly Gly Glu Ala Ile Thr His Thr Gly Ala


        435                 440                 445


Ser Leu Arg Asn Thr Val Asn Asn Leu Arg Gln Arg Pro Ala Arg Glu


    450                 455                 460


Ala Asp Ile Glu Glu Gly Gly Thr Ala Ala Ser Pro Ser Glu Ile Pro


465                 470                 475                 480


Phe Arg Pro Met Arg Ser


                485






The DNA molecule of ORF6 from the Pseudomonas syringae pv. tomato DC3000 CEL has a nucleotide sequence (SEQ. ID. No. 8) as follows:









atgtctggtc ctttcgagaa aaaatggcgg tgtttcaccc gaaccgtgac ctacgttggc
60



tggtcgctgt tctggcttct gctctgggac gtggccgtca ccgtggacgt catgctgata
120


gaaggcaaag gcatcgactt ccccctgatg cccctcacgt tgctttgctc ggcactgatc
180


gtgctgatca gctttcgcaa ctcgagtgcc tataaccgtt ggtgggaagc gcgcaccttg
240


tggggcgcaa tggtcaacac ttcacgcagt tttggccggc aggtactgac gctgatcgat
300


ggcgaacggg atgacctcaa caaccctgtc aaagccatac tctttcaacg tcatgtggct
360


tacttgcgtg ccctgcgcgc gcacctcaaa ggcgacgtca aaacagcaaa actcgacggg
420


ttactgtcgc ccgacgagat tcagcgcgcc agccagagca acaacttccc caatgacatc
480


ctcaatggct ctgctgcggt tatctcgcaa gcctttgccg ccggccagtt cgacagcatc
540


cgtctgaccc gcctggaatc gaccatggtc gatctgtcca actgtcaggg cggcatggag
600


cgcatcgcca acacgccact gccctacccc tacgtttatt tcccacggct gttcagcacg
660


ctgttctgca tcctgatgcc gctgagcatg gtcaccaccc tgggctggtt caccccggcg
720


atctccacgg tggtaggctg catgctgctg gcaatggacc gcatcggtac agacctgcaa
780


gccccgttcg gcaacagtca gcaccggatc cgcatggaag acctgtgcaa caccatcgaa
840


aagaacctgc aatcgatgtt ctcttcgcca gagaggcagc cgctgctggc tgacctgaaa
900


agccccgtac cgtggcgcgt ggccaacgca tcaattggcg gtctgagcag gcagaaaaac
960


aggttagggg aaggcgcgag gcttatcgca agtgaaagtc tgctctgggc accatttcgc
1020


tcagttgcag acgttgctcc gtgccacgcc agtgcgtacc tacgtcgcgc ttga
1074






The protein or polypeptide encoded by Pto DC3000 CEL ORF6 has an amino acid sequence (SEQ. ID. No. 9) as follows:









Met Ser Gly Pro Phe Glu Lys Lys Trp Arg Cys Phe Thr Arg Thr Val



  1               5                  10                  15


Thr Tyr Val Gly Trp Ser Leu Phe Trp Leu Leu Leu Trp Asp Val Ala


             20                  25                  30


Val Thr Val Asp Val Met Leu Ile Glu Gly Lys Gly Ile Asp Phe Pro


         35                  40                  45


Leu Met Pro Leu Thr Leu Leu Cys Ser Ala Leu Ile Val Leu Ile Ser


     50                  55                  60


Phe Arg Asn Ser Ser Ala Tyr Asn Arg Trp Trp Glu Ala Arg Thr Leu


 65                  70                  75                  80


Trp Gly Ala Met Val Asn Thr Ser Arg Ser Phe Gly Arg Gln Val Leu


                 85                  90                  95


Thr Leu Ile Asp Gly Glu Arg Asp Asp Leu Asn Asn Pro Val Lys Ala


            100                 105                 110


Ile Leu Phe Gln Arg His Val Ala Tyr Leu Arg Ala Leu Arg Ala His


        115                 120                 125


Leu Lys Gly Asp Val Lys Thr Ala Lys Leu Asp Gly Leu Leu Ser Pro


    130                 135                 140


Asp Glu Ile Gln Arg Ala Ser Gln Ser Asn Asn Phe Pro Asn Asp Ile


145                 150                 155                 160


Leu Asn Gly Ser Ala Ala Val Ile Ser Gln Ala Phe Ala Ala Gly Gln


                165                 170                 175


Phe Asp Ser Ile Arg Leu Thr Arg Leu Glu Ser Thr Met Val Asp Leu


            180                 185                 190


Ser Asn Cys Gln Gly Gly Met Glu Arg Ile Ala Asn Thr Pro Leu Pro


        195                 200                 205


Tyr Pro Tyr Val Tyr Phe Pro Arg Leu Phe Ser Thr Leu Phe Cys Ile


    210                 215                 220


Leu Met Pro Leu Ser Met Val Thr Thr Leu Gly Trp Phe Thr Pro Ala


225                 230                 235                 240


Ile Ser Thr Val Val Gly Cys Met Leu Leu Ala Met Asp Arg Ile Gly


                245                 250                 255


Thr Asp Leu Gln Ala Pro Phe Gly Asn Ser Gln His Arg Ile Arg Met


            260                 265                 270


Glu Asp Leu Cys Asn Thr Ile Glu Lys Asn Leu Gln Ser Met Phe Ser


        275                 280                 285


Ser Pro Glu Arg Gln Pro Leu Leu Ala Asp Leu Lys Ser Pro Val Pro


    290                 295                 300


Trp Arg Val Ala Asn Ala Ser Ile Gly Gly Leu Ser Arg Gln Lys Asn


305                 310                 315                 320


Arg Leu Gly Glu Gly Ala Arg Leu Ile Ala Ser Glu Ser Leu Leu Trp


                325                 330                 335


Ala Pro Phe Arg Ser Val Ala Asp Val Ala Pro Cys His Ala Ser Ala


            340                 345                 350


Tyr Leu Arg Arg Ala


        355






The DNA molecule of ORF7 from the Pseudomonas syringae pv. tomato DC3000 CEL has a nucleotide sequence (SEQ. ID. No. 10) as follows:









atgtatatcc agcaatctgg cgcccaatca ggggttgccg ctaagacgca acacgataag
60



ccctcgtcat tgtccggact cgcccccggt tcgtcggatg cgttcgcccg ttttcatccc
120


gaaaaggcgg gcgcctttgt cccattggag gggcatgaag aggtcttttt cgatgcgcgc
180


tcttcctttt cgtcggtcga tgccgctgat cttcccagtc ccgagcaggt acaaccccag
240


cttcattcgt tgcgtaccct gctaccggat ctgatggtct ctatcgcctc attacgtgac
300


ggcgccacgc aatacatcaa gaccagaatc aaggctatgg cggacaacag cataggcgcg
360


actgcgaaca tcgaagccaa aagaaagatt gcccaagagc acggctgtca gcttgtccac
420


ccgtttcacc agagcaaatt tctatttgaa aaaactatcg atgatagagc gtttgctgct
480


gactatggcc gcgcgggtgg cgacgggcac gcttgtctgg ggctatcagt aaattggtgt
540


cagagccgtg caaaagggca gtcggatgag gccttctttc acaaactgga ggactatcag
600


ggcgatgcat tgctacccag ggtaatgggc ttccagcata tcgagcagca ggcctattca
660


aacaagttgc agaacgcagc acctatgctt ctggacacac ttcccaagtt gggcatgaca
720


cttggaaaag ggctgggcag agcacagcac gcgcactatg cggttgctct ggaaaacctt
780


gatcgcgatc tcaaagcagt gttgcagccc ggtaaagacc agatgcttct gtttttgagt
840


gatagccatg cgatggctct gcatcaggac agtcagggat gtctgcattt ttttgatcct
900


ctttttggcg tggttcaggc agacagcttc agcaacatga gccattttct tgctgatgtg
960


ttcaagcgcg acgtaggtac gcactggcgt ggcacggagc aacgtctgca actgagcgaa
1020


atggtgccca gagcagactt tcacttgcga taa
1053






The protein or polypeptide encoded by Pto DC3000 CEL ORF7 has an amino acid sequence (SEQ. ID. No. 11) as follows:









Met Tyr Ile Gln Gln Ser Gly Ala Gln Ser Gly Val Ala Ala Lys Thr



  1               5                  10                  15


Gln His Asp Lys Pro Ser Ser Leu Ser Gly Leu Ala Pro Gly Ser Ser


             20                  25                  30


Asp Ala Phe Ala Arg Phe His Pro Glu Lys Ala Gly Ala Phe Val Pro


         35                  40                  45


Leu Glu Gly His Glu Glu Val Phe Phe Asp Ala Arg Ser Ser Phe Ser


     50                  55                  60


Ser Val Asp Ala Ala Asp Leu Pro Ser Pro Glu Gln Val Gln Pro Gln


 65                  70                  75                  80


Leu His Ser Leu Arg Thr Leu Leu Pro Asp Leu Met Val Ser Ile Ala


                 85                  90                  95


Ser Leu Arg Asp Gly Ala Thr Gln Tyr Ile Lys Thr Arg Ile Lys Ala


            100                 105                 110


Met Ala Asp Asn Ser Ile Gly Ala Thr Ala Asn Ile Glu Ala Lys Arg


        115                 120                 125


Lys Ile Ala Gln Glu His Gly Cys Gln Leu Val His Pro Phe His Gln


    130                 135                 140


Ser Lys Phe Leu Phe Glu Lys Thr Ile Asp Asp Arg Ala Phe Ala Ala


145                 150                 155                 160


Asp Tyr Gly Arg Ala Gly Gly Asp Gly His Ala Cys Leu Gly Leu Ser


                165                 170                 175


Val Asn Trp Cys Gln Ser Arg Ala Lys Gly Gln Ser Asp Glu Ala Phe


            180                 185                 190


Phe His Lys Leu Glu Asp Tyr Gln Gly Asp Ala Leu Leu Pro Arg Val


        195                 200                 205


Met Gly Phe Gln His Ile Glu Gln Gln Ala Tyr Ser Asn Lys Leu Gln


    210                 215                 220


Asn Ala Ala Pro Met Leu Leu Asp Thr Leu Pro Lys Leu Gly Met Thr


225                 230                 235                 240


Leu Gly Lys Gly Leu Gly Arg Ala Gln His Ala His Tyr Ala Val Ala


                245                 250                 255


Leu Glu Asn Leu Asp Arg Asp Leu Lys Ala Val Leu Gln Pro Gly Lys


            260                 265                 270


Asp Gln Met Leu Leu Phe Leu Ser Asp Ser His Ala Met Ala Leu His


        275                 280                 285


Gln Asp Ser Gln Gly Cys Leu His Phe Phe Asp Pro Leu Phe Gly Val


    290                 295                 300


Val Gln Ala Asp Ser Phe Ser Asn Met Ser His Phe Leu Ala Asp Val


305                 310                 315                 320


Phe Lys Arg Asp Val Gly Thr His Trp Arg Gly Thr Glu Gln Arg Leu


                325                 330                 335


Gln Leu Ser Glu Met Val Pro Arg Ala Asp Phe His Leu Arg


            340                 345                 350






The DNA molecule of ORF8 from the Pseudomonas syringae pv. tomato DC3000 CEL has a nucleotide sequence (SEQ. ID. No. 12) as follows:









atgcggcctg tcgaggcaaa agatcggctt tatcagtggc tgcgcaatcg aggcatcgat
60



gcgcaggagg gtcaacgcca caacgtaagg accgcgaatg gaagcgagtg tctgctctgg
120


ttgccagaac aggacacttc gttgttcatc ttcacacaga tcgaaaggct gacgatgccg
180


caggacaacg tcattttgat tctggcaatg gcgctgaatc tggagcctgc tcgcacaggt
240


ggcgctgcgc ttggctataa ccctgattca agggaactgt tgttgcgcag tgtgcactca
300


atggcggatc tggatgagac cggacttgat cacctcatga cgcgaattag cacattggcc
360


gtctcgttgc agcgctatct ggaagattat cgacgccagg agcaagccgg aaaaaccgcc
420


cagaaagagc ctcggttctt accggctgtc catctgaccc cacgaacgtt catgacctga
480






The protein or polypeptide encoded by Pto DC3000 CEL ORF8 has an amino acid sequence (SEQ. ID. No. 13) as follows:









Met Arg Pro Val Glu Ala Lys Asp Arg Leu Tyr Gln Trp Leu Arg Asn



  1               5                  10                  15


Arg Gly Ile Asp Ala Gln Glu Gly Gln Arg His Asn Val Arg Thr Ala


             20                  25                  30


Asn Gly Ser Glu Cys Leu Leu Trp Leu Pro Glu Gln Asp Thr Ser Leu


         35                  40                  45


Phe Ile Phe Thr Gln Ile Glu Arg Leu Thr Met Pro Gln Asp Asn Val


     50                  55                  60


Ile Leu Ile Leu Ala Met Ala Leu Asn Leu Glu Pro Ala Arg Thr Gly


 65                  70                  75                  80


Gly Ala Ala Leu Gly Tyr Asn Pro Asp Ser Arg Glu Leu Leu Leu Arg


                 85                  90                  95


Ser Val His Ser Met Ala Asp Leu Asp Glu Thr Gly Leu Asp His Leu


            100                 105                 110


Met Thr Arg Ile Ser Thr Leu Ala Val Ser Leu Gln Arg Tyr Leu Glu


        115                 120                 125


Asp Tyr Arg Arg Gln Glu Gln Ala Gly Lys Thr Ala Gln Lys Glu Pro


    130                 135                 140


Arg Phe Leu Pro Ala Val His Leu Thr Pro Arg Thr Phe Met Thr


145                 150                 155






The DNA molecule of ORF9 from the Pseudomonas syringae pv. tomato DC3000 CEL has a nucleotide sequence (SEQ. ID. No. 14) as follows:









atgcttaaaa aatgcctgct actggttata tcaatgtcac ttggcggctg ctggagcctg
60



atgattcatc tggacggcga gcgttgcatc tatcccggca ctcgccaagg ttgggcgtgg
120


ggaacccata acggagggca gagttggccc atacttatag acgtgccgtt ttccctcgcg
180


ttggacacac tgctgctgcc ctacgacctc accgcttttc tgcccgaaaa tcttggcggt
240


gatgaccgca aatgtcagtt cagtggagga ttgaacgtgc tcggttga
288






The protein or polypeptide encoded by Pto DC3000 CEL ORF9 has an amino acid sequence (SEQ. ID. No. 15) as follows:









Met Leu Lys Lys Cys Leu Leu Leu Val Ile Ser Met Ser Leu Gly Gly



  1               5                  10                  15


Cys Trp Ser Leu Met Ile His Leu Asp Gly Glu Arg Cys Ile Tyr Pro


             20                  25                  30


Gly Thr Arg Gln Gly Trp Ala Trp Gly Thr His Asn Gly Gly Gln Ser


         35                  40                  45


Trp Pro Ile Leu Ile Asp Val Pro Phe Ser Leu Ala Leu Asp Thr Leu


     50                  55                  60


Leu Leu Pro Tyr Asp Leu Thr Ala Phe Leu Pro Glu Asn Leu Gly Gly


 65                  70                  75                  80


Asp Asp Arg Lys Cys Gln Phe Ser Gly Gly Leu Asn Val Leu Gly


                 85                  90                  95






The DNA molecule of ORF10 from the Pseudomonas syringae pv. tomato DC3000 CEL has a nucleotide sequence (SEQ. ID. No. 16) as follows:









atgaaacagg tagaagtcca gatcattact gaattgcctt gtcaggttct gatcctggag
60



caagaggcag tagcagaggg cttcaggttt cttacccgct tgatcgagga gtggaggtcc
120


ggaaagaatc gattcgaggc caagggtgaa tgcctcatgg tcgtacttct ggacggcgct
180


ctggcaggta tcggaggcct ttcgcgtgat ccgcatgccc ggggtgatat gggcaggcta
240


cgacggttat acgtcgcaag cgcatcaaga ggtcaaggcc ttggaaagac tctggtgaat
300


cgacttgtgg agcatgcggc gcaggaattt ttcgccgtgc gcctgttcac tgatactccg
360


agcggagcaa aattttactt acgttgcggc tttcaggcag ttgacgaggt gcatgccacg
420


catataaagc ttttaaggcg ggtttga
447






The protein or polypeptide encoded by Pto DC3000 CEL ORF10 has an amino acid sequence (SEQ. ID. No. 17) as follows:









Met Lys Gln Val Glu Val Gln Ile Ile Thr Glu Leu Pro Cys Gln Val



  1               5                  10                  15


Leu Ile Leu Glu Gln Glu Ala Val Ala Glu Gly Phe Arg Phe Leu Thr


             20                  25                  30


Arg Leu Ile Glu Glu Trp Arg Ser Gly Lys Asn Arg Phe Glu Ala Lys


         35                  40                  45


Gly Glu Cys Leu Met Val Val Leu Leu Asp Gly Ala Leu Ala Gly Ile


     50                  55                  60


Gly Gly Leu Ser Arg Asp Pro His Ala Arg Gly Asp Met Gly Arg Leu


 65                  70                  75                  80


Arg Arg Leu Tyr Val Ala Ser Ala Ser Arg Gly Gln Gly Leu Gly Lys


                 85                  90                  95


Thr Leu Val Asn Arg Leu Val Glu His Ala Ala Gln Glu Phe Phe Ala


            100                 105                 110


Val Arg Leu Phe Thr Asp Thr Pro Ser Gly Ala Lys Phe Tyr Leu Arg


        115                 120                 125


Cys Gly Phe Gln Ala Val Asp Glu Val His Ala Thr His Ile Lys Leu


    130                 135                 140


Leu Arg Arg Val


145






A DNA molecule which contains the EEL of Pseudomonas syringae pv. tomato DC3000 has a nucleotide sequence (SEQ. ID. No. 18) as follows:









ggatccagcg gcgtattgtc gtggcgatgg aacgcgttac ggattttcag cacaccggta
60



tcgatgaaca ggtggccgtt gcgggcgttg cgggtcggca tgacacaatc gaacatatca
120


acgccacggc gcacaccttc gaccagatct tcgggcttgc ctacacccat caagtaacga
180


ggtttgtctg ctggcataag gcccggcagg taatccagca ccttgatcat ctcgtgcttg
240


ggctcgccca ccgacagacc gccaatcgcc aggccgtcaa agccgatctc atccaggcct
300


tcgagcgaac gcttgcgcag gttctcgtgc atgccaccct gaacaatgcc gaacagcgcg
360


gcagtgtttt cgccgtgcgc gaccttggag cgcttggccc agcgcaacga cagctccatg
420


gagacacgtg ctacgtcttc gtcggccggg tacggcgtgc actcatcgaa aatcatcacg
480


acgtccgaac ccaggtcacg ctggacctgc atcgactctt ccgggcccat gaacaccttg
540


gcaccatcga ccggagaggc gaaggtcacg ccctcctcct tgatcttgcg catggcgccc
600


aggctgaaca cctgaaaacc gccagagtcg gtcagaatcg gccctttcca ctgcatgaaa
660


tcgtgcaggt cgccgtggcc cttgatgacc tcggtgcccg gacgcagcca caagtggaag
720


gtgttgccca gaatcatctg cgcaccggtg gcctcgatat cacgcggcaa catgcccttg
780


accgtgccgt aggtgcccac cggcatgaac gccggggtct cgaccacgcc acgcggaaag
840


gtcaggcgac cgcgacgggc cttgccgtcg gtggccaaca actcgaaaga catacgacag
900


gtgcgactca tgcgtgatcc tctggtgccg attcctgtgg ggccgtcggc gcgggattgc
960


gggtgatgaa catggcatca ccgtaactga agaagcggta cccgtgttcg atggccgccg
1020


cgtaggccgc catggtttcg ggataaccgg cgaacgccga aaccagcatc aacagcgtgg
1080


attcaggcaa atgaaaatta gtcaccaggg catcgaccac atgaaacggc cgccccggat
1140


agatgaagat gtcggtgtcg ccgctaaacg gcttcaactg gccatcacgc gcggcactct
1200


ccagcgaacg cacgctggtg gtcccgaccg caatcacccg cccgccccgc gcacggcacg
1260


ccgccacggc atcgaccacg tcctggctga cttccagcca ttcgctgtgc atgtggtgat
1320


cttcgatctg ctcgacacgc accggctgga acgtacccgc gccgacgtgc agagtgacaa
1380


aagcagtctc gacgcccttg gcggcaattg cttccatcaa cggctggtcg aaatgcaggc
1440


cggcagtcgg cgccgccaca gcaccggcgc gctgggcgta aacggtctga taacgctcgc
1500


ggtcggcacc ttcgtccggg cggtctatat aaggaggcaa cggcatatgg ccgacacgat
1560


ccagcaacgg cagcacttct tcggcaaagc gcaactcgaa cagcgcgtca tgccgcgcca
1620


ccatctcggc ctcgccgccg ccatcgatca ggatcgacga gcccggcttt ggcgacttgc
1680


tggcacgcac gtgcgccagc acacgatggc tgtccagcac gcgctcgacc agaatctcca
1740


gcttgccgcc ggacgccttc tgcccgaaca aacgtgcggg aatgacacgg gtattgttga
1800


acaccatcaa gtcgcccgag cgcaaatgct cgagcaaatc ggtgaattga cgatgtgcca
1860


gcgcgcccgt cggcccatca agggtcaaca gacgactgct gcgacgctcg gccaacgggt
1920


gacgagcaat cagggaatcg gggagttcga aggtaaagtc agcgacgcgc atgatcgggt
1980


tcgtttagca gggccgggaa gtttatccgg tttgacggca ttagtaaaaa acctgcgtaa
2040


atccctgttg accaacggaa aactcatcct tatacttcgc cgccattgag ccctgatggc
2100


ggaattggta gacgcggcgg attcaaaatc cgttttcgaa agaagtggga gttcgattct
2160


ccctcggggc accaccattg agaaaagacc ttgaaattca aggtcttttt tttcgtctgg
2220


tggaaagtgg tctgactgag gctgcgatct accccacctg cccggaattg gccgcggagc
2280


gcccaggact gccttccagc gcagagcgtc ggtacccgga tcacacgacc aaggataacg
2340


ctatgaacaa gatcgtctac gtaaaagctt acttcaaacc cattggggag gaagtctcgg
2400


ttaaagtacc tacaggcgaa attaaaaagg gctttttcgg cgacaaggaa atcatgaaaa
2460


aagagaccca gtggcagcaa accgggtggt ctgattgtca gatagacggt gaacggctat
2520


cgaaagacgt cgaagacgca gtggcgcaac tcaatgctga cggttatgag attcaaacgg
2580


tattgcctat attgtccggg gcttatgatt atgcgctcaa ataccgatac gaaatacgtc
2640


acaatagaac tgaactaagc ccaggagacc agtcctatgt cttcggctat ggctacagct
2700


tcaccgaagg cgtgacgctg gtggcgaaaa aatttcagtc gtctgcaagc tgaataatag
2760


tgacctcgtg ccacggacgc cgctctgccc cctgatacga aaacgccttc ctcaacaaga
2820


ggcaggcgta ctaacgtgca caagacctgc ccgtatcagc aagcgcaaga cgctcgcctc
2880


cacgaaataa cacggtaggt cgcgttgcta ctttttagcg gcagacggcg tgccgttgta
2940


gttgtcggtg ttgttgtcgt tatcaagatc gcggtcattt ccaccgaaag ccgcatcggt
3000


tttgttgtcg ttgtcgagat ctttgtcgtt accgccaaac gctgcatccg tatggtgatc
3060


gttgtccagg tccttgtcgt tacccccaaa tgccgcgtcg gtgtggtggt cattgtccat
3120


atccttgtcg ttgccgccaa atgccgcgtc agtcacgttg tcgttatcca gatccttgtc
3180


gttgccgcca cacgtggcac cggtgctgtt gtcgttgtcc agatcacaat cgtttacggc
3240


aaatgcaggt agcgaagtgc caatgatcgt cagcgcaagc agaaagccgc cgatctttgc
3300


cgtcaggttt ttatacgcgc gcatcaggtt ttcccggata agtgaaaatg atgaagcaag
3360


ggttactgaa cacgttcgat cagtgactaa aacagtatgt aactgcagcc ttctgcaaga
3420


ccgacagagg tcgaccaaac tgcagcctgt ttcataccca tcaatttcta tagcgaccgt
3480


tcacacgact ctcctaccga tgctgggagt accaaaaaac ttccgcactg catttttttg
3540


cagtgtcgga tggtttgacc ggttttgggg agaattgctc aaacggagaa cgatgagttt
3600


tttgttgcgt ggcatgctaa tcgatacatt tatcagtgtg tgatgcggta tggcagcttc
3660


atgcctccgt caaatagtgg acgccagtca cgttgcataa aacctgacgt cactccaaaa
3720


aaggctacgc acgaggacat tgctgagatt cggctgggca ttttcgctgt ttacacaggg
3780


atcgagcaga acgcccccat gccagccacc cgttaactca attgtctttt gccctgaaaa
3840


caacaatccc tggcttttcc gatacatagt ccagaaaagg caaatccatc acctttctgt
3900


tttcttttcg tgaagatgca tttcgcaaga cagggccttt atccgtcacg ataaagaaac
3960


cgacgtgtgt cacatccagc ccgggaagcg ggggtgtaaa tgccaatgta atcaccggtg
4020


cgcaggtggc tcaccacctg actgtcgaca aggcggctcg ggatatacgt catgctacgc
4080


tcaaccacag gcaaccctgg cagatagact ttgcctttgg ccctttcatt aaggcgtttt
4140


ctgacactta ccgcaccggg gcttatctgc gcggtaatgt catccgccac agggtatgcc
4200


gttccgtaag cccaatccgt gaaaaagtgc ttgcgattca aaaagtcaac atcgccaccc
4260


ttgtaacgaa cctgaacgag attcctcaca aaatcctgct gcgatgttga tcttcgaaac
4320


gcttcgacgt aatccagata agcaaaacaa tccagacctc tgaagtcgat gactaattgt
4380


tcaggtacat tcgctgagcc caccaacatg tttgagcggt acggtgttcc taaaaacgct
4440


cctgatacaa ggtcgatcag ctgaccttta ttcatataac ttttgttggt gcgggcttcc
4500


agcacagcat ccagtttttt tgaggtgtag gcatccagat ttagtttaac gggtgttttc
4560


atctctgcct gggcaccctg aatatcactt cccggcgccg gccccgaaac cccacaccct
4620


gccaacattg caaaggctaa agcccatagg gtcgtctttt gcatctgatt caccgtaatt
4680


ccaaagcgtc gtcggacctg attgtggctc gcgatacgcg agcaggctgc tccattcctt
4740


cgagatgccg cattggttag ctcaatcacg gcgcactatt taccacgtgt catcggttgc
4800


gtcatcggct gggagcatca gttggcaatg cattcgcggt ctcggcctca gcagacgctg
4860


gtagtgccca gagtgcagct gaccagcgtg ccgccatcga ggccgccgca gaggccgccc
4920


agcgatacgg attcgtttgc ggcaggggcc atgcccgcta ttgaatcggc tgactggccc
4980


gtgataaagg cctgatgcct cagtacgcca cctggcttac aggcgggttg cattgcaata
5040


ggtctatacc ttttgcaagg ttaacgaact gtcatcaaaa aacatggaag cacaatcaga
5100


aaaaagacct tgagtttcaa ggtctttttt cgtttggtga aaagtgatct gactcaaccc
5160


gcgatcttac cctcctctac tcgggttggc cgttagcacc caaagctacc ttcctgcgcg
5220


aatgcttgtt tcgttatggg catggcgtga tacaagcggt aggcgtacag caggtccatg
5280


agtctcggga acctgattga gagccgctct gcgctgtacc cccctggcct gagccactgt
5340


tcaaggcaac gcttccctga ccttgagcac cacttagctg ggcgccacca tcggcatgca
5400


ccaaaggcat ttgcagagag aggacagcaa agctggccaa tgcaatgaat tttgttttag
5460


agcagatatc tttaagtttc ataacaacca cctttgttga tcagaattgt tgaagaaatc
5520


atgagtcacg cttatgtgtg gcgactcatc gaaatcggtt ccaatgcaag atgggatttt
5580


tacgtccggc ctatccgctg atggcgatgc tgcggattca cctgatgcag aactggtttg
5640


attacagcga tccggcgatg gaggaagcac tttacgagac aacgatcctg cgccagttcg
5700


cagggttgag tctggatcga atcgccgatg aaaccacgat tctcaatttc cggcgcctgc
5760


tggaaaagca tgagttggca ggcgggattt tgcaggtcat caatggctat ctgggtgatc
5820


gaggtttgat gctgcgccaa ggtatggtgg tcgatgcgac gatcattcat gcgccgagct
5880


cgaccaagaa caaggacggc aaacgcgatc ccgaaatgca tcagacgaag aaaggaaacc
5940


agtatttctt cggcatgaaa gcgcatatcg gcgtcgatgc cgagtcgggt ttagtccata
6000


gcctggtggg tactgcggcg aatgtggcgg acgtgactca ggtcgatcaa ctgctgcaca
6060


gtgaggaaac ctatgtcagc ggtgatgcgg gctacaccgg cgtggacaag cgtgcggagc
6120


atcaggatcg ccagatgatc tggtcaattg cggcacgccc aagccgttat aaaaagcatg
6180


gcgagaaaag tttgatcgca cgggtctatc gcaaaatcga gttcacgaaa gcccagttgc
6240


gggcgaaggt tgaacatccg cttcgcgtga tcaagcgcca gtttggttat acgaaagtcc
6300


ggtttcgcgg gctggctaaa aacaccgcgc aacaggctac tctgtttgcc ttgtcgaacc
6360


tttggatggt gcgaaaacgg ctgctggcga tgggagaggt gcgcctgtaa tgcggaaaaa
6420


cgccttggaa aggtgctgtt tgaaggaaaa tcgatgagtt aacagcgcaa aaacgtctga
6480


ctatctgatc gggcgagttt ttttgaacct caggccatga aggcatcaaa aatcgatgct
6540


tacttcagac cttccttaac ctcagtagcg aggccggata aacgagtccc tttctatgat
6600


gctgtttcca gtaaactgac aaatttcatg cactgccgcc cgcgtgttca agcgctcaga
6660


ccttatagga aagcctcacg tctggattca gcttgccgcc gtagtttttc acattgatat
6720


cgacggtcgc tcgggacttg aggcccagat catcgatcac cagactgcgt accccatgca
6780


actctgccaa ccctgggact ccgtcacagg aagtggcgtg cgttgccccg acaaaagcga
6840


cccacttacc ttccggtttg ctcagcctta ttttttctgc tgcgtagtaa ttcatggctt
6900


gggcacgctt tatctcagct ttctccgggg ccatataggt ggacgttgta tccagcgaga
6960


caacgcgcaa cccggcgtgc ttggccgctt ccaccaaggt ggtgaagtta tatttcgtgt
7020


ggagctcttc cggggcctga tgaccctgac tctgcaaatc gaggtagttt ttcagcctgg
7080


caggcatcgg actgcctttg ggcgcgctca ggtaattatt gagcgccttg tcatgtgact
7140


cggcgcagag gtgctccata aaaagcgtgg tcacgccact ggccttcaag ctcttcatgt
7200


tattgatcag ttcacgcttg ctggacgttg aattgtgacc ctcaccaata acaagccccg
7260


gcgcatcacg taacagctcg cgcatgacac cgagactgtc cttgcttttc atcttcgtca
7320


acggcgccag ctcaggtaac ttttgcgcgt tgaaatcatc aaaataacgc gctgccttgg
7380


caatcagttt cttgtcatta ctgtcaggtg cccataaacc cttggacgtc cccagacaac
7440


tgtccatttc aaggtaattg agatttatat gaaggtggtc ccgaccttcc gagacaacaa
7500


cgtcggccag cttgagacct tgagcctcaa ggcgctgttc aagggcgtgc ttgccttctt
7560


gcaacaggat gctcacaaca tttgcagaca gttggctgct tttccccgct gcttttgagg
7620


gtgccagcgc ataggggtgc gggctctcac accagcgcgc gagctcggca agatcgctcg
7680


ccttgaagtt cgtatcctgc aatgctttgc tttgagctga agccgaggtc gaggccacgc
7740


tctggccgcc gtgcacatga ctgctgcctg ctgcgtccgg cttacgcctt ctggtgtgct
7800


ttacgccatc ctttccgcca ggctcctgcc cctcgatttt cagccggata ttttctacct
7860


tcatatccgg atagcgcccg gctggaaagc gcttcaggtc ccccagcatt ggagtctctg
7920


gcgcaacgct ggctgctgga gaggaactgg cctgtgaaga tcgggcgcga tcgtttcctg
7980


cagcttgcgc agtgggacgc tcagcttcat aggttggcgg ataatagcct ggagccggtc
8040


caccgacggg tctcatgatt gaatctccgc gtacgaaaaa tagtgccgag cccgggcgtg
8100


acgctgcccg ggccccgaca tttcagtcaa tcaatgcgcc ttcgcaatcc cgaactgatc
8160


aagcaccgga tcaacgttat ggtcgaacgc cttctgcgcc ttatgctttt tcacagcatc
8220


aatgatcatg gaaataccga aacctaccgc cagggcgcca tcgattgccc agccgaccac
8280


tggaatcgcg gcgcctaggg cggcacctgc ggcaaggccg gtggcttcac cggcaaccat
8340


gccgacggcg cgaccgatca tctgtccgcc cagacgccct aggccggctg aggcttcgcg
8400


gcccatcatc ttcgccccgg cgtcgatgcc acctttaatg gcctcggcgc ccatcctcgt
8460


gctgtcgtaa atggcctggg ttgcgccaag cttgtcgcca tgagcgatca ggctggacac
8520


tgaagcaaag cccacgatcg agttgagcgc cttgccgccg acgcccgcct cggcgagctg
8580


agtcaacatg gacggtccgc cctcatcgct tttgccttcc agaagcttgc ggcctttttt
8640


ggagtcttgc agcgtaccca acgtgctgtt catgtagttt tcatgctgat tttcggtgaa
8700


atcagggggc agcacgctgt cgtaaatggc tttctggtta tcggcggttt gcagagactg
8760


gctggcatca gactttttct ggccaagcag ctgcttcagt gcaccgcctt cgctgaagtt
8820


ggtcacgtag gacgtggcaa tcttgtcttg cagatcgggt ttgttttcaa gcacctgatt
8880


ggtagtgggt actttggaat cggggaacag gtctttttgc agttgcaact gggcggacaa
8940


accgctgatg gcgccgctgt aatcggcatt cggattatgt ttgttgacgg ccttgtccgc
9000


cttgtccata tcagtctgca gcgcttgacc gctattgacg tttttcgtct gctcgacgac
9060


tgccttttgc agcgaggcat cactgcggac cagattgcgc tcctgctcgg gaatgctttt
9120


attgaggtac gcttgtacgt caggatcagc ctgtagctgg gaaatccggt cgttcaaacc
9180


ctgctcggtc ttgtcggtgt tgcgcaggct gcgcccggcg ataacgcttt gctgggtctg
9240


ctgcaacttg accatgacgg ccgctttctg tgcaccgctg taagacttgg gtttgtcgaa
9300


tacgtccttg tccagcttgc tgatatcaat cccggccacc gcattgagcg tcgcagaatc
9360


gctgagcatg ctggcgaact ggccgccgtt ggtgggtgcg cttttcttga tccactcact
9420


cagatttttc gcgtcgaaca tcttatcagg gctgtgcgca gccttcttgc gccccgacat
9480


gcccgcttcg tctacctgac ccaaaaagcc tggttgcgac caggtgctgc aggactgttt
9540


gagcgctccg gacaaccctg ggttactttg tgccaacccc ttcaggtctt ctgcgtcgac
9600


attaccgtca actttggtct tgtccgctgc atccactgca tgatgtgggt cggcagcaat
9660


cgccagtggc atattggctc gcatcactgc cgcgctgcgc accatttcca gtgactgcgg
9720


gtcagcgtcg gggttgtcct tggtgtagtt ggccaagtcc ttgtcggcac tgtctgcggc
9780


cttttccata ttttttgcga aggtcttgag atctttgttc gtgatcttgc catctgcgtt
9840


gccaccaccc tgagcaacgt ccacggcggt cttcagcgcc gggttggcgt tgatgaaatc
9900


catggccttg ccggcatcgg ggccatcatc acgcgccatc catgccgctg caatcgggcg
9960


attgagctct ttcgccgcct gctcgcgctc ttcgggcggc agatgggcaa ccatcggctc
10020


ccaacgtttc agagcttctg gcgaggagta ttcagaattg tcgagaaagg ctgcgtctgc
10080


ggctttgggg gcgttggaag cgtcggttgc atctgtgttc gtgggagctg cgacctgttc
10140


aaccggagcg gccggggcag tcgcttcagt cggtgaagcc tcggcaggag aatctgcgca
10200


gggttgcggc tggacctgat tattcacatt ggcattggca gctgccccgc cactgccctg
10260


gagcaaaaga gccaggatag acgacgcggt ctgctcggct cctgtcggcg cgccttgcgt
10320


gttgccggcc ggctgaccga actgcacgcc ggcttgccca ccgccaccca caggtgtcgg
10380


caaggctttg gcaagaggcg actcaacagc cagagccagt tcgccaggag tgggttggtt
10440


cacgataacg aagggagaac tggatatacg catggtgagt tgccatccga gagtgagcga
10500


tggcaactgt gtggttgaag gtgcaagttg gttccagaaa aaatgatcga gatcgccatt
10560


caggcgaacg ggtcgatttg ctgcttgagc tgaacccgcg cgcgggacag gcgtgagcga
10620


acggtgccaa tcggcacgcc gaggctgttc gctgtttcct gataattgcc gtccatctcc
10680


agcgacactt ccagcacttt ttgcatgttc gacggcaggc aatcaatggc ctgaatgact
10740


cgcgccagtt gccgatgccc ctctacctga tgactgacat caccgtgccc ttccagctcg
10800


gaatgcactt cgtcttccca gctttcctga tacggctgac gatacatttt gcggaagtga
10860


ttgcggatca ggttcagcgc gatgccacac agccaggtct gcggtttgct ggcatgttga
10920


aacttgtgct cgttacgcan ggcttcaaga aacacgcact ggagaatgtc atccacatca
10980


tcagggttca tacccgcttt ttggataaac gccctgagca tctgaatctg atcgggcggc
11040


atttggcgaa ataccgcgga cnaaaatggc tgacngggct gggttgagtc nangatcaca
11100


atcttttgaa acatgggctt accctgatta atggngtaca aaccctatag cgataaccat
11160


gccnncttaa aaaaanaaaa aactggntga tttatnaaaa aattttaaaa anngaaattt
11220


tttgtataca aaacttgggc naccgntttt gcccaaaact tttgggcaaa aanatnggan
11280


ctttcanggg antgatccng gaccgnaacc cttannggaa taatccggtt aaancggcta
11340


tnaaanagng ttccnctata tggnaaaatt cgggggccca cccnttngaa ccttttggna
11400


accctttcaa tgttgatttg ncaaataagg gattnnccca aaaggtttng ctttnggg
11458






Several undefined nucleotides exist in SEQ. ID. No. 18, however these appear to be present in intergenic regions. The EEL of Pseudomonas syringae pv. tomato DC3000 contains a number of ORFs. One of the products encoded by the EEL is a homolog of TnpA′ from P. stutzeri. An additional four products are produced by ORF1-4, respectively. The nucleotide sequences for a number of these ORFs and their encoded protein or polypeptide products are provided below.

The DNA molecule of ORF1 from the Pseudomonas syringae pv. tomato DC3000 EEL has a nucleotide sequence (SEQ. ID. No. 19) as follows:









atgagacccg tcggtggacc ggctccaggc tattatccgc caacctatga agctgagcgt
60



cccactgcgc aagctgcagg aaacgatcgc gcccgatctt cacaggccag ttcctctcca
120


gcagccagcg ttgcgccaga gactccaatg ctgggggacc tgaagcgctt tccagccggg
180


cgctatccgg atatgaaggt agaaaatatc cggctgaaaa tcgaggggca ggagcctggc
240


ggaaaggatg gcgtaaagca caccagaagg cgtaagccgg acgcagcagg cagcagtcat
300


gtgcacggcg gccagagcgt ggcctcgacc tcggcttcag ctcaaagcaa agcattgcag
360


gatacgaact tcaaggcgag cgatcttgcc gagctcgcgc gctggtgtga gagcccgcac
420


ccctatgcgc tggcaccctc aaaagcagcg gggaaaagca gccaactgtc tgcaaatgtt
480


gtgagcatcc tgttgcaaga aggcaagcac gcccttgaac agcgccttga ggctcaaggt
540


ctcaagctgg ccgacgttgt tgtctcggaa ggtcgggacc accttcatat aaatctcaat
600


taccttgaaa tggacagttg tctggggacg tccaagggtt tatgggcacc tgacagtaat
660


gacaagaaac tgattgccaa ggcagcgcgt tattttgatg atttcaacgc gcaaaagtta
720


cctgagctgg cgccgttgac gaagatgaaa agcaaggaca gtctcggtgt catgcgcgag
780


ctgttacgtg atgcgccggg gcttgttatt ggtgagggtc acaattcaac gtccagcaag
840


cgtgaactga tcaataacat gaagagcttg aaggccagtg gcgtgaccac gctttttatg
900


gagcacctct gcgccgagtc acatgacaag gcgctcaata attacctgag cgcgcccaaa
960


ggcagtccga tgcctgccag gctgaaaaac tacctcgatt tgcagagtca gggtcatcag
1020


gccccggaag agctccacac gaaatataac ttcaccacct tggtggaagc ggccaagcac
1080


gccgggttgc gcgttgtctc gctggataca acgtccacct atatggcccc ggagaaagct
1140


gagataaagc gtgcccaagc catgaattac tacgcagcag aaaaaataag gctgagcaaa
1200


ccggaaggta agtgggtcgc ttttgtcggg gcaacgcacg ccacttcctg tgacggagtc
1260


ccagggttgg cagagttgca tggggtacgc agtctggtga tcgatgatct gggcctcaag
1320


tcccgagcga ccgtcgatat caatgtgaaa aactacggcg gcaagctgaa tccagacgtg
1380


aggctttcct ataaggtctg a
1401






The protein or polypeptide encoded by Pto DC3000 EEL ORF1 has an amino acid sequence (SEQ. ID. No. 20) as follows:









Met Arg Pro Val Gly Gly Pro Ala Pro Gly Tyr Tyr Pro Pro Thr Tyr



  1               5                  10                  15


Glu Ala Glu Arg Pro Thr Ala Gln Ala Ala Gly Asn Asp Arg Ala Arg


             20                  25                  30


Ser Ser Gln Ala Ser Ser Ser Pro Ala Ala Ser Val Ala Pro Glu Thr


         35                  40                  45


Pro Met Leu Gly Asp Leu Lys Arg Phe Pro Ala Gly Arg Tyr Pro Asp


     50                  55                  60


Met Lys Val Glu Asn Ile Arg Leu Lys Ile Glu Gly Gln Glu Pro Gly


 65                  70                  75                  80


Gly Lys Asp Gly Val Lys His Thr Arg Arg Arg Lys Pro Asp Ala Ala


                 85                  90                  95


Gly Ser Ser His Val His Gly Gly Gln Ser Val Ala Ser Thr Ser Ala


            100                 105                 110


Ser Ala Gln Ser Lys Ala Leu Gln Asp Thr Asn Phe Lys Ala Ser Asp


        115                 120                 125


Leu Ala Glu Leu Ala Arg Trp Cys Glu Ser Pro His Pro Tyr Ala Leu


    130                 135                 140


Ala Pro Ser Lys Ala Ala Gly Lys Ser Ser Gln Leu Ser Ala Asn Val


145                 150                 155                 160


Val Ser Ile Leu Leu Gln Glu Gly Lys His Ala Leu Glu Gln Arg Leu


                165                 170                 175


Glu Ala Gln Gly Leu Lys Leu Ala Asp Val Val Val Ser Glu Gly Arg


            180                 185                 190


Asp His Leu His Ile Asn Leu Asn Tyr Leu Glu Met Asp Ser Cys Leu


        195                 200                 205


Gly Thr Ser Lys Gly Leu Trp Ala Pro Asp Ser Asn Asp Lys Lys Leu


    210                 215                 220


Ile Ala Lys Ala Ala Arg Tyr Phe Asp Asp Phe Asn Ala Gln Lys Leu


225                 230                 235                 240


Pro Glu Leu Ala Pro Leu Thr Lys Met Lys Ser Lys Asp Ser Leu Gly


                245                 250                 255


Val Met Arg Glu Leu Leu Arg Asp Ala Pro Gly Leu Val Ile Gly Glu


            260                 265                 270


Gly His Asn Ser Thr Ser Ser Lys Arg Glu Leu Ile Asn Asn Met Lys


        275                 280                 285


Ser Leu Lys Ala Ser Gly Val Thr Thr Leu Phe Met Glu His Leu Cys


    290                 295                 300


Ala Glu Ser His Asp Lys Ala Leu Asn Asn Tyr Leu Ser Ala Pro Lys


305                 310                 315                 320


Gly Ser Pro Met Pro Ala Arg Leu Lys Asn Tyr Leu Asp Leu Gln Ser


                325                 330                 335


Gln Gly His Gln Ala Pro Glu Glu Leu His Thr Lys Tyr Asn Phe Thr


            340                 345                 350


Thr Leu Val Glu Ala Ala Lys His Ala Gly Leu Arg Val Val Ser Leu


        355                 360                 365


Asp Thr Thr Ser Thr Tyr Met Ala Pro Glu Lys Ala Glu Ile Lys Arg


    370                 375                 380


Ala Gln Ala Met Asn Tyr Tyr Ala Ala Glu Lys Ile Arg Leu Ser Lys


385                 390                 395                 400


Pro Glu Gly Lys Trp Val Ala Phe Val Gly Ala Thr His Ala Thr Ser


                405                 410                 415


Cys Asp Gly Val Pro Gly Leu Ala Glu Leu His Gly Val Arg Ser Leu


            420                 425                 430


Val Ile Asp Asp Leu Gly Leu Lys Ser Arg Ala Thr Val Asp Ile Asn


        435                 440                 445


Val Lys Asn Tyr Gly Gly Lys Leu Asn Pro Asp Val Arg Leu Ser Tyr


    450                 455                 460


Lys Val


465






The DNA molecule of ORF2 from the Pseudomonas syringae pv. tomato DC3000 EEL has a nucleotide sequence (SEQ. ID. No. 21) as follows:









atgcaaaaga cgaccctatg ggctttagcc tttgcaatgt tggcagggtg tggggtttcg
60



gggccggcgc cgggaagtga tattcagggt gcccaggcag agatgaaaac acccgttaaa
120


ctaaatctgg atgcctacac ctcaaaaaaa ctggatgctg tgctggaagc ccgcaccaac
180


aaaagttata tgaataaagg tcagctgatc gaccttgtat caggagcgtt tttaggaaca
240


ccgtaccgct caaacatgtt ggtgggctca gcgaatgtac ctgaacaatt agtcatcgac
300


ttcagaggtc tggattgttt tgcttatctg gattacgtcg aagcgtttcg aagatcaaca
360


tcgcagcagg attttgtgag gaatctcgtt caggttcgtt acaagggtgg cgatgttgac
420


tttttgaatc gcaagcactt tttcacggat tgggcttacg gaacggcata ccctgtggcg
480


gatgacatta ccgcgcagat aagccccggt gcggtaagtg tcagaaaacg ccttaatgaa
540


agggccaaag gcaaagtcta tctgccaggg ttgcctgtgg ttgagcgtag catgacgtat
600


atcccgagcc gccttgtcga cagtcaggtg gtgagccacc tgcgcaccgg tgattacatt
660


ggcatttaca cccccgcttc ccgggctgga tgtgacacac gtcggtttct ttatcgtgac
720


ggataa
726






The protein or polypeptide encoded by Pto DC3000 EEL ORF2 has amino acid sequence (SEQ. ID. No. 22) as follows:









Met Gln Lys Thr Thr Leu Trp Ala Leu Ala Phe Ala Met Leu Ala Gly



  1               5                  10                  15


Cys Gly Val Ser Gly Pro Ala Pro Gly Ser Asp Ile Gln Gly Ala Gln


             20                  25                  30


Ala Glu Met Lys Thr Pro Val Lys Leu Asn Leu Asp Ala Tyr Thr Ser


         35                  40                  45


Lys Lys Leu Asp Ala Val Leu Glu Ala Arg Thr Asn Lys Ser Tyr Met


     50                  55                  60


Asn Lys Gly Gln Leu Ile Asp Leu Val Ser Gly Ala Phe Leu Gly Thr


 65                  70                  75                  80


Pro Tyr Arg Ser Asn Met Leu Val Gly Ser Ala Asn Val Pro Glu Gln


                 85                  90                  95


Leu Val Ile Asp Phe Arg Gly Leu Asp Cys Phe Ala Tyr Leu Asp Tyr


            100                 105                 110


Val Glu Ala Phe Arg Arg Ser Thr Ser Gln Gln Asp Phe Val Arg Asn


        115                 120                 125


Leu Val Gln Val Arg Tyr Lys Gly Gly Asp Val Asp Phe Leu Asn Arg


    130                 135                 140


Lys His Phe Phe Thr Asp Trp Ala Tyr Gly Thr Ala Tyr Pro Val Ala


145                 150                 155                 160


Asp Asp Ile Thr Ala Gln Ile Ser Pro Gly Ala Val Ser Val Arg Lys


                165                 170                 175


Arg Leu Asn Glu Arg Ala Lys Gly Lys Val Tyr Leu Pro Gly Leu Pro


            180                 185                 190


Val Val Glu Arg Ser Met Thr Tyr Ile Pro Ser Arg Leu Val Asp Ser


        195                 200                 205


Gln Val Val Ser His Leu Arg Thr Gly Asp Tyr Ile Gly Ile Tyr Thr


    210                 215                 220


Pro Ala Ser Arg Ala Gly Cys Asp Thr Arg Arg Phe Leu Tyr Arg Asp


225                 230                 235                 240


Gly






The DNA molecule of ORF3 from the Pseudomonas syringae pv. tomato DC3000 EEL has a nucleotide sequence (SEQ. ID. No. 23) as follows:









   atgcgcgcgt ataaaaacct gacggcaaag atcggcggct ttctgcttgc gctgacgatc
60



attggcactt cgctacctgc atttgccgta aacgattgtg atctggacaa cgacaacagc
120


accggtgcca cgtgtggcgg caacgacaag gatctggata acgacaacgt gactgacgcg
180


gcatttggcg gcaacgacaa ggatatggac aatgaccacc acaccgacgc ggcatttggg
240


ggtaacgaca aggacctgga caacgatcac catacggatg cagcgtttgg cggtaacgac
300


aaagatctcg acaacgacaa caaaaccgat gcggctttcg gtggaaatga ccgcgatctt
360


gataacgaca acaacaccga caactacaac ggcacgccgt ctgccgctaa aaagtag
417






The protein or polypeptide encoded by Pto DC3000 EEL ORF3 has an amino acid sequence (SEQ. ID. No. 24) as follows:









Met Arg Ala Tyr Lys Asn Leu Thr Ala Lys Ile Gly Gly Phe Leu Leu



  1               5                  10                  15


Ala Leu Thr Ile Ile Gly Thr Ser Leu Pro Ala Phe Ala Val Asn Asp


             20                  25                  30


Cys Asp Leu Asp Asn Asp Asn Ser Thr Gly Ala Thr Cys Gly Gly Asn


         35                  40                  45


Asp Lys Asp Leu Asp Asn Asp Asn Val Thr Asp Ala Ala Phe Gly Gly


     50                  55                  60


Asn Asp Lys Asp Met Asp Asn Asp His His Thr Asp Ala Ala Phe Gly


 65                  70                  75                  80


Gly Asn Asp Lys Asp Leu Asp Asn Asp His His Thr Asp Ala Ala Phe


                 85                  90                  95


Gly Gly Asn Asp Lys Asp Leu Asp Asn Asp Asn Lys Thr Asp Ala Ala


            100                 105                 110


Phe Gly Gly Asn Asp Arg Asp Leu Asp Asn Asp Asn Asn Thr Asp Asn


        115                 120                 125


Tyr Asn Gly Thr Pro Ser Ala Ala Lys Lys


    130                 135






P.s. syringae pv. tomato DC3000 EEL ORF3 has now been shown to significantly reduce virulence when mutated. Perhaps more interestingly, overexpression strongly increases lesion size. Hence, this effector is biologically active and appears to have a key role in symptom production.

The DNA molecule of ORF4 from the Pseudomonas syringae pv. tomato DC3000 EEL has a nucleotide sequence (SEQ. ID. No. 25) as follows:









   atgaacaaga tcgtctacgt aaaagcttac ttcaaaccca ttggggagga agtctcggtt
60



aaagtaccta caggcgaaat taaaaagggc tttttcggcg acaaggaaat catgaaaaaa
120


gagacccagt ggcagcaaac cgggtggtct gattgtcaga tagacggtga acggctatcg
180


aaagacgtcg aagacgcagt ggcgcaactc aatgctgacg gttatgagat tcaaacggta
240


ttgcctatat tgtccggggc ttatgattat gcgctcaaat accgatacga aatacgtcac
300


aatagaactg aactaagccc aggagaccag tcctatgtct tcggctatgg ctacagcttc
360


accgaaggcg tgacgctggt ggcgaaaaaa tttcagtcgt ctgcaagctg a
411






The protein or polypeptide encoded by Pto DC3000 EEL ORF4 has an amino acid sequence (SEQ. ID. No. 26) as follows:









   Met Asn Lys Ile Val Tyr Val Lys Ala Tyr Phe Lys Pro Ile Gly Glu



  1               5                  10                  15


Glu Val Ser Val Lys Val Pro Thr Gly Glu Ile Lys Lys Gly Phe Phe


             20                  25                  30


Gly Asp Lys Glu Ile Met Lys Lys Glu Thr Gln Trp Gln Gln Thr Gly


         35                  40                  45


Trp Ser Asp Cys Gln Ile Asp Gly Glu Arg Leu Ser Lys Asp Val Glu


     50                  55                  60


Asp Ala Val Ala Gln Leu Asn Ala Asp Gly Tyr Glu Ile Gln Thr Val


 65                  70                  75                  80


Leu Pro Ile Leu Ser Gly Ala Tyr Asp Tyr Ala Leu Lys Tyr Arg Tyr


                 85                  90                  95


Glu Ile Arg His Asn Arg Thr Glu Leu Ser Pro Gly Asp Gln Ser Tyr


            100                 105                 110


Val Phe Gly Tyr Gly Tyr Ser Phe Thr Glu Gly Val Thr Leu Val Ala


        115                 120                 125


Lys Lys Phe Gln Ser Ser Ala Ser


    130                 135






The EEL of Pseudomonas syringae pv. syringae B728a contains a number of ORFs. Two of the open reading frames appear to be mobile genetic elements without comparable homologs in EELs of other Pseudomonas syringae variants. An additional four products are produced by ORF1-2 and ORF5-6, respectively. The nucleotide sequences for a number of these ORFs and their encoded protein or polypeptide products are provided below.
The DNA molecule of ORF1 from the Pseudomonas syringae pv. syringae B728a EEL has a nucleotide sequence (SEQ. ID. No. 27) as follows:









   atgggttgcg tatcgtcaaa agcatctgtc atttcttcgg acagctttcg cgcatcatat
60



acaaactctc cagaggcatc ctcagtccat caacgagcca ggacgccaag gtgcggtgag
120


cttcaggggc cccaagtgag cagattgatg ccttaccagc aggcgttagt aggtgtggcc
180


cgatggccta atccgcattt taacagggac gatgcgcccc accagatgga gtatggagaa
240


tcgttctacc ataaaagccg agagcttggt gcgtcggtcg ccaatggaga gatagaaacg
300


tttcaggagc tctggagtga agctcgtgat tggagagctt ccagagcagg ccaagatgct
360


cggcttttta gttcatcgcg tgatcccaac tcttcacggg cgtttgttac gcctataact
420


ggaccatacg aatttttaaa agatagattc gcaaaccgta aagatggaga aaagcataag
480


atgatggatt ttctcccaca cagcaatacg tttaggtttc atgggaaaat tgacggtgag
540


cgacttcctc tcacctggat ctcgataagt tctgatcgtc gtgccgacag aacaaaggat
600


ccttaccaaa ggttgcgcga ccaaggcatg aacgatgtgg gtgagcctaa tgtgatgttg
660


cacacccaag ccgagtatgt gcccaaaatt atgcaacatg tggagcatct ttataaggcc
720


gctacggatg ctgcattgtc cgatgccaat gcgctgaaaa aactcgcaga gatacattgg
780


tggacggtac aagctgttcc cgactttcgt ggaagtgcag ctaaggctga gctctgcgtg
840


cgctccattg cccaggcaag gggcatggac ctgccgccga tgagactcgg catcgtgccg
900


gatctggaag cgcttacgat gcctttgaaa gactttgtga aaagttacga agggttcttc
960


gaacataact ga
972






The protein or polypeptide encoded by Psy B728a EEL ORF1 has an amino acid sequence (SEQ. ID. No. 28) as follows:









    Met Gly Cys Val Ser Ser Lys Ala Ser Val Ile Ser Ser Asp Ser Phe



  1               5                  10                  15


Arg Ala Ser Tyr Thr Asn Ser Pro Glu Ala Ser Ser Val His Gln Arg


             20                  25                  30


Ala Arg Thr Pro Arg Cys Gly Glu Leu Gln Gly Pro Gln Val Ser Arg


         35                  40                  45


Leu Met Pro Tyr Gln Gln Ala Leu Val Gly Val Ala Arg Trp Pro Asn


     50                  55                  60


Pro His Phe Asn Arg Asp Asp Ala Pro His Gln Met Glu Tyr Gly Glu


 65                  70                  75                  80


Ser Phe Tyr His Lys Ser Arg Glu Leu Gly Ala Ser Val Ala Asn Gly


                 85                  90                  95


Glu Ile Glu Thr Phe Gln Glu Leu Trp Ser Glu Ala Arg Asp Trp Arg


            100                 105                 110


Ala Ser Arg Ala Gly Gln Asp Ala Arg Leu Phe Ser Ser Ser Arg Asp


        115                 120                 125


Pro Asn Ser Ser Arg Ala Phe Val Thr Pro Ile Thr Gly Pro Tyr Glu


    130                 135                 140


Phe Leu Lys Asp Arg Phe Ala Asn Arg Lys Asp Gly Glu Lys His Lys


145                 150                 155                 160


Met Met Asp Phe Leu Pro His Ser Asn Thr Phe Arg Phe His Gly Lys


                165                 170                 175


Ile Asp Gly Glu Arg Leu Pro Leu Thr Trp Ile Ser Ile Ser Ser Asp


            180                 185                 190


Arg Arg Ala Asp Arg Thr Lys Asp Pro Tyr Gln Arg Leu Arg Asp Gln


        195                 200                 205


Gly Met Asn Asp Val Gly Glu Pro Asn Val Met Leu His Thr Gln Ala


    210                 215                 220


Glu Tyr Val Pro Lys Ile Met Gln His Val Glu His Leu Tyr Lys Ala


225                 230                 235                 240


Ala Thr Asp Ala Ala Leu Ser Asp Ala Asn Ala Leu Lys Lys Leu Ala


                245                 250                 255


Glu Ile His Trp Trp Thr Val Gln Ala Val Pro Asp Phe Arg Gly Ser


            260                 265                 270


Ala Ala Lys Ala Glu Leu Cys Val Arg Ser Ile Ala Gln Ala Arg Gly


        275                 280                 285


Met Asp Leu Pro Pro Met Arg Leu Gly Ile Val Pro Asp Leu Glu Ala


    290                 295                 300


Leu Thr Met Pro Leu Lys Asp Phe Val Lys Ser Tyr Glu Gly Phe Phe


305                 310                 315                 320


Glu His Asn






As indicated in Table 1 (see Example 2), the DNA molecule encoding this protein or polypeptide bears significant homology to the nucleotide sequence from Pseudomonas syringae pv. phaseolicola which encodes AvrPphC.

The DNA molecule of ORF2 from the Pseudomonas syringae pv. syringae B728a EEL has a nucleotide sequence (SEQ. ID. No. 29) as follows:









   atgagaattc acagttccgg tcatggcatc tccggaccag tatcctctgc agaaaccgtt
60



gaaaaggccg tgcaatcatc ggcccaagcg cagaatgaag cgtctcacag cggtccatca
120


gaacatcctg aatcccgctc ctgtcaggca cgcccgaact acccttattc gtcagtcaaa
180


acacggttac cccctgttgc gtctgcaggg cagtcgctgt ctgagacacc ctcttcattg
240


cctggctacc tgctgttacg tcggcttgat cgtcgtccgc tggaccagga cgcaataaag
300


gggcttattc ctgctgatga agcagtgggc gaagcgcgcc gcgcgttgcc cttcggcagg
360


ggcaacattg atgtggatgc gcaacgctcc aacctggaaa gcggggcccg cacgctcgcc
420


gcaagacgcc tgagaaaaga cgccgagacg gcgggtcatg agccgatgcc cgagaacgaa
480


gacatgaact ggcatgtgct ggttgccatg tcgggtcagg tgttcggggc tggcaactgt
540


ggcgaacatg cccgtatagc gagctttgcc tacggtgcat cggctcagga aaaaggacgc
600


gctggcgatg aaaatattca tctggctgcg cagagcgggg aagatcatgt ctgggctgaa
660


acggatgatt ccagcgctgg ctcttcgcct attgtcatgg acccctggtc aaacggtcct
720


gccgtttttg cagaggacag tcggtttgct aaagataggc gcgcggtaga gcgaacggat
780


tcgttcacgc tttcaaccgc tgccaaagca ggcaagatta cacgagagac agccgagaag
840


gcgctgaccc aagcgaccag ccgtttgcag caacgtcttg ctgatcagca ggcgcaagtc
900


tcgccggttg aaggtggtcg ctatcggcaa gaaaactcgg tgcttgatga tgcgttcgcc
960


cgacgagtca gtgacatgtt gaacaatgcc gatccacggc gtgcattgca ggtggaaatc
1020


gaggcgtccg gagttgcaat gtcgctgggt gcccaaggcg tcaagacggt cgtccgacag
1080


gcgccaaaag tggtcaggca agccagaggc gtcgcatctg ctaaaggtat gtctccgcga
1140


gcaacctga
1149






The protein or polypeptide encoded by psy B728a EEL ORF2 has an amino acid sequence (SEQ. ID. No. 30) as follows:









   Met Arg Ile His Ser Ser Gly His Gly Ile Ser Gly Pro Val Ser Ser



  1               5                  10                  15


Ala Glu Thr Val Glu Lys Ala Val Gln Ser Ser Ala Gln Ala Gln Asn


             20                  25                  30


Glu Ala Ser His Ser Gly Pro Ser Glu His Pro Glu Ser Arg Ser Cys


         35                  40                  45


Gln Ala Arg Pro Asn Tyr Pro Tyr Ser Ser Val Lys Thr Arg Leu Pro


     50                  55                  60


Pro Val Ala Ser Ala Gly Gln Ser Leu Ser Glu Thr Pro Ser Ser Leu


 65                  70                  75                  80


Pro Gly Tyr Leu Leu Leu Arg Arg Leu Asp Arg Arg Pro Leu Asp Gln


                 85                  90                  95


Asp Ala Ile Lys Gly Leu Ile Pro Ala Asp Glu Ala Val Gly Glu Ala


            100                 105                 110


Arg Arg Ala Leu Pro Phe Gly Arg Gly Asn Ile Asp Val Asp Ala Gln


        115                 120                 125


Arg Ser Asn Leu Glu Ser Gly Ala Arg Thr Leu Ala Ala Arg Arg Leu


    130                 135                 140


Arg Lys Asp Ala Glu Thr Ala Gly His Glu Pro Met Pro Glu Asn Glu


145                 150                 155                 160


Asp Met Asn Trp His Val Leu Val Ala Met Ser Gly Gln Val Phe Gly


                165                 170                 175


Ala Gly Asn Cys Gly Glu His Ala Arg Ile Ala Ser Phe Ala Tyr Gly


            180                 185                 190


Ala Ser Ala Gln Glu Lys Gly Arg Ala Gly Asp Glu Asn Ile His Leu


        195                 200                 205


Ala Ala Gln Ser Gly Glu Asp His Val Trp Ala Glu Thr Asp Asp Ser


    210                 215                 220


Ser Ala Gly Ser Ser Pro Ile Val Met Asp Pro Trp Ser Asn Gly Pro


225                 230                 235                 240


Ala Val Phe Ala Glu Asp Ser Arg Phe Ala Lys Asp Arg Arg Ala Val


                245                 250                 255


Glu Arg Thr Asp Ser Phe Thr Leu Ser Thr Ala Ala Lys Ala Gly Lys


            260                 265                 270


Ile Thr Arg Glu Thr Ala Glu Lys Ala Leu Thr Gln Ala Thr Ser Arg


        275                 280                 285


Leu Gln Gln Arg Leu Ala Asp Gln Gln Ala Gln Val Ser Pro Val Glu


    290                 295                 300


Gly Gly Arg Tyr Arg Gln Glu Asn Ser Val Leu Asp Asp Ala Phe Ala


305                 310                 315                 320


Arg Arg Val Ser Asp Met Leu Asn Asn Ala Asp Pro Arg Arg Ala Leu


                325                 330                 335


Gln Val Glu Ile Glu Ala Ser Gly Val Ala Met Ser Leu Gly Ala Gln


            340                 345                 350


Gly Val Lys Thr Val Val Arg Gln Ala Pro Lys Val Val Arg Gln Ala


        355                 360                 365


Arg Gly Val Ala Ser Ala Lys Gly Met Ser Pro Arg Ala Thr


    370                 375                 380






As indicated in Table 1 (see Example 2), the DNA molecule encoding this protein or polypeptide bears significant homology to the nucleotide sequence from Pseudomonas syringae pv. phaseolicola which encodes AvrPphE.

The DNA molecule of ORF5 from the Pseudomonas syringae pv. syringae B728a EEL has a nucleotide sequence (SEQ. ID. No. 31) as follows:









atgaatatct caggtccgaa cagacgtcag gggactcagg cagagaacac tgaaagcgct
60



tcgtcatcat cggtaactaa cccaccgcta cagcgtggcg agggcagacg tctgcgacgt
120


caggatgcgc tgccaacgga tatcagatac aacgccaacc agacagcgac atcaccgcaa
180


aacgcgcgcg cggcaggaag atatgaatca ggggccagct catccggcgc gaatgatact
240


ccgcaggctg aaggttcaat gccttcgtcg tccgcccttt tacaatttcg cctcgccggc
300


gggcggaacc attctgagct ggaaaatttt catactatga tgctgaactc accgaaagca
360


tcacggggag atgctatacc tgagaagccc gaagcaatac ctaagcgcct actggagaag
420


atggaaccga ttaacctggc ccagttagct ttgcgtgata aggatctgca tgaatatgcc
480


gtaatggtct gtaaccaagt gaaaaagggt gaaggtccga actccaatat tacgcaagga
540


gatatcaagt tactgccgct gttcgccaaa gcggaaaata caagaaatcc cggcttgaat
600


ctgcatacat tcaaaagtca taaagactgt taccaggcga taaaagagca aaacagggat
660


attcaaaaaa acaagcaatc gctgagtatg cgggttgttt accccccatt caaaaagatg
720


ccagaccacc atatagcctt ggatatccaa ctgagatacg gccatcgacc gtcgattgtc
780


ggctttgagt ctgcccctgg gaacattata gatgctgcag aaagggaaat actttcagca
840


ttaggcaacg tcaaaatcaa aatggtagga aattttcttc aatactcgaa aactgactgc
900


accatgtttg cgcttaataa cgccctgaaa gcttttaaac atcacgaaga atataccgcc
960


cgtctgcaca atggagaaaa gcaggtgcct atcccggcga ccttcttgaa acatgctcag
1020


tcaaaaagct tagtggagaa tcacccggaa aaagatacca ccgtcactaa agaccagggc
1080


ggtctgcata tggaaacgct attacacaga aaccgtgcct accgggcgca acgatctgcc
1140


ggtcagcacg ttacctctat tgaaggtttc agaatgcagg aaataaagag agcaggtgac
1200


ttccttgccg caaacagggt ccgggccaag ccttga
1236






The protein or polypeptide encoded by Psy B728a EEL ORF5 has an amino acid sequence (SEQ. ID. No. 32) as follows:









Met Asn Ile Ser Gly Pro Asn Arg Arg Gln Gly Thr Gln Ala Glu Asn



  1               5                  10                  15


Thr Glu Ser Ala Ser Ser Ser Ser Val Thr Asn Pro Pro Leu Gln Arg


             20                  25                  30


Gly Glu Gly Arg Arg Leu Arg Arg Gln Asp Ala Leu Pro Thr Asp Ile


         35                  40                  45


Arg Tyr Asn Ala Asn Gln Thr Ala Thr Ser Pro Gln Asn Ala Arg Ala


     50                  55                  60


Ala Gly Arg Tyr Glu Ser Gly Ala Ser Ser Ser Gly Ala Asn Asp Thr


 65                  70                  75                  80


Pro Gln Ala Glu Gly Ser Met Pro Ser Ser Ser Ala Leu Leu Gln Phe


                 85                  90                  95


Arg Leu Ala Gly Gly Arg Asn His Ser Glu Leu Glu Asn Phe His Thr


            100                 105                 110


Met Met Leu Asn Ser Pro Lys Ala Ser Arg Gly Asp Ala Ile Pro Glu


        115                 120                 125


Lys Pro Glu Ala Ile Pro Lys Arg Leu Leu Glu Lys Met Glu Pro Ile


    130                 135                 140


Asn Leu Ala Gln Leu Ala Leu Arg Asp Lys Asp Leu His Glu Tyr Ala


145                 150                 155                 160


Val Met Val Cys Asn Gln Val Lys Lys Gly Glu Gly Pro Asn Ser Asn


                165                 170                 175


Ile Thr Gln Gly Asp Ile Lys Leu Leu Pro Leu Phe Ala Lys Ala Glu


            180                 185                 190


Asn Thr Arg Asn Pro Gly Leu Asn Leu His Thr Phe Lys Ser His Lys


        195                 200                 205


Asp Cys Tyr Gln Ala Ile Lys Glu Gln Asn Arg Asp Ile Gln Lys Asn


    210                 215                 220


Lys Gln Ser Leu Ser Met Arg Val Val Tyr Pro Pro Phe Lys Lys Met


225                 230                 235                 240


Pro Asp His His Ile Ala Leu Asp Ile Gln Leu Arg Tyr Gly His Arg


                245                 250                 255


Pro Ser Ile Val Gly Phe Glu Ser Ala Pro Gly Asn Ile Ile Asp Ala


            260                 265                 270


Ala Glu Arg Glu Ile Leu Ser Ala Leu Gly Asn Val Lys Ile Lys Met


        275                 280                 285


Val Gly Asn Phe Leu Gln Tyr Ser Lys Thr Asp Cys Thr Met Phe Ala


    290                 295                 300


Leu Asn Asn Ala Leu Lys Ala Phe Lys His His Glu Glu Tyr Thr Ala


305                 310                 315                 320


Arg Leu His Asn Gly Glu Lys Gln Val Pro Ile Pro Ala Thr Phe Leu


                325                 330                 335


Lys His Ala Gln Ser Lys Ser Leu Val Glu Asn His Pro Glu Lys Asp


            340                 345                 350


Thr Thr Val Thr Lys Asp Gln Gly Gly Leu His Met Glu Thr Leu Leu


        355                 360                 365


His Arg Asn Arg Ala Tyr Arg Ala Gln Arg Ser Ala Gly Gln His Val


    370                 375                 380


Thr Ser Ile Glu Gly Phe Arg Met Gln Glu Ile Lys Arg Ala Gly Asp


385                 390                 395                 400


Phe Leu Ala Ala Asn Arg Val Arg Ala Lys Pro


                405                 410






The DNA molecule of ORF6 from the pseudomonas syringae pv. syringae B728a EEL has a nucleotide sequence (SEQ. ID. No. 33) as follows:









atgacgctgg aacggattga acagcaaaat acgctgtttg tttatctgtg cgtgggcacg
60



ctttctactc cagccagcag cacacttctg agcgatattc tggccgccaa cctctttcat
120


tatgggtcca gcgatggggc ggccttcggg ctggacgaaa aaaataatga agtgctgctt
180


tttcagcggt ttgatccgtt acggattgat gaggatcact ttgtcagcgc ctgcgttcag
240


atgatcgaag tggcgaaaat atggcgggca aagttactgc atggccattc tgctccgctc
300


gcctcctcaa ccaggctgac gaaagccggt ttaatgctaa ccatggcggg gactattcga
360


tga
363






The protein or polypeptide encoded by Psy B728a EEL ORF6 has an amino acid sequence (SEQ. ID. No. 34) as follows:









Met Thr Leu Glu Arg Ile Glu Gln Gln Asn Thr Leu Phe Val Tyr Leu



  1               5                  10                  15


Cys Val Gly Thr Leu Ser Thr Pro Ala Ser Ser Thr Leu Leu Ser Asp


             20                  25                  30


Ile Leu Ala Ala Asn Leu Phe His Tyr Gly Ser Ser Asp Gly Ala Ala


         35                  40                  45


Phe Gly Leu Asp Glu Lys Asn Asn Glu Val Leu Leu Phe Gln Arg Phe


     50                  55                  60


Asp Pro Leu Arg Ile Asp Glu Asp His Phe Val Ser Ala Cys Val Gln


 65                  70                  75                  80


Met Ile Glu Val Ala Lys Ile Trp Arg Ala Lys Leu Leu His Gly His


                 85                  90                  95


Ser Ala Pro Leu Ala Ser Ser Thr Arg Leu Thr Lys Ala Gly Leu Met


            100                 105                 110


Leu Thr Met Ala Gly Thr Ile Arg


        115                 120






The EEL of Pseudomonas syringae pv. syringae 61 contains a number of ORFs. One of the open reading frames encodes the outer membrane protein HopPsyA. The DNA molecule which encodes HopPsyA has a nucleotide sequence (SEQ. ID. No. 35) as follows:









gtgaacccta tccatgcacg cttctccagc gtagaagcgc tcagacattc aaacgttgat
60



attcaggcaa tcaaatccga gggtcagttg gaagtcaacg gcaagcgtta cgagattcgt
120


gcggccgctg acggctcaat cgcggtcctc agacccgatc aacagtccaa agcagacaag
180


ttcttcaaag gcgcagcgca tcttattggc ggacaaagcc agcgtgccca aatagcccag
240


gtactcaacg agaaagcggc ggcagttcca cgcctggaca gaatgttggg cagacgcttc
300


gatctggaga agggcggaag tagcgctgtg ggcgccgcaa tcaaggctgc cgacagccga
360


ctgacatcaa aacagacatt tgccagcttc cagcaatggg ctgaaaaagc tgaggcgctc
420


gggcgatacc gaaatcggta tctacatgat ctacaagagg gacacgccag acacaacgcc
480


tatgaatgcg gcagagtcaa gaacattacc tggaaacgct acaggctctc gataacaaga
540


aaaaccttat catacgcccc gcagatccat gatgatcggg aagaggaaga gcttgatctg
600


ggccgataca tcgctgaaga cagaaatgcc agaaccggct tttttagaat ggttcctaaa
660


gaccaacgcg cacctgagac aaactcggga cgacttacca ttggtgtaga acctaaatat
720


ggagcgcagt tggccctcgc aatggcaacc ctgatggaca agcacaaatc tgtgacacaa
780


ggtaaagtcg tcggtccggc aaaatatggc cagcaaactg actctgccat tctttacata
840


aatggtgatc ttgcaaaagc agtaaaactg ggcgaaaagc tgaaaaagct gagcggtatc
900


cctcctgaag gattcgtcga acatacaccg ctaagcatgc agtcgacggg tctcggtctt
960


tcttatgccg agtcggttga agggcagcct tccagccacg gacaggcgag aacacacgtt
1020


atcatggatg ccttgaaagg ccagggcccc atggagaaca gactcaaaat ggcgctggca
1080


gaaagaggct atgacccgga aaatccggcg ctcagggcgc gaaactga
1128






HopPsyA has an amino acid sequence (SEQ. ID. No. 36) as follows:









Val Asn Pro Ile His Ala Arg Phe Ser Ser Val Glu Ala Leu Arg His



  1               5                  10                  15


Ser Asn Val Asp Ile Gln Ala Ile Lys Ser Glu Gly Gln Leu Glu Val


             20                  25                  30


Asn Gly Lys Arg Tyr Glu Ile Arg Ala Ala Ala Asp Gly Ser Ile Ala


         35                  40                  45


Val Leu Arg Pro Asp Gln Gln Ser Lys Ala Asp Lys Phe Phe Lys Gly


     50                  55                  60


Ala Ala His Leu Ile Gly Gly Gln Ser Gln Arg Ala Gln Ile Ala Gln


 65                  70                  75                  80


Val Leu Asn Glu Lys Ala Ala Ala Val Pro Arg Leu Asp Arg Met Leu


                 85                  90                  95


Gly Arg Arg Phe Asp Leu Glu Lys Gly Gly Ser Ser Ala Val Gly Ala


            100                 105                 110


Ala Ile Lys Ala Ala Asp Ser Arg Leu Thr Ser Lys Gln Thr Phe Ala


        115                 120                 125


Ser Phe Gln Gln Trp Ala Glu Lys Ala Glu Ala Leu Gly Arg Tyr Arg


    130                 135                 140


Asn Arg Tyr Leu His Asp Leu Gln Glu Gly His Ala Arg His Asn Ala


145                 150                 155                 160


Tyr Glu Cys Gly Arg Val Lys Asn Ile Thr Trp Lys Arg Tyr Arg Leu


                165                 170                 175


Ser Ile Thr Arg Lys Thr Leu Ser Tyr Ala Pro Gln Ile His Asp Asp


            180                 185                 190


Arg Glu Glu Glu Glu Leu Asp Leu Gly Arg Tyr Ile Ala Glu Asp Arg


        195                 200                 205


Asn Ala Arg Thr Gly Phe Phe Arg Met Val Pro Lys Asp Gln Arg Ala


    210                 215                 220


Pro Glu Thr Asn Ser Gly Arg Leu Thr Ile Gly Val Glu Pro Lys Tyr


225                 230                 235                 240


Gly Ala Gln Leu Ala Leu Ala Met Ala Thr Leu Met Asp Lys His Lys


                245                 250                 255


Ser Val Thr Gln Gly Lys Val Val Gly Pro Ala Lys Tyr Gly Gln Gln


            260                 265                 270


Thr Asp Ser Ala Ile Leu Tyr Ile Asn Gly Asp Leu Ala Lys Ala Val


        275                 280                 285


Lys Leu Gly Glu Lys Leu Lys Lys Leu Ser Gly Ile Pro Pro Glu Gly


    290                 295                 300


Phe Val Glu His Thr Pro Leu Ser Met Gln Ser Thr Gly Leu Gly Leu


305                 310                 315                 320


Ser Tyr Ala Glu Ser Val Glu Gly Gln Pro Ser Ser His Gly Gln Ala


                325                 330                 335


Arg Thr His Val Ile Met Asp Ala Leu Lys Gly Gln Gly Pro Met Glu


            340                 345                 350


Asn Arg Leu Lys Met Ala Leu Ala Glu Arg Gly Tyr Asp Pro Glu Asn


        355                 360                 365


Pro Ala Leu Arg Ala Arg Asn


    370                 375






The remaining open reading frame, designated shcA, is a DNA molecule having a nucleotide sequence (SEQ. ID. No. 37) as follows:









atggagatgc ccgccttggc gtttgacgat aagggtgcgt gcaacatgat catcgacaag
60



gcattcgctc tgacgctgtt gcgcgacgac acgcatcaac gtttgttgct gattggtctg
120


cttgagccac acgaggatct acccttgcag cgcctgttgg ctggcgctct caaccccctt
180


gtgaatgccg gccccggcat tggctgggat gagcaaagcg gcctgtacca cgcttaccaa
240


agcatcccgc gggaaaaagt cagcgtggag atgctgaagc tcgaaattgc aggattggtc
300


gaatggatga agtgttggcg agaagcccgc acgtga
336






The encoded protein or polypeptide, ShcA, has an amino acid sequence (SEQ. ID. No. 38) as follows:









Met Glu Met Pro Ala Leu Ala Phe Asp Asp Lys Gly Ala Cys Asn Met



  1               5                  10                  15


Ile Ile Asp Lys Ala Phe Ala Leu Thr Leu Leu Arg Asp Asp Thr His


             20                  25                  30


Gln Arg Leu Leu Leu Ile Gly Leu Leu Glu Pro His Glu Asp Leu Pro


         35                  40                  45


Leu Gln Arg Leu Leu Ala Gly Ala Leu Asn Pro Leu Val Asn Ala Gly


     50                  55                  60


Pro Gly Ile Gly Trp Asp Glu Gln Ser Gly Leu Tyr His Ala Tyr Gln


 65                  70                  75                  80


Ser Ile Pro Arg Glu Lys Val Ser Val Glu Met Leu Lys Leu Glu Ile


                 85                  90                  95


Ala Gly Leu Val Glu Trp Met Lys Cys Trp Arg Glu Ala Arg Thr


            100                 105                 110






In addition to the above DNA molecules and proteins or polypeptides, the present invention also relates to homologs of various DNA molecules of the present invention which have been isolated from other Pseudomonas syringae pathovars. For example, a number of AvrPphE, AvrPphF, and HopPsyA homologs have been identified from Pseudomonas syringae pathovars.
The DNA molecule from Pseudomonas syringae pv. angulata which encodes an AvrPphE homolog has a nucleotide sequence (SEQ. ID. No. 39) as follows:









atgagaattc acagtgctgg tcacagcctg cctgcgccag gccctagcgt ggaaaccact
60



gaaaaggctg ttcaatcatc atcggcccag aaccccgctt cttacagttc acaaacagaa
120


cgtcctgaag ccggttcgac tcaagtgcga ctgaactacc cttactcatc agtcaagaca
180


cgcttgccac ccgtttcttc tacagggcag gccatttctg ccacgccatc ttcattgccc
240


ggttacctgc tgttacgtcg gctcgaccga cgtccactgg atgaagacag tatcaaggct
300


ctggttccgg cagacgaagc ggtgcgtgaa gcacgccgcg cgttgccctt cggcaggggc
360


aacattgatg tggatgcaca acgtacccac ctgcaaagcg gcgctcgcgc agtcgctgca
420


aagcgcttga gaaaagatgc cgagcgcgct ggccatgagc cgatgcccgg gaatgatgag
480


atgaactggc atgttcttgt cgccatgtca gggcaggtgt ttggcgctgg caactgtggc
540


gaacatgctc gtatagcaag cttcgcttac ggggccctgg ctcaggaaag cgggcgtagt
600


ccccgcgaaa agattcattt ggccgagcag cccggaaaag atcacgtctg ggctgaaacg
660


gataattcca gcgctggctc ttcgcccatc gtcatggacc cgtggtctaa cggcgcagcc
720


attttggcgg aggacagccg gtttgccaaa gatcgcagta cggtagagcg aacatattca
780


ttcacccttg caatggcagc tgaagccggc aaggttacgc gtgaaaccgc cgagaacgtt
840


ctgacccaca cgacaagccg tctgcagaaa cgtcttgctg atcagttgcc gaacgtctca
900


ccgcttgaag gaggccgcta tcagcaggaa aagtcggtgc ttgatgaggc gttcgcccga
960


cgagtgagcg acaagttgaa tagtgacgat ccacggcgtg cgttgcagat ggaaattgaa
1020


gctgttggtg ttgcaatgtc gctgggtgcc gaaggcgtca agacggtcgc ccgacaggcg
1080


ccaaaggtgg tcaggcaagc cagaagcgtc gcgtcgtcta aaggcatgcc tccacgaaga
1140


taa
1143






The amino acid sequence (SEQ. ID. No. 40) for the AvrPphE homolog of Pseudomonas syringae pv. angulata is as follows:









Met Arg Ile His Ser Ala Gly His Ser Leu Pro Ala Pro Gly Pro Ser



  1               5                  10                  15


Val Glu Thr Thr Glu Lys Ala Val Gln Ser Ser Ser Ala Gln Asn Pro


             20                  25                  30


Ala Ser Tyr Ser Ser Gln Thr Glu Arg Pro Glu Ala Gly Ser Thr Gln


         35                  40                  45


Val Arg Leu Asn Tyr Pro Tyr Ser Ser Val Lys Thr Arg Leu Pro Pro


     50                  55                  60


Val Ser Ser Thr Gly Gln Ala Ile Ser Ala Thr Pro Ser Ser Leu Pro


 65                  70                  75                  80


Gly Tyr Leu Leu Leu Arg Arg Leu Asp Arg Arg Pro Leu Asp Glu Asp


                 85                  90                  95


Ser Ile Lys Ala Leu Val Pro Ala Asp Glu Ala Val Arg Glu Ala Arg


            100                 105                 110


Arg Ala Leu Pro Phe Gly Arg Gly Asn Ile Asp Val Asp Ala Gln Arg


        115                 120                 125


Thr His Leu Gln Ser Gly Ala Arg Ala Val Ala Ala Lys Arg Leu Arg


    130                 135                 140


Lys Asp Ala Glu Arg Ala Gly His Glu Pro Met Pro Gly Asn Asp Glu


145                 150                 155                 160


Met Asn Trp His Val Leu Val Ala Met Ser Gly Gln Val Phe Gly Ala


                165                 170                 175


Gly Asn Cys Gly Glu His Ala Arg Ile Ala Ser Phe Ala Tyr Gly Ala


            180                 185                 190


Leu Ala Gln Glu Ser Gly Arg Ser Pro Arg Glu Lys Ile His Leu Ala


        195                 200                 205


Glu Gln Pro Gly Lys Asp His Val Trp Ala Glu Thr Asp Asn Ser Ser


    210                 215                 220


Ala Gly Ser Ser Pro Ile Val Met Asp Pro Trp Ser Asn Gly Ala Ala


225                 230                 235                 240


Ile Leu Ala Glu Asp Ser Arg Phe Ala Lys Asp Arg Ser Thr Val Glu


                245                 250                 255


Arg Thr Tyr Ser Phe Thr Leu Ala Met Ala Ala Glu Ala Gly Lys Val


            260                 265                 270


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


        275                 280                 285


Gln Lys Arg Leu Ala Asp Gln Leu Pro Asn Val Ser Pro Leu Glu Gly


    290                 295                 300


Gly Arg Tyr Gln Gln Glu Lys Ser Val Leu Asp Glu Ala Phe Ala Arg


305                 310                 315                 320


Arg Val Ser Asp Lys Leu Asn Ser Asp Asp Pro Arg Arg Ala Leu Gln


                325                 330                 335


Met Glu Ile Glu Ala Val Gly Val Ala Met Ser Leu Gly Ala Glu Gly


            340                 345                 350


Val Lys Thr Val Ala Arg Gln Ala Pro Lys Val Val Arg Gln Ala Arg


        355                 360                 365


Ser Val Ala Ser Ser Lys Gly Met Pro Pro Arg Arg


    370                 375                 380






This protein or polypeptide has GC content of about 57 percent, an estimated isoelectric point of about 9.5, and an estimated molecular weight of about 41 kDa.

The DNA molecule from Pseudomonas syringae pv. glycinea which encodes an AvrPphE homolog has a nucleotide sequence (SEQ. ID. No. 41) as follows:









atgagaattc acagtgctgg tcacagcctg cccgcgccag gccctagcgt ggaaaccact
60



gaaaaggctg ttcaatcatc atcggcccag aaccccgctt cttgcagttc acaaacagaa
120


cgtcctgaag ccggttcgac tcaagtgcga ccgaactacc cttactcatc agtcaagaca
180


cgcttgccac ccgtttcttc cacagggcag gccatttctg acacgccatc ttcattgtcc
240


ggttacctgc tgttacgtcg gctcgaccga cgtccactgg atgaagacag tatcaaggct
300


ctggttccgg cagacgaagc gttgcgtgaa gcacgccgcg cgttgccctt cggcaggggc
360


aacattgatg tggatgcaca acgtacccac ctgcaaagcg gcgctcgcgc agtcgctgca
420


aagcgcttga gaaaagatgc cgagcgcgct ggccatgagc cgatgcccga gaatgatgag
480


atgaactggc atgttcttgt cgccatgtca gggcaggtgt ttggcgctgg caactgtggc
540


gaacatgctc gtatagcaag cttcgcttac ggggccctgg ctcaggaaag cgggcgtagt
600


ccccgcgaaa agattcattt ggccgagcag cccggaaaag atcacgtctg ggctgaaacg
660


gataattcca gcgctggctc ttcgcccatc gtcatggacc cgtggtctaa cggcgtagcc
720


attttggcgg aggacagccg gtttgccaaa gatcgcagtg cggtagagcg aacatattca
780


ttcacccttg caatggcagc tgaagccggc aaggttgcgc gtgaaaccgc cgagaacgtt
840


ctgacccaca cgacaagccg tctgcagaaa cgtcttgctg atcagttgcc gaacgtctca
900


ccgcttgaag gaggccgcta tcagccggaa aagtcggtgc ttgatgaggc gttcgcccga
960


cgagtgagcg acaagttgaa tagtgacgat ccacggcgtg cgttgcagat ggaaattgaa
1020


gctgttggtg ttgcaatgtc gctgggtgcc gaaggcgtca agacggtcgc ccgacaggcg
1080


ccaaaggtgg tcaggcaagc cagaagcgtc gcgtcgtcta aaggcatgcc tccacgaaga
1140


taa
1143






The amino acid sequence (SEQ. ID. No. 42) for the AvrPphE homolog of Pseudomonas syringae pv. glycinea is as follows:









Met Arg Ile His Ser Ala Gly His Ser Leu Pro Ala Pro Gly Pro Ser



  1               5                  10                  15


Val Glu Thr Thr Glu Lys Ala Val Gln Ser Ser Ser Ala Gln Asn Pro


             20                  25                  30


Ala Ser Cys Ser Ser Gln Thr Glu Arg Pro Glu Ala Gly Ser Thr Gln


         35                  40                  45


Val Arg Pro Asn Tyr Pro Tyr Ser Ser Val Lys Thr Arg Leu Pro Pro


     50                  55                  60


Val Ser Ser Thr Gly Gln Ala Ile Ser Asp Thr Pro Ser Ser Leu Ser


 65                  70                  75                  80


Gly Tyr Leu Leu Leu Arg Arg Leu Asp Arg Arg Pro Leu Asp Glu Asp


                 85                  90                  95


Ser Ile Lys Ala Leu Val Pro Ala Asp Glu Ala Leu Arg Glu Ala Arg


            100                 105                 110


Arg Ala Leu Pro Phe Gly Arg Gly Asn Ile Asp Val Asp Ala Gln Arg


        115                 120                 125


Thr His Leu Gln Ser Gly Ala Arg Ala Val Ala Ala Lys Arg Leu Arg


    130                 135                 140


Lys Asp Ala Glu Arg Ala Gly His Glu Pro Met Pro Glu Asn Asp Glu


145                 150                 155                 160


Met Asn Trp His Val Leu Val Ala Met Ser Gly Gln Val Phe Gly Ala


                165                 170                 175


Gly Asn Cys Gly Glu His Ala Arg Ile Ala Ser Phe Ala Tyr Gly Ala


            180                 185                 190


Leu Ala Gln Glu Ser Gly Arg Ser Pro Arg Glu Lys Ile His Leu Ala


        195                 200                 205


Glu Gln Pro Gly Lys Asp His Val Trp Ala Glu Thr Asp Asn Ser Ser


    210                 215                 220


Ala Gly Ser Ser Pro Ile Val Met Asp Pro Trp Ser Asn Gly Val Ala


225                 230                 235                 240


Ile Leu Ala Glu Asp Ser Arg Phe Ala Lys Asp Arg Ser Ala Val Glu


                245                 250                 255


Arg Thr Tyr Ser Phe Thr Leu Ala Met Ala Ala Glu Ala Gly Lys Val


            260                 265                 270


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


        275                 280                 285


Gln Lys Arg Leu Ala Asp Gln Leu Pro Asn Val Ser Pro Leu Glu Gly


    290                 295                 300


Gly Arg Tyr Gln Pro Glu Lys Ser Val Leu Asp Glu Ala Phe Ala Arg


305                 310                 315                 320


Arg Val Ser Asp Lys Leu Asn Ser Asp Asp Pro Arg Arg Ala Leu Gln


                325                 330                 335


Met Glu Ile Glu Ala Val Gly Val Ala Met Ser Leu Gly Ala Glu Gly


            340                 345                 350


Val Lys Thr Val Ala Arg Gln Ala Pro Lys Val Val Arg Gln Ala Arg


        355                 360                 365


Ser Val Ala Ser Ser Lys Gly Met Pro Pro Arg Arg


    370                 375                 380






This protein or polypeptide has GC content of about 57 percent, an estimated isoelectric point of about 9.1, and an estimated molecular weight of about 41 kDa.

The DNA molecule from Pseudomonas syringae pv. tabaci which encodes an AvrPphE homolog has a nucleotide sequence (SEQ. ID. No. 43) as follows:









atgagaattc acagtgctgg tcacagcctg cctgcgccag gccctagcgt ggaaaccact
60



gaaaaggctg ttcaatcatc atcggcccag aaccccgctt cttgcagttc acaaacagaa
120


cgtcctgaag ccggttcgac tcaagtgcga ccgaactacc cttactcatc agtcaagaca
180


cgcttgccac ccgtttcttc tacagggcag gccatttctg acacgccatc ttcattgccc
240


ggttacctgc tgttacgtcg gctcgaccga cgtccactgg atgaagacag tatcaaggct
300


ctggttccgg cagacgaagc ggtgcgtgaa gcacgccgcg cgttgccctt cggcaggggc
360


aacattgatg tggatgcaca acgtacccac ctgcaaagcg gcgctcgcgc agtcgctgca
420


aagcgcttga gaaaagatgc cgagcgcgct ggccatgagc cgatgcccgg gaatgatgag
480


atgaactggc atgttcttgt cgccatgtca gggcaggtgt ttggcgctgg caactgtggc
540


gaacatgctc gtatagcaag cttcgcttac ggggccctgg ctcaggaaag cgggcgtagt
600


ccccgcgaaa agattcattt ggccgagcag cccggaaaag atcacgtctg ggctgaaacg
660


gataattcca gcgctggctc ttcgcccatc gtcatggacc cgtggtctaa cggcgcagcc
720


attttggcgg aggacagccg gtttgccaaa gatcgcagtg cggtagagcg aacatattca
780


ttcacccttg caatggcagc tgaagccggc aaggttacgc gtgaaactgc cgagaacgtt
840


ctgacccaca cgacaagccg tctgcagaaa cgtcttgctg atcagttgcc gaacgtctca
900


ccgcttgaag gaggccgcta tcagcaggaa aagtcggtgc ttgatgaggc gttcgcccga
960


cgagtgagcg acaagttgaa tagtgacgat ccacggcgtg cgttgcagat ggaaattgaa
1020


gctgttggtg ttgcaatgtc gctgggtgcc gaaggcgtca agacggtcgc ccgacaggcg
1080


ccaaaggtgg tcaggcaagc cagaagcgtc gcgtcgtcta aaggcatgcc tccacgaaga
1140


taa
1143






The amino acid sequence (SEQ. ID. No. 44) for the AvrPphE homolog of Pseudomonas syringae pv. tabaci is as follows:









Met Arg Ile His Ser Ala Gly His Ser Leu Pro Ala Pro Gly Pro Ser



  1               5                  10                  15


Val Glu Thr Thr Glu Lys Ala Val Gln Ser Ser Ser Ala Gln Asn Pro


             20                  25                  30


Ala Ser Cys Ser Ser Gln Thr Glu Arg Pro Glu Ala Gly Ser Thr Gln


         35                  40                  45


Val Arg Pro Asn Tyr Pro Tyr Ser Ser Val Lys Thr Arg Leu Pro Pro


     50                  55                  60


Val Ser Ser Thr Gly Gln Ala Ile Ser Asp Thr Pro Ser Ser Leu Pro


 65                  70                  75                  80


Gly Tyr Leu Leu Leu Arg Arg Leu Asp Arg Arg Pro Leu Asp Glu Asp


                 85                  90                  95


Ser Ile Lys Ala Leu Val Pro Ala Asp Glu Ala Val Arg Glu Ala Arg


            100                 105                 110


Arg Ala Leu Pro Phe Gly Arg Gly Asn Ile Asp Val Asp Ala Gln Arg


        115                 120                 125


Thr His Leu Gln Ser Gly Ala Arg Ala Val Ala Ala Lys Arg Leu Arg


    130                 135                 140


Lys Asp Ala Glu Arg Ala Gly His Glu Pro Met Pro Gly Asn Asp Glu


145                 150                 155                 160


Met Asn Trp His Val Leu Val Ala Met Ser Gly Gln Val Phe Gly Ala


                165                 170                 175


Gly Asn Cys Gly Glu His Ala Arg Ile Ala Ser Phe Ala Tyr Gly Ala


            180                 185                 190


Leu Ala Gln Glu Ser Gly Arg Ser Pro Arg Glu Lys Ile His Leu Ala


        195                 200                 205


Glu Gln Pro Gly Lys Asp His Val Trp Ala Glu Thr Asp Asn Ser Ser


    210                 215                 220


Ala Gly Ser Ser Pro Ile Val Met Asp Pro Trp Ser Asn Gly Ala Ala


225                 230                 235                 240


Ile Leu Ala Glu Asp Ser Arg Phe Ala Lys Asp Arg Ser Ala Val Glu


                245                 250                 255


Arg Thr Tyr Ser Phe Thr Leu Ala Met Ala Ala Glu Ala Gly Lys Val


            260                 265                 270


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


        275                 280                 285


Gln Lys Arg Leu Ala Asp Gln Leu Pro Asn Val Ser Pro Leu Glu Gly


    290                 295                 300


Gly Arg Tyr Gln Gln Glu Lys Ser Val Leu Asp Glu Ala Phe Ala Arg


305                 310                 315                 320


Arg Val Ser Asp Lys Leu Asn Ser Asp Asp Pro Arg Arg Ala Leu Gln


                325                 330                 335


Met Glu Ile Glu Ala Val Gly Val Ala Met Ser Leu Gly Ala Glu Gly


            340                 345                 350


Val Lys Thr Val Ala Arg Gln Ala Pro Lys Val Val Arg Gln Ala Arg


        355                 360                 365


Ser Val Ala Ser Ser Lys Gly Met Pro Pro Arg Arg


    370                 375                 380






This protein or polypeptide has GC content of about 57 percent, an estimated isoelectric point of about 9.3, and an estimated molecular weight of about 41 kDa.

Another DNA molecule from Pseudomonas syringae pv. tabaci which encodes a AvrPphE homolog has a nucleotide sequence (SEQ. ID. No. 45) as follows:









atgagaattc acagtgctgg tcacagcctg cctgcgccag gccctagcgt ggaaaccact
60



gaaaaggctg ttcaatcatc atcggcccag aaccccgctt cttgcagttc acaaacagaa
120


cgtcctgaag ccggttcgac tcaagtgcga ccgaactacc cttactcatc agtcaagaca
180


cgcttgccac ccgtttcttc tacagggcag gccatttctg acacgccatc ttcattgccc
240


ggttacctgc tgttacgtcg gctcgaccga cgtccactgg atgaagacag tatcaaggct
300


ctggttccgg cagacgaagc ggtgcgtgaa gcacgccgcg cgttgccctt cggcaggggc
360


aacattgatg tggatgcaca acgtacccac ctgcaaagcg gcgctcgcgc agtcgctgca
420


aagcgcttga gaaaagatgc cgagcgcgct ggccatgagc cgatgcccgg gaatgatgag
480


atgaactggc atgttcttgt cgccatgtca gggcaggtgt ttggcgctgg caactgtggc
540


gaacatgctc gtatagcaag cttcgcttac ggggccctgg ctcaggaaag cgggcgtagt
600


ccccgcgaaa agattcattt ggccgagcag cccggaaaag atcacgtctg ggctgaaacg
660


gataattcca gcgctggctc ttcgcccatc gtcatggacc cgtggtctaa cggcgcagcc
720


attttggcgg aggacagccg gtttgccaaa gatcgcagtg cggtagagcg aacatattca
780


ttcacccttg caatggcagc tgaagccggc aaggttacgc gtgaaactgc cgagaacgtt
840


ctgacccaca cgacaagccg tctgcagaaa cgtcttgctg atcagttgcc gaacgtctca
900


ccgcttgaag gaggccgcta tcagcaggaa aagtcggtgc ttgatgaggc gttcgcccga
960


cgagtgagcg acaagttgaa tagtgacgat ccacggcgtg cgttgcagat ggaaattgaa
1020


gctgttggtg ttgcaatgtc gctgggtgcc gaaggcgtca agacggtcgc ccgacaggcg
1080


ccaaaggtgg tcaggcaagc cagaagcgtc gcgtcgtcta aaggcatgcc tccacgaaga
1140


taa
1143






The encoded AvrPphE homolog has an amino acid sequence according to SEQ. ID. No. 46 as follows:









Met Arg Ile His Ser Ala Gly His Ser Leu Pro Ala Pro Gly Pro Ser



  1               5                  10                  15


Val Glu Thr Thr Glu Lys Ala Val Gln Ser Ser Ser Ala Gln Asn Pro


             20                  25                  30


Ala Ser Cys Ser Ser Gln Thr Glu Arg Pro Glu Ala Gly Ser Thr Gln


         35                  40                  45


Val Arg Pro Asn Tyr Pro Tyr Ser Ser Val Lys Thr Arg Leu Pro Pro


     50                  55                  60


Val Ser Ser Thr Gly Gln Ala Ile Ser Asp Thr Pro Ser Ser Leu Pro


 65                  70                  75                  80


Gly Tyr Leu Leu Leu Arg Arg Leu Asp Arg Arg Pro Leu Asp Glu Asp


                 85                  90                  95


Ser Ile Lys Ala Leu Val Pro Ala Asp Glu Ala Val Arg Glu Ala Arg


            100                 105                 110


Arg Ala Leu Pro Phe Gly Arg Gly Asn Ile Asp Val Asp Ala Gln Arg


        115                 120                 125


Thr His Leu Gln Ser Gly Ala Arg Ala Val Ala Ala Lys Arg Leu Arg


    130                 135                 140


Lys Asp Ala Glu Arg Ala Gly His Glu Pro Met Pro Gly Asn Asp Glu


145                 150                 155                 160


Met Asn Trp His Val Leu Val Ala Met Ser Gly Gln Val Phe Gly Ala


                165                 170                 175


Gly Asn Cys Gly Glu His Ala Arg Ile Ala Ser Phe Ala Tyr Gly Ala


            180                 185                 190


Leu Ala Gln Glu Ser Gly Arg Ser Pro Arg Glu Lys Ile His Leu Ala


        195                 200                 205


Glu Gln Pro Gly Lys Asp His Val Trp Ala Glu Thr Asp Asn Ser Ser


    210                 215                 220


Ala Gly Ser Ser Pro Ile Val Met Asp Pro Trp Ser Asn Gly Ala Ala


225                 230                 235                 240


Ile Leu Ala Glu Asp Ser Arg Phe Ala Lys Asp Arg Ser Ala Val Glu


                245                 250                 255


Arg Thr Tyr Ser Phe Thr Leu Ala Met Ala Ala Glu Ala Gly Lys Val


            260                 265                 270


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


        275                 280                 285


Gln Lys Arg Leu Ala Asp Gln Leu Pro Asn Val Ser Pro Leu Glu Gly


    290                 295                 300


Gly Arg Tyr Gln Gln Glu Lys Ser Val Leu Asp Glu Ala Phe Ala Arg


305                 310                 315                 320


Arg Val Ser Asp Lys Leu Asn Ser Asp Asp Pro Arg Arg Ala Leu Gln


                325                 330                 335


Met Glu Ile Glu Ala Val Gly Val Ala Met Ser Leu Gly Ala Glu Gly


            340                 345                 350


Val Lys Thr Val Ala Arg Gln Ala Pro Lys Val Val Arg Gln Ala Arg


        355                 360                 365


Ser Val Ala Ser Ser Lys Gly Met Pro Pro Arg Arg


    370                 375                 380






A DNA molecule from Pseudomonas syringae pv. glycinea race 4 which encodes an avrPphE homolog has a nucleotide sequence (SEQ. ID. No. 47) as follows:









atgagaattc acagtgctgg tcacagcctg cccgcgccag gccctagcgt ggaaaccact
60



gaaaaggctg ttcaatcatc atcggcccag aaccccgctt cttgcagttc acaaacagaa
120


cgtcctgaag ccggttcgac tcaagtgcga ccgaactacc cttactcatc agtcaagaca
180


cgcttgccac ccgtttcttc cacagggcag gccatttctg acacgccatc ttcattgtcc
240


ggttacctgc tgttacgtcg gctcgaccga cgtccactgg atgaagacag tatcaaggct
300


ctggttccgg cagacgaagc gttgcgtgaa gcacgccgcg cgttgccctt cggcaggggc
360


aacattgatg tggatgcaca acgtacccac ctgcaaagcg gcgctcgcgc agtcgctgca
420


aagcgcttga gaaaagatgc cgagcgcgct ggccatgagc cgatgcccga gaatgatgag
480


atgaactggc atgttcttgt cgccatgtca gggcaggtgt ttggcgctgg caactgtggc
540


gaacatgctc gtatagcaag cttcgcttac ggggccctgg ctcaggaaag cgggcgtagt
600


ccccgcgaaa agattcattt ggccgagcag cccggaaaag atcacgtctg ggctgaaacg
660


gataattcca gcgctggctc ttcgcccatc gtcatggacc cgtggtctaa cggcgtagcc
720


attttggcgg aggacagccg gtttgccaaa gatcgcagtg cggtagagcg aacatattca
780


ttcacccttg caatggcagc tgaagccggc aaggttgcgc gtgaaaccgc cgagaacgtt
840


ctgacccaca cgacaagccg tctgcagaaa cgtcttgctg atcagttgcc gaacgtctca
900


ccgcttgaag gaggccgcta tcagccggaa aagtcggtgc ttgatgaggc gttcgcccga
960


cgagtgagcg acaagttgaa tagtgacgat ccacggcgtg cgttgcagat ggaaattgaa
1020


gctgttggtg ttgcaatgtc gctgggtgcc gaaggcgtca agacggtcgc ccgacaggcg
1080


ccaaaggtgg tcaggcaagc cagaagcgtc gcgtcgtcta aaggcatgcc tccacgaaga
1140


taa
1143






The encoded AvrPphE homolog has an amino acid sequence according to SEQ. ID. No. 48 as follows:









Met Arg Ile His Ser Ala Gly His Ser Leu Pro Ala Pro Gly Pro Ser



  1               5                  10                  15


Val Glu Thr Thr Glu Lys Ala Val Gln Ser Ser Ser Ala Gln Asn Pro


             20                  25                  30


Ala Ser Cys Ser Ser Gln Thr Glu Arg Pro Glu Ala Gly Ser Thr Gln


         35                  40                  45


Val Arg Pro Asn Tyr Pro Tyr Ser Ser Val Lys Thr Arg Leu Pro Pro


     50                  55                  60


Val Ser Ser Thr Gly Gln Ala Ile Ser Asp Thr Pro Ser Ser Leu Ser


 65                  70                  75                  80


Gly Tyr Leu Leu Leu Arg Arg Leu Asp Arg Arg Pro Leu Asp Glu Asp


                 85              90                      95


Ser Ile Lys Ala Leu Val Pro Ala Asp Glu Ala Leu Arg Glu Ala Arg


            100                 105                 110


Arg Ala Leu Pro Phe Gly Arg Gly Asn Ile Asp Val Asp Ala Gln Arg


        115                 120                 125


Thr His Leu Gln Ser Gly Ala Arg Ala Val Ala Ala Lys Arg Leu Arg


    130                 135                 140


Lys Asp Ala Glu Arg Ala Gly His Glu Pro Met Pro Glu Asn Asp Glu


145                 150                 155                 160


Met Asn Trp His Val Leu Val Ala Met Ser Gly Gln Val Phe Gly Ala


                165                 170                 175


Gly Asn Cys Gly Glu His Ala Arg Ile Ala Ser Phe Ala Tyr Gly Ala


            180                 185                 190


Leu Ala Gln Glu Ser Gly Arg Ser Pro Arg Glu Lys Ile His Leu Ala


        195                 200                 205


Glu Gln Pro Gly Lys Asp His Val Trp Ala Glu Thr Asp Asn Ser Ser


    210                 215                 220


Ala Gly Ser Ser Pro Ile Val Met Asp Pro Trp Ser Asn Gly Val Ala


225                 230                 235                 240


Ile Leu Ala Glu Asp Ser Arg Phe Ala Lys Asp Arg Ser Ala Val Glu


                245                 250                 255


Arg Thr Tyr Ser Phe Thr Leu Ala Met Ala Ala Glu Ala Gly Lys Val


            260                 265                 270


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


        275                 280                 285


Gln Lys Arg Leu Ala Asp Gln Leu Pro Asn Val Ser Pro Leu Glu Gly


    290                 295                 300


Gly Arg Tyr Gln Pro Glu Lys Ser Val Leu Asp Glu Ala Phe Ala Arg


305                 310                 315                 320


Arg Val Ser Asp Lys Leu Asn Ser Asp Asp Pro Arg Arg Ala Leu Gln


                325                 330                 335


Met Glu Ile Glu Ala Val Gly Val Ala Met Ser Leu Gly Ala Glu Gly


            340                 345                 350


Val Lys Thr Val Ala Arg Gln Ala Pro Lys Val Val Arg Gln Ala Arg


        355                 360                 365


Ser Val Ala Ser Ser Lys Gly Met Pro Pro Arg Arg


    370                 375                 380






A DNA molecule from Pseudomonas syringae pv. phaseolicola strain B130 which encodes AvrPphE has a nucleotide sequence (SEQ. ID. No. 49) as follows:









atgagaattc acagtgctgg tcacagcctg cccgcgccag gccctagcgt ggaaaccact
60



gaaaaggctg ttcaatcatc atcggcccag aaccccgctt cttgcagttc acaaacagaa
120


cgtcctgaag ccggttcgac tcaagtgcga ccgaactacc cttactcatc agtcaagaca
180


cgcttgccac ccgtttcttc cacagggcag gccatttctg acacgccatc ttcattgccc
240


ggttacctgc tgttacgtcg gctcgaccga cgtccactgg atgaagacag tatcaaggct
300


ctggttccgg cagacgaagc gttgcgtgaa gcacgccgcg cgttgccctt cggcaggggc
360


aacattgatg tggatgcaca acgtacccac ctgcaaagcg gcgctcgcgc agtcgctgca
420


aagcgcttga gaaaagatgc cgagcgcgct ggccatgagc cgatgcccga gaatgatgag
480


atgaactggc atgttcttgt cgccatgtca gggcaggtgt ttggcgctgg caactgtggc
540


gaacatgctc gtatagcaag cttcgcttac ggggccctgg ctcaggaaag cgggcgtagt
600


ccccgcgaaa agattcattt ggccgagcag cccggaaaag atcacgtctg ggctgaaacg
660


gataattcca gcgctggctc ttcgcccatc gtcatggacc cgtggtctaa cggcgcagcc
720


attttggcgg aggacagccg gtttgccaaa gatcgcagtg cggtagagcg aacatattca
780


ttcacccttg caatggcagc tgaagccggc aaggttgcgc gtgaaaccgc cgagaacgtt
840


ctgacccaca cgacaagccg tctgcagaag cgtcttgctg atcagttgcc gaacgtctca
900


ccgcttgaag gaggccgcta tcagccggaa aagtcggtgc ttgatgaggc gttcgcccga
960


cgagtgagcg acaagttgaa tagtgacgat ccacggcgtg cgttgcagat ggaaattgaa
1020


gctgttggtg ttgcaatgtc gctgggtgcc gaaggcgtca agacggtcgc ccgacaggcg
1080


ccaaaggtgg tcaggcaagc cagaagcgtc gcgtcgtcta aaggcatgcc tccacgaaga
1140


taa
1143






The encoded AvrPphE homolog has an amino acid sequence according to SEQ. ID. No. 50 as follows:









Met Arg Ile His Ser Ala Gly His Ser Leu Pro Ala Pro Gly Pro Ser



  1               5                  10                  15


Val Glu Thr Thr Glu Lys Ala Val Gln Ser Ser Ser Ala Gln Asn Pro


             20                  25                  30


Ala Ser Cys Ser Ser Gln Thr Glu Arg Pro Glu Ala Gly Ser Thr Gln


         35                  40                  45


Val Arg Pro Asn Tyr Pro Tyr Ser Ser Val Lys Thr Arg Leu Pro Pro


     50                  55                  60


Val Ser Ser Thr Gly Gln Ala Ile Ser Asp Thr Pro Ser Ser Leu Pro


 65                  70                  75                  80


Gly Tyr Leu Leu Leu Arg Arg Leu Asp Arg Arg Pro Leu Asp Glu Asp


                 85                  90                  95


Ser Ile Lys Ala Leu Val Pro Ala Asp Glu Ala Leu Arg Glu Ala Arg


            100                 105                 110


Arg Ala Leu Pro Phe Gly Arg Gly Asn Ile Asp Val Asp Ala Gln Arg


        115                 120                 125


Thr His Leu Gln Ser Gly Ala Arg Ala Val Ala Ala Lys Arg Leu Arg


    130                 135                 140


Lys Asp Ala Glu Arg Ala Gly His Glu Pro Met Pro Glu Asn Asp Glu


145                 150                 155                 160


Met Asn Trp His Val Leu Val Ala Met Ser Gly Gln Val Phe Gly Ala


                165                 170                 175


Gly Asn Cys Gly Glu His Ala Arg Ile Ala Ser Phe Ala Tyr Gly Ala


            180                 185                 190


Leu Ala Gln Glu Ser Gly Arg Ser Pro Arg Glu Lys Ile His Leu Ala


        195                 200                 205


Glu Gln Pro Gly Lys Asp His Val Trp Ala Glu Thr Asp Asn Ser Ser


    210                 215                 220


Ala Gly Ser Ser Pro Ile Val Met Asp Pro Trp Ser Asn Gly Ala Ala


225                 230                 235                 240


Ile Leu Ala Glu Asp Ser Arg Phe Ala Lys Asp Arg Ser Ala Val Glu


                245                 250                 255


Arg Thr Tyr Ser Phe Thr Leu Ala Met Ala Ala Glu Ala Gly Lys Val


            260                 265                 270


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


        275                 280                 285


Gln Lys Arg Leu Ala Asp Gln Leu Pro Asn Val Ser Pro Leu Glu Gly


    290                 295                 300


Gly Arg Tyr Gln Pro Glu Lys Ser Val Leu Asp Glu Ala Phe Ala Arg


305                 310                 315                 320


Arg Val Ser Asp Lys Leu Asn Ser Asp Asp Pro Arg Arg Ala Leu Gln


                325                 330                 335


Met Glu Ile Glu Ala Val Gly Val Ala Met Ser Leu Gly Ala Glu Gly


            340                 345                 350


Val Lys Thr Val Ala Arg Gln Ala Pro Lys Val Val Arg Gln Ala Arg


        355                 360                 365


Ser Val Ala Ser Ser Lys Gly Met Pro Pro Arg Arg


    370                 375                 380






A DNA molecule from Pseudomonas syringae pv. angulata strain Pa9 which encodes AvrPphE homolog has a nucleotide sequence (SEQ. ID. No. 51) as follows:









atgagaattc acagtgctgg tcacagcctg cctgcgccag gccctagcgt ggaaaccact
60



gaaaaggctg ttcaatcatc atcggcccag aaccccgctt cttacagttc acaaacagaa
120


cgtcctgaag ccggttcgac tcaagtgcga ctgaactacc cttactcatc agtcaagaca
180


cgcttgccac ccgtttcttc tacagggcag gccatttctg ccacgccatc ttcattgccc
240


ggttacctgc tgttacgtcg gctcgaccga cgtccactgg atgaagacag tatcaaggct
300


ctggttccgg cagacgaagc ggtgcgtgaa gcacgccgcg cgttgccctt cggcaggggc
360


aacattgatg tggatgcaca acgtacccac ctgcaaagcg gcgctcgcgc agtcgctgca
420


aagcgcttga gaaaagatgc cgagcgcgct ggccatgagc cgatgcccgg gaatgatgag
480


atgaactggc atgttcttgt cgccatgtca gggcaggtgt ttggcgctgg caactgtggc
540


gaacatgctc gtatagcaag cttcgcttac ggggccctgg ctcaggaaag cgggcgtagt
600


ccccgcgaaa agattcattt ggccgagcag cccggaaaag atcacgtctg ggctgaaacg
660


gataattcca gcgctggctc ttcgcccatc gtcatggacc cgtggtctaa cggcgcagcc
720


attttggcgg aggacagccg gtttgccaaa gatcgcagta cggtagagcg aacatattca
780


ttcacccttg caatggcagc tgaagccggc aaggttacgc gtgaaaccgc cgagaacgtt
840


ctgacccaca cgacaagccg tctgcagaaa cgtcttgctg atcagttgcc gaacgtctca
900


ccgcttgaag gaggccgcta tcagcaggaa aagtcggtgc ttgatgaggc gttcgcccga
960


cgagtgagcg acaagttgaa tagtgacgat ccacggcgtg cgttgcagat ggaaattgaa
1020


gctgttggtg ttgcaatgtc gctgggtgcc gaaggcgtca agacggtcgc ccgacaggcg
1080


ccaaaggtgg tcaggcaagc cagaagcgtc gcgtcgtcta aaggcatgcc tccacgaaga
1140


taa
1143






The encoded AvrPphE homolog has an amino acid sequence according to SEQ. ID. No. 52 as follows:









Met Arg Ile His Ser Ala Gly His Ser Leu Pro Ala Pro Gly Pro Ser



  1               5                  10                  15


Val Glu Thr Thr Glu Lys Ala Val Gln Ser Ser Ser Ala Gln Asn Pro


             20                  25                  30


Ala Ser Tyr Ser Ser Gln Thr Glu Arg Pro Glu Ala Gly Ser Thr Gln


         35                  40                  45


Val Arg Leu Asn Tyr Pro Tyr Ser Ser Val Lys Thr Arg Leu Pro Pro


     50                  55                  60


Val Ser Ser Thr Gly Gln Ala Ile Ser Ala Thr Pro Ser Ser Leu Pro


 65                  70                  75                  80


Gly Tyr Leu Leu Leu Arg Arg Leu Asp Arg Arg Pro Leu Asp Glu Asp


                 85                  90                  95


Ser Ile Lys Ala Leu Val Pro Ala Asp Glu Ala Val Arg Glu Ala Arg


            100                 105                 110


Arg Ala Leu Pro Phe Gly Arg Gly Asn Ile Asp Val Asp Ala Gln Arg


        115                 120                 125


Thr His Leu Gln Ser Gly Ala Arg Ala Val Ala Ala Lys Arg Leu Arg


    130                 135                 140


Lys Asp Ala Glu Arg Ala Gly His Glu Pro Met Pro Gly Asn Asp Glu


145                 150                 155                 160


Met Asn Trp His Val Leu Val Ala Met Ser Gly Gln Val Phe Gly Ala


                165                 170                 175


Gly Asn Cys Gly Glu His Ala Arg Ile Ala Ser Phe Ala Tyr Gly Ala


            180                 185                 190


Leu Ala Gln Glu Ser Gly Arg Ser Pro Arg Glu Lys Ile His Leu Ala


        195                 200                 205


Glu Gln Pro Gly Lys Asp His Val Trp Ala Glu Thr Asp Asn Ser Ser


    210                 215                 220


Ala Gly Ser Ser Pro Ile Val Met Asp Pro Trp Ser Asn Gly Ala Ala


225                 230                 235                 240


Ile Leu Ala Gln Asp Ser Arg Phe Ala Lys Asp Arg Ser Thr Val Glu


                245                 250                 255


Arg Thr Tyr Ser Phe Thr Leu Ala Met Ala Ala Glu Ala Gly Lys Val


            260                 265                 270


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


        275                 280                 285


Gln Lys Arg Leu Ala Asp Gln Leu Pro Asn Val Ser Pro Leu Glu Gly


    290                 295                 300


Gly Arg Tyr Gln Gln Glu Lys Ser Val Leu Asp Glu Ala Phe Ala Arg


305                 310                 315                 320


Arg Val Ser Asp Lys Leu Asn Ser Asp Asp Pro Arg Arg Ala Leu Gln


                325                 330                 335


Met Glu Ile Glu Ala Val Gly Val Ala Met Ser Leu Gly Ala Glu Gly


            340                 345                 350


Val Lys Thr Val Ala Arg Gln Ala Pro Lys Val Val Arg Gln Ala Arg


        355                 360                 365


Ser Val Ala Ser Ser Lys Gly Met Pro Pro Arg Arg


    370                 375                 380






A DNA molecule from Pseudomonas syringae pv. delphinii strain PDDCC529 which encodes a AvrPphE homolog has a nucleotide sequence (SEQ. ID. No. 53) as follows:









atgaaaatac ataacgctgg cccaagcatt ccgatgcccg ctccatcgat tgagagcgct
60



ggcaagactg cgcaatcatc attggctcaa ccgcagagcc aacgagccac ccccgtctcg
120


ccatcagaga cttctgatgc ccgtccgtcc agtgtgcgta cgaactaccc ttattcatca
180


gtcaaaacac ggttgcctcc cgttgcgtct gcagggcagc cactgtccgg gatgccgtct
240


tcattacccg gctacttgct gttacgtcgg cttgaccatc gtccactgga tcaagacggt
300


atcaaaggtt tgattccagc agatgaagcg gtgggtgaag cacgtcgcgc gttgcctttc
360


ggcaggggca atatcgacgt ggatgcgcaa cgctccaact tggaaagcgg agcccgcaca
420


ctcgcggcta ggcgtttgag aaaagatgcc gaggccgcgg gtcacgaacc aatgcctgca
480


aatgaagata tgaactggca tgttcttgtt gcgatgtcag gacaggtttt tggcgcaggt
540


aactgcgggg aacatgcccg catagcgagt ttcgcctacg gtgcactggc tcaggaaaaa
600


gggcggaacg ccgatgagac tattcatttg gctgcgcaac gcggtaaaga ccacgtctgg
660


gctgaaacgg acaattcaag cgctggatct tcaccggttg tcatggatcc gtggtcgaac
720


ggtcctgcca tttttgcgga ggatagtcgg tttgccaaag atcgaagtac ggtagaacga
780


acggattcct tcacgcttgc aactgctgct gaagcaggca agatcacgcg agagacggcc
840


gagaatgctt tgacacaggc gaccagccgt ttgcagaaac gtcttgctga tcagaaaacg
900


caagtctcgc cgcttgcagg agggcgctat cggcaagaaa attcggtgct tgatgacgcg
960


ttcgcccgac gggcaagtgg caagttgagc aacaaggatc cgcggcatgc attacaggtg
1020


gaaatcgagg cggccgcagt tgcaatgtcg ctgggcgccc aaggcgtaaa agcggttgcg
1080


gaacaggccc ggacggtagt tgaacaagcc aggaaggtcg catctcccca aggcacgcct
1140


cagcgagata cgtga
1155






The encoded avrPphE homolog has an amino acid sequence according to SEQ. ID. No. 54 as follows:









Met Lys Ile His Asn Ala Gly Pro Ser Ile Pro Met Pro Ala Pro Ser



  1               5                  10                  15


Ile Glu Ser Ala Gly Lys Thr Ala Gln Ser Ser Leu Ala Gln Pro Gln


             20                  25                  30


Ser Gln Arg Ala Thr Pro Val Ser Pro Ser Gln Thr Ser Asp Ala Arg


         35                  40                  45


Pro Ser Ser Val Arg Thr Asn Tyr Pro Tyr Ser Ser Val Lys Thr Arg


     50                  55                  60


Leu Pro Pro Val Ala Ser Ala Gly Gln Pro Leu Ser Gly Met Pro Ser


 65                  70                  75                  80


Ser Leu Pro Gly Tyr Leu Leu Leu Arg Arg Leu Asp His Arg Pro Leu


                 85                  90                  95


Asp Gln Asp Gly Ile Lys Gly Leu Ile Pro Ala Asp Glu Ala Val Gly


            100                 105                 110


Glu Ala Arg Arg Ala Leu Pro Phe Gly Arg Gly Asn Ile Asp Val Asp


        115                 120                 125


Ala Gln Arg Ser Asn Leu Glu Ser Gly Ala Arg Thr Leu Ala Ala Arg


    130                 135                 140


Arg Leu Arg Lys Asp Ala Glu Ala Ala Gly His Glu Pro Met Pro Ala


145                 150                 155                 160


Asn Glu Asp Met Asn Trp His Val Leu Val Ala Met Ser Gly Gln Val


                165                 170                 175


Phe Gly Ala Gly Asn Cys Gly Glu His Ala Arg Ile Ala Ser Phe Ala


            180                 185                 190


Tyr Gly Ala Leu Ala Gln Glu Lys Gly Arg Asn Ala Asp Glu Thr Ile


        195                 200                 205


His Leu Ala Ala Gln Arg Gly Lys Asp His Val Trp Ala Glu Thr Asp


    210                 215                 220


Asn Ser Ser Ala Gly Ser Ser Pro Val Val Met Asp Pro Trp Ser Asn


225                 230                 235                 240


Gly Pro Ala Ile Phe Ala Glu Asp Ser Arg Phe Ala Lys Asp Arg Ser


                245                 250                 255


Thr Val Glu Arg Thr Asp Ser Phe Thr Leu Ala Thr Ala Ala Glu Ala


            260                 265                 270


Gly Lys Ile Thr Arg Glu Thr Ala Glu Asn Ala Leu Thr Gln Ala Thr


        275                 280                 285


Ser Arg Leu Gln Lys Arg Leu Ala Asp Gln Lys Thr Gln Val Ser Pro


    290                 295                 300


Leu Ala Gly Gly Arg Tyr Arg Gln Glu Asn Ser Val Leu Asp Asp Ala


305                 310                 315                 320


Phe Ala Arg Arg Ala Ser Gly Lys Leu Ser Asn Lys Asp Pro Arg His


                325                 330                 335


Ala Leu Gln Val Glu Ile Glu Ala Ala Ala Val Ala Met Ser Leu Gly


            340                 345                 350


Ala Gln Gly Val Lys Ala Val Ala Glu Gln Ala Arg Thr Val Val Glu


        355                 360                 365


Gln Ala Arg Lys Val Ala Ser Pro Gln Gly Thr Pro Gln Arg Asp Thr


    370                 375                 380






A DNA molecule from Pseudomonas syringae pv. delphinii strain PDDCC529 which encodes a homolog of P. syringae pv. tomato DC3000 EEL ORF2 has a nucleotide sequence (SEQ. ID. No. 55) as follows:









gtggttgagc gaaccggcac tgcatatcga aggcgtggag cagcctgctc gcgtatcacg
60



agccaaaatc aggtccgacg acgctttgga attacggtga atcagatgca aaagacgtcc
120


ctattggctt tggcctttgc aatcctggca gggtgtgggg gttcggggca ggcgccgggg
180


agtgatattc agggtgccca ggcagagatg aaaacaccca ttaaagtaga tctggatgcc
240


tacacctcaa aaaaacttga tgctgtgttg gaagctcggg ccaataaaag ctatgtgaat
300


aaaggtcaac tgatcgacct tgtgtcaggg gcgtttttgg gaacaccgta ccgctcaaac
360


atgttggtgg gcacagagga aatacctgaa cagttagtca tcgactttag aggtctggat
420


tgttttgctt atctggatta cgtagaggcg ttgcgaagat caacatcgca gcaggatttt
480


gtgaggaatc tcgttcaggt tcgttacaag ggtggtgatg ttgacttttt gaatcgcaag
540


cactttttca cggattgggc ttatggcact acacacccgg tggcggatga catcaccacg
600


cagataagcc ccggtgcggt aagtgtcaga aaacgcctta atgaaagggc caaaggcaaa
660


gtctatctgc caggtttgcc tgtggttgag cgcagcatga cctatatccc gagccgcctt
720


gtcgacagtc aggtggtaag ccacttgcgc acaggtgatt acatcggcat ttacaccccg
780


cttcccgggc tggatgtgac gcacgtcggt ttctttatca tgacggataa aggccctgtc
840


ttgcgaaatg catcttcacg aaaagaaaac agaaaggtaa tggatttgcc ttttctggac
900


tatgtatcgg aaaagccagg gattgttgtt ttcagggcaa aagacaattg a
951






The encoded protein or polypeptide has an amino acid sequence according to SEQ. ID. No. 56 as follows:









Val Val Glu Arg Thr Gly Thr Ala Tyr Arg Arg Arg Gly Ala Ala Cys



  1               5                  10                  15


Ser Arg Ile Thr Ser Gln Asn Gln Val Arg Arg Arg Phe Gly Ile Thr


             20                  25                  30


Val Asn Gln Met Gln Lys Thr Ser Leu Leu Ala Leu Ala Phe Ala Ile


         35                  40                  45


Leu Ala Gly Cys Gly Gly Ser Gly Gln Ala Pro Gly Ser Asp Ile Gln


     50                  55                  60


Gly Ala Gln Ala Glu Met Lys Thr Pro Ile Lys Val Asp Leu Asp Ala


 65                  70                  75                  80


Tyr Thr Ser Lys Lys Leu Asp Ala Val Leu Glu Ala Arg Ala Asn Lys


                 85                  90                  95


Ser Tyr Val Asn Lys Gly Gln Leu Ile Asp Leu Val Ser Gly Ala Phe


            100                 105                 110


Leu Gly Thr Pro Tyr Arg Ser Asn Met Leu Val Gly Thr Glu Glu Ile


        115                 120                 125


Pro Glu Gln Leu Val Ile Asp Phe Arg Gly Leu Asp Cys Phe Ala Tyr


    130                 135                 140


Leu Asp Tyr Val Glu Ala Leu Arg Arg Ser Thr Ser Gln Gln Asp Phe


145                 150                 155                 160


Val Arg Asn Leu Val Gln Val Arg Tyr Lys Gly Gly Asp Val Asp Phe


                165                 170                 175


Leu Asn Arg Lys His Phe Phe Thr Asp Trp Ala Tyr Gly Thr Thr His


            180                 185                 190


Pro Val Ala Asp Asp Ile Thr Thr Gln Ile Ser Pro Gly Ala Val Ser


        195                 200                 205


Val Arg Lys Arg Leu Asn Glu Arg Ala Lys Gly Lys Val Tyr Leu Pro


    210                 215                 220


Gly Leu Pro Val Val Glu Arg Ser Met Thr Tyr Ile Pro Ser Arg Leu


225                 230                 235                 240


Val Asp Ser Gln Val Val Ser His Leu Arg Thr Gly Asp Tyr Ile Gly


                245                 250                 255


Ile Tyr Thr Pro Leu Pro Gly Leu Asp Val Thr His Val Gly Phe Phe


            260                 265                 270


Ile Met Thr Asp Lys Gly Pro Val Leu Arg Asn Ala Ser Ser Arg Lys


        275                 280                 285


Glu Asn Arg Lys Val Met Asp Leu Pro Phe Leu Asp Tyr Val Ser Glu


    290                 295                 300


Lys Pro Gly Ile Val Val Phe Arg Ala Lys Asp Asn


305                 310                 315






A DNA molecule from Pseudomonas syringae pv. delphinii strain PDDCC529 ORF1 encodes a homolog of AvrPphF and has a nucleotide sequence (SEQ. ID. No. 57) as follows:









atgaaaaact catttgatct tcttgtcgac ggtttggcga aagactacag catgccgaat
60



ttgccgaaca agaaacacga caatgaagtc tattgcttca cattccagag cgggctcgaa
120


gtaaacattt atcaggacga ctgtcgatgg gtgcatttct ccgccacaat cggacaattt
180


caagacgcca gcaatgacac gctcagccac gcacttcaac tgaacaattt cagtcttgga
240


aagcccttct tcacctttgg aatgaacgga gaaaaggtcg gcgtacttca cacacgcgtt
300


ccgttgattg aaatgaatac cgttgaaatg cgcaaggtat tcgaggactt gctcgatgta
360


gcaggcggca tcagagcgac attcaagctc agttaa
396






The encoded AvrPphF homolog has an amino acid sequence according to SEQ. ID. No 58 as follows:









Met Lys Asn Ser Phe Asp Leu Leu Val Asp Gly Leu Ala Lys Asp Tyr



  1               5                  10                  15


Ser Met Pro Asn Leu Pro Asn Lys Lys His Asp Asn Glu Val Tyr Cys


             20                  25                  30


Phe Thr Phe Gln Ser Gly Leu Glu Val Asn Ile Tyr Gln Asp Asp Cys


         35                  40                  45


Arg Trp Val His Phe Ser Ala Thr Ile Gly Gln Phe Gln Asp Ala Ser


     50                  55                  60


Asn Asp Thr Leu Ser His Ala Leu Gln Leu Asn Asn Phe Ser Leu Gly


 65                  70                  75                  80


Lys Pro Phe Phe Thr Phe Gly Met Asn Gly Glu Lys Val Gly Val Leu


                 85                  90                  95


His Thr Arg Val Pro Leu Ile Glu Met Asn Thr Val Glu Met Arg Lys


            100                 105                 110


Val Phe Glu Asp Leu Leu Asp Val Ala Gly Gly Ile Arg Ala Thr Phe


        115                 120                 125


Lys Leu Ser


    130






A DNA molecule from Pseudomonas syringae pv. delphinii strain PDDCC529 ORF1 encodes a homolog of AvrPphF and has a nucleotide sequence (SEQ. ID. No. 59) as follows:









atgagtacta tacctggcac ctcgggcgct cacccgattt atagctcaat ttccagccca
60



cgaaatatgt ctggctcgcc cacaccgagt caccgtattg gcggggaaac cctgacctct
120


attcatcagc tctctgccag ccagagagaa caatttctga atactcatga ccccatgaga
180


aaactcagga ttaacaatga tacgccactg tacagaacaa ccgagaagcg ttttatacag
240


gaaggcaaac tggccggcaa tccaaagtct attgcacgtg tcaacttgca cgaagaactg
300


cagcttaatc cgctcgccag tattttaggg aacttacctc acgaggcaag cgcttacttt
360


ccgaaaagcg cccgcgctgc ggatctgaaa gacccttcat tgaatgtaat gacaggctct
420


cgggcaaaaa atgctattcg cggctacgct catgacgacc atgtggcggt caagatgcga
480


ctgggcgact ttcttgaaaa aggcggcaag gtgtacgcgg acacttcatc agtcattgac
540


ggcggagacg aggcgagcgc gctgatcgtt acattgccta aaggacaaaa agttccagtc
600


gagattatcc ctacccataa cgacaacagc aataaaggca gaggctga
648






The encoded AvrPphF homolog has an amino acid sequence according to SEQ. ID. No. 60 as follows:









Met Ser Thr Ile Pro Gly Thr Ser Gly Ala His Pro Ile Tyr Ser Ser



  1               5                  10                  15


Ile Ser Ser Pro Arg Asn Met Ser Gly Ser Pro Thr Pro Ser His Arg


             20                  25                  30


Ile Gly Gly Glu Thr Leu Thr Ser Ile His Gln Leu Ser Ala Ser Gln


         35                  40                  45


Arg Glu Gln Phe Leu Asn Thr His Asp Pro Met Arg Lys Leu Arg Ile


     50                  55                  60


Asn Asn Asp Thr Pro Leu Tyr Arg Thr Thr Glu Lys Arg Phe Ile Gln


 65                  70                  75                  80


Glu Gly Lys Leu Ala Gly Asn Pro Lys Ser Ile Ala Arg Val Asn Leu


                 85                  90                  95


His Glu Glu Leu Gln Leu Asn Pro Leu Ala Ser Ile Leu Gly Asn Leu


            100                 105                 110


Pro His Glu Ala Ser Ala Tyr Phe Pro Lys Ser Ala Arg Ala Ala Asp


        115                 120                 125


Leu Lys Asp Pro Ser Leu Asn Val Met Thr Gly Ser Arg Ala Lys Asn


    130                 135                 140


Ala Ile Arg Gly Tyr Ala His Asp Asp His Val Ala Val Lys Met Arg


145                 150                 155                 160


Leu Gly Asp Phe Leu Glu Lys Gly Gly Lys Val Tyr Ala Asp Thr Ser


                165                 170                 175


Ser Val Ile Asp Gly Gly Asp Glu Ala Ser Ala Leu Ile Val Thr Leu


            180                 185                 190


Pro Lys Gly Gln Lys Val Pro Val Glu Ile Ile Pro Thr His Asn Asp


        195                 200                 205


Asn Ser Asn Lys Gly Arg Gly


    210                 215






A DNA molecule from Pseudomonas syringae pv. syringae strain 226 encodes a homolog of HopPsyA and has a nucleotide sequence (SEQ. ID. No. 61) as follows:









gtgaacccta tccatgcacg cttctccagc gtagaagcgc tcagacattc aaacgttgat
60



attcaggcaa tcaaatccga gggtcagttg gaagtcaacg gcaagcgtta cgagattcgt
120


gcggccgctg acggctcaat cgcggtcctc agacccgatc aacagtccaa agcagacaag
180


ttcttcaaag gcgcagcgca tcttattggc ggacaaagcc agcgtgccca aatagcccag
240


gtactcaacg agaaagcggc ggcagttcca cgcctggaca gaatgttggg cagacgcttc
300


gatctggaga agggcggaag tagcgctgtg ggcgccgcaa tcaaggctgc cgacagccga
360


ctgacatcaa aacagacatt tgccagcttc cagcaatggg ctgaaaaagc tgaggcgctc
420


gggcgcgata ccgaaatcgg tatctacatg atctacaaga gggacacgcc agacacaacg
480


cctatgaatg cggcagagca agaacattac ctggaaacgc tacaggctct cgataacaag
540


aaaaacctta tcatacgccc gcagatccat gatgatcggg aagaggaaga gcttgatctg
600


ggccgataca tcgctgaaga cagaaatgcc agaaccggct tttttagaat ggttcctaaa
660


gaccaacgcg cacctgagac aaactcggga cgacttacca ttggtgtaga acctaaatat
720


ggagcgcagt tggccctcgc aatggcaacc ctgatggaca agcacaaatc tgtgacacaa
780


ggtaaagtcg tcggtccggc aaaatatggc cagcaaactg actctgccat tctttacata
840


aatggtgatc ttgcaaaagc agtaaaactg ggcgaaaagc tgaaaaagct gagcggtatc
900


cctcctgaag gattcgtcga acatacaccg ctaagcatgc agtcgacggg tctcggtctt
960


tcttatgccg agtcggttga agggcagcct tccagccacg gacaggcgag aacacacgtt
1020


atcatggatg ccttgaaagg ccagggcccc atggagaaca gactcaaaat ggcgctggca
1080


gaaagaggct atgacccgga aaatccggcg ctcagggcgc gaaactga
1128






The encoded HopPsyA homolog has an amino acid sequence according to SEQ. ID No. 62 as follows:









Val Asn Pro Ile His Ala Arg Phe Ser Ser Val Glu Ala Leu Arg His



  1               5                  10                  15


Ser Asn Val Asp Ile Gln Ala Ile Lys Ser Glu Gly Gln Leu Glu Val


             20                  25                  30


Asn Gly Lys Arg Tyr Glu Ile Arg Ala Ala Ala Asp Gly Ser Ile Ala


         35                  40                  45


Val Leu Arg Pro Asp Gln Gln Ser Lys Ala Asp Lys Phe Phe Lys Gly


     50                  55                  60


Ala Ala His Leu Ile Gly Gly Gln Ser Gln Arg Ala Gln Ile Ala Gln


 65                  70                  75                  80


Val Leu Asn Glu Lys Ala Ala Ala Val Pro Arg Leu Asp Arg Met Leu


                 85                  90                  95


Gly Arg Arg Phe Asp Leu Glu Lys Gly Gly Ser Ser Ala Val Gly Ala


            100                 105                 110


Ala Ile Lys Ala Ala Asp Ser Arg Leu Thr Ser Lys Gln Thr Phe Ala


        115                 120                 125


Ser Phe Gln Gln Trp Ala Glu Lys Ala Glu Ala Leu Gly Arg Asp Thr


    130                 135                 140


Glu Ile Gly Ile Tyr Met Ile Tyr Lys Arg Asp Thr Pro Asp Thr Thr


145                 150                 155                 160


Pro Met Asn Ala Ala Glu Gln Glu His Tyr Leu Glu Thr Leu Gln Ala


                165                 170                 175


Leu Asp Asn Lys Lys Asn Leu Ile Ile Arg Pro Gln Ile His Asp Asp


            180                 185                 190


Arg Glu Glu Glu Glu Leu Asp Leu Gly Arg Tyr Ile Ala Glu Asp Arg


        195                 200                 205


Asn Ala Arg Thr Gly Phe Phe Arg Met Val Pro Lys Asp Gln Arg Ala


    210                 215                 220


Pro Glu Thr Asn Ser Gly Arg Leu Thr Ile Gly Val Glu Pro Lys Tyr


225                 230                 235                 240


Gly Ala Gln Leu Ala Leu Ala Met Ala Thr Leu Met Asp Lys His Lys


                245                 250                 255


Ser Val Thr Gln Gly Lys Val Val Gly Pro Ala Lys Tyr Gly Gln Gln


            260                 265                 270


Thr Asp Ser Ala Ile Leu Tyr Ile Asn Gly Asp Leu Ala Lys Ala Val


        275                 280                 285


Lys Leu Gly Glu Lys Leu Lys Lys Leu Ser Gly Ile Pro Pro Glu Gly


    290                 295                 300


Phe Val Glu His Thr Pro Leu Ser Met Gln Ser Thr Gly Leu Gly Leu


305                 310                 315                 320


Ser Tyr Ala Glu Ser Val Glu Gly Gln Pro Ser Ser His Gly Gln Ala


                325                 330                 335


Arg Thr His Val Ile Met Asp Ala Leu Lys Gly Gln Gly Pro Met Glu


            340                 345                 350


Asn Arg Leu Lys Met Ala Leu Ala Glu Arg Gly Tyr Asp Pro Glu Asn


        355                 360                 365


Pro Ala Leu Arg Ala Arg Asn


    370                 375






A DNA molecule from Pseudomonas syringae pv. atrofaciens strain B143 encodes a homolog of HopPsyA and has a nucleotide sequence (SEQ. ID. No. 63) as follows:









atgaacccga tacaaacgcg tttctctaac gtcgaagcac ttagacattc agaggtggat
60



gtacaggagc tcaaagcaca cggtcaaata gaagtgggtg gcaaatgcta cgacattcgc
120


gcggctgcca ataacgacct gactgtccag cgttctgaca aacagatggc gatgagcaag
180


tttttcaaaa aagcagggtt aagtgggagt tccggcagtc agtccgatca aattgcgcag
240


gtactgaatg acaagcgcgg ctcttccgtt ccccgtctta tacgccaggg gcagacccat
300


ctgggccgta tgcaattcaa catcgaagag gggcaaggca gttcggccgc cacgtccgtc
360


cagaacagca ggctgcccaa tggccgcttg gtaaacagca gtattttgca atgggtcgaa
420


aaggcgaaag ccaatggcag cacaagtacc agtgctcttt atcagatcta cgcaaaagaa
480


ctcccgcgtg tagaactgct gccacgcact gagcaccggg cgtgtctggc gcatatgtat
540


aagctgaacg gtaaggacgg tatcagtatt tggccgcagt ttctggatgg cgtgcgcggg
600


ttgcagctaa aacatgacac aaaagtgttc atgatgaaca accccaaagc agcggacgag
660


ttctacaaga tcgaacgttc gggcacgcaa tttccggatg aggctgtcaa ggcgcgcctg
720


acgataaatg tcaaacctca attccagaag gccatggtcg acgcagcggt caggttgacc
780


gctgagcgtc acgatatcat tactgccaaa gtggcaggtc ctgcaaagat tggcacgatt
840


acagatgcag cggttttcta tgtaagcgga gatttttccg ctgcgcagac acttgcaaaa
900


gagcttcagg cactgctccc tgacgatgcg tttatcaatc atacgccagc tggaatgcaa
960


tccatgggca aggggctgtg ttacgccgag cgtacaccgc aggacaggac aagccacgga
1020


atgtcgcgcg ccagcataat cgagtcggca ctggcagaca ccagcaggtc gtcactggag
1080


aagaagctgc gcaatgcttt caagagcgcc ggatacaatc ccgacaaccc ggcattcagg
1140


ttggaatga
1149






The encoded HopPsyA homolog has an amino acid sequence according to SEQ. ID. No. 64 as follows:









Met Asn Pro Ile Gln Thr Arg Phe Ser Asn Val Glu Ala Leu Arg His



  1               5                  10                  15


Ser Glu Val Asp Val Gln Glu Leu Lys Ala His Gly Gln Ile Glu Val


             20                  25                  30


Gly Gly Lys Cys Tyr Asp Ile Arg Ala Ala Ala Asn Asn Asp Leu Thr


         35                  40                  45


Val Gln Arg Ser Asp Lys Gln Met Ala Met Ser Lys Phe Phe Lys Lys


     50                  55                  60


Ala Gly Leu Ser Gly Ser Ser Gly Ser Gln Ser Asp Gln Ile Ala Gln


 65                  70                  75                  80


Val Leu Asn Asp Lys Arg Gly Ser Ser Val Pro Arg Leu Ile Arg Gln


                 85                  90                  95


Gly Gln Thr His Leu Gly Arg Met Gln Phe Asn Ile Glu Glu Gly Gln


            100                 105                 110


Gly Ser Ser Ala Ala Thr Ser Val Gln Asn Ser Arg Leu Pro Asn Gly


        115                 120                 125


Arg Leu Val Asn Ser Ser Ile Leu Gln Trp Val Glu Lys Ala Lys Ala


    130                 135                 140


Asn Gly Ser Thr Ser Thr Ser Ala Leu Tyr Gln Ile Tyr Ala Lys Glu


145                 150                 155                 160


Leu Pro Arg Val Glu Leu Leu Pro Arg Thr Glu His Arg Ala Cys Leu


                165                 170                 175


Ala His Met Tyr Lys Leu Asn Gly Lys Asp Gly Ile Ser Ile Trp Pro


            180                 185                 190


Gln Phe Leu Asp Gly Val Arg Gly Leu Gln Leu Lys His Asp Thr Lys


        195                 200                 205


Val Phe Met Met Asn Asn Pro Lys Ala Ala Asp Glu Phe Tyr Lys Ile


    210                 215                 220


Glu Arg Ser Gly Thr Gln Phe Pro Asp Glu Ala Val Lys Ala Arg Leu


225                 230                 235                 240


Thr Ile Asn Val Lys Pro Gln Phe Gln Lys Ala Met Val Asp Ala Ala


                245                 250                 255


Val Arg Leu Thr Ala Glu Arg His Asp Ile Ile Thr Ala Lys Val Ala


            260                 265                 270


Gly Pro Ala Lys Ile Gly Thr Ile Thr Asp Ala Ala Val Phe Tyr Val


        275                 280                 285


Ser Gly Asp Phe Ser Ala Ala Gln Thr Leu Ala Lys Glu Leu Gln Ala


    290                 295                 300


Leu Leu Pro Asp Asp Ala Phe Ile Asn His Thr Pro Ala Gly Met Gln


305                 310                 315                 320


Ser Met Gly Lys Gly Leu Cys Tyr Ala Glu Arg Thr Pro Gln Asp Arg


                325                 330                 335


Thr Ser His Gly Met Ser Arg Ala Ser Ile Ile Glu Ser Ala Leu Ala


            340                 345                 350


Asp Thr Ser Arg Ser Ser Leu Glu Lys Lys Leu Arg Asn Ala Phe Lys


        355                 360                 365


Ser Ala Gly Tyr Asn Pro Asp Asn Pro Ala Phe Arg Leu Glu


    370                 375                 380






A DNA molecule from pseudomonas syringae pv. tomato strain DC3000 encodes a homolog of HopPtoA, identified herein as HopPtoA2, and has a nucleotide sequence (SEQ. ID. No. 65) as follows:









atgcacatca accaatccgc ccaacaaccg cctggcgttg caatggagag ttttcggaca
60



gcttccgacg cgtcccttgc ttcgagttct gtgcggtctg tcagcactac ctcgtgccgc
120


gatctacaag ctattaccga ttatctgaaa catcacgtgt tcgctgcgca caggttttcg
180


gtaataggct caccggatga gcgtgatgcc gctcttgcac acaacgagca gatcgatgcg
240


ttggtagaga cacgcgccaa ccgcctgtac tccgaagggg agacccccgc aaccatcgcc
300


gaaacattcg ccaaggcgga aaagttcgac cgtttggcga cgaccgcatc aagtgctttt
360


gagaacacgc catttgccgc tgcctcggtg cttcagtaca tgcagcctgc gatcaacaag
420


ggcgattggc tagcaacgcc gctcaagccg ctgaccccgc tcatttccgg agcgctgtcg
480


ggagccatgg accaggtggg caccaaaatg atggatcgtg cgaggggtga tctgcattac
540


ctgagcactt cgccggacaa gttgcatgat gcgatggccg tatcggtgaa gcgccactcg
600


cctgcgcttg gtcgacaggt tgtggacatg gggattgcag tgcagacgtt ctcggcgcta
660


aatgtggtgc gtaccgtatt ggctccagca ctagcgtcca gaccgtcggt gcagggtgct
720


gttgattttg gcgtatctac ggcgggtggc ttggttgcga atgcaggctt tggcgaccgc
780


atgctcagtg tgcaatcgcg cgatcaactg cgtggggggg cattcgtact tggcatgaaa
840


gataaagagc ccaaggccgc gttgagtgaa gaaactgatt ggcttgatgc ttacaaagcg
900


atcaagtcgg ccagctactc aggtgcggcg ctcaatgcgg gcaagcggat ggccggcctg
960


ccactggacg tcgcgaccga cgggctcaag gcggtgagaa gtctggtgtc ggccaccagc
1020


ctgacaaaaa atggcctggc cctagccggt ggttacgccg gggtaagtaa gttgcagaaa
1080


atggcgacga aaaatatcac tgattcggcg accaaggctg cggttagtca gctgagcaac
1140


ctggtgggtt cggtaggcgt tttcgcaggc tggaccaccg ctggactggc gactgaccct
1200


gcggttaaga aagccgagtc gtttatacag gataaggtga aatcgaccgc atctagtacc
1260


acaagctatg ttgccgacca gaccgtcaaa ctggcgaaaa cagtcaagga catgagcggg
1320


gaggcgatct ccagcaccgg tgccagctta cgcagtactg tcaataacct gcgtcatcgc
1380


tccgctccgg aagctgatat cgaagaaggt gggatttcgg cgttttctcg aagtgaaaca
1440


ccgtttcagc tcaggcgttt gtaa
1464






Although hopPtoA2 does not lie within the CEL, it is included here as a homolog of hopPtoA, which corresponds to CEL ORF5 as noted above. The encoded HopPtoA2 protein or polypeptide has an amino acid sequence according to SEQ. ID. No. 66 as follows:









Met His Ile Asn Gln Ser Ala Gln Gln Pro Pro Gly Val Ala Met Glu



  1               5                  10                  15


Ser Phe Arg Thr Ala Ser Asp Ala Ser Leu Ala Ser Ser Ser Val Arg


             20                  25                  30


Ser Val Ser Thr Thr Ser Cys Arg Asp Leu Gln Ala Ile Thr Asp Tyr


         35                  40                  45


Leu Lys His His Val Phe Ala Ala His Arg Phe Ser Val Ile Gly Ser


     50                  55                  60


Pro Asp Glu Arg Asp Ala Ala Leu Ala His Asn Glu Gln Ile Asp Ala


 65                  70                  75                  80


Leu Val Glu Thr Arg Ala Asn Arg Leu Tyr Ser Glu Gly Glu Thr Pro


                 85                  90                  95


Ala Thr Ile Ala Glu Thr Phe Ala Lys Ala Glu Lys Phe Asp Arg Leu


            100                 105                 110


Ala Thr Thr Ala Ser Ser Ala Phe Glu Asn Thr Pro Phe Ala Ala Ala


        115                 120                 125


Ser Val Leu Gln Tyr Met Gln Pro Ala Ile Asn Lys Gly Asp Trp Leu


    130                 135                 140


Ala Thr Pro Leu Lys Pro Leu Thr Pro Leu Ile Ser Gly Ala Leu Ser


145                 150                 155                 160


Gly Ala Met Asp Gln Val Gly Thr Lys Met Met Asp Arg Ala Arg Gly


                165                 170                 175


Asp Leu His Tyr Leu Ser Thr Ser Pro Asp Lys Leu His Asp Ala Met


            180                 185                 190


Ala Val Ser Val Lys Arg His Ser Pro Ala Leu Gly Arg Gln Val Val


        195                 200                 205


Asp Met Gly Ile Ala Val Gln Thr Phe Ser Ala Leu Asn Val Val Arg


    210                 215                 220


Thr Val Leu Ala Pro Ala Leu Ala Ser Arg Pro Ser Val Gln Gly Ala


225                 230                 235                 240


Val Asp Phe Gly Val Ser Thr Ala Gly Gly Leu Val Ala Asn Ala Gly


                245                 250                 255


Phe Gly Asp Arg Met Leu Ser Val Gln Ser Arg Asp Gln Leu Arg Gly


            260                 265                 270


Gly Ala Phe Val Leu Gly Met Lys Asp Lys Glu Pro Lys Ala Ala Leu


        275                 280                 285


Ser Glu Glu Thr Asp Trp Leu Asp Ala Tyr Lys Ala Ile Lys Ser Ala


    290                 295                 300


Ser Tyr Ser Gly Ala Ala Leu Asn Ala Gly Lys Arg Met Ala Gly Leu


305                 310                 315                 320


Pro Leu Asp Val Ala Thr Asp Gly Leu Lys Ala Val Arg Ser Leu Val


                325                 330                 335


Ser Ala Thr Ser Leu Thr Lys Asn Gly Leu Ala Leu Ala Gly Gly Tyr


            340                 345                 350


Ala Gly Val Ser Lys Leu Gln Lys Met Ala Thr Lys Asn Ile Thr Asp


        355                 360                 365


Ser Ala Thr Lys Ala Ala Val Ser Gln Leu Ser Asn Leu Val Gly Ser


    370                 375                 380


Val Gly Val Phe Ala Gly Trp Thr Thr Ala Gly Leu Ala Thr Asp Pro


385                 390                 395                 400


Ala Val Lys Lys Ala Glu Ser Phe Ile Gln Asp Lys Val Lys Ser Thr


                405                 410                 415


Ala Ser Ser Thr Thr Ser Tyr Val Ala Asp Gln Thr Val Lys Leu Ala


            420                 425                 430


Lys Thr Val Lys Asp Met Ser Gly Glu Ala Ile Ser Ser Thr Gly Ala


        435                 440                 445


Ser Leu Arg Ser Thr Val Asn Asn Leu Arg His Arg Ser Ala Pro Glu


    450                 455                 460


Ala Asp Ile Glu Glu Gly Gly Ile Ser Ala Phe Ser Arg Ser Glu Thr


465                 470                 475                 480


Pro Phe Gln Leu Arg Arg Leu


                485






Fragments of the above-identified proteins or polypeptides as well as fragments of full length proteins from the EELs and CELs of other bacteria, in particular Gram-negative pathogens, can also be used according to the present invention.
Suitable fragments can be produced by several means. Subclones of the gene encoding a known protein can be produced using conventional molecular genetic manipulation for subcloning gene fragments, such as described by Sambrook et al., 1989, and Ausubel et al., 1994. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or polypeptide that can be tested for activity, e.g., as a product required for pathogen virulence.
In another approach, based on knowledge of the primary structure of the protein, fragments of the protein-coding gene may be synthesized using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein (Erlich et al., 1991). These can then be cloned into an appropriate vector for expression of a truncated protein or polypeptide from bacterial cells as described above.
As an alternative, fragments of a protein can be produced by digestion of a full-length protein with proteolytic enzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin. Different proteolytic enzymes are likely to cleave different proteins at different sites based on the amino acid sequence of the particular protein. Some of the fragments that result from proteolysis may be active virulence proteins or polypeptides.
Chemical synthesis can also be used to make suitable fragments. Such a synthesis is carried out using known amino acid sequences for the polyppetide being produced. Alternatively, subjecting a full length protein to high temperatures and pressures will produce fragments. These fragments can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE).
Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
The proteins or polypeptides used in accordance with the present invention are preferably produced in purified form (preferably at least about 80%, more preferably 90%, pure) by conventional techniques. Typically, the protein or polypeptide of the present invention is secreted into the growth medium of recombinant host cells (discussed infra). Alternatively, the protein or polypeptide of the present invention is produced but not secreted into growth medium. In such cases, to isolate the protein, the host cell (e.g., E. coli) carrying a recombinant plasmid is propagated, lysed by sonication, heat, or chemical treatment, and the homogenate is centrifuged to remove bacterial debris. The supernatant is then subjected to sequential ammonium sulfate precipitation. The fraction containing the protein or polypeptide of interest is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC.
DNA molecules encoding other EEL and CEL protein or polypeptides can be identified using a PCR-based methodology for cloning portions of the pathogenicity islands of a bacterium. Basically, the PCR-based strategy involves the use of conserved sequences from the hrpK and tRNAleu genes (or other conserved border sequences) as primers for cloning EEL intervening regions of the pathogenicity island. As shown in FIGS. 2B-C, the hrpK and tRNAleu genes are highly conserved among diverse Pseudomonas syringae variants. Depending upon the size of EEL, additional primers can be prepared from the originally obtained cDNA sequence, allowing for recovery of clones and walking through the EEL in a step-wise fashion. If full-length coding sequences are not obtained from the PCR steps, contigs can be assembled to prepare full-length coding sequences using suitable restriction enzymes. Similar PCR-based procedures can be used for obtaining clones that encode open reading frames in the CEL. As shown in FIG. 3, the CEL of diverse Pseudomonas syringae pathovars contain numerous conserved domains. Moreover, known sequences of the hrp/hrc domain, hrp W, AvrE, or gstA can be used to prepare primers.
Using the above-described PCR-based methods, a number of DNA sequences were utilized as the source for primers. One such DNA molecule is isolated from the tRNAleu gene of Pseudomonas syringae pv. tomato DC3000, which has a nucleotide sequence (SEQ. ID. No. 67) as follows:









gccctgatgg cggaattggt agacgcggcg gattcaaaat ccgttttcga aagaagtggg
60



agttcgattc tccctcgggg caccacca
88






An additional DNA molecule which can be used to supply suitable primers is from the tRNAleu gene of Pseudomonas syringae pv. syringae B728a, which has a nucleotide sequence (SEQ. ID. No. 68) as follows:










gccctgatgg cggaattggt agacgcggcg gattcaaaat ccgttttcga aagaagtggg
60



agttcgattc tccctcgggg cacca
85






Another DNA molecule is isolated from the queA gene of Pseudomonas syringae pv. tomato DC3000, which has a nucleotide sequence (SEQ. ID. No. 69) as follows:










atgcgcgtcg ctgactttac cttcgaactc cccgattccc tgattgctcg tcacccgttg
60



gccgagcgtc gcagcagtcg tctgttgacc cttgatgggc cgacgggcgc gctggcacat
120


cgtcaattca ccgatttgct cgagcatttg cgctcgggcg acttgatggt gttcaacaat
180


acccgtgtca ttcccgcacg tttgttcggg cagaaggcgt ccggcggcaa gctggagatt
240


ctggtcgagc gcgtgctgga cagccatcgt gtgctggcgc acgtgcgtgc cagcaagtcg
300


ccaaagccgg gctcgtcgat cctgatcgat ggcggcggcg aggccgagat ggtggcgcgg
360


catgacgcgc tgttcgagtt gcgctttgcc gaagaagtgc tgccgttgct ggatcgtgtc
420


ggccatatgc cgttgcctcc ttatatagac cgcccggacg aaggtgccga ccgcgagcgt
480


tatcagaccg tttacgccca gcgcgccggt gctgtggcgg cgccgactgc cggcctgcat
540


ttcgaccagc cgttgatgga agcaattgcc gccaagggcg tcgagactgc ttttgtcact
600


ctgcacgtcg gcgcgggtac gttccagccg gtgcgtgtcg agcagatcga agatcaccac
660


atgcacagcg aatggctgga agtcagccag gacgtggtcg atgccgtggc ggcgtgccgt
720


gcgcggggcg ggcgggtgat tgcggtcggg accaccagcg tgcgttcgct ggagagtgcc
780


gcgcgtgatg gccagttgaa gccgtttagc ggcgacaccg acatcttcat ctatccgggg
840


cggccgtttc atgtggtcga tgccctggtg actaattttc atttgcctga atccacgctg
900


ttgatgctgg tttcggcgtt cgccggttat cccgaaacca tggcggccta cgcggcggcc
960


atcgaacacg ggtaccgctt cttcagttac ggtgatgcca tgttcatcac ccgcaatccc
1020


gcgccgacgg ccccacagga atcggcacca gaggatcacg catga
1065






This DNA molecule encodes QueA, which has an amino acid sequence (SEQ. ID. No. 70) as follows:









Met Arg Val Ala Asp Phe Thr Phe Glu Leu Pro Asp Ser Leu Ile Ala



  1               5                  10                  15


Arg His Pro Leu Ala Glu Arg Arg Ser Ser Arg Leu Leu Thr Leu Asp


             20                 25                  30


Gly Pro Thr Gly Ala Leu Ala His Arg Gln Phe Thr Asp Leu Leu Glu


         35                  40                  45


His Leu Arg Ser Gly Asp Leu Met Val Phe Asn Asn Thr Arg Val Ile


     50                  55                  60


Pro Ala Arg Leu Phe Gly Gln Lys Ala Ser Gly Gly Lys Leu Glu Ile


 65                  70                  75                  80


Leu Val Glu Arg Val Leu Asp Ser His Arg Val Leu Ala His Val Arg


                 85                  90                  95


Ala Ser Lys Ser Pro Lys Pro Gly Ser Ser Ile Leu Ile Asp Gly Gly


            100                 105                 110


Gly Glu Ala Glu Met Val Ala Arg His Asp Ala Leu Phe Glu Leu Arg


        115                 120                 125


Phe Ala Glu Glu Val Leu Pro Leu Leu Asp Arg Val Gly His Met Pro


    130                 135                 140


Leu Pro Pro Tyr Ile Asp Arg Pro Asp Glu Gly Ala Asp Arg Glu Arg


145                 150                 155                 160


Tyr Gln Thr Val Tyr Ala Gln Arg Ala Gly Ala Val Ala Ala Pro Thr


                165                 170                 175


Ala Gly Leu His Phe Asp Gln Pro Leu Met Glu Ala Ile Ala Ala Lys


            180                 185                 190


Gly Val Glu Thr Ala Phe Val Thr Leu His Val Gly Ala Gly Thr Phe


        195                 200                 205


Gln Pro Val Arg Val Glu Gln Ile Glu Asp His His Met His Ser Glu


    210                 215                 220


Trp Leu Glu Val Ser Gln Asp Val Val Asp Ala Val Ala Ala Cys Arg


225                 230                 235                 240


Ala Arg Gly Gly Arg Val Ile Ala Val Gly Thr Thr Ser Val Arg Ser


                245                 250                 255


Leu Glu Ser Ala Ala Arg Asp Gly Gln Leu Lys Pro Phe Ser Gly Asp


            260                 265                 270


Thr Asp Ile Phe Ile Tyr Pro Gly Arg Pro Phe His Val Val Asp Ala


        275                 280                 285


Leu Val Thr Asn Phe His Leu Pro Glu Ser Thr Leu Leu Met Leu Val


    290                 295                 300


Ser Ala Phe Ala Gly Tyr Pro Glu Thr Met Ala Ala Tyr Ala Ala Ala


305                 310                 315                 320


Ile Glu His Gly Tyr Arg Phe Phe Ser Tyr Gly Asp Ala Met Phe Ile


                325                 330                 335


Thr Arg Asn Pro Ala Pro Thr Ala Pro Gln Glu Ser Ala Pro Glu Asp


            340                 345                 350


His Ala






DNA molecules encoding other EEL and CEL proteins or polypeptides can also be identified by determining whether such DNA molecules hybridize under stringent conditions to a DNA molecule as identified above. An example of suitable stringency conditions is when hybridization is carried out at a temperature of about 37° C. using a hybridization medium that includes 0.9M sodium citrate (“SSC”) buffer, followed by washing with 0.2×SSC buffer at 37° C. Higher stringency can readily be attained by increasing the temperature for either hybridization or washing conditions or decreasing the sodium concentration of the hybridization or wash medium. Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase. Wash conditions are typically performed at or below stringency. Exemplary high stringency conditions include carrying out hybridization at a temperature of about 42° C. to about 65° C. for up to about 20 hours in a hybridization medium containing 1M NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 50 μg/ml E. coli DNA, followed by washing carried out at between about 42° C. to about 65° C. in a 0.2×SSC buffer.
Also encompassed by the present invention are nucleic acid molecules which contain conserved substitutions as compared to the above identified DNA molecules and, thus, encode the same protein or polypeptides identified above. Further, complementary sequences are also encompassed by the present invention.
The nucleic acid of the present invention can be either DNA or RNA, which can readily be prepared using the above identified DNA molecules of the present invention.
The delivery of effector proteins or polypeptides can be achieved in several ways, depending upon the host being treated and the materials being used: (1) as a stable or plasmid-encoded transgene; (2) transiently expressed via Agrobacterium or viral vectors; (3) delivered by the type III secretion systems of disarmed pathogens or recombinant nonpathogenic bacteria which express a functional, heterologous type III secretion system; or (4) delivered via topical application followed by TAT protein transduction domain-mediated spontaneous uptake into cells. Each of these is discussed infra.
The DNA molecule encoding the protein or polypeptide can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
U.S. Pat. No.4,237,224 to Cohen and Boyer describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture.
Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK+/− or KS+/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et al., 1990). Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., 1989.
A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include, but are not limited to, the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation).
Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters. Eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.
Similarly, translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals which differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, 1979.
Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires an SD sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
Once the isolated DNA molecule encoding the polypeptide or protein has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
Because it is desirable for recombinant host cells to secrete the encoded protein or polypeptide, it is preferable that the host cell also possess a functional type III secretion system. The type III secretion system can be heterologous to host cell (Ham et al., 1998) or the host cell can naturally possess a type III secretion system. Host cells which naturally contain a type III secretion system include many pathogenic Gram-negative bacterium, such as numerous Erwinia species, Pseudomonas species, Xanthomonas species, etc. Other type III secretion systems are known and still others are continually being identified. Pathogenic bacteria that can be utilized to deliver effector proteins or polypeptides are preferably disarmed according to known techniques, i.e., as described above. Alternatively, isolation of the effector protein or polypeptide from the host cell or growth medium can be carried out as described above.
Another aspect of the present invention relates to a transgenic plant which express a protein or polypeptide of the present invention and methods of making the same.
In order to express the DNA molecule in isolated plant cells or tissue or whole plants, a plant expressible promoter is needed. Any plant-expressible promoter can be utilized regardless of its origin, i.e., viral, bacterial, plant, etc. Without limitation, two suitable promoters include the nopaline synthase promoter (Fraley et al., 1983) and the cauliflower mosaic virus 35S promoter (O'Dell et al., 1985). Both of these promoters yield constitutive expression of coding sequences under their regulatory control.
While constitutive expression is generally suitable for expression of the DNA molecule, it should be apparent to those of skill in the art that temporally or tissue regulated expression may also be desirable, in which case any regulated promoter can be selected to achieve the desired expression. Typically, the temporally or tissue regulated promoters will be used in connection with the DNA molecule that are expressed at only certain stages of development or only in certain tissues.
In some plants, it may also be desirable to use promoters which are responsive to pathogen infiltration or stress. For example, it may be desirable to limit expression of the protein or polypeptide in response to infection by a particular pathogen of the plant. One example of a pathogen-inducible promoter is the gst1 promoter from potato, which is described in U.S. Pat. Nos. 5,750,874 and 5,723,760 to Strittmayer et al., which are hereby incorporated by reference.
Expression of the DNA molecule in isolated plant cells or tissue or whole plants also requires appropriate transcription termination and polyadenylation of mRNA. Any 3′ regulatory region suitable for use in plant cells or tissue can be operably linked to the first and second DNA molecules. A number of 3′ regulatory regions are known to be operable in plants. Exemplary 3′ regulatory regions include, without limitation, the nopaline synthase 3′ regulatory region (Fraley et al., 1983) and the cauliflower mosaic virus 3′ regulatory region (Odell et al., 1985).
The promoter and a 3′ regulatory region can readily be ligated to the DNA molecule using well known molecular cloning techniques described in Sambrook et al., 1989.
One approach to transforming plant cells with a DNA molecule of the present invention is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford, et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells. Other variations of particle bombardment, now known or hereafter developed, can also be used.
Another method of introducing the DNA molecule into plant cells is fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies that contain the DNA molecule (Fraley et al., 1982).
The DNA molecule may also be introduced into the plant cells by electroporation (Fromm, et al., 1985). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the DNA molecule. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.
Another method of introducing the DNA molecule into plant cells is to infect a plant cell with Agrobacterium tumefaciens or Agrobacterium rhizogenes previously transformed with the DNA molecule. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots or roots, and develop further into plants. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
Agrobacterium is a representative genus of the Gram-negative family Rhizobiaceae. Its species are responsible for crown gall (A. tumefaciens) and hairy root disease (A. rhizogenes). The plant cells in crown gall tumors and hairy roots are induced to produce amino acid derivatives known as opines, which are catabolized only by the bacteria. The bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes. In addition, assaying for the presence of opines can be used to identify transformed tissue.
Heterologous genetic sequences such as a DNA molecule of the present invention can be introduced into appropriate plant cells by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome (Schell, 1987).
Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers.
After transformation, the transformed plant cells can be selected and regenerated.
Preferably, transformed cells are first identified using, e.g., a selection marker simultaneously introduced into the host cells along with the DNA molecule of the present invention. Suitable selection markers include, without limitation, markers coding for antibiotic resistance, such as kanamycin resistance (Fraley et al., 1983). A number of antibiotic-resistance markers are known in the art and other are continually being identified. Any known antibiotic-resistance marker can be used to transform and select transformed host cells in accordance with the present invention. Cells or tissues are grown on a selection media containing an antibiotic, whereby generally only those transformants expressing the antibiotic resistance marker continue to grow.
Once a recombinant plant cell or tissue has been obtained, it is possible to regenerate a full-grown plant therefrom. Thus, another aspect of the present invention relates to a transgenic plant that includes a DNA molecule of the present invention, wherein the promoter induces transcription of the first DNA molecule in response to infection of the plant by an oomycete. Preferably, the DNA molecule is stably inserted into the genome of the transgenic plant of the present invention.
Plant regeneration from cultured protoplasts is described in Evans et al., 1983, and Vasil, 1984 and 1986.
It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.
Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
After the DNA molecule is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing or by preparing cultivars. With respect to sexual crossing, any of a number of standard breeding techniques can be used depending upon the species to be crossed. Cultivars can be propagated in accord with common agricultural procedures known to those in the field.
Diseases caused by the vast majority of bacterial pathogens result in limited lesions. That is, even when everything is working in the pathogen's favor (e.g., no triggering of the hypersensitive response because of R-gene detection of one of the effectors), the parasitic process still triggers defenses after a couple of days, which then stops the infection from spreading. Thus, the very same effectors that enable parasitism to proceed must also eventually trigger defenses. Therefore, premature expression of these effectors is believed to “turn on” plant defenses earlier (i.e., prior to infection) and make the plant resistant to either the specific bacteria from which the effector protein was obtained or many pathogens. An advantage of this approach is that it involves natural products and plants seem highly sensitive to pathogen effector proteins.
According to one embodiment, a transgenic plant is provided that contains a heterologous DNA molecule of the present invention. Preferably, the heterologous DNA molecule is derived from a plant pathogen EEL. When the heterologous DNA molecule is expressed in the transgenic plant, plant defenses are activated, imparting disease resistance to the transgenic plant. The transgenic plant can also contain an R-gene which is activated by the protein or polypeptide product of the heterologous DNA molecule. The R gene can be naturally occurring in the plant or heterologously inserted therein. A number of R genes have been identified in various plant species, including without limitation: RPS2, RPM1, and RPP5 from Arabidopsis thaliana; Cf2, Cf9, I2, Pto, and Prf from tomato; N from tobacco; L6 and M from flax; Xa2l from rice; and Hs1pro-1 from sugar beet. In addition to imparting disease resistance, it is believed that stimulation of plant defenses in transgenic plants of the present invention will also result in a simultaneous enhancement in growth and resistance to insects.
According to another embodiment, a plant, transgenic or non-transgenic, is treated with a protein or polypeptide of the present invention. By treating, it is intended to include various forms of applying the protein or polypeptide to the plant. The embodiments of the present invention where the effector polypeptide or protein is applied to the plant can be carried out in a number of ways, including: 1) application of an isolated protein (or composition containing the same) or 2) application of bacteria which do not cause disease and are transformed with a gene encoding the effector protein of the present invention. In the latter embodiment, the effector protein can be applied to plants by applying bacteria containing the DNA molecule encoding the effector protein. Such bacteria are preferably capable of secreting or exporting the protein so that the protein can contact plant cells. In these embodiments, the protein is produced by the bacteria in planta.
Such topical application is typically carried out using an effector fusion protein which includes a transduction domain, which will afford transduction domain-mediated spontaneous uptake of the effector protein into cells. Basically, this is carried out by fusing an 11-amino acid peptide (YGRKKRRQRRR, SEQ. ID. No. 91) by standard rDNA techniques to the N-terminus of the effector protein, and the resulting tagged protein is taken up into cells by a poorly understood process. This peptide is the protein transduction domain (PTD) of the human immunodeficiency virus (HIV) TAT protein (Schwarze et al., 2000). Other PTDs are known and may possibly be used for this purpose (Prochiantz, 2000).
When the effector protein is topically applied to plants, it can be applied as a composition, which includes a carrier in the form, e.g., of water, aqueous solutions, slurries, or dry powders. In this embodiment, the composition contains greater than about 5 nM of the protein of the present invention.
Although not required, this composition may contain additional additives including fertilizer, insecticide, fungicide, nematicide, and mixtures thereof. Suitable fertilizers include (NH4)2NO3. An example of a suitable insecticide is Malathion. Useful fungicides include Captan.
Other suitable additives include buffering agents, wetting agents, coating agents, and, in some instances, abrading agents. These materials can be used to facilitate the process of the present invention.
According to another aspect of the present invention, a transgenic plant is provided that contains a heterologous DNA molecule that encodes a transcript or a protein or polypeptide capable of disrupting function of a plant pathogen CEL product. Because the genes in the CEL are particularly important in pathogenesis, disrupting the function of their products in plants can result in broad resistance since CEL genes are highly conserved among Gram negative pathogens, particularly along species lines. An exemplary protein or polypeptide which can disrupt function of a CEL product is an antibody, polyclonal or monoclonal, raised against the CEL product using conventional techniques. Once isolated, the antibody can be sequenced and nucleic acids synthesized for encoding the same. Such nucleic acids, e.g., DNA, can be used to transform plants.
Transgenic plants can also be engineered so that they are hypersusceptible and, therefore, will support the growth of nonpathogenic bacteria for biotechnological purposes. It is known that many plant pathogenic bacteria can alter the environment inside plant leaves so that nonpathogenic bacteria can grow. This ability is presumably based on changes in the plant caused by pathogen effector proteins. Thus, transgenic plants expressing the appropriate effector genes can be used for these purposes.
According to one embodiment, a transgenic plant including a heterologous DNA molecule of the present invention expresses one or more effector proteins, wherein the transgenic plant is capable of supporting growth of compatible nonpathogenic bacteria (i.e., non-pathogenic endophytes such as various Clavibacter ssp.). The compatible nonpathogenic bacteria can be naturally occurring or it can be recombinant. Preferably, the nonpathogenic bacteria is recombinant and expresses one or more useful products. Thus, the transgenic plant becomes a green factory for producing desirable products. Desirable products include, without limitation, products that can enhance the nutritional quality of the plant or products that are desirable in isolated form. If desired in isolated form, the product can be isolated from plant tissues. To prevent competition between the non-pathogenic bacteria which express the desired product and those that do not, it is possible to tailor the needs of recombinant, non-pathogenic bacteria so that only they are capable of living in plant tissues expressing a particular effector protein or polypeptide of the present invention.
The effector proteins or polypeptides of the present invention are believed to alter the plant physiology by shifting metabolic pathways to benefit the parasite and by activating or suppressing cell death pathways. Thus, they may also provide useful tools for efficiently altering the nutrient content of plants and delaying or triggering senescence. There are agricultural applications for all of these possible effects.
A further aspect of the present invention relates to diagnostic uses of the CEL and EEL. The CEL genes are universal to species of Gram negative bacteria, particularly pathogenic Gram negative bacteria (such as P. syringae), whereas the EEL sequences are strain-specific and provide a “virulence gene fingerprint” that could be used to track the presence, origins, and movement (and restrict the spread through quarantines) of strains that are particularly threatening. Although the CEL and EEL have been identified in various pathovars of Pseudomonas syringae, it is expected that most all Gram-negative pathogens can be identified, distinguished, and classified based upon the homology of the CEL and EEL genes.
According to one embodiment, a method of determining relatedness between two bacteria is carried out by comparing a nucleic acid alignment or amino acid alignment for a CEL of the two bacteria and then determining the relatedness of the two bacteria, wherein a higher sequence identity indicates a closer relationship. The CEL is particularly useful for determining the relatedness of two distinct bacterial species.
According to another embodiment, a method of determining relatedness between two bacteria which is carried out by comparing a nucleic acid alignment or amino acid alignment for an EEL of the two bacteria and then determining the relatedness of the two bacteria, wherein a higher sequence identity indicates a closer relationship. The EEL is particularly useful for determining the relatedness of two pathovars of a single bacterial species.
Given the methods of determining relatedness of bacteria species and/or pathovars, these methods can be utilized in conjunction with plant breeding programs. By detecting the “virulence gene fingerprint” of pathogens which are prevalent in a particular growing region, it is possible either to develop transgenic cultivars as described above or to identify existing plant cultivars which are resistant to the prevalent pathogens.
In addition to the above described uses, another aspect of the present invention relates to gene- and protein-based therapies for animals, preferably mammals including, without limitation, humans, dogs, mice, rats. The P. syringae pv. syringae B728a EEL ORF5 protein (SEQ. ID. No. 32) is a member of the AvrRxv/YopJ protein family. YopJ is injected into human cells by the Yersinia type III secretion system, where it disrupts the function of certain protein kinases to inhibit cytokine release and promote programmed cell death. It is believed that the targets of many pathogen effector proteins (i.e., P. syringae effector proteins) will be universal to eukaryotes and therefore have a variety of potentially useful functions. In fact, two of the proteins in the P. syringae Hrp pathogenicity islands are toxic when expressed in yeast. They are HopPsyA from the P. syringae pv. syringae EEL and HopPtoA from the P. syringae pv. tomato DC3000 CEL. This supports the concept of universal eukaryote targets.
Thus, a further aspect of the present invention relates to a method of causing eukaryotic cell death which is carried out by introducing into a eukaryotic cell a cytotoxic Pseudomonas protein. The cytotoxic Pseudomonas protein is preferably HopPsyA (e.g., SEQ. ID. Nos. 36 (Psy 61), 62 (Psy 226), or 64 (Psy B143)) HopPtoA (SEQ. ID. No. 7), or HopPtoA2 (SEQ. ID. No. 66). The eukaryotic cell which is treated can be either in vitro or in vivo. When treating eukaryotic cells in vivo, a number of different protein- or DNA-delivery systems can be employed to introduce the effector protein into the target eukaryotic cell.
Without being bound by theory, it is believed that at least the HopPsyA effector proteins exert their cytotoxic effects through Mad2 interactions, disrupting cell checkpoint of spindle formation (see infra).
The protein- or DNA-delivery systems can be provided in the form of pharmaceutical compositions which include the delivery system in a pharmaceutically acceptable carrier, which may include suitable excipients or stabilizers. The dosage can be in solid or liquid form, such as powders, solutions, suspensions, or emulsions. Typically, the composition will contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active compound(s), together with the carrier, excipient, stabilizer, etc.
The compositions of the present invention are preferably administered in injectable or topically-applied dosages by solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical carrier. Such carriers include sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier, including adjuvants, excipients or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.
Alternatively, the effector proteins can also be delivered via solution or suspension packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
Depending upon the treatment being effected, the compounds of the present invention can be administered orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes.
Compositions within the scope of this invention include all compositions wherein the compound of the present invention is contained in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
One approach for delivering an effector protein into cells involves the use of liposomes. Basically, this involves providing a liposome which includes that effector protein to be delivered, and then contacting the target cell with the liposome under conditions effective for delivery of the effector protein into the cell.
Liposomes are vesicles comprised of one or more concentrically ordered lipid bilayers which encapsulate an aqueous phase. They are normally not leaky, but can become leaky if a hole or pore occurs in the membrane, if the membrane is dissolved or degrades, or if the membrane temperature is increased to the phase transition temperature. Current methods of drug delivery via liposomes require that the liposome carrier ultimately become permeable and release the encapsulated drug at the target site. This can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body. Every liposome composition will have a characteristic half-life in the circulation or at other sites in the body and, thus, by controlling the half-life of the liposome composition, the rate at which the bilayer degrades can be somewhat regulated.
In contrast to passive drug release, active drug release involves using an agent to induce a permeability change in the liposome vesicle. Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., Proc. Natl. Acad. Sci. USA 84:7851 (1987); Biochemistry 28:908 (1989), which are hereby incorporated by reference). When liposomes are endocytosed by a target cell, for example, they can be routed to acidic endosomes which will destabilize the liposome and result in drug release.
Alternatively, the liposome membrane can be chemically modified such that an enzyme is placed as a coating on the membrane which slowly destabilizes the liposome. Since control of drug release depends on the concentration of enzyme initially placed in the membrane, there is no real effective way to modulate or alter drug release to achieve “on demand” drug delivery. The same problem exists for pH-sensitive liposomes in that as soon as the liposome vesicle comes into contact with a target cell, it will be engulfed and a drop in pH will lead to drug release.
This liposome delivery system can also be made to accumulate at a target organ, tissue, or cell via active targeting (e.g., by incorporating an antibody or hormone on the surface of the liposomal vehicle). This can be achieved according to known methods.
Different types of liposomes can be prepared according to Bangham et al., (1965); U.S. Pat. No. 5,653,996 to Hsu et al., U.S. Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613 to Holland et al.; U.S. Pat. No. 5,631,237 to Dzau et al.; and U.S. Pat. No. 5,059,421 to Loughrey et al.
An alternative approach for delivery of effector proteins involves the conjugation of the desired effector protein to a polymer that is stabilized to avoid enzymatic degradation of the conjugated effector protein. Conjugated proteins or polypeptides of this type are described in U.S. Pat. No. 5,681,811 to Ekwuribe.
Yet another approach for delivery of proteins or polypeptides involves preparation of chimeric proteins according to U.S. Pat. No. 5,817,789 to Heartlein et al. The chimeric protein can include a ligand domain and, e.g., an effector protein of the present invention. The ligand domain is specific for receptors located on a target cell. Thus, when the chimeric protein is delivered intravenously or otherwise introduced into blood or lymph, the chimeric protein will adsorb to the targeted cell, and the targeted cell will internalize the chimeric protein, which allows the effector protein to de-stabilize the cell checkpoint control mechanism, affording its cytotoxic effects.
When it is desirable to achieve heterologous expression of an effector protein of the present invention in a target cell, DNA molecules encoding the desired effector protein can be delivered into the cell. Basically, this includes providing a nucleic acid molecule encoding the effector protein and then introducing the nucleic acid molecule into the cell under conditions effective to express the effector protein in the cell. Preferably, this is achieved by inserting the nucleic acid molecule into an expression vector before it is introduced into the cell.
When transforming mammalian cells for heterologous expression of an effector protein, an adenovirus vector can be employed. Adenovirus gene delivery vehicles can be readily prepared and utilized given the disclosure provided in Berkner, 1988, and Rosenfeld et al., 1991. Adeno-associated viral gene delivery vehicles can be constructed and used to deliver a gene to cells. The use of adeno-associated viral gene delivery vehicles in vitro is described in Chatterjee et al. 1992; Walsh et al. 1992; Walsh et al., 1994; Flotte et al., 1993a; Ponnazhagan et al., 1994; Miller et al., 1994; Einerhand et al., 1995; Luo et al., 1995; and Zhou et al., 1996. In vivo use of these vehicles is described in Flotte et al., 1993b and Kaplitt et al., 1994. Additional types of adenovirus vectors are described in U.S. Pat. No. 6,057,155 to Wickham et al.; U.S. Pat. No. 6,033,908 to Bout et al.; U.S. Pat. No. 6,001,557 to Wilson et al.; U.S. Pat. No. 5,994,132 to Chamberlain et al.; U.S. Pat. No. 5,981,225 to Kochanek et al.; U.S. Pat. No. 5,885,808 to Spooner et al.; and U.S. Pat. No. 5,871,727 to Curiel.
Retroviral vectors which have been modified to form infective transformation systems can also be used to deliver nucleic acid encoding a desired effector protein into a target cell. One such type of retroviral vector is disclosed in U.S. Pat. No. 5,849,586 to Kriegler et al.
Regardless of the type of infective transformation system employed, it should be targeted for delivery of the nucleic acid to a specific cell type. For example, for delivery of the nucleic acid into tumor cells, a high titer of the infective transformation system can be injected directly within the tumor site so as to enhance the likelihood of tumor cell infection. The infected cells will then express the desired effector protein, e.g., HopPtoA, HopPsyA, or HopPtoA2, disrupting cellular functions and producing cytotoxic effects.
Particularly preferred is use of the effector proteins of the present invention to treat a cancerous condition (i.e., the eukaryotic cell which is affected is a cancer cell). This can be carried out by introducing a cytotoxic Pseudomonas protein into cancer cells of a patient under conditions effective to inhibit cancer cell division, thereby treating the cancerous condition.
By introducing, it is intended that the effector protein is administered to the patient, preferably in the form of a composition which will target delivery to the cancer cells. Alternatively, when using DNA-based therapies, it is intended that the introducing be carried out by administering a target DNA delivery system to the patient such that the cancer cells are targeted and the effector protein is expressed therein.
EXAMPLES
The following Examples are intended to be illustrative and in no way are intended to limit the scope of the present invention.
Materials and Methods
Bacterial Strains, Culture Conditions, Plasmids, and DNA Manipulation Techniques
Three experimentally amenable strains that represent different levels of diversity in P. syringae were investigated: Psy 61, Psy B728a, and Pto DC3000. (i) Psy 61 is a weak pathogen of bean whose hrp gene cluster, cloned on cosmid pHIR11, contains all of the genes necessary for nonpathogenic bacteria like Pseudomonas fluorescens and Escherichia coli to elicit the HR in tobacco and to secrete in culture the HrpZ harpin, a protein with unknown function that is secreted abundantly by the Hrp system (Alfano et al., 1996). The pHIR11 hip cluster has been completely sequenced (FIG. 1) (Alfano and Collmer, 1997), and the hopPsyA gene in the hypervariable region at the left edge of the cluster was shown to encode a protein that has an Avr phenotype, travels the Hrp pathway, and elicits cell death when expressed in tobacco cells (Alfano and Collmer, 1997; Alfano et al., 1997; van Dijk et al., 1999). (ii) Psy B728a is in the same pathovar as strain 61 but is highly virulent and is a model for studying the role of the Hrp system in epiphytic fitness and pathogenicity (brown spot of bean) in the field (Hirano et al., 1999). (iii) Pto DC3000 is a well-studied pathogen of Arabidopsis and tomato (causing bacterial speck) that is highly divergent from pathovar syringae strains. Analysis of rRNA operon RFLP patterns has indicated that Pto and Psy are distantly related and could be considered separate species (Manceau and Horvais, 1997). Thus, we were able to compare two strains in the same pathovar with a strain from a highly divergent pathovar.
Conditions for culturing E. coli and P. syringae strains have been described (van Dijk et al., 1999), as have the sources for Psy 61 (Preston et al., 1995), Psy B728a (Hirano et al., 1999), and Pto DC3000 (Preston et al., 1995). Cloning and DNA manipulations were done in E. coli DH5α using pBluescript II (Stratagene, La Jolla, Calif.), pRK415 (Keen et al., 1988), and cosmid pCPP47 (Bauer and Collmer, 1997), according to standard procedures (Ausubel et al., 1994). Cosmid libraries of Pto DC3000 and Psy B728a genomic DNA were previously constructed (Charkowski et al., 1998). Oligonucleotide synthesis and DNA sequencing were performed at the Cornell Biotechnology Center. The nucleotide sequence of the Pto DC3000 hrp/hrc cluster was determined using subclones of pCPP2473, a cosmid selected from a genomic cosmid library based on hybridization with the hrpK gene of Psy 61. The nucleotide sequence of the Psy B728a hrp/hrc cluster was determined using subclones of pCPP2346 and pCPP3017. These cosmids were selected from a genomic library based on hybridization with the hrpC operon of 61. The left side of the Psy 61 EEL region was cloned by PCR into pBSKSII+ Xhol and EcoRI sites using the following primers:

    
    
        SEQ. ID. NO. 71, which primes within queA and contains an Xhol site:
        SEQ. ID. NO. 72, which primes within hopPsyA and contains an EcoRI site:
        Pfu polymerase was used for all PCR experiments. DNA sequence data were managed and analyzed with the DNAStar Program (Madison, Wis.), and databases were searched with the BLASTX, BLASTP, and BLASTN programs (Altschul et al., 1997).

Mutant Construction and Analysis

    
    


Large deletions in the Pto DC3000 Hrp Pai were constructed by subcloning border fragments into restriction sites on either side of an ΩSpR cassette in pRK415, electroporating the recombinant plasmids into DC3000, and then selecting and screening for marker exchange mutants as described (Alfano et al., 1996). The following left and right side (FIGS. 2 and 3) deletion border fragments were used (with residual gene fragments indicated): for CUCPB5110 left tgt-gueA-tRNA-Leu-ORF4′ (27 bp of ORF4) and right ORF1′-hrpK (396 bp of ORF1); and for CUCPB5115 left hrpS′-avrE′ (2569 bp of avrE) and right ORF6 (156 bp upstream of ORF6 start codon). The later fragment was PCR-amplified using the following primers:

    
    
        SEQ. ID. NO. 73, which primes in the ORF5-ORF6 intergenic region and contains an XbaI site:
        SEQ. ID. NO. 74, which primes in ORF6 and contains a HindIII site:
        Mutant constructions were confirmed by Southern hybridizations using previously described conditions (Charkowski et al., 1998). The ability of mutants to secrete AvrPto was determined with anti-AvrPto antibodies and immunoblot analysis of cell fractions as previously described (van Dijk et al., 1999). Mutant CUCPB5 115 was complemented with pCPP3016, which carries ORF2 through ORF10 in cosmid pCPP47, and was introduced from E. coli DH5α by triparental mating using helper strain E. coli DH5α(pRK600), as described (Charkowski et al., 1998).

T7 Expression Analysis

    
    


Protein products of the Pto DC3000 EEL were analyzed by T7 polymerase-dependent expression using vector pET21 and E. coli BL21(DE3) as previously described (Huang et al., 1995). The following primer sets were used to PCR each ORF from pCPP3091, which carries in pBSKSII+ a BamH1 fragment containing tgt to hrcV:

    
    
        ORF1, SEQ. ID. Nos. 75 and 76, respectively:
    
    












agtaggatcc tgaaatgtag gggcccgg
28



agtaaagctt atgatgctgt ttccagta
28






    
    
        ORF2, SEQ. ID. Nos. 77 and 78, respectively:
    
    












agtaggatcc tctcgaagga atggagca
28



agtaaagctt cgtgaagatg catttcgc
28






    
    
        ORF3, SEQ. ID. Nos. 79 and 80, respectively:
    
    












agtaggatcc tagtcactga tcgaacgt
28



agtactcgag ccacgaaata acacggta
28






    
    
        ORF4, SEQ. ID. Nos. 81 and 82, respectively:
    
    












agtaggatcc caggactgcc ttccagcg
28



agtactcgag cagagcggcg tccgtggc
28






    
    
        tnpA, SEQ. ID. Nos. 83 and 84, respectively:
    
    












agtaggatcc agaattgttg aagaaatc
28



agtaaagctt tgcgctgtta actcatcg
28






Plant Bioassays

Tobacco (Nicotiana tabacum L. cv. Xanthi) and tomato (Lycopersicon esculentum Mill. cvs. Moneymaker and Rio Grande) were grown under greenhouse conditions and then maintained at 25° C. with daylight and supplemental halide illumination for HR and virulence assays. Bacteria were grown overnight on King's medium B agar supplemented with appropriate antibiotics, suspended in 5 mM MES pH 5.6, and then infiltrated with a needleless syringe into the leaves of test plants at 108 cfu/ml for HR assays and 104 cfu/ml for pathogenicity assays (Charkowski et al., 1998). All assays were repeated at least four times on leaves from different plants. Bacterial growth in tomato leaves was assayed by excising disks from infiltrated areas with a cork borer, comminuting the tissue in 0.5 ml of 5 mM MES, pH 5.6, with a Kontes Pellet Pestle (Fisher Scientific, Pittsburgh, Pa.), and then dilution plating the homogenate on King's medium B agar with 50 μg/ml rifampicin and 2 μg/ml cycloheximide to determine bacterial populations. The mean and SD from three leaf samples were determined for each time point. The relative growth in planta of DC3000 and CUCPB5110 was similarly assayed in 4 independent experiments and the relative growth of DC3000, CUCPB5115, and CUCPB5115(pCPP3016) in 3 independent experiments. Although the final population levels achieved by DC3000 varied between experiments, the populations levels of the mutants relative to the wild type were the same as in the representative experiments presented below.
Example 1
Comparison of hrp/hrc Gene Clusters of Psy 61, Psy B728a, and Pto DC3000
To determine if the hrp/hrc clusters from Psy B728a and Pto DC3000 were organized similarly to the previously characterized hrp/hrc cluster of Psy 61, two cosmids carrying hrp/hrc inserts were partially characterized. pCPP2346 carries the entire hrp/hrc cluster of B728a, and pCPP2473 carries the left half of the hrp/hrc cluster of DC3000. The right half of the DC3000 hrp/hrc cluster had been characterized previously (Preston et al., 1995). Sequencing the ends of several subclones derived from these cosmids provided fingerprints of the B728a and DC3000 hrp/hrc clusters, which indicated that both are arranged like that of strain 61 (FIG. 1). However, B728a contains between hrcU and hrpV a 3.6-kb insert with homologs of bacteriophage lambda genes Ea59 (23% amino-acid identity; E=2e-7) and Ea3l (30% amino-acid identity; E=6e-8) (Hendrix et al., 1983), and the B728a hrcU ORF has 36 additional codons. A possible insertion of this size in several Psy strains that are highly virulent on bean was suggested by a previous RFLP analysis (Legard et al., 1993). Cosmid pCPP2346, which contains the B728a hrp/hrc region and flanking sequences (4 kb on the left and 13 kb on the right), enabled P. fluorescens to secrete the B728a HrpZ harpin in culture and to elicit the HR in tobacco leaves, however, confluent necrosis developed more slowly than with P. fluorescens (pHIR11) (data not shown). To further test the relatedness of the Psy 61 and B728a hrp/hrc gene clusters using an internal reference, the B728a hrpA gene was sequenced. Of the hrp/hrc genes that have been sequenced in Psy and Pto, hrpA, which encodes the major subunit of the Hrp pilus (Roine et al., 1997), is the least conserved (28% amino-acid identity) (Preston et al., 1995). However, the hrpA genes of strains 61 and B728a were 100% identical, which further supports the close relationship of these strains and their Hrp systems.
Example 2
Identification of an Exchangeable Effector Locus (EEL) in the Hrp Pai between hrpK and tRNALeu 
Sequence analysis of the left side of the Psy 61, Psy B728a, and Pto DC3000 Hrp Pais revealed that the high percentage identity in hrpK sequences in these strains abruptly terminates three nucleotides after the hrpK stop codon and then is restored near tRNALeu, queA, and tgt sequences after 2.5 kb (Psy 61), 7.3 kb (Psy B728a), or 5.9 kb (Pto DC3000) of dissimilar, intervening DNA (FIG. 2). The difference between Psy strains 61 and B728a in this region was particularly surprising. This region of the P. syringae Hrp Pai was given the EEL designation because it contained completely different effector protein genes (Table 1 below), which appear to be exchanged at this locus at a high frequency. In this regard, it is noteworthy that (i) ORF2 in the B728a EEL is a homolog of avrPphE, which is in a different location, immediately downstream of hrpK (hrpY), in Pph 1302A (Mansfield et al., 1994), (ii) hopPsyA (hrmA) is present in only a few Psy strains (Heu and Hutcheson, 1993; Alfano et al., 1997), (iii) and ORF5 in the B728a EEL predicts a protein that is similar to Xanthomonas AvrBsT and possesses multiple motifs characteristic of the AvrRxv family (Ciesiolka et al., 1999). G+C content different from the genomic average is a hallmark of horizontally transferred genes, and the G+C contents of the ORFs in the three EELs are considerably lower than the average of 59-61% for P. syringae (Palleroni et al., 1984) (Table 1 below). They are also lower than hrpK (60%) and queA (63-64%). The ORFs in the Pto DC3000 EEL predict no products with similarity to known effector proteins, however T7 polymerase-dependent expression revealed products in the size range predicted for ORF 1, ORF3, and ORF4. Furthermore, the ORF1 protein is secreted in a hrp-dependent manner by E. coli(pCPP2156), which expresses an Erwinia chrysanthemi Hrp system that secretes P. syringae Avr proteins (Ham et al., 1998). Several ORFs in these EELs are preceded by Hrp boxes indicative of HrpL-activated promoters (FIG. 1) (Xiao and Hutcheson, 1994), and the lack of intervening Rho-independent terminator sequences or promoters suggests that ORF1 in DC3000 and ORF1 and ORF2 in B728a are expressed from HrpL-activated promoters upstream of the respective hrpK genes.
The EELs of these three strains also contain sequences homologous to insertion sequences, transposases, phage integrase genes, and plasmids (FIG. 2 and Table 1 below). The Psy B728a ORF5 and ORF6 operon is bordered on the left side linked to any type III secretion system genes or other genes in the Hrp Pai (FIG. 2). Thus, this is the apparent point of insertion of the Hrp Pai in the ancestral Pseudomonas genome.
Example 3
Identification of a Conserved Effector Locus (CEL) Located on the Right Side of the Hrp Pai in Psy B728a and Pto DC3000
Previous studies of the region to the right of hrpR in DC3000 had revealed the existence of the avrE locus, which is comprised of two transcriptional units (Lorang and Keen, 1995), the 5′ sequences for the first 4 transcriptional units beyond hrpR (Lorang and Keen, 1995), and the identity of the fourth transcriptional unit as the hrpW gene encoding a second harpin (Charkowski et al., 1998). The DNA sequence of the first 14 ORFs to the right of hrpR in Pto DC3000 was completed in this investigation and the corresponding region in Psy B728a was partially sequenced (FIG. 3). Like the EEL, this region contains putative effector genes, e.g., avrE (Lorang and Keen, 1995). Unlike the EEL, the ORFs in this region have an average G+C content of 58.0%, which is close to that of the hrp/hrc genes, the region contains no sequences similar to known mobile genetic elements, and it appears conserved between Psy and Pto (FIG. 3). Comparison of the regions sequenced in B728a and DC3000 revealed that the first 7 ORFs are arranged identically and have an average DNA sequence identity of 78%. Hence, this region was given the CEL designation.
The precise border of the CEL remains undefined, and no sequences that were repeated in the EEL border of the Hrp Pai were found. ORF7 and ORF8 are likely to be part of the CEL, based on the presence of an upstream Hrp box (FIG. 3). However, the region beyond ORF10 probably is not in the CEL because the product of the next ORF shows homology to a family of bacterial GstA proteins (e.g., 28% identity with E. coli GstA over 204 amino acids; E=1e-8)(Blattner et al., 1997), and glutathione-S-transferase activity is common in nonpathogenic fluorescent pseudomonads (Zablotowicz et al., 1995). The presence of a galP homolog (38% identity over 256 amino acids, based on incomplete sequence, to E. coli GalP; E=2e-42) (Blattner et al., 1997) in this region further suggests that it is beyond the CEL.
Several other features of this region in B728a and DC3000 are noteworthy. (i) Both strains have a 1-kb intergenic region between hrpR and ORF1 that is distinguished by low sequence identity (44%) but which contains three inverted repeats that could form stem loop structures affecting expression of the hrpRS operon. (ii) ORF1 is most similar to E. coli murein lytic transglycosylase MltD (38% identity over 324 amino acids; E=4e-56). (iii) ORF2 is 42% identical over 130 amino acids with E. amylovora DspF (E=9e-24), a candidate chaperone (Bogdanove et al., 1998a; Gaudriault et al., 1997). (iv) The ORF5 protein is secreted in a hrp-dependent manner by E. coli(pCPP2156), but mutation with an ΩSpr cassette has little effect on either HR elicitation in tobacco or pathogenicity in tomato (Charkowski, unpublished). (v) Finally, six operons in this region are preceded by Hrp boxes (Lorang and Keen, 1995) (FIG. 3), which is characteristic of known avr genes in P. syringae (Alfano et al., 1996). Thus, the CEL carries multiple candidate effectors.
Example 4
Investigation of EEL and CEL Roles in Pathogenicity
A mutation was constructed in DC3000 that replaced all of the ORFs between hrpK and tRNALeu (EEL) with an ΩSpr cassette (FIG. 2). This Pto mutant, CUCPB5110, was tested for its ability to elicit the HR in tobacco and to cause disease in tomato. The mutant retained the ability to elicit the HR and to produce disease symptoms, but it failed to reach population levels as high as the parental strain in tomato (FIG. 4A).
A mutation was constructed in DC3000 that replaced avrE through ORF5 (CEL) with an ΩSpr cassette. This deleted all of the CEL ORFs that were both partially characterized and likely to encode effectors. This Pto mutant, CUCPB5115, still elicited the HR in tobacco, but tissue collapse was delayed ca. 5 h (FIG. 4C). The mutant no longer elicited disease symptoms in tomato when infiltrated at a concentration of 104 cfu/ml, and growth in planta was strongly reduced (FIG. 4B). However, the mutant elicited an HR dependent on the tomato Pto R gene that was indistinguishable from the wild-type in tests involving PtoS (susceptible) and PtoR (resistant) Rio Grande tomato lines. Plasmid pCPP3016, which carries ORF2 through ORF10, fully restored the ability of CUCPB5115 to cause disease symptoms and partially restored the ability of the mutant to multiply in tomato leaves (FIGS. 4B and 4E). Deletion of the hrp/hrc cluster abolishes HR and pathogenicity phenotypes in Pto DC3000 (Collmer et al., 2000). To confirm that the large deletions in Pto mutants CUCPB5110 and CUCPB5115 did not disrupt Hrp secretion functions, we compared the ability of these mutants, the DC3000 hrp/hrc deletion mutant, and wild-type DC3000 to make and secrete AvrPto in culture while retaining a cytoplasmic marker comprised of β-lactamase lacking its signal peptide. AvrPto provided an ideal subject for this test because it is a well-studied effector protein that is secreted in culture and injected into host cells in planta (Alfano and Collmer, 1997; van Dijk et al., 1999). Only the hrp/hrc deletion cluster mutant was impaired in AvrPto production and secretion (FIG. 5).
Based on the above studies, the P. syringae hrp/hrc genes are part of a Hrp Pai that has three distinct loci: an EEL, the hrp/hrc gene cluster, and a CEL. The EEL harbors exchangeable effector genes and makes only a quantitative contribution to parasitic fitness in host plants. The hrp/hrc locus encodes the Hrp secretion system and is required for effector protein delivery, parasitism, and pathogenicity. The CEL makes no discernible contribution to Hrp secretion functions but contributes strongly to parasitic fitness and is required for Pto pathogenicity in tomato. The Hrp Pai of P. syringae has several properties of Pais possessed by animal pathogens (Hacker et al., 1997), including the presence of many virulence-associated genes (several with relatively low G+C content) in a large (ca. 50-kb) chromosomal region linked to a tRNA locus and absent from the corresponding locus in a closely related species. In addition, the EEL portion of the Hrp Pai is unstable and contains many sequences related to mobile genetic elements.
The EEL is a novel feature of known Pais, which is likely involved in fine-tuning the parasitic fitness of P. syringae strains with various plant hosts. By comparing closely- and distantly-related strains of P. syringae, we were able to establish the high instability of this locus and the contrasting high conservation of its border sequences. No single mechanism can explain the high instability, as we found fragments related to phages, insertion sequences, and plasmids in the Psy and Pto EELs, and insertion sequences were recently reported in the corresponding region of three other P. syringae strains (Inoue and Takikawa, 1999). The mechanism or significance of the localization of the EELs between tRNALeu and hrpK sequences in the Hrp Pais also is unclear. Pto DC3000 carries at least one other effector gene, avrPto, that is located elsewhere in the genome (Ronald et al., 1992), many P. syringae avr genes are located on plasmids (Leach and White, 1996), and the EEL ORFs represent a mix of widespread, (e.g., avrRxv family) and seemingly rare (e.g., hopPsyA), effector genes. The G+C content of the EEL ORFs is significantly lower than that of the rest of the Hrp Pai and the P. syringae genome. Although certain genes in the non-EEL portions of the Hrp Pai, such as hrpA, are highly divergent, they have a high G+C content, and there is no evidence that they have been horizontally transferred separately from the rest of the Hrp Pai. The relatively low G+C content of the ORFs in the EELs (and of other P. syringae avr genes) suggests that these genes may be horizontally acquired from a wider pool of pathogenic bacteria than just P. syringae (Kim et al., 1998). Indeed, the avrRxv family of genes is found in a wide range of plant and animal pathogens (Ciesiolka et al., 1999). The weak effect on parasitic fitness of deleting the Pto DC3000 EEL, or of mutating hopPsyA (hrmA) in Psy 61 (Huang et al., 1991), is typical of mutations in individual avr genes and presumably results from redundancy in the effector protein system (Leach and White, 1996).
The functions of hrpK and of the CEL ORF1 are unclear but warrant discussion. These two ORFs reside just outside the hrpL and hrpR delimited cluster of operons containing both hrp and hrc genes and thereby spatially separate the three regions of the Hrp Pai (FIGS. 1-3). hrpK mutants have a variable Hrp phenotype (Mansfield et al., 1994; Bozso et al., 1999), and a Psy B728a hrpK mutant still secretes HrpZ (Alfano, unpublished), which suggests that HrpK may be an effector protein. Nevertheless, the HrpK proteins of Psy 61 and Pto DC3000 are 79% identical and therefore are more conserved than many Hrp secretion system components. It is also noteworthy that hrpK appears to be in an operon with other effector genes in Psy B728a and Pto DC3000. In contrast, the CEL ORF1 may contribute (weakly or redundantly) to Hrp secretion functions by promoting penetration of the system through the bacterial peptidoglycan layer. The ORF1 product has extensive homology with E. coli MltD and shares a lysozyme-like domain with the product of ipgF (Mushegian et al., 1996), a Shigella flexneri gene that is also located between loci encoding a type III secretion system and effector proteins (Allaoui et al., 1993). Mutations in these genes in Pto and S. flexneri have no obvious phenotype (Lorang and Keen, 1995; Allaoui et al., 1993), as is typical for genes encoding peptidoglycan hydrolases (Dijkstra and Keck, 1996).
The loss of pathogenicity in Pto mutant CUCPB5115, with an avrE-ORF5 deletion in the CEL, was surprising because pathogenicity is retained in DC3000 mutants in which the corresponding operons are individually disrupted (Lorang and Keen, 1995; Charkowski et al., 1998). In assessing the possible function of this region and the conservation of its constituent genes, it should be noted that avrE is unlike other avr genes found in Pto in that it confers avirulence to P. syringae pv glycinea on all tested soybean cultivars and it has a homolog (dspE) in E. amylovora that is required for pathogenicity (Lorang and Keen, 1995; Bogdanove et al., 1998b). Although the CEL is required for pathogenicity, it is not essential for type III effector protein secretion because the mutant still secretes AvrPto. It also appears to play no essential role in type III translocation of effector proteins into plant cells because the mutant still elicits the HR in nonhost tobacco and in a PtoR-resistance tomato line, and pHIR11, which lacks this region, appears capable of translocating several Avr proteins (Gopalan et al., 1996; Pirhonen et al., 1996). The conservation of this region in the divergent pathovars Psy and Pto, and its importance in disease, suggests that the products of the CEL may be redundantly involved in a common, essential aspect of pathogenesis.
The similar G+C content and codon usage of the hrp/hrc genes, the genes in the CEL, and total P. syringae genomic DNA suggests that the Hrp Pai was acquired early in the evolution of P. syringae. Although, the EEL region may have similarly developed early in the radiation of P. syringae into its many pathovars, races, and strains, the apparent instability that is discussed above suggests ongoing rapid evolution at this locus. Indeed, many P. syringae avr genes are associated with mobile genetic elements, regardless of their location (Kim et al., 1998). Thus, it appears that Hrp-mediated pathogenicity in P. syringae is collectively dependent on a set of genes that are universal among divergent pathovars and on another set that varies among strains even in the same pathovar. The latter are presumably acquired and lost in response to opposing selection pressures to promote parasitism while evading host R-gene surveillance systems.
Example 5
Role of ShcA as a Type III Chaperone for the HopPsyA Effector
The ORF upstream of hopPsyA, tentatively named shcA, encodes a protein product of the predicted molecular mass. The ORF upstream of the hopPsyA gene in P. s. syringae 61 (originally designated ORF1) shares sequence identity with exsC and ORF7, which are genes adjacent to type III effector genes in P. aeruginosa and Yersinia pestis, respectively (Frank and Iglewski, 1991; Perry et al., 1998). Although neither of these ORFs have been shown experimentally to encode chaperones, they have been noted to share properties that type III chaperones often possess (Cornellis et al., 1998). One of these properties is the location of the chaperone gene itself (FIGS. 1 and 6). Chaperone genes are often adjacent to a gene that encodes the effector protein with which the chaperone interacts. Furthermore, shcA also shares other common characteristics of type III chaperones: its protein product is relatively small (about 14 kDa), it has an acidic pI, and it has a C-terminal region that is predicted to be an amphipathic α-helix. To begin assessing the function of shcA, it was first determined whether shcA encodes a protein product. A construct was prepared using PCR that fused shcA in-frame to a sequence encoding the FLAG epitope. This construct, pLV26, contains the nucleotide sequence upstream of shcA, including a putative ribosome binding site (RBS). DH5αF′IQ(pLV26) cultures were grown in rich media and induced at the appropriate density with IPTG. Whole cell lysates were separated by SDS-PAGE and analyzed with immunoblots using anti-FLAG antibodies. By comparing the ShcA-FLAG encoded by pLV26 to a construct that made ShcA-FLAG from a vector RBS, it was concluded that the native RBS upstream of shcA was competent for translation (FIG. 7). Thus, the shcA ORF is a legitimate gene that encodes a protein product.
To test the effects of shcA on bacterial-plant interactions, an shcA mutation was constructed in the minimalist hrp/hrc cluster carried on cosmid pHIR11. There are distinct advantages to having the shcA mutation marker-exchanged into pHIR11. The main one is that the HR assay can be used as a screen to determine if HopPsyA is being translocated into plant cells because the pHIR11-dependent HR requires the delivery of HopPsyA into plant cells (Alfano et al., 1996; Alfano et al., 1997). With the chromosomal shcA mutant, other Hop proteins would probably be delivered to the interior of plant cells. Some of these proteins would be recognized by the R gene-based plant surveillance system and initiate an HR masking any defect in HopPsyA delivery. E. coli MC4100 carrying pLV10, a pHIR11 derivative, which contains a nonpolar nptII cartridge within shcA, was unable to elicit an HR on tobacco (FIG. 8). This indicates that shcA is required for the translocation of HopPsyA into plant cells. To determine if HopPsyA was secreted in culture, cultures of the nonpathogen P. fluorescens 55 were grown. This bacterium carried either pHIR11, pCPP2089 (a pHIR11 derivative defective in type III secretion), or pLV10. The representative results can be seen in FIG. 8. shcA was required for the in-culture type III secretion of the HopPsyA effector protein, but not for HrpZ secretion, another protein secreted by the pHIR11 encoded Hrp system. These results indicate that the defect in type III secretion is specific to HopPsyA and are consistent with shcA encoding a chaperone for HopPsyA. It was after these results that the ORF upstream of the hopPsyA gene was named shcA for specific hop chaperone for HopPsyA, a naming system consistent with the naming system researchers have employed for chaperones in the archetypal Yersinia type III system.
Example 6
Cytotoxic Effects of hopPsyA Expressed in Plants
Transient expression of hopPsyA DNA in planta induces cell death in Nicotiana tabacum, but not in N. benthamiana, bean, or in Arabidopsis. To determine whether HopPsyA induced cell death on tobacco leaves as it did when produced in tobacco suspension cells, a transformation system that delivers the hopPsyA gene on T-DNA of Agrobacterium tumefaciens was used (Rossi et al., 1993; van den Ackerveken et al., 1996). This delivery system works better than biolistics for transiently transforming whole plant leaves. For these experiments, vector pTA7002, kindly provided by Nam-Hai Chua and his colleagues at Rockefeller University, was used. The unique property of this vector is that it contains an inducible expression system that uses the regulatory mechanism of the glucocorticoid receptor (Picard et al., 1988; Aoyama and Chua, 1997; McNellis et al., 1998). pTA7002 encodes a chimeric transcription factor consisting of the DNA-binding domain of GAL4, the transactivating domain of the herpes viral protein VP16, and the receptor domain of the rat glucocorticoid receptor. Also contained on this vector is a promoter containing GAL4 upstream activating sequences (UAS) upstream of a multiple cloning site. Thus, any gene cloned downstream of the promoter containing the GAL4-UAS is induced by glucocorticoids, of which a synthetic glucocorticoid, dexamethasone (DEX), is available commercially. hopPsyA was PCR-cloned downstream of the GAL4-UAS. Plant leaves from several different test plants were infiltrated with Argrobacterium carrying pTA7002::hopPsyA and after 48 hours these plants were sprayed with DEX. Only N. tabacum elicited an HR in response to the DEX-induced transient expression of hopPsyA (FIG. 13A). In contrast, N. benthamiana produced no obvious response after DEX induction (FIG. 13B). Moreover, transient expression of hopPsyA in bean plants (Phaseolus vulgaris L. ‘Eagle’)(data not shown) and Arabidopsis thaliana ecotype Col-1 (FIG. 13) did not result in a HR. These results suggest that bean cv. Eagle, Arabidopsis Col-1, and N. benthamiana lack a resistance protein that can recognize HopPsyA. The lack of an apparent defense response for HopPsyA transiently expressed in bean was predicted, because HopPsyA is normally produced in P. s. syringae 61, a pathogen of bean. But, it was somewhat unknown how transient expression of HopPsyA would effect Arabidopsis. However, since P. s. tomato DC3000, a pathogen of Arabidopsis, appears to have a hopPsyA homolog based on DNA gel blots using hopPsyA as a probe, it was expected that HopPsyA would not to be recognized by an R protein in Arabidopsis (i.e., no HR produced) (Alfano et al., 1997). Thus, these plants (bean, Arabidopsis, and N. benthamiana) should represent ideal plants to explore the bacterial-intended role of HopPsyA in plant pathogenicity.
P.s. pv. syringae 61 secretes HopPsyA in culture via the Hrp (type III) protein secretion system. Because the P. syringae Avr proteins AvrB and AvrPto were found to be secreted by the type III secretion system encoded by the functional E. chrysanthemi hrp cluster carried on cosmid pCPP2 156 expressed in E. coli (Ham et al., 1998), detection of HopPsyA secretion in culture directly via the native Hrp system carried in P. s. syringae 61 was tested. P. s. syringae 61 cultures grown in hrp-derepressing fructose minimal medium at 22° C. were separated into cell-bound and supernatant fractions by centrifugation. Proteins present in the supernatant fractions were concentrated by TCA precipitation, and the cell-bound and supernatant samples were resolved with SDS-PAGE and analyzed with immunoblots using anti-HopPsyA antibodies. A HopPsyA signal was detected in supernatant fractions from wild type P. s. syringae 61 (FIG. 14). Importantly, HopPsyA was not detected in supernatant fractions from P. s. syringae 61-2089, which is defective in Hrp secretion, indicating that the HopPsyA signal in the supernatant was due specifically to type III protein secretion (FIG. 14). As a second control, both strains contained pCPP2318, which encodes the mature β-lactamase lacking its N-terminal signal peptide, and provides a marker for cell lysis. β-lactamase was detected only in the cell-bound fractions of these samples, clearly showing that cell lysis did not occur at a significant level (FIG. 14). The fact that HopPsyA is secreted via the type III secretion system in culture and that the avirulence activity of HopPsyA occurs only when it is expressed in plant cells strongly support that HopPsyA is delivered into plant cells via the type III pathway.
HopPsyA contributes in a detectable, albeit minor, way to growth of P. s. syringae 61 in bean. The effect of a HopPsyA mutation on the multiplication of P. s. syringae 61 in bean tissue has been reported (Huang et al., 1991). These data essentially indicate that HopPsyA contributes little to the ability of P. s. syringae 61 to multiply in bean. The P. s. syringae 61 hopPsyA mutant does not grow as well in bean leaves as the wild-type strain (FIG. 15). This was unexpected, because these results are in direct conflict with previously reported data. One rationale for the discrepancy is that the previous reports focused primarily on the major phenotype that a hrp mutant exhibits on in planta growth and predated the discovery that HopPsyA was a type III-secreted protein. Thus, it is quite possible that the earlier experiments missed the more subtle effect that HopPsyA appears to have on the multiplication of P. s. syringae 61 in bean tissue (Huang et al., 1991). The data presented here supports that HopPsyA contributes to the pathogenicity of P. s. syringae and are consistent with the hypothesis that the majority of Hops from P. syringae contribute subtly to pathogenicity. The lack of strong pathogenicity phenotypes for mutants defective in different avr and hop genes may be due to possible avr/hop gene redundancy or a decreased dependence on any one Hop protein through coevolution with the plant. Indeed, the type III-delivered proteins of plant pathogens that are delivered into plant cells may not be virulence proteins per se, but rather they may suppress responses of the plant that are important for pathogenicity to proceed (Jakobek et al., 1993). These responses may be defense responses or other more general processes that maintain the status quo within the plant (e.g., the cell cycle).
Example 7
Molecular Interactions of HopPsyA
HopPsyA interacts with the Arabidopsis Mad2 protein in the yeast 2-hybrid system. To determine a pathogenic target for HopPsyA, the yeast 2-hybrid system was used with cDNA libraries made from Arabidopsis (Fields and Song, 1989; Finley and Brent, 1994). In the yeast 2-hybrid system, a fusion between the protein of interest (the “bait”) and the LexA DNA-binding domain was transformed into a yeast tester strain. A cDNA expression library was constructed in a vector that creates fusions to a transcriptional activator domain. This library was transformed into the tester strain en masse, and clones encoding partners for the “bait” are selected via their ability to bring the transcriptional activator domain into proximity with the DNA binding domain, thus initiating transcription of the LEU2 selectable marker gene. A second round screening of candidates, that activate the LEU2 marker, relies on their ability to also activate a lacZ reporter gene. Bait constructs were initially made with hopPsyA in the yeast vector pEG202 that corresponded to a full-length HopPsyA-LexA fusion, the carboxy-terminal half of HopPsyA fused to LexA, and the amino-terminal half of HopPsyA fused to LexA, and named these constructs pLV23, pLV24, and pLV25, respectively. However, pLV23 was lethal to yeast and pLV25 activated the lacZ reporter gene in relatively high amounts on its own (i.e., without the activation domain present). Thus, both pLV23 and pLV25 were not used to screen for protein interactors via the yeast 2-hybrid system. pLV24, which contains the 3′ portion of hopPsyA fused to lexA, proved to be an appropriate construct to use for bait in the yeast 2-hybrid system, because it did not autoactivate the lacZ reporter gene and, based on the lacZ repression assay using pJK101, the 'HopPsyA-LexA fusion produced by pLV24 appeared to localize to the nucleus. In addition, it was confirmed that pLV24 made a protein of the appropriate size that corresponds to HopPsyA by performing immunoblots with anti-HopPsyA antibodies on yeast cultures carrying this vector.
Initial screens with pLV24 and Arabidopsis cDNA libraries in the yeast 2-hybrid vector pJG4-5. From three independent screens, several hundred by sequences similar to those in a Pph plasmid that carries several avr genes (Jackson et al., 1999) and by a sequence homologous to insertion elements that are typically found on plasmids, suggesting plasmid integration via an IS element in this region (Szabo and Mills, 1984). Psy B728a ORF3 and ORF4 show similarity to sequences implicated in the horizontal acquisition of the LEE Pai by pathogenic E. coli strains (Perna et al., 1998). These Psy B728a ORFs are not preceded by Hrp boxes and are unlikely to encode effector proteins.







TABLE 1







ORFs and fragments of genetic elements in the EELs of Pto


DC3000, Psy B728a, and Psy 61 and similarities with known


avr genes and mobile genetic elements.













BLAST E value with representative


ORF or
%

similar sequence(s) in


sequence
G + C
Size
database, or relevant feature





Pto DC3000a





ORF1
55
466 aa
Hrp-secreted (Alfano, unpublished)


TnpA′
55
279 aa
1e-125 P. stutzeri TnpA1





(Bosch et al., 1999)


ORF2
51
241 aa
None


ORF3
53
138 aa
None


ORF4
47
136 aa
None


Psy B728a


ORF1
51
323 aa
9e-40 Pph AvrPphC (Yucel et al., 1994)


ORF2
58
382 aa
1e-154 Pph AvrPphE (Mansfield et al.,





1994)


ORF3
55
507 aa
2e-63 E. coli L0015 (Perna et al., 1998)


ORF4
55
118 aa
9e-9 E. coli L0014 (Perna et al., 1998)


ORF5
49
411 aa
1e-4 Xcv AvrBsT (Ciesiolka et al.,





1999)


ORF6
52
120 aa
None


B plasmid
46
 96 nt
1e-25 Pph pAV511 (Jackson et al.,





1999)


IntA′
59
 49 aa
3e-5 E. coli CP4-like integrase





(Perna et al., 1998)


Psy 61


HopPsyA
53
375 aa
Hrp-secreted Avr (Alfano et al., 1997;





van Dijk et al., 1999)


ShcA
57
112 aa
6e-4 Y0008 (Perry et al., 1998)





aPathovar abbreviations correspond to the recommendations of Vivian and Mansfield (1993) for uniform avr nomenclature. 






The left border of the EELs contains sequences similar to many tRNALeu genes and to E. coli queA and tgt queuosine biosynthesis genes (ca. 70% amino-acid identity in predicted products). The EEL sequences terminate at the 3′ end of the P. syringae tRNA sequences, as is typical for Pais (Hou, 1999). Virtually identical tgt-queA-tRNALeu sequences are found in the genome of P. aeruginosa PAO1 (www.pseudomonas.com), which is also in the fluorescent pseudomonad group. But PAO1 is not a plant pathogen, and this tRNALeu in P. aeruginosa is not putative interactors with HopPsyA were identified, each activating the two reporter systems to varying degrees. When these putative positive yeast strains were rescreened and criteria were limited to interactors that strongly induced both the lacZ reporter and LEU2 gene in the presence of galactose, about 50 yeast strains were identified that appeared to contain pJG4-5 derivatives that encoded proteins that could interact with the C-terminal half of HopPsyA. DNA gel blots using PCR-amplified inserts from selected pJG4-5 derivatives as probes allowed each of these putative positives to be grouped. Approximately 50% of the pJG4-5 derivatives that encoded strong HopPsyA interactors belonged to the same group. A pJG4-5 derivative containing this insert, pLV116 was sequenced. The predicted amino acid sequence of the insert contained within pLV116 shared high amino acid identity to Mad2 homologs (for mitotic arrest deficient) found in yeast, humans, frogs, and corn. Moreover, based on amino acid comparison with the other Mad2 proteins, pLV116 contains a cDNA insert that corresponds to the full-length mad2 mRNA. Table 2 below shows the amino acid percent identity of all of the Mad2 homologs currently in the databases.







TABLE 2







Percent Amino Acid Sequence Identity Between Different Mad2 Homologs*














Mad2





Fission
Budding


Homolog
Arabidopsis
Corn
Human
Mouse
Frog
Yeast
Yeast





Arabidopsis
—








Corn
81.3
—


Human
44.4
44.9
—


Mouse
45.4
45.9
94.6
—


Frog
43.3
42.9
78.3
77.3
—


Fission
40.4
41.9
43.8
43.8
46.3
—


Yeast


Budding
38.3
38.8
39.3
39.3
39.8
45.4
—


Yeast





*Comparisons were made with the MEGALIGN program at DNAStar (Madison, WI) using sequences present in Genbank. Abbreviations and accession numbers are as follows: Arabidopsis, A. thaliana Col-0 (this work); Corn, Zea mays (AAD30555); Human, Homo sapiens (NP_002349); Mouse, Mus musculus (AAD09238); Frog, Xenopus laevis, (AAB41527); Fission yeast, Schizosaccharomyces pombe (AAB68597); Budding yeast, Saccharamoyces cerevisiae (P40958). 






Not unexpectedly, the sequence of the Arabidopsis Mad2 protein is more closely related to the corn Mad2, the only plant Mad2 homolog represented in the databases.

The corn Mad2 is about 82% identical to the Arabidopsis Mad2. FIGS. 16A-B show yeast strains containing either pLV24 and pJG4-5, pEG202 and pLV116, or pLV24 and pLV116 on leucine drop-out plates and plates containing X-Gal, showing that only when both HopPsyA and Mad2 are present, β-galactosidase and LEU2 activity are induced. It is important to note that the cDNA library that yielded mad2 has been used for many different yeast 2-hybrid screens and a mad2 clone has never been isolated from it before. Thus, the results shown in FIGS. 16A-B are unlikely to represent an artifact produced by the nature of the cDNA library. Moreover, different Mad2 homologs are known to interact with specific proteins and one of these homologs was isolated with a yeast 2-hybrid screen using a protein of the spindle checkpoint as bait (Kim et al., 1998). This is reassuring for two reasons. First, other Mad2 homologs do not appear to be nonspecifically “sticky” proteins. Second, they appear to modulate cellular processes through protein-protein interactions.
The above results are very promising, because Mad2 is a regulator controlling the transition from metaphase to anaphase during mitosis, a key step in the cell cycle of eukaryotes. The eukaryotic cell cycle is dependent on the completion of earlier events before another phase of the cell cycle can be initiated. For example, before mitosis can occur DNA replication has to be completed. Some of these dependencies in the cell cycle can be relieved by mutations and represent checkpoints that insure the cell cycle is proceeding normally (Hartwell and Weinert, 1989). In pioneering work, Hoyt et al. and Li and Murray independently discovered that there is a checkpoint in place in Saccharomyces cerevisiae to monitor whether the spindle assembly required for chromosome segregation is completed (Hoyt et al., 1991; Li and Murray, 1991). This so-called spindle checkpoint was discovered when the observation was made that wild-type yeast cells plated onto media containing drugs that disrupt microtubule polymerization arrested in mitosis, whereas certain mutants proceeded into anaphase. These initial reports identified 6 different nonessential genes that are involved in the spindle checkpoint: bub1-3 named for budding uninhibited by benzimidazole and mad1-3 for mitotic arrest deficient. Mutations in these genes ignore spindle assembly abnormalities and attempt mitosis regardless. In the years since, the spindle checkpoint has been shown to be conserved in other eukaryotes and many advances have occurred resulting in a better picture of what is taking place at the spindle checkpoint (Glotzer, 1996; Rudner and Murray, 1996).
Required for the transition from metaphase to anaphase (as well as other cell cycle transitions) is the ubiquitin proteolysis pathway. Proteins that inhibit entry into anaphase (e.g., Pds1 in S. cerevisiae) are tagged for degradation via the ubiquitin pathway by the anaphase-promoting complex (APC) (King et al., 1996). Only when these proteins are degraded by the 26S proteosome are the cells allowed to cycle to anaphase. Although it is not well understood how the APC knows when to tag the anaphase inhibitors for degradation, there have been several important advances (Elledge, 1996; Elledge, 1998; Hardwick, 1998). The Mad2 protein and the Bub1 protein kinase have been shown to bind to kinetochores when these regions are not attached to microtubules (Chen et al., 1996; Li and Benezra, 1996; Taylor and McKeon, 1997; Yu et al., 1999). Thus, these proteins appear to somehow relay a signal that all of the chromosomes are not bound to spindle fibers ready to separate. Mad1 encodes a phosphoprotein, which becomes hyperphosphorylated when the spindle checkpoint is activated and the hyperphosphorylation of Mad1 is dependent on functional Bub1, Bub3, and Mad2 proteins (Hardwick and Murray, 1995). Another required protein in this checkpoint is Mps1, a protein kinase that activates the spindle checkpoint when overexpressed in a manner that is dependent on all of the Bub and Mad proteins, indicating that Mps1 acts very early in the spindle checkpoint (Hardwick et al., 1996).
Based on data from the different Mad2 homologs that have been studied, Mad2 appears to have a central role in the spindle checkpoint. Addition of Mad2 to Xenopus egg extracts results in inhibition of cyclin B degradation and mitotic arrest due to the inhibition of the ubiquitin ligase activity of the APC (Li et al., 1997). The overexpression of Mad2 from fission yeast causes mitotic arrest by activating the spindle checkpoint (He et al., 1997). Whereas, introducing anti-Mad2 antibodies into mammalian cell cultures causes early transition to anaphase in the absence of microtubule drugs, indicating that Mad2 is involved in the normal cell cycle. Several reports suggest that different Mad2 homologs directly interact with the APC (Li et al., 1997; Fang et al., 1998; Kallio et al., 1998). Another protein called Cdc20 in S. cerevisiae binds to the APC, is required for activation of the APC during certain cell cycles, and Mad2 binds to it (Hwang et al., 1998; Kim et al., 1998; Lorca et al., 1998; Wassmann and Benezra, 1998). The picture that is emerging from all of these exciting findings is that Mad2 acts as an inhibitor of the APC, probably by binding to Cdc20. When Mad2 is not present, the Cdc20 binds to the APC, which activates the APC to degrade inhibitors of the transition to anaphase. FIG. 12 shows a summary of the spindle checkpoint focusing on Mad2's involvement and using the names of the spindle checkpoint proteins from S. cerevisiae. 
The plant spindle checkpoint: A possible target of bacterial pathogens. Many of the cell cycle proteins from animals have homologs in plants (Mironov et al., 1999). In fact, one of the early clues that there existed a spindle checkpoint was first made in plants. The observation noted was that chromosomes that lagged behind in their attachment to the spindle caused a delay in the transition to anaphase (Bajer and Mole-Bajer, 1956). Moreover, mad2 has been recently isolated from corn and the Mad2 protein localization in plant cells undergoing mitosis is consistent with the localization of Mad2 in other systems (Yu et al., 1999). Based on a published meeting report, genes that encode components of the APC from Arabidopsis have been recently cloned (Inze et al., 1999). Thus, it appears that a functional spindle checkpoint probably is conserved in plants. The data presented above shows that the P. syringae HopPsyA protein interacts with the Arabidopsis Mad2 protein in the yeast 2-hybrid system.
It is possible that a pathogenic strategy of a bacterial plant pathogen is to alter the plant cell cycle. Duan et al. recently reported that pthA, a member of the avrBs3 family of avr genes from X. citri, is expressed in citrus and causes cell enlargement and cell division, which may implicate the plant cell cycle (Duan et al., 1999). If HopPsyA does target Mad2, at least two possible benefits to pathogenicity can be envisioned. Since plant cells in mature leaves are quiescent, one benefit of delivering HopPsyA into these cells may be that it may trigger cell division through its interaction with Mad2. This is consistent with the observation that anti-Mad2 antibodies cause an early onset of anaphase in mammalian cells (Gorbsky et al., 1998). More plant cells near the pathogen may increase the nutrients available in the apoplast. A second possible benefit may occur if HopPsyA is delivered into plant cells actively dividing in young leaves. Delivery of HopPsyA into plant cells of these leaves may derail the spindle checkpoint through its interaction with Mad2. These cells would be prone to more mistakes segregating their chromosomes; in some cells this would result in death and the cellular contents would ultimately leak into the apoplast providing nutrients for the pathogen.
Example 8
Cytotoxic Effects of HopPtoA and HopPsyA Expressed in Yeast
Both hopPtoA (SEQ. ID. No. 6) and hopPsyA (SEQ. ID. No. 35) were first cloned into pFLAG-CTC (Kodak) to generate an in-frame fusion with the FLAG epitope, which permitted monitoring of protein production with anti-FLAG monoclonal antibodies. The FLAG-tagged genes were then cloned under the control of the GALL promoter in the yeast shuttle vector p415GAL1(Mumberg et al., 1994). These regulatable promoters of Saccharomyces cerevisiae allowed comparison of transcriptional activity and heterologous expression. The recombinant plasmids were transformed into uracil auxotrophic yeast strains FY833/4, selecting for growth on SC-Ura (synthetic complete medium lacking uracil) based on the presence of the URA3 gene on the plasmid. The transformants were then streaked onto SC-Ura medium plates containing either 2% galactose (which will induce expression of HopPsyA and HopPtoA) or 2% glucose. No growth was observed on the plates supplemented with 2% galactose. This effect was observed with repeated testing and was not observed with empty vector controls, with four other effectors similarly cloned into p415GAL1, or when raffinose was used instead of galactose. FLAG-tagged nontoxic Avr proteins were used to confirm that the genes were differentially expressed, as expected, on plates containing galactose. Importantly, the toxic effect with HopPsyA was observed when the encoding gene was recloned into p416GALS, which expresses foreign genes at a substantially lower level than p415GAL1.
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Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

## Claims

1. An isolated protein or polypeptide selected from the group consisting of (i) a protein or polypeptide comprising an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 66, and (ii) a protein or polypeptide encoded by a nucleic acid molecule whose complement hybridizes, at a temperature of at least about 37° C. in a medium comprising at most about 0.9M SSC, to a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 65.

2. The isolated protein or polypeptide according to claim 1, wherein the protein or polypeptide comprises an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 66.

3. A composition comprising:
a carrier and 
a protein or polypeptide according to claim 1.

4. The isolated protein or polypeptide according to claim 1, wherein the protein or polypeptide is a recombinant protein or polypeptide.

5. The isolated protein or polypeptide according to claim 1, wherein the protein or polypeptide is at least about 80% pure.

6. The isolated protein or polypeptide according to claim 1, wherein the protein or polypeptide is at least about 90% pure.

7. The isolated protein or polypeptide according to claim 1, wherein the protein or polypeptide is encoded by a nucleic acid molecule whose complement hybridizes, at a temperature of at least about 37° C. in a medium comprising at most about 0.9M SSC, to a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 65.

8. The isolated protein or polypeptide according to claim 1, wherein the protein or polypeptide is encoded by a nucleic acid molecule whose complement hybridizes, at a temperature of at least about 42° C. in a medium comprising at most about 0.9M SSC, to a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 65.

9. The isolated protein or polypeptide according to claim 1, wherein the protein or polypeptide is encoded by a nucleic acid molecule whose complement hybridizes, at a temperature of about 65° C. in a medium comprising at most about 0.9M SSC, to a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 65.

10. The composition according to claim 3, wherein the protein or polypeptide comprises an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 66.

11. The composition according to claim 3, wherein the protein or polypeptide is encoded by a nucleic acid molecule whose complement hybridizes, at a temperature of at least about 37° C. in a medium comprising at most about 0.9M SSC, to a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 65.