# Patent

## Title
Pseudomonas  avr and hop proteins, their encoding nucleic acids, and use thereof

## Abstract

One aspect of the present invention relates to isolated nucleic acid molecules encoding avirulence proteins or polypeptides of Pseudomonas syringae pv. syringae DC 3000, or nucleic acid molecules which are complementary thereto. 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 various nucleic acid molecules and proteins of the present invention can be used to impart disease resistance to a plant, make a plant hypersusceptible to colonization by nonpathogenic bacteria, modify a metabolic pathway in a cell, cause eukaryotic cell death and treat a cancerous condition, as well as inhibit programmed cell death.

## Background
This application claims benefit of U.S. Provisional Patent Application Ser. Nos. 60/356,408, filed Feb. 12, 2002, and 60/380,185, filed May 10, 2002, each of which is hereby incorporated by reference in its entirety.


This work was supported by National Science Foundation Grant Nos. MCB-9982646 and IBN-0096348, National Science Foundation Plant Genome Research Program Cooperative Agreement DBI-0077622, and National Research Initiative Competitive Grants Program, U.S. Department of Agriculture, Grant No. 01-35319-10019. 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 of Pseudomonas syringae pv. tomato DC3000, the isolated avirulence effector proteins and hrp-dependent outer proteins encoded thereby, as well as their various uses.
BACKGROUND OF THE INVENTION
The plant pathogenic bacterium Pseudomonas syringae is noted for its diverse and host-specific interactions with plants. 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 & Collmer, J. Bacteriol. 179:5655–5662 (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 & Collmer, J. Bacteriol. 179:5655–5662 (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, Annu. Rev. Genet. 24:447–463 (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 & Collmer, J. Bacteriol. 179:5655–5662 (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 & Collmer, J. Bacteriol. 179:5655–5662 (1997)).
Virulence effector proteins delivered to or into host cells by type III secretion systems are key factors in the pathogenicity of many bacteria, including animal pathogens in the genera Salmonella, Yersinia, Shigella, and Escherichia, and plant pathogens in the genera Pseudomonas, Erwinia, Xanthomonas, Ralstonia, and Pantoea (Galán & Collmer, Science 284:1322–1328 (1999)). In plant pathogens, the type III secretion machinery is referred to as the hypersensitive response and pathogenicity (Hrp) system because secretion mutants typically lose their ability to elicit the defense-associated hypersensitive response in nonhost plants and to grow parasitically or be pathogenic in host plants (Alfano & Collmer, J. Bacteriol. 179:5655–5662 (1997)). These phenotypes demonstrate the importance of the Hrp system in bacterium-plant interactions, and global identification of effectors will be important for understanding the pathogenesis of bacteria that use type III secretion systems. Unfortunately, several factors have hindered searches for type III effector genes. These factors include: (i) effectors are often redundant with mutants having only subtle phenotypes; (ii) with few exceptions (see e.g., Miao & Miller, Proc. Natl. Acad. Sci. USA 97:7539–7544 (2000)) motifs that can identify proteins as substrates for type III secretion have not been recognized (Lloyd et al., Mol. Microbiol. 39:520–523) (2001); (iii) many effectors show no similarity to known proteins; and (iv) some pathogens have multiple type III secretion systems which deliver different sets of effectors (Cornelis & Van Gijsegem, Annu. Rev. Microbiol. 54:735–774 (2000)). Thus, a complete inventory of type III effector genes is lacking for any pathogen, although it seems that pathogens such as Salmonella may have many such genes (Worley et al., Mol. Microbiol. 36:749–761 (2000)).
Plant pathogen type III effector proteins are mostly designated Avr or Hop, depending on whether their primary phenotype involves plant reaction or secretion behavior. Many effectors were initially discovered through their ability to betray the pathogen to the host R (resistance) gene surveillance system, thereby rendering the pathogen avirulent on a test plant (Keen, Annu. Rev. Genet. 24:447–463 (1990)). Over 25 effector genes have been identified by Avr or Hop phenotypes in various P. syringae pathovars and races (Vivian & Arnold, J. Plant Pathol. 82:163–178 (2000); Alfano et al., Proc. Natl. Acad. Sci. USA 97:4856–4861 (2000)). The encoded effectors seem to determine both basic pathogenicity and host range, but the number of such proteins produced by any single strain has not been systematically investigated. P. s. tomato DC3000 is known to carry at least three avr genes, avrPto (Ronald et al., J. Bacteriol. 174:1604–1611 (1992)), avrPtoB (Kim et al., Cell 109:589–598 (2002)), and avrE (Lorang & Keen, Mol. Plant-Microbe Interact. 8:49–57 (1995)), with the latter being in the Hrp pathogenicity island along with five other candidate effector genes (Alfano et al., Proc. Natl. Acad. Sci. USA 97:4856–486 (2000); Lorang & Keen, Mol. Plant-Microbe Interact. 8:49–57 (1995)).
The present invention is a further advance in the effort to identify, clone, and sequence Avr and Hop proteins or polypeptides from plant pathogens.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to an isolated nucleic acid molecule that includes a nucleotide sequence which (i) encodes a protein or polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, or SEQ ID NO: 209; or (ii) hybridizes, under stringency conditions comprising a hybridization medium which includes 0.9×SSC at a temperature of 42° C., to a DNA molecule complementary to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, OR SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, or SEQ ID NO: 208; or (iii) 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 effector proteins or polypeptides encoded by the nucleic acid molecules of the present invention. Compositions which contain the proteins or polypeptides 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 further aspect of the present invention relates to a method of causing eukaryotic cell death which includes: introducing into a eukaryotic cell a cytotoxic Pseudomonas protein of the present invention, said introducing being performed under conditions effective to cause cell death.
A still further aspect of the present invention relates to a method of treating a cancerous condition which includes introducing a cytotoxic Pseudomonas protein of the present invention into cancer cells of a patient under conditions effective to cause death of cancer cells, thereby treating the cancerous condition.
Yet another aspect of the present invention relates to a method of inhibiting programmed cell death which includes introducing into a eukaryotic cell susceptible to programmed cell death, a protein of the present invention that is a hypersensitive response suppressor, said introducing being performed under conditions effective to inhibit programmed cell death of the eukaryotic cell.
Yet another aspect of the present invention relates to a method of modifying a metabolic pathway in a cell which includes: introducing into a cell a protein or polypeptide of the present invention which interacts with a native cellular protein involved in a metabolic pathway, wherein the protein or polypeptide modifies the metabolic pathway through its interaction with the native cellular protein.
It is believed that bacteria have evolved effector proteins to make exquisite alterations in host metabolism. While plant disease resistance, suppression of programmed cell death, and cancer cell toxicity are important uses, as mentioned above, it is believed that these effector proteins can be used to modify or effect metabolic targets in eukaryotes, including both yeasts and higher order species, such as plants and animals. It is noteworthy that several of the effector proteins disclosed herein have homologs in other phytopathogenic bacteria. Thus, these proteins appear to represent a set of effectors that are conserved among Pseudomonas, Erwinia, Xanthomonas, and Ralstonia spp. By disrupting or augmenting the function of these effectors through, for example, transgenic expression thereof in a host plant, it is believed that use of these effectors may lead to widely applicable means for controlling diseases of plants.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A–C illustrate assays for Hrp system-dependent secretion in culture or translocation in planta of candidate effector proteins. P. s. tomato DC3000 and a Hrp secretion mutant derivative were used for tests of newly identified candidate effectors (1A–B). DC3000 or a DC3000 hrcC mutant (Yuan & He, J. Bacteriol. 178:6399–6402 (1996), which is hereby incorporated by reference in its entirety) carrying test ORFs (i.e., candidate effectors) fused to either the FLAG (F) or hemagglutinin (HA) epitopes were grown in Hrp-inducing media, and cultures were separated into cell (lanes 1–3) and supernatant (lanes 4 and 5) fractions and analyzed by SDS/PAGE and immunobloting. Lanes 1 and 4, wild-type DC3000; lanes 2 and 5, wild-type DC3000 (pTestORF); lanes 3 and 6, DC3000 hrcC mutant (pTestORF). As an additional control against leakage, pCPP2318 was included in all strains, which encodes the mature form of β-lactamase (β-lac). The presence of an epitope-tagged protein in the supernatant fraction of the wild type (lane 5), but absence in the hrcC secretion mutant (lane 6), indicated that the test ORF encoded a secreted product. In FIG. 1C, an AvrRpt2 translocation assay was performed with DC3000 HopPtoG. Test strains were infiltrated into A. thaliana Col-0 (RPS2) and Col-0 rps2-201 (rps2) plants. Plant responses were scored 18 h after inoculation for hypersensitive response (HR) or no visible response (N).
FIGS. 2A–C illustrate pHIR11-dependent HR is suppressed by HopPtoE, and a P. s. tomato DC3000 hopPtoE mutant exhibits an enhanced HR. FIG. 2A is a schematic representation of the pHIR11-based suppression assay in P. fluorescens (Pf) 55. When DC3000 effectors are individually expressed in trans in Pf(pHIR11), they can potentially suppress the HopPsyA-dependent HR. FIG. 2B shows N. tabacum cv. xanthi leaves that were infiltrated with Pf(pHIR11) (left panel) or Pf(pHIR11, phopPtoE)(right panel). ‘N’ denotes no HR. FIG. 2C shows quantitative differences in the ability of DC3000 wild-type (WT), hopPtoE mutant UNL139, and complemented mutant UNL139(phopPtoE) to elicit the HR in N. tabacum cv. xanthi leaves. Different dilutions of bacterial cells/ml (1, 108 cells/ml; 2, 107 cells/ml; 3, 106 cells/ml; and 4, 105 cells/ml) were infiltrated into leaves, then leaves were photographed after 24 hr.
FIGS. 3A–C illustrate that HR suppression is not due to blocking TTSS, and the TTSS is functional. FIG. 3A is an image of an immunoblot showing that AvrPto is type III-secreted from DC3000 (WT), but not from a secretiondefective DC3000 hrcC mutant (hrcC). β-Lactamase (β-Lac) was used as a lysis control. C, cell pellet fractions; S, supernatant fractions. FIG. 3B is an image showing that the HR elicited in N. tabacum cv. xanthi by DC3000 is inhibited when hopPtoE is expressed in trans. FIG. 3C shows the results of P. fluorescens(pHIR11) mixing experiments in N. tabacum cv. Xanthi, demonstrating that HR suppression can occur when HopPtoE and HopPsyA are TTSS-delivered by different bacteria. pLN18 is a pHIR11 derivative that lacks hopPsyA, but encodes a functional TTSS. pCPP2089 (Huang et al., Mol. Plant-Microbe Interact. 4:469–476 (1991), which is hereby incorporated by reference in its entirety) is a pHIR11 derivative encoding a defective TTSS.
FIGS. 4A–C identify P. s. tomato DC3000 effectors that suppress the HR on tobacco and Arabidopsis. FIG. 4A lists DC3000 effectors that were tested in the pHIR11 assay. A ‘y’ indicates that the effector inhibited the HR, an ‘n’ indicates that it did not, and a ‘y*’ indicates that it partially suppressed the HR. Refer to the Materials and Methods for information regarding effector constructs. The nucleic acid and amino acid sequences of AvrPphEPto, AvrPpiB1Pto, HopPtoB, HopPtoC, HopPtoD1, HopPtoD2, HopPtoF (previously designated AvrPphFPto ORF2), HopPtoJ, and HopPtoK are disclosed in U.S. patent application Ser. No. 09/825,414 to Collmer et al., filed Apr. 2, 2002, which is hereby incorporated by reference in its entirety. The nucleic acid and amino acid sequence of AvrPto is reported at Genbank Accession L20425; Salmeron & Staskawicz, Mol. Gen. Genet. 239:6–16 (1993), each of which is hereby incorporated by reference in its entirety. The nucleic acid and amino acid sequence of AvrPtoB is reported at Genbank Accession AY074795 and Kim et al., Cell 109:589–598 (2002), each of which is hereby incorporated by reference in its entirety. AvrPtoB was independently shown to suppress the programmed cell death elicited by AvrPto or by heterologously-expressed BAX in Nicotiana benthamiana (Abramovitch et al., EMBO J. 22:60–69 (2003), which is hereby incorporated by reference in its entirety). FIG. 4B is an image of N. tabacum cv. xanthi leaves that were infiltrated with P. fluorescens(pHIR11) with different effector constructs (noted above each picture). Complete suppression of the HR is denoted with ‘N’. FIG. 4C is an image of the same strains (as illustrated in FIG. 4B) infiltrated into Arabidopsis Ws-0, producing identical results.
FIGS. 5A–B illustrate that the HR elicited by HopPsyA can be suppressed via Agrobacterium transient expression of effectors. In FIG. 5A, N. tabacum cv. xanthi leaves were co-infiltrated with A. tumefaciens C58C1 carrying phopPsyA and another strain carrying each candidate suppressor. All of the suppressive effectors identified in the pHIR11 screen also suppressed the HR elicited by HopPsyA in this test. In FIG. 5B, an immunoblot of plant tissues with different agroinfiltrations shows that each HA epitope-tagged effector was made in planta. The asterisks indicates a protein of the predicted size of the effector in that lane.
FIG. 6 is an image showing that Pseudomonas syringae pv. tomato DC3000 suppressor mutants display an enhanced ability to elicit the HR. N. tabacum cv. xanthi leaves were infiltrated with P. syringae strains that were 10-fold serially diluted from 108 cells/ml. The last dilution (106 cells/ml) that resulted in an HR is shown. In all cases, the mutants exhibit more HR at this dilution than the wild type, and this phenotype was complemented when the suppressors were provided in trans. The following strains were infiltrated: DC3000 wild type, WT; avrPphEPto mutant, UNL113; avrPpiB1Pto mutant, UNL114; avrPtoB mutant, UNL127; hopPtoF mutant, UNL109; hopPtoG mutant, UNL124. HR was scored for each sample: spotty HR (HR−); strong HR (HR+); or no HR.
FIGS. 7A–B illustrate that Pseudomonas syringae pv. tomato DC3000 HR suppressors inhibit the PCD initiated by Bax in plants and yeast. In FIG. 7A, Agrobacterium C58C1 strains carrying binary vectors that encode Bax or a specific effector were co-infiltrated into N. benthamiana leaves. Leaves were photographed after 7 days. N* indicates that the HR was nearly absent. Effector constructs were the same as in FIG. 4. In FIG. 7B, yeast strain EGY48 carrying plasmids that encoded for Bax (pJG4-5-Bax) and a specific effector were spotted on plates at 5-fold dilutions. Expression of Bax was induced by galactose, whereas effector expression was constitutive. Only AvrPpiB1 was unable to suppress Bax-induced killing. Bcl-xL (pGilda-Bcl-xL), an animal protein known to inhibit Bax-induced PCD, was used as a positive control.



DETAILED DESCRIPTION OF THE INVENTION
One aspect of the present invention relates to Pseudomonas syringae pv. syringae DC 3000 nucleic acid molecules which encode Avr or Hop effector proteins.
A first nucleic acid molecule encodes HopPtoI (ORF1) and has a nucleotide sequence according to SEQ ID NO: 1 as follows:








atgcttatcgggcacagcttgcatcacatgcgacccactgctgtggattctagcctaccaacttccgca



 


actagccagactatcagcaataccaaaagtcggctggatccgcatcgtgtccgtgaacttacattcatc


 


ggagtgggtagtagtgttgcctacctactcaatgagcttaatggtcgctttgccgatagcggggtaaca


 


acgccgtttttaggaaaagtcagtattgtaggcaaggacgactcttgggccgagaatgttcgtgggaaa


 


ggttatattaaccaccagactgaaattataagccaatgggaccaacaggttccaaaatatgatcctaac


 


tatgctgctcgtgccgaattttctgcgagtaaccgaagacagttgacgcgaacagtggagttaggcgca


 


gaacatttgaaagcacaggtaacaggcatttcgcgattggatgacggttgttttcgaataaatctggac


 


aatggccagattttgcaaagccgacagattgtactggggactggtgccggaccccataccagtatctgg


 


aacagcgttacatcacacactcaagcagaaaaacgactggacaacatcaaattgcatgagcagaaagcc


 


ttgcgtggcaaggtgctggacctggatgagtttatgcgagcgagtgatgcctctccccagacgtttgct


 


ggaaaaacggtggtgatacatggaccaaatgcaggcattgatgcagctgaacgtgccggggagcttggg


 


gcaaatgcggtttggtttacccgcagtacgaatccggtattgctggatggcaatcaactaaaattcgcg


 


ccagagctggccaaaagcgctatacataaagttgacaaattagatattcgcccaacaaaactagagaat


 


ggtttcgcattgcgactacattacagttcgctaggacaagactcacgggagccaaagaaggtgctagat


 


gcggactattatgtgtacgccatgggtcaagatattcataagccgggtagcgcagcggccatactaggc


 


agtcttcttgaccacctagaacctatatatgactacgatcaagtctatagcgaccagcctttcaagaca


 


gtaataggcttgcaaagtcgcggctccaatagcgataatggtttaattattgtcggggcggcagttgct


 


cagctggccactaatgttcagcatagctataaggaccacgcgttggatcgtatacttgaggaaatgacc


 


aggctccccgaaaagcaaacagaaaagctatcacaaatgctgttagaaggtgcgccatcagtacagatc


 


cagacatatctaaaaacctggcagttagatagcggtcaaccgccagataaacaggtactgcagaatcaa


 


gtagaaaactatctggcggcccgagactacttccagcggcaaaccaacgaacaaaagggcaacctggac


 


ggggttgccgcagaggtaaaaaatcaaaccttaaccgaggttgcatcggtcatcgtgtcaccacagtta


 


ggcacgatcaaggcctccgctgcagcattgtcgggacttatgccagcatatgtggctaacggcgaaaat


 


aactttaccaccgataatcgaactatgctccgtgccggcattgcagcaagatatccgaatataggtaac


 


gctgaagccagtgcatttatcgatgaagtagtaactttgcgtcaccttaatagtcagcgttttattgag


 


aaggtagcaggcgaaatgatggacaaaggagctcaaccactggtgtcgttacgccccccggtcctaggt


 


gtcccggcgtcggtcaggactgcttatgaggcttacttgcacgcgctgaattctggagcgcacgatggt


 


acgccgttaagtcagcgctggctgcccaaaaaatag






The HopPtoI protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 2 as follows:









MLIGHSLHHMRPTAVDSSLPTSATSQTISNTKSRLDPHRVRELTFIGVGSSVAYLLNELNGRFADSGVT



 


TPFLGKVSIVGKDDSWAENVRGKGYINHQTEIISQWDQQVPKYDPNYAARAEFSASNRRQLTRTVELGA


 


EHLKAQVTGISRLDDGCFRINLDNGQILQSRQIVLGTGAGPHTSIWNSVTSHTQAEKRLDNIKLHEQKA


 


LRGKVLDLDEFMRASDASPQTFAGKTVVIHGPNAGIDAAERAGELGANAVWFTRSTNPVLLDGNQLKFA


 


PELAKSAIHKVDKLDIRPTKLENGFALRLHYSSLGQDSREPKKVLDADYYVYAMGQDIHKPGSAAAILG


 


SLLDHLEPIYDYDQVYSDQPFKTVIGLQSRGSNSDNGLIIVGAAVAQLATNVQHSYKDHALDRILEEMT


 


RLPEKQTEKLSQMLLEGAPSVQIQTYLKTWQLDSGQPPDKQVLQNQVENYLAARDYFQRQTNEQKGNLD


 


GVAAEVKNQTLTEVASVIVSPQLGTIKASAAALSGLMPAYVANGENNFTTDNRTMLRAGIAARYPNIGN


 


AEASAFIDEVVTLRHLNSQRFIEKVAGEMMDKGAQPLVSLRPPVLGVPASVRTAYEAYLHALNSGAHDG


 


TPLSQRWLPKK






HopPtoI has been shown to be a protein that is secreted by DC3000.

A second nucleic acid molecule encodes HopPtoH (ORF2) and has a nucleotide sequence according to SEQ ID NO: 3 as follows:








atgatcactccgtctcgatatccaggcatctatatcgcccccctcagtaacgaaccgacagcagctcac



 


acatttaaagaacaagcagaggaagcacttgaccatatcagcgccgcaccctctggcgataagctattg


 


cgaaaaatatccactcttgccagtcaaaaagatagaaaagtcacgctaaaagagattgaaataaataac


 


cagtgttataccgaagctgttctgagcagraggcaactggaaaagtacgaaccagaaaactttaacgag


 


aaccggcacattgcatcacagctatcacgaaaggggacctttaccaaaggtgaaggaagcaacgcgatt


 


attggctggtcaccagacaaagcaagcatacgcttaaatcagaatggctcaccgttacaccttggaatg


 


gataacgacgacaaaatcacgaccctagctcatgagctcgttcatgctcgacatgtgttaggtggcagc


 


tccttagcggatggcggagatcgctataatccacgtacgggatctggcaaagaggaacttagggccgtt


 


ggattagataagtaccgctattcacttacaaaaaaaccgtcagagaactccatccgagctgaacacggc


 


ctgcctctgcgcatgaagtacagggcacatcaatag






The HopPtoH protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 4 as follows:









MITPSRYPGIYIAPLSNEPTAAHTFKEQAEEALDHISAAPSGDKLLRKISTLASQKDRKVTLKEIEINN



 


QCYTEAVLSRRQLEKYEPENFNENRHIASQLSRKGTFTKGEGSNAIIGWSPDKASIRLNQNGSPLHLGM


 


DNDDKITTLAHELVHARHVLGGSSLADGGDRYNPRTGSGKEELRAVGLDKYRYSLTKKPSENSIRAEHG


 


LPLRMKYRAHQ






HopPtoH has been shown to be a protein that is secreted by DC3000. HopPtoH has significant homology (1e-114), as detected by BLAST search, to ORF3 from Pseudomonas syringae pv. pisi avrPpiC2 locus (Arnold et al., Microbiology 147:1171–1182 (2001); GenBank Accession No. CAC16702, each of which is hereby incorporated by reference in its entirety.

A third nucleic acid molecule encodes HopPtoE (ORF3) and has a nucleotide sequence according to SEQ ID NO: 5 as follows:








atgaatagagtttccggtagctcgtcagcgacttggcaggcagtcaacgatcttgtggagcaagtaagc



 


gagagaaccacgttgtctacgacaggttatcagacggcaatgggccgcttgaacaaaccggaaaaatca


 


gatgcggatgcgctgatgactatgaggagggcgcaacagtacacggatagcgcgaagcgaacttatatt


 


tcggaaacgctgatgaatctggcagatttgcagcaaaggaaaatctatcgcaccaacagcgggaacttg


 


cgtggcgcgattgagatgacgcctacgcaactcacagattgcgtacagaagtgccgcgaagaggggttc


 


tccaattgtgacatacaggcgctggaaatcggcttgcaccttcgacataagttaggaatctcagatttc


 


accatctacagcaaccgtaagttaagccataactatgtggtcatccaccccagcaatgcatttccgaaa


 


ggagcgattgtagactcttggacgggacagggcgtggtggagctggacttcaagacccgattgaaattc


 


aagcaccgggaagagaactacgcagtgaacgccaatatgcacgagtggatcgagagatacggccaagcg


 


catgtgattgactga






The HopPtoE protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 6 as follows:









MNRVSGSSSATWQAVNDLVEQVSERTTLSTTGYQTAMGRLNKPEKSDADALMTMRRAQQYTDSAKRTYI



 


SETLMNLADLQQRKIYRTNSGNLRGAIEMTPTQLTDCVQKCREEGFSNCDIQALEIGLHLRHKLGISDF


 


TIYSNRKLSHNYVVIHPSNAFPKGAIVDSWTGQGVVELDFKTRLKFKHREENYAVNANMHEWIERYGQA


 


HVID






HopPtoE has been shown to be a protein that is secreted by DC3000 as well as translocated in planta.

A fourth nucleic acid molecule encodes HopPtoG (ORF4) and has a nucleotide sequence according to SEQ ID NO: 7 as follows:








atgcaaataaagaacagtcatctctattcagcttcaagaatggtgcagaatacttttaatgcctcgcct



 


aagatggaagtaactaatgcaatagcaaaaaataatgaacctgctgcgctgagcgctacgcaaactgca


 


aagacacacgaaggcgattcaaaaggccaatccagcaataactctaaattgcccttccgcgccatgagg


 


tacgctgcataccttgcaggcagcgcctacctctacgataaaactgccaataatttttttctttctacc


 


acttctctgcatgatggcaaaggtggttttaccagcgatgccaggcttaacgatgcacaagataaagcg


 


cgaaagcgctaccaaaacaaccatagcagcactcttgaaaataaaaactcgcttttaagcccgcttagg


 


ctttgcggagagaatcagttcttaacgatgattgattatcgtgcagcaactaagatttacctctccgac


 


ctagttgacacggagcaagcgcacacatcaattctgaagaatattatgtgcctgaaaggtgagcttacc


 


aatgaagaggcaataaaaaaactcaacccggaaaaaacaccaaaagactatgaccttacaaatagcgaa


 


gcctatataagcaagaacaaatattctttgaccggcgttaaaaatgaggagacgggatctactggttat


 


acatctcgttctatcacaaagccatttgtggaaaaaggcctgaaacactttataaaagcgactcatggc


 


gaaaaagctctcacgcccaagcagtgtatggaaactcttgataacttacttcgaaaaagtatcacgctc


 


aacagtgattcccaattcgcagcaggccaggcacttttggttttcagacaggtctatgcgggtgaagac


 


gcttggggggatgcggaacgggtcatattgaaaagccattataatcggggcactgtactccaagatgaa


 


gctgataaaatagaactaagtaggccgttctcagagcaagatttagcaaagaacatgtttaagaggaat


 


accagcattgcagggccagtgctctaccacgcatatatttatatacaagaaaaaatcttcaagctaccc


 


cccgacaaaatagaagatttgaaacataaatcaatggcagacttgaaaaacctgcctttgactcatgtt


 


aagcttagcaattccggtgtgggatttgaagacgcctcagggttaggagactcgtttacagctctcaac


 


gcgacgtcctgtgttaatcacgcaagaataatgagtggtgagcctcccttgtcaaaagatgatgttgtg


 


attctgataggttgcctcaacgccgtatacgacaattcgagcggaataaggcattctctccgcgaaatt


 


gcacgagggtgctttgtgggtgctggttttacggtccaggacggtgacgacttctacaaacagatctgc


 


aaaaacgcctctaagcagttttacaacggctaa






The HopPtoG protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 8 as follows:









MQIKNSHLYSASRMVQNTFNASPKMEVTNAIAKNNEPAALSATQTAKTHEGDSKGQSSNNSKLPFRAMR



 


YAAYLAGSAYLYDKTANNFFLSTTSLHDGKGGFTSDARLNDAQDKARKRYQNNHSSTLENKNSLLSPLR


 


LCGENQFLTMIDYRAATKIYLSDLVDTEQAHTSILKNIMCLKGELTNEEAIKKLNPEKTPKDYDLTNSE


 


AYISKNKYSLTGVKNEETGSTGYTSRSITKPFVEKGLKHFIKATHGEKALTPKQCMETLDNLLRKSITL


 


NSDSQFAAGQALLVFRQVYAGEDAWGDAERVILKSHYNRGTVLQDEADKIELSRPFSEQDLAKNMFKRN


 


TSIAGPVLYHAYIYIQEKIFKLPPDKIEDLKHKSMADLKNLPLTHVKLSNSGVGFEDASGLGDSFTALN


 


ATSCVNHARIMSGEPPLSKDDVVILIGCLNAVYDNSSGIRHSLREIARGCFVGAGFTVQDGDDFYKQIC


 


KNASKQFYNG






HopPtoG has been shown to be a protein that is secreted by DC3000 as well as translocated in planta by recombinant Pseudomonas syringae pv. pisi. Thus, HopPtoG appears to be a Hrp-injected effector protein. HopPtoG has significant homology, as detected by BLAST search (1e-137), to a hypothetical protein of Ralstonia solanacearum (see GenBank Accession No. NP—521884, which is hereby incorporated by reference in its entirety).

A fifth nucleic acid molecule encodes HopPtoS1 (ORF5) and has a nucleotide sequence according to SEQ ID NO: 9 as follows:








atgggtaatatttgtggtacttctggctccaatcatgtgtatagtccgcctattagccctcaacatgca



 


tctggttcgtccacaccagtgcccagtgcttctgggacgatgctttctctcagtcatgaacaaatatta


 


agccagaactatgctagcaatataaaggggaaatatcgcacgaacccccgaaaaggaccatctcctagg


 


ctttctgatacgctgatgaagcaggcgctgtcttcagtgatcacacaagagaaaaagcgacttaaaagt


 


caaccaaagtcaatagcccaagatattcagcctccaaacagcatgatcaaaaatgcacttgatgaaaaa


 


gacagccacccttttggtgattgcttttcagacgatgaatttcttgcgatccatctctatacgagttgt


 


ctttacagaccgatcaaccatcatctgcggtatgccccgaaaaatgatgtcgcgcctgttgtggaggca


 


atgaatagcggtttggccaaacttgctcaataccctgattatcaggtgtctggtcagctgcatagaggc


 


atcaagcaaaagatggatgatggtgaagttatgagtcgcttcaagccgggtaatacttatcgtgatgac


 


gcgttcatgageacatcgactagaatggatgttacagaagaatttacttccgatgtcacgttacatctg


 


cagtcctcatcagccgtcaatataggtcccttttcaaaaaacccatacgaggacgaagcgctcatcccg


 


cccctgacgcctttcaaagtaaccggtctgcacaagcaggacgataggtggcacgtccacttgaacgag


 


atcgcagagagctctgacgagtga






The HopPtoS1 protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 10 as follows:









MGNICGTSGSNHVYSPPISPQHASGSSTPVPSASGTMLSLSHEQILSQNYASNIKGKYRTNPRKGPSPR



 


LSDTLMKQALSSVITQEKKRLKSQPKSIAQDIQPPNSMIKNALDEKDSHPFGDCFSDDEFLAIHLYTSC


 


LYRPINHHLRYAPKNDVAPVVEAMNSGLAKLAQYPDYQVSGQLHRGIKQKMDDGEVMSRFKPGNTYRDD


 


AFMSTSTRMDVTEEFTSDVTLHLQSSSAVNIGPFSKNPYEDEALIPPLTPFKVTGLHKQDDRWHVHLNE


 


IAESSDE






HopPtoS1 has been shown to be a protein that is secreted by DC3000 as well as translocated in planta. HopPtoS1 has significant homology, as detected by BLAST search (1e-5), to a chicken ADP-ribosyltransferases (Tsuchiya, J. Biol. Chem. 269:27451–27457 (1994); GenBank Accession No. P55807, each of which is hereby incorporated by reference in its entirety), as well as significant homology to a type III-secreted ADP-ribosyltransferase from P. aeruginosa (Yahr et al., Mol. Microbiol. 22:991–1003 (1996), which is hereby incorporated by reference in its entirety). Further confirming its similarity to ADP-ribosyltransferases, HopPtoS1 has been determined to possess an ART domain (pfam1129).

A sixth nucleic acid molecule encodes ORF6 and has a nucleotide sequence according to SEQ ID NO: 11 as follows:








atgagcttatcgccgacgctgcaaaagctaactaatatattgggcccgac



 


aaaaaatgccaagcctgtcacagaggctatccagtggcaggaaggcatgg


 


atataacgctgcatgtcagcggcgacagccttaccttactagctaaaatc


 


atagaactgcgtacagaccctaaagacgacattttattgcgcaagctgct


 


tacccatacgtttccgggcctgcgtctgcgccgtggcgcgcttaccatca


 


accctgatggaagtgccctggttttctcttatgaacacgattttcacctt


 


ctggacaaagcccgttttgagagcctgctggccaactttgctgaaacggc


 


gcaggagcttcgagacacagcgacacattttcgttttaactga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 12 as follows:









MSLSPTLQKLTNILGPTKNAKPVTEAIQWQEGMDITLHVSGDSLTLLAKI



 


IELRTDPKDDILLRKLLTHTFPGLRLRRGALTINPDESALVFSYEHDFHL


 


LDKARFEsLLANFAETAQELRDTATHFRFN






Although the protein of SEQ ID NO: 12 possesses N-terminal Hop features and features shared by type III chaperones, this protein was shown not to be secreted by DC3000. Because ORF6 is located directly upstream of ORF17 (described infra), it is believed that the protein of SEQ ID NO: 12 is a type III chaperone for the protein encoded by ORF17.

A seventh nucleic acid molecule encodes ORF7 and has a nucleotide sequence according to SEQ ID NO: 13 as follows:








atgaaacaacgagcgacagtcatctgcaaacgtgacggccaggtgcttta



 


cgtacgcaaaccaaaatcccgctgggctttgccaggtggcaagattgaag


 


ccggggaaacgcctttccaggctgccgtgcgcgagctttgcgaagaaacc


 


ggtctggaaaatctcgatctgttgtacctggcggtgtacgagaaaggtga


 


ggtcacgcactacgtgttcaccactcaggttcctgcctacagcgagcctt


 


cgccccagaacgagatttctgcctgcaaatggcttgcgcccaaaaatctt


 


ggcgaccttaaggccagcagcgcgaccaaggctatcgtcaagtcgtatgg


 


ccgccaggctgaagacggtttactcagcgctaactag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 14 as follows:









MKQRATVICKRDGQVLYVRKPKSRWALPGGKIEAGETPFQAAVRELCEET



 


GLENLDLLYLAVYEKGEVTHYVFTTQVPAYSEPSPQNEISACKWLAPKNL


 


GDLKASSATKAIVKSYGRQAEDGLLSAN






This protein shares significant homology, as detected by BLAST analysis (3e-7), to MutT mutator of Mesorhizobium loti (Genbank Accession No. NP—104556, which is hereby incorporated by reference in its entirety). The protein of SEQ ID NO: 12 was shown not to be secreted by DC3000. Although this protein is not secreted, it may still be an effector protein, because AvrB similarly is not secreted in culture although it is translocated in planta (see van Dijk et al., J. Bacteriol. 181:4790–4797 (1999); Gopalan et al., Plant Cell 8:1095–1105 (1996), each of which is hereby incorporated by reference in its entirety).

An eighth nucleic acid molecule encodes ORF8 and has a nucleotide sequence according to SEQ ID NO: 15 as follows:








gtgctcgcttttgcatacgtcagcctgattagagagcagaaattggacatcaaaaaacgttggccttcc



 


agtgagcaggagttggtagaagtccgacggtttaacaaaaccctcgcccggctgccgcgtttccaggtt


 


cgcaatcgcctcacgccccgcttgattcdggcgctgctgcgggcggctcagattggtcgcgcgttgaaa


 


ccggtcaaacatgacctgcggattgaaacaaccatcgtcagcaccggtaacgtccctgtttcagtgcga


 


atcataaggcccaaaggcaaacccaaaggcgtggtgtttgatattcacggcggcggttgggtgatcggc


 


aacgcccagatgaacgatgacctcaatatcggtatcgttaacgcgtgcaacgtggcggtcgtgtccgtt


 


gattacagattggctttatcgacccccgtcgaagggctgatggatgactgcttttctgccgcatgctgg


 


ctgctgggtagcgactgtaaggagtttgccggcctgccggttattgtcgtcggtgagtccgcgggcggg


 


catcttgccgcagccactttgctcaaattgaaagccaggcccgacttgctcaagcgcgtagtcggcacg


 


gttctgtattacggcgtgtacgacctgaccgggacaaaaagcgttcgtaccgcaggcccggaaacgctg


 


gtgctcgacggcccgggcatggtcggcgcaatgcgcttgctcgccccggacagaaccgacgagaagcgc


 


cgcgagccgccgttatcgcccttgtatggcgacctcacggatctgccgcccgccctgatgtttgtcggc


 


gaactcgacccgctgctggacgacacgctggaaatggccgagcgatggaaaaactcggcagacgttgaa


 


atgcatcttctgcccgagtctccacatgggttcatccacttcccgactgccttggcgcgcaaggtactt


 


gcgcgcagccacgagtggataaacgcgaggatggaaggacggccttaa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 16 as follows:









VLAFAYVSLIREQKLDIKKRWPSSEQELVEVRRFNKTLARLPRFQVRNRL



 


TPRLIQALLRAAQIGRALKPVKHDLRIETTIVSTGNVPVSVRIIRPKGKP


 


KGVVFDIHGGGWVIGNAQMNDDLNIGIVNACNVAVVSVDYRLALSTPVEG


 


LMDDCESAAcWLLGSDCKEFAGLPVIVVGESAGGHLAAATLLKLKARPDL


 


LKRVVGTVLYYGVYDLTGTKSVRTAGPETLVLDGPGMVGAMRLLAPDRTD


 


EKRREPPLSPLYGDLTDLPPALMFVGELDPLLDDTLEMAERWKNSADVEM


 


HLLPESPHGFIHFPTALARKVLARSHEWINARMEGRP






This protein shares significant homology, as detected by BLAST analysis (1e-12), to a putative esterase/lipase of Mesorhizobium loti (Genbank Accession No. NP—105776, which is hereby incorporated by reference in its entirety). The protein of SEQ ID NO: 16 was shown not to be secreted by DC3000. Although this protein is not secreted, it may still be an effector protein, because AvrB similarly is not secreted in culture although it is translocated in planta (see van Dijk et al., J. Bacteriol. 181:4790–4797 (1999); Gopalan et al., Plant Cell 8:1095–1105 (1996), each of which is hereby incorporated by reference in its entirety).

A ninth nucleic acid molecule encodes ORF9 and has a nucleotide sequence according to SEQ ID NO: 17 as follows:








atgcaaacctatataccctatccaaaaaaccctcccaccgttggtacagttctgctgacttcctatggc



 


tcattcgcccatgaaaacgagatacctaaatcttgtgctgccgacgctttaagagtaggcaaagagctc


 


gctgatggtttcgatggcgaggttcatcatctaggcgctctgatgctgatgatttccgactttccagca


 


gagccgctgctgaaagcatctgctgctaagaaaggttctttgctaggaattacttcgcttggctaccta


 


ttatcctatggatctactggtgaaaaagcgaagcgaatcatcgaagcaggttgtggtatttttctcgtc


 


agagtgagtggtgatattgaaaaccctaaagcaaaaattgaagtttatagctcttggtctgaataccag


 


aagttccttgaacccattttgaagacaggtgacttttatccagtgaaaacgtcgtcgttttccgaataa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 18 as follows:









MQTYIPYPKNPPTVGTVLLTSYGSFAHENETPKSCAADALRVGKELADGF



 


DGEVHHLGALMLMISDFPAEPLLKASAAKKGSLLGITSLGYLLSYGSTGE


 


KAKRIIEAGCGIFLVRVSGDIENPKAKIEVYSSWSEYQKFLEPILKTGDF


 


YPVKTSSFSE






This protein shares significant homology, as detected by BLAST analysis (3e-50), to ORF4 of Pseudomonas serioboryae (Genbank Accession No. BAA87063, which is hereby incorporated by reference in its entirety).

A tenth nucleic acid molecule encodes ORF10 and has a nucleotide sequence according to SEQ ID NO: 19 as follows:








atgatcaacctcacccacattgcgtcttcattggcgcgggcagcgctcagcgattcgacaaagccgaag



 


atggagcgcgcgataaacgtcgcgagccacatcgctggcaaagtcgcgttgcaggtcaccagctcatta


 


ctggagcagaaaggtctgcttaacgagcgtcagcagaaagggctctcgatgattctgaaggccttgagc


 


ggcaaggagccggtgaacaatgtcgagacgcacgaagggggaggccgattcaatctggcgcgagccgcc


 


ttcgacgtggccagcgttgtctgggagcgcgacaagtcgatgcataacgtgatgagctttctgggcgtc


 


agcgacagcaagggcaagatgttgttctctctgggcaagaagctggcggatgcaatggccaagcctgag


 


cctggcaaggacaacagtgaggccacaaatgcgcgccatgcctatttctccagcaacttgaaactgaac


 


aagttgatgaacgacctcactgaccaggttttcaacaagattcgccagtcgaacggtgatcgcgtgcga


 


cgacccatgccagaaccattctggagaccttacggcgcccaacagcaagcgcgcccgcaaacgcctccc


 


ggcactcgcccacaagccaacagcgccccgccaccgccgccgaaagcagagccacgacctgcgtcgggc


 


cggcctgacggcgcccaacagcaggcgcgcccggaaacgccgcctcgtactcgaccgcaggccaatagc


 


actccgccaccgccgccgaaagcagagccacgacctgcgtcgggccggcctgacggcgcccagcagcaa


 


gcacgcccggaaacgccgccgcgcactcgcccgcaggcgaacagcacgccgccaccgccgcccaaggca


 


gagccacgacctgcgtccggccggcctgacggcgcccaacagcaagcacgcccggaaacgccacctcgc


 


actcgcccccaagcgaacagcgcgccgcctccgccgcccaaagcagagccacgacctgcgtccggccgg


 


cctgacggcacccaacagcaagcacgcccggaaacgccacctcgcactcgcccccaagcgaacagcgcg


 


ccgcctccgccgcccaaagcagaacccagcgcaggcggcgaacggccttcaacggcgcggcccaataac


 


acatcggctgctgacgcatctgccagggtgggcgattccgcacctgccaagccgcccgtcaagccgttg


 


tacgagcacttgggcctcactgacatgtcggtagacttatccgccgttaaaaaggcttacagagatgcc


 


gcgatgaagaaccaccctgataaaaaccgcggcaacgaggccgaggcggccgagcgcttcaaagtcatt


 


tcaaatgcgtacaagattttgtccgacccggagttgcgcaaagcatacgacaacggccgtatcaatgag


 


gctggtaatagggcatga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 20 as follows:









MINLTHIASSLARAALSDSTKPKMERAINVASHIAGKVALQVTSSLLEQKGLLNERQQKGLSMILKALS



 


GKEPVNNVETHEGGGRFNLAPAAFDVASVVWERDKSMHNVMSFLGVSDSKGKMLFSLGKKLADAMAKPE


 


PGKDNSEATNARHAYFSSNLKLNKLMNDLTDQVFNKIRQSNGDRVRRPMPEPFWRPYGAQQQARPQTPP


 


GTRPQANSAPPPPPKAEPRPASGRPDGAQQQARPETPPRTRPQANSTPPPPPKAEPRPASGRPDGAQQQ


 


ARPETPPRTRPQANSTPPPPPKAEPRPASGRPDGAQQQARPETPPRTRPQANSAPPPPPKAEPRPASGR


 


PDGTQQQARPETPPRTRPQANSAPPPPPKAEPSAGGERPSTARPNNTSAADASARVGDSAPAKPPVKPL


 


YEHLGLTDMSVDLSAVKKAYRDAAMKNHPDKNRGNEAEAAERFKVISNAYKILSDPELRKAYDNGRINE


 


AGNRA.






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted, and shares significant homology, as detected by BLAST analysis (2e-11), to DnaJ protein (Genbank Accession No. BAB17689, which is hereby incorporated by reference in its entirety).

An eleventh nucleic acid molecule encodes ORF11 and has a nucleotide sequence according to SEQ ID NO: 21 as follows:








atgaacattacgccgctcacgtcagccgcgggcaagggctcgtccgcacaaggcacagacaaaatttcc



 


attcccaactccacgcgcatgatcaatgccgcttcaatcaagtggttgaataaggtgcgtagcgccatc


 


agtgaccacatccgcaccagcatcgagaaagggaaactgttcgagctcgcctccttgggcagcaacatg


 


ttcggtgtcccggctctttcagcgcgcccctcgacgctccaacctgtgttggcgtttgaggctgacccc


 


aatcacgacctgaaccttgtcagggtctatatgcaggacagcgccggaaagctcactccctgggacccg


 


acgcccaacgcggtcacgacgacgtcgaatccatcagagcctgatgcgcagagcgatacggcttcgtca


 


tcattacctcggcggcctcccgcaggctcggtgctgagtttgctgggcattgcgctggatcacgcgcaa


 


cgccacagtcctcgcgcggacaggtctgccaagggacgacctggccgagaggagaggaacggggcaagg


 


ttcaatgccaagcaaacaaagccgacagaggctgaagcctacggtgatcatcagacacccaatcctgat


 


ttgcacaggcaaaaagagacagctcaacgcgttgctgaaagcatcaacagcatgcgagagcagcaaaat


 


ggaatgcaacgcgccgaagggcttctcagagccaaagaagcgttgcaagctcgggaagccgcgcgcaag


 


cagcttctggacgtgctcgaggccatccaggctggccgtgaagactccaccgacaagaagatcagcgcc


 


actgaaaagaacgccacgggcatcaactaccagtga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 22 as follows:









MNITPLTSAAGKGSSAQGTDKISIPNSTRMINAASIKWLNKVRSAISDHIRTSIEKGKLFELASLGSNM



 


FGVPALSARPSTLQPVLAFEADPNHDLNLVRVYMQDSAGKLTPWDPTPNAVTTTSNPSEPDAQSDTASS


 


SLPRRPPAGSVLSLLGIALDHAQRHSPRADRSAKGRPGREERNGARFNAKQTKPTEAEAYGDHQTPNPD


 


LHRQKETAQRVAESINSMREQQNGMQRAEGLLRAKEALQAREAARKQLLDVLEAIQAGREDSTDKKISA


 


TEKNATGINYQ






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted, and has significant homology, as detected by BLAST analysis (5e-7), to a HrpA-like protein (Genbank Accession No. AAB00126.1, which is hereby incorporated by reference in its entirety).

A twelfth nucleic acid molecule encodes ORF13 and has a nucleotide sequence according to SEQ ID NO: 23 as follows:








atgcgcacatccgttaatggtctgcttgagcacagcctgaagaccctgggctttgatacttcggcattg



 


caggccttgcgcgacgacggttatttactgtggcaaggcaaggataagcaagccagtcttctggttccc


 


tctactgacggcgacgcgcttttcgctatctgtaccttgagccgtgtcgatcccgagcacgacggacgt


 


ctgctggcgcttgcattgcacctgaacctgtctcctgtccacacgatgagcgcatgtatagcdcttgat


 


gtcgagcaaaacacgttgtgtcttcgctacacccatgaccttggcgggaacggggcagataccctgttg


 


cttgcgctcgaaaacgcccaagcgcttgctgaacagatcaagcaggtaatcgaaaactttaggcacgat


 


cagggacgccgatag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 24 as follows:









MRTSVNGLLEHSLKTLGFDTSALQALRDDGYLLWQGKDKQASLLVPSTDGDALEAICTLSRVDPEHDGR



 


LLALALHLNLSPVHTMSACIALDVEQNTLCLRYTHDLGGNGADTLLLALENAQALAEQIKQVIENFRHD


 


QGRR






Because ORF13 shares features common to type III chaperones and is located directly upstream of hopPtoS1 (ORF5), it is believed that the protein of SEQ ID NO: 24 is a type III chaperone for HopPtoS1.

A thirteenth nucleic acid molecule encodes ORF14 and has a nucleotide sequence according to SEQ ID NO: 25 as follows:








atgatcgcgttcgcaaccggactgctagaacacagcctgaaacggcttggatacgacgccgcagatttg



 


caatcccttcgggatgaagggtatttgctgtggcacgggaaaaacggtcacaccagcctgttggtgccc


 


gctgctggcgggatgcgctttttgtcatcagcaccctgagctacatcgatcctgaacaggacgggcgg


 


ctgctggcgcttgcgctgcatttgaacttgtcgccagcccacactctgggcgccagtatcgcgctggat


 


atcgagcaaaataccttgtgcctgcgttacacgcacgacctcactgggcacggcacagacaatttgtcc


 


cgcgcgcttgaaagcactcaggcacttgccgagcagatcaagcaggtcatcgaaacttccgcagtgaa


 


ttcggacgcccgccaatgcccgcccacacagcccgacggccagatgccctggcgctttag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 26 as follows:









MIAFATGLLEHSLKRLGYDAADLQSLRDEGYLLWHGKNGHTSLLVPAAGGDALFVISTLSYIDPEQDGR



 


LLALALHLNLSPAHTLGASIALDIEQNTLCLRYTHDLTGHGTDNLSRALESTQALAEQIKQVIETFRSE


 


FGRPPMPAHTARRPDAVAL






ORF14 shares features common to type III chaperones and shares weak similarity with ORF8 of the DC3000 Conserved Effector Locus (“CEL”) (U.S. patent application Ser. No. 09/825,414 to Collmer et al., filed Apr. 3, 2001, which is hereby incorporated by reference in its entirety), which is a candidate chaperone for the protein encoded by CEL ORF7. Thus, the protein of SEQ ID NO: 26 is likely a chaperone for the protein of SEQ ID NO: 28.

A fourteenth nucleic acid molecule encodes ORF15 and has a nucleotide sequence according to SEQ ID NO: 27 as follows:








gtgaaaaagtctggcgctggaactcaagcctatgcgttgttcgcctctgcgacgggaagctcgtcgaag



 


ggcgttctaagtaccattgccaggcacctgacgggatgttttgcacccaacaagactgcgcttcattca


 


gcaacagccgtttcgtatgagctattgccgggcaattattctgtcgccgccagtgtgcatggcttgtcg


 


gttgatcaccgccagccggcgctgacacgactgagtaacgtgctgttcaatcaggcactggcgctggac


 


ctggagcgttttgacgagggcgcgccagccgacgaaatgttcaggccttcactgaaacgcgaacgtgcc


 


catccccgattggccgactcactgggtggcgagcaactggctgtgcaaaccatggagaagggccttaaa


 


cggctggcagaggatcctgcgcagtcctttgcgcgatgccattcatttttttacccgatcagtagtgat


 


accacttcacctcaagcatcacttcattctgtggcgagctcatctggctga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 28 as follows:









VKKSGAGTQAYALFASATGSSSKGVLSTIARHLTGCFAPNKTALHSATAVSYELLPGNYSVAASVHGLS



 


VDHRQPALTRLSNVLFNQALALDLERFDEGAPADEMFRPSLKREGAHPRLADSLGGEQLAVQTMEKGLK


 


RLAEDPAQSFARCHSFFYPISSDTTSPQASLHSVASSSG






The protein of SEQ ID NO: 28 does not share all of the N-terminal features associated with known Hops, however, it is 34% identical with the product of ORF 26, which does.

A fifteenth nucleic acid molecule encodes HopPtoT1 (ORF16) and has a nucleotide sequence according to SEQ ID NO: 29 as follows:








atgaaaacagtcagcaatcactcgatacccagtacaaatctcgtcgtggatgcgggaacggaaacttcg



 


gcgcagaaatcccagccggtttgcagcgaaatccagcgtaacagcaagatcgaaaaagcagtcatcgaa


 


cacattgccgaccacccggcagcgaaaatgacaataagcgcgctggttgacacgttgacagacgttttt


 


gtcagggctcatggggaggttaaggggtgggccgaaatcgtccaggcagtctctcgccctcatgacagt


 


aatcgacacggcagtggagtgctcagcccgcgctttgatgtaatggggagtgttggttggaatgcggca


 


gctatccgggccaccagtcgcgtcgggacgcttcgagagaaaggtacactgttcactaaccttatgctc


 


agtaacaactttaaacatttgcttaaacgagtggttaacgatccagccttgcagcaaaagctcgacggt


 


gggttagacctcaactatctgaaggcttgtgaaggcgatctttatgtcdtgtcagggtgggctgcacgg


 


gctagcgaaagtcgtgaacaaattggcaaagcccggtatgaaacggcatcaaatcttagccagacgctg


 


atcagtgcacgtgagttggcttttcatcgtcacaatccggttaatcatccgtctgcccaaacgaaagtg


 


ggcttcgataagggtttgcctgaggaatctgatctgcaggttctgagaggccatggcagcagtgtatgg


 


agtgtaaaaccgggcagcgatttcgcaaagcgtgctgaagtttctggaaagcctattatcgccggcccg


 


tccggtaccgcttcgcgcatggtcgctgttgcgcgttttctggcaccggcttgtttgaaaagcctgggt


 


attgagagtgagcagaacctgaaagagcttgtgcggtatgcctgctatgcctatttcggtcaggacagc


 


caccattcgatgcttgaagtgaatcttggtgtcgcttcccatggaatgccggaacaatgggacgacacg


 


ctttataacgagcctttcagtaattcaattaaaggtcgcgggtttggtatagacaatctcgcgcatagg


 


caagtcgtcaggcaggcggctcaaaagtcatga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 30 as follows:









MKTVSNHSIPSTNLVVDAGTETSAQKSQPVCSEIQRNSKIEKAVIEHIADHPAAKMTISALVDTLTDVF



 


VRAHGFVKGWAEIVQAVSRPHDSNRHGSGVLSPRFDVMGSVGWNAAAIRATSRVGTLREKGTLFTNLML


 


SNNFKHLLKRVVNDPALQQKLDGGLDLNYLKACEGDLYVMSGWAARASESREQIGKARYETASNLSQTL


 


ISARELAFHRHNPVNHPSAQTKVGFDKGLPEESDLQVLRGHGSSVWSVKPGSDFAKRAEVSGKPIIAGP


 


SGTASRMVAVARFLAPAcLKSLGIESEQNLKELVRYACYAYEGQDSHHSMLEVNLGVASHGMPEQWDDT


 


LYNEPFSNSIKGRGFGIDNLAHRQVVRQAAQKS






HopPtoT1 has been shown to be translocated by DC3000 in planta.

A sixteenth nucleic acid molecule encodes ORF17 and has a nucleotide sequence according to SEQ ID NO: 31 as follows:








atgcggtttgatgctgcccgaggccagaagcccaaagcccctatggatgcaccgtcatcattacgtttg



 


cgagcgatagcaggtggcatgcccagtgaagaagcaggaacgactgcacctgctgacgtgaatcagcct


 


ccacctgctgatgttcgtccagaaatgggtgtaggtcctgtgagactcttcgttaaactgatggtagga


 


actctggcgctgtcgacaggagtccgttttgcaagatacccaggtgatttcgcgaaggatccgggaggc


 


agtgtatgggcagcaatcaatctgcagcatcgctcgagcgtcacacatcttgaacaaggcaataagacg


 


gttcttgagcgtttcggtgcacatattccaaaagacagtgcgtgtttcaaagctcgcgctgacgtcaca


 


cacgatgttccctcaggcgtggcagggcagtggaaccacaaaacccaacgggtaaaactgaaccctaac


 


attcatttcgagagccatccggcacaggtcgccggacatgagttcatacactgttacacgcatcctgag


 


tttgtcgaacgccatataaaacatccgcactggaaagccctgaacgaagggttgacgactcgtttgaca


 


gagaaactgccagaccctaagcgtctcttgcccattcccttggcaaaggatccctatcatggtttcaag


 


ctgtccaccggggactcctggccggatgcggccaggcgaatcgaagacgaagttggcgaagatgtgttg


 


ttgaaagcgttctttggcggcgatgaccaggctattagtgaagtagctaaagccgctgctcagatctac


 


cccaagattgcctcacgtattaccgagagggagttgtatcaagcgggcagcatgcgtggaggacaacag


 


ctggccgagtgttacgtaggtgctttgctcaaaaacggtcagaaactgcctgacagttttacgaattat


 


ctgctacctgtatttagctattcagatataagccctggtcacgcgaaaaaaatacaggcgcaagcggaa


 


aaaagtcaaaagcggatgggaattgtgttcgatacagcgtttttttcacctgacctgaagacccagaga


 


ctggcacttggcatgctacgggaggacctgctgatgcactggaaaaaagttattccggatagaaagtaa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 32 as follows:









MRFDAARGQKPKAPMDAPSSLRLRAIAGGMPSEEAGTTAPADVNQPPPADVRPEMGVGPVRLFVKLMVG



 


TLALSTGVRFARYPGDFAKDPGGSVWAAINLQHRSSVTHLEQGNKTVLERFGAHIPKDSACFKARADVT


 


HDVPSGVAGQWNHKTQRVKLNPNIHFESHPAQVAGHEFIHCYTHPEFVERHIKHPHWKALNEGLTTRLT


 


EKLPDPKRLLPIPLAKDPYHGFKLSTGDSWPDAARRIEDEVGEDVLLKAFFGGDDQAISEVAKAAAQIY


 


PKIASRITERELYQAGSMRGGQQLAECYVGALLKNGQKLPDSFTNYLLPVFSYSDISPGHAKKIQAQAE


 


KSQKRMGIVFDTAFFSPDLKTQRLALGMLREDLLMHWKKVIPDRK






While the protein of SEQ ID NO: 32 does not possess several N-terminal features associated with known Hops, ORF17 is preceded by a good candidate chaperone protein, encoded by ORF6. Furthermore, the protein of SEQ ID NO: 32 has been shown to be translocated by DC3000 in planta.

A seventeenth nucleic acid molecule encodes ORF18 and has a nucleotide sequence according to SEQ ID NO: 33 as follows:








atgaacaggcttcacaagaccagtctgctggcggctatattgaccgcatccccctgcattatggcagct



 


aacgctcatgctatgagttgtcctgtcccgcaaagcgtgaagtacgttaatggtatctatatcgcgccg


 


gaaacgtttgctggttgggaggggaactgggtttctcaaccacacaagaaacactccattaaagagttt


 


tccactgctttatatctttcagtggataaaagtcagaagggaggaacattgactaactgtagttattca


 


ctaagcggagataatggcgtaatagatcttgagtatcgaaaatcaggaaatgagaatagactaaagaca


 


cttatcgtttccattgaaggtcagcacaattggattaaagagcgtggcgcggttggaattcaaggatat


 


gaatgtacaaagtcagcatctgagtgtcagttcgttccgctgcggctaaacgaggactga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 34 as follows:









MNRLHKTSLLAAILTASPCIMAANAHAMSCPVPQSVKYVNGIYIAPETFA



 


GWEGNWVSQPHKKHSIKEFSTALYLSVDKSQKGGTLTNCSYSLSGDNGVI


 


DLEYRKSGNENRLKTLIVSIEGQHNWIKERGAVGIQGYECTKSASECQFV


 


PLRLNED






This protein has significant homology, as detected by BLAST analysis (1e-6), to a putative Yersinia pestis exported protein (Genbank Accession No. NP—406993, which is hereby incorporated by reference in its entirety).

An eighteenth nucleic acid molecule encodes ORF19 and has a nucleotide sequence according to SEQ ID NO: 35 as follows:








atgcatcgtcctatcaccgcaggccataccacctcacgtctcatcctagatcagtcaaaacaaatatca



 


cgtaccccatcggaaagtagcgcgcaatcagcgctttctcagcaagcaagcatgagcagcccagttttg


 


gagcggtcgaaaagtgcgccagctttattgactgcggcacagcgcacgatgcttgcacaagtgggagcc


 


tgtaacgctcatctgacctcagatgaaaacatggccatcaacgaactgacatcacacaagcccctttta


 


cctaaggatacgtggtttttcactgatcctaacaaggacccagatgatgtcgtgacctacaccttgggc


 


aagcaattgcaggctgagggctttgtgcacatcacggatgtagtggcgacactgggtgatgctgaagtt


 


cgctctcaacgtgccgagatggccaaaggcgtgttcaacaagcttgagttgcatgacgtgcatgtgtcg


 


cgtggtcgggattacgcaatgaattcgcttcagtcgaaggaacatgccaaatttttactggaaggtcat


 


gctttaagggctggacctggtgaaatacaccgcgacagcttgcaggacatgagcaggcgcctggcccgt


 


gcgccacatggagtcggtattgtcgtaattgcaggcatgagtgatatcaatgcgctcatcactacctgc


 


ccggatatggtgcgcgaacgggttgatgacatcaccatcatgggcggcgtcgagcctttaaaggacgca


 


gatggttttgtacagcctgatgcacgcgcttacaacaatgcgaccgacatggacgctgcgcgcagtctt


 


tatcggaaagcgcaggagcttggcattccacttcgtatagtgacaaaggaggcggcctataaaacggcg


 


gtttcgccttcattttacgaagggatagcggggagcggacatccagtaggccactacctgagagacgtt


 


cagaagagtgcgttgaaaggcctctgggaaggtattcaagctggattgcttcccgggttggatgactca


 


tggttctttcggacgttcatgccgaatgcacagattgaagcagcacaactggataaaaataaagagagt


 


tcgtttgaagatatctggcctaaggtgacgaagctaaacctgtatgatcctctgacattactggcctca


 


gtgccaggggcggcaaaactgctatttaaaccaaaagctatacacacagaaggatttggtgttgtagag


 


caagtaggtccagatgatgtgacgcatccagagaaagcaaagttattgatgtccgctttagccaaatct


 


gcgcttgtccagtcgacggtagccccagattga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 36 as follows:









MHRPITAGHTTSRLILDQSKQISRTPSESSAQSALSQQASMSSPVLERSKSAPALLTAAQRTMLAQVGA



 


CNAHLTSDENMAINELRSHKPLLPKDTWFFTDPNKDPDDVVTYTLGKQLQAEGFVHITDVVATLGDAEV


 


RSQRAEMAKGVFNKLELHDVHVSRGRDYAMNSLQSKEHAKFLLEGHALRAGPGEIHRDSLQDMSRRLAR


 


APHGVGIVVIAGMSDINALITTCPDMVRERVDDITIMGGVEPLKDADGFVQPDARAYNNATDMDAARSL


 


YRKAQELGIPLRIVTKEAAYKTAVSPSFYEGIAGSGHPVGHYLRDVQKSALKGLWEGIQAGLLPGLDDS


 


WFFRTFMPNAQIEAAQLDKNKESSFEDIWPKVTKLNLYDPLTLLASVPGAAKLLFKPKAIHTEGFGVVE


 


QVGPDDVTHPEKAKLLMSALAKSALVQSTVAPD






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted, and has significant homology, as detected by BLAST analysis (2e-92), to a putative protein of Ralstonia solacearum (Genbank Accession No. NP—518366, which is hereby incorporated by reference in its entirety). Furthermore, the protein of SEQ ID NO: 36 has been shown to be translocated by DC3000 in planta.

A nineteenth nucleic acid molecule encodes ORF20 and has a nucleotide sequence according to SEQ ID NO: 37 as follows:








gtgaaaatcaatctccccgcgctcagaacaacgtcttcacgcgtgcagat



 


ctgcttgaccgcagtcctgctgtgcacaccgctgctgttttccgcgcatg


 


cccaggcagccggcacggcttctgaacaagccaatgtggaagtgatgatt


 


cgtcagctcaacgcgctcgaggccgtcgcccagcgcagtgtcgatcttcc


 


acaagacccggcccaacgctatcacctggactatccccggttggtcagcg


 


acatcgcgcgcatccgccagggcttgcaagactacctgtcgccgtcccgc


 


gcacagccccgcgaccccgtggagctatcaggccattacaacgtcagcgg


 


tgatcacacgccatga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 38 as follows:









VKINLPALRTTSSRVQICLTAVLLCTPLLFSAHAQAAGTASEQANVEVMI



 


RQLNALEAVAQRSVDLPQDPAQRYHLDYPRLVSDIARIRQGLQDYLSPSR


 


AQPRDPVELSGHYNVSGDHTP






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A twentieth nucleic acid molecule encodes ORF21 and has a nucleotide sequence according to SEQ ID NO: 39 as follows:








atgcgttccagggttataactacatcgctggtagtcattatgctctcatgtgcatcagccgctccagct



 


tgcttctccgcagacatgactcccagcgtgtcgaacgagagcacgtcggaggcggattttcagcaatgg


 


ctggctactttccgcagcaatgcaactactaagggcatcgacacagccacactcgatcttgctttccaa


 


aacatcacgcttgacccgactgtgcaccagttggatatggcgcaaccagagttcacgacggccatctgg


 


gattatttgtctgaacgtctgactccgaagaatatccagcaagggcaggagcttctgcaaaaagagcca


 


gttctgaacgaggtagagcgtcactacggtgtggatgcgaagattatcgcggccatctggtgtattgaa


 


agcggctacggtaaggatattggtagtcgcgatgtgattcgttccttggccacgcttgcttacaagggc


 


cggcggatggattacggggctacacagttgatggccgcccttcatatcgtgcaaaacaaagacatcgcc


 


cgtgcgcaattgattggctcgtgggctggcgcgatggggcagacgcaattcatcccgacgacctatctc


 


gactatgcagttgattttaaccacgacaatcggcgcgacgtttggagttcccgggccgatgcgctggcc


 


tccactgcctcttatttacaacgcagcgcttggaactcgcgcgtctcttggggacaggaggtgcagttg


 


cccgagaatttcgattacgctcaggctgacatgtcgatcaagaagcccgttgccgaatggcaacggctc


 


ggggtgatgggaacgaagcaagcgattccgggcgagctcgcacaggagcaagcatcggtcctgctgccc


 


gcaggttatcgcgggccagcatttatggtcctaagtaatttccgtagcatcctgcgctataacaactcc


 


actgcctatgcgctaacgatcgggctactagccgacagttatgctggcgggaccggcgtgtctcacccg


 


tggccaactgataatcctcccttgggcagcattgcgcaggtaaccgatttgcagaaactgctgactgct


 


aagggctactccctgggtgctgctgacggtgttataggggcgatgacccgggcggccatccgggcttac


 


cagaaggatcagcatttgccacccgacggttacgccagcactgtactactggagagcctgcgccgatag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 40 as follows:









MRSRVITTSLVVIMLSCASAAPACFSADMTPSVSNESTSEADFQQWLATF



 


RSNATTKGIDTATLDLAFQNITLDPTVHQLDMAQPEFTTAIWDYLSERLT


 


PKNIQQGQELLQKEPVLNEVERHYGVDAKIIAAIWCIESGYGKDIGSRDV


 


IRSLATLAYKGRRMDYGATQLMAALHIVQNKDIARAQLIGSWAGAMGQTQ


 


FIPTTYLDYAVDFNHDNRRDVWSSRADALASTASYLQRSAWNSRVSWGQE


 


VQLPENFDYAQADMSIKKPVAEWQRLGVMGTKQAIPGELAQEQASVLLPA


 


GYRGPAFMVLSNFRSILRYNNSTAYALTIGLLADSYAGGTGVSHPWPTDN


 


PPLGSIAQVTDLQKLLTAKGYSLGAADGVIGAMTRAAIRAYQKDQHLPPD


 


GYASTVLLESLRR






This protein has significant homology, as detected by BLAST analysis (1e-106), to a putative transglycolase from Pseudomonas aeruginosa and Ralstonia solanacearum (respectively, Genbank Accession Nos. NP—252681 and NP—522801, each of which is hereby incorporated by reference in its entirety).

A twenty-first nucleic acid molecule encodes ORF22 and has a nucleotide sequence according to SEQ ID NO: 41 as follows:








atgcttgctcctgacggcgtagaaatcgatatcgtgctatcaggtatatgcggaactgatctggcggta



 


ttgtcgggccgtgaaggtggagaggtgggcattatacgcgggcacgaagcagttggcattattatcgat


 


gtaggtaaggatgtagtacacctacaaaaagggatgcgggtggtggttgatcccaacgaatactgtggc


 


gtttgcgaaccttgccgtcttgctaaaacgcacctatgcaatgggggggtgaacgctgggttggatatc


 


gcaggtgtcaacaaacatggaacttttgccgagcgcttcgttactcgtgagcgttttgtgtatcaattg


 


ccagacgatatgagctgggcagctggtgtgttggttgagcctgttgcctgcattctgaataatatagac


 


caggcgttcattcgagcgggagagcgtgtgttgatcctagggtctggccctatgagtctgattgcgcag


 


atcgttctgcgctcaatgggagttgacacgctcgccactgatcgaaacacacatcgcatacagttcggc


 


cgctcacaaagtcttgatgttatacatgccgatgatcttgagttgcagatgcagcaccaagaaaagttt


 


gatgttgttatcgatactgtcggtaatcagatcgatacagcttcacgctacatcggtcgcggtgggaga


 


attgtactttttggatttgatagtgactatcactacatgctgcctgtaaagtacttcctggttaacgct


 


atcagtattatttctgctggagaatacaatcagcactttcctagagcaattcgtcttgtgcaaaaactt


 


cctgagctagggcggctggtaacgcatcgctacgtactagaaaatcactcggaggttttcgatgcactt


 


ctgaacgatgcttccgcccccaatataaaaagcgtattcacaccaaatctcgcttatctttaa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 42 as follows:









MLAPDGVEIDIVLSGICGTDLAVLSGREGGEVGIIRGHEAVGIIIDVGKD



 


VVHLQKGMRVVVDPNEYCGVCEPCRLAKTHLCNGGVNAGLDIAGVNKHGT


 


FAERFVTRERFVYQLPDDMSWAAGVLVEPVACILNNIDQAFIRAGERVLI


 


LGSGPMSLIAQIVLRSMGVDTLATDRNTHRIQFGRSQSLDVIHADDLELQ


 


MQHQEKFDVVIDTVGNQIDTASRYIGRGGRIVLFGFDSDYHYMLPVKYFL


 


VNAISIISAGEYNQHFPRAIRLVQKLPELGRLVTHRYVLENHSEVFDALL


 


NDASAPNIKSVFTPNLAYL






This protein has significant homology, as detected by BLAST analysis (2e-18), to a putative sorbitol dehydrogenase (Genbank Accession No. NP—389115, which is hereby incorporated by reference in its entirety).

A twenty-second nucleic acid molecule encodes ORF23 and has a nucleotide sequence according to SEQ ID NO: 43 as follows:








atgaaagttactgtattcagtcagatatcaattgatggcaagttgacgatgggcaaaggcgcatccagc



 


aagccgttgtttcagaactttgatgatgatgacatgcgttttattcataagttccgcggcgaagtcgac


 


gcaatcatggtagggcgcaatacaattgttactgacgatccacaattgaccaatcgctatgagtcgggt


 


cgtaacccaatacgtatcattcccaccacctccttagatctgcctacttccgccagtattttcaaatca


 


ccagagaaaactattatcgcaactagcgaacaggctcgtgatcatgaaatggtcaaacatatccgtgct


 


tgtggaaaggaggtgctctttgccggtgcaaagcatgtcgactttacacgacttttccctatgctggag


 


gcgcgcggaataaaccacatcatggttgagggcggtggccacctgaactggcaggtattcaatctcgat


 


ctggtagatgaaattatactcatgcaggtgcctatcatcataggtggtgcggcaactgcaacgcttgct


 


gacggggtggggtatcgggatatcaacatggccaattcgtttacgctgcatgctttagaagcacgcccc


 


cattacaatctcatgcacttcaagcgcgaatcgaacaatcggagcccgtactga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 44 as follows:









MKVTVFSQISIDGKLTMGKGASSKPLFQNFDDDDMRFIHKFRGEVDAIMV



 


GRNTIVTDDPQLTNRYESGRNPIRIIPTTSLDLPTSASIFKSPEKTIIAT


 


SEQARDHEMVKHIRACGKEVLFAGAKHVDFTRLFPMLEARGINHIMVEGG


 


GHLNWQVFNLDLVDEIILMQVPIIIGGAATATLADGVGYRDINMANSFTL


 


HALEARPHYNLMHFKRESNNRSPY






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted, and has significant homology, as detected by BLAST analysis (8e-38), to a riboflavin specific deaminase (Genbank Accession No. NP—213307, which is hereby incorporated by reference in its entirety).

A twenty-third nucleic acid molecule encodes ORF24 and has a nucleotide sequence according to SEQ ID NO: 45 as follows:








atggagcaggaaaagagttcctgtttgcgctacggcgtgacccttaatga



 


aaaagatctgtcacgttttttgggaactacacagcactacatgtggagca


 


cgattaaaaatgagtacgcgctcactgaatccatcgaccacttgatggca


 


cagcatcaacagcaattaatgcgctcaatcagttttgaattgtttcaatc


 


catgcctggcgtggaggcgcttctcaatttactggagcataccggagtgc


 


cctgtgccgtagcctcttcgtctccacgtaatttggtcgagcttatattg


 


aagaaaacgaaattgcgtcgatttttcaaagaggttatttgtggtactga


 


tgttaaagagagtaaaccgaatccggagatttttcttacggcggccaagg


 


gacttggagtgtcacctcgtgcatgtctggttattgaagactcccatcac


 


ggtgttaccgctgcgaaggccgcccatatgttttgtataggtttgcgtca


 


ttccagctcatttcagcaggatctgagcgctgctgatctgatcgccaata


 


atcattatgacatcaagcaatggtttgcagaaaaatag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 46 as follows:









MEQEKSSCLRYGVTLNEKDLSRFLGTTQHYMWSTIKNEYALTESIDHLMA



 


QHQQQLMRSISFELFQSMPGVEALLNLLEHTGVPCAVASSSPRNLVELIL


 


KKTKLRRFFKEVICGTDVKESKPNPEIFLTAAKGLGVSPRACLVIEDSHH


 


GVTAAKAAHMFCIGLRHSSSFQQDLSAADLIANNHYDIKQWFAEK






This protein has significant homology, as detected by BLAST analysis (5e-32), to a putative phosphatase from Clostridium (Genbank Accession No. NP—347269, which is hereby incorporated by reference in its entirety).

A twenty-fourth nucleic acid molecule encodes ORF25 and has a nucleotide sequence according to SEQ ID NO: 47 as follows:








atgaatgcgttcgcaaccggtcagcttgaatacagcctgaaaaagctggg



 


atacgatgccgccgctttgcaggccctgcgcgaagaagggtacttgctgt


 


ggaaagggaaaaacgaccagaccagcttgctggtgccctcggccgatctg


 


gatgcacttttcgttatcaacacgttgagctacatcgaccccgagcatga


 


cgcacgtctgctggcgcttgcattgcaccttaacctgtcccctgtccata


 


cgatgagcgcctgcatagccctcgatgtcgagcaaaacacgttatgcctg


 


cgctacacccatgaccttggcgggagcggggctgataccctgttgcttgc


 


gctcgaaaacgcccaggcgctggccgaacaggtcaggcaggtgatcgaaa


 


ccttcaggcgtgaccaagggcgtccgtccgggcaaacgtctttgtcccgg


 


caatccagtgctctgatgcgataa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 48 as follows:









MNAFATGQLEYSLKKLGYDAAALQALREEGYLLWKGKNDQTSLLVPSADL



 


DALFVINTLSYTDPEHDGRLLALALHLNLSPVHTMSACIALDVEQNTLCL


 


RYTHDLGGSGADTLLLALENAQALAEQVRQVIETFRRDQGRPSGQTSLSR


 


QSSALMR






This protein shares features common to type III chaperones and is a putative chaperone for the product of ORF26 (described below).

A twenty-fifth nucleic acid molecule encodes ORF26 and has a nucleotide sequence according to SEQ ID NO: 49 as follows:








atgaaaatatccggctccacatcgcctgcacacacttcaacgaattccgc



 


gcagaagtcctcttcaaaagggctgctgagtggtttggccaagcatttca


 


aggggatgctcgtttctggcaacacttctggtcattcggcgctcgggcat


 


tacgcgtcatccagcagcggctccaaaggcaaggcaccggtacgggacga


 


ttacagcaacggaccgcaaacacgccttaacaacacacctctgaaacgag


 


cactggcccgagagcttgatcgctttggctacggttcatcggcgaccgag


 


tcttttgaccgctcattgcagcgtaaggataaaaatccagagcttgggaa


 


ggtctga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 50 as follows:









MKISGSTSPAHTSTNSAQKSSSKGLLSGLAKHFKGMLVSGNTSGHSALGH



 


YASSSSGSKGKAPVRDDYSNGPQTRLNNTPLKRALARELDRFGYGSSATE


 


SFDRSLQRKDKNPELGKV






The protein of SEQ ID NO: 50 has been shown to be translocated by DC3000 in planta.

A twenty-sixth nucleic acid molecule encodes ORF27 and has a nucleotide sequence according to SEQ ID NO: 51 as follows:








atgaaaaaatgtattgctctgctccttactctggtcgtctgcgaaggtgcattggcaggdacggcacgt



 


gatgaacagaacatcacgtcttacatcgacagtcacggcaccgaacagatcgcgttgcttgagaagctg


 


gtcaacatcaacagcgggacagacaacgttgagggtgtcgtcaaggtcggtaacctgatcaagccggag


 


ctggaggcgttgggtttcgagaccgcctggcacgacctgccctcggcaatgaaccatgccggcagcctt


 


gtcgctgtgcatgacggcagcaagtctgcaaaacgtattctgctgataggccatctggatacggtcttt


 


cctcaaacaagccgctttcagacgttcgcttacctggacggcggcaaaaaagccaagggccccggcgtc


 


attgatgacaaaggcggcgtggtcacgatgctttatgcattgcaggcgctcaagcacagcggcgcgctg


 


gaaaagatgaacatctcggtagtcttgataggcgatgaagagctggcggccaaaccgaccgagatttcc


 


agagagtggctgatcgccgaagccaaaagaagcgacattgcgctgggcttcgaattcgccttgtcgccc


 


aatcaactgatcaccgagcgaagagggctgagcgaatggtttttgaccagcaccggcatcgacaaacat


 


tcagcgacgatctttcagcctgagaccggttttggtgcgatgtacgagtcggcccgagtgcttgacgag


 


attcgtcagaaactgtcgaacgagcagggcctgaccatcaatccgggactcattctgggcggctcaacg


 


gctgtggaagatagcgccagtgggcaaggcacggcttctggaagaaagacaacagttgcccggatcacg


 


tcggtgcatggtgatttgcgcttcagcagtgaagaccagagggcctctgcggaaacccgaatgaaggac


 


atagccagtcacccgctgccgcagaccaacagcgacctgaaaataaaagccatcatgccggtcatggcg


 


gatcgcgaaagcaatcgccaactactggcagcctacagtcaggtcagccaggatctcgacggacctgct


 


ttggagtcggcgccttcagcagaacgaggcggcgcagatatttcctatgtgaacaagtatgtgactgcg


 


agcctggacggtcttggtgcgtggggggcaggtgcgcacagtgaaaatgaaaccatcgagttgggctcc


 


ttgcccgtggtgacgaaacgggcggctattttcctgagccgctatggtaaccagtga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 52 as follows:









MKKCIALLLTLVVCEGALAGTARDEQNITSYIDSHGTEQIALLEKLVNINSGTDNVEGVVKVGNLIKPE



 


LEALGFETAWHDLPSAMNHAGSLVAVHDGSKSAKRILLIGHLDTVFPQTSRFQTFAYLDGGKKAKGPGV


 


IDDKGGVVTMLYALQALKHSGALEKMNISVVLIGDEELAAKPTEISREWLIAEAKRSDIALGFEFALSP


 


NQLITERRGLSEWFLTSTGIDKHSATIFQPETGFGAMYESARVLDEIRQKLSNEQGLTINPGLILGGST


 


AVEDSASGQGTASGRKTTVARITSVHGDLRFSSEDQRASAETRMKDIASHPLPQTNSDLKIKAIMPVMA


 


DRESNRQLLAAYSQVSQDLDGPALESAPSAERGGADISYVNKYVTASLDGLGAWGAGAHSENETTELGS


 


LPVVTKPAAIFLSRYGNQ






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted, and has significant homology, as detected by BLAST analysis, to a carboxypeptidase from Bacillus (4e-29) and a hydrolase from Ralstonia (5e-22) (Genbank Accession Nos. NP—241218 and NP—521834, respectively, each of which is hereby incorporated by reference in its entirety).

A twenty-seventh nucleic acid molecule encodes ORF28 and has a nucleotide sequence according to SEQ ID NO: 53 as follows:








atgaaccctataacacacagctttagtcatcttgggttttcaaacgctcaaagtacgtcagcgctggcg



 


cccggcggtaataaagtgccgaactttgtttcgcgagggcgaggcaaaggagtcccgcttgagcatttc


 


aacaccgctgatgagtatcgtttggcacgccagcagggcggcgtgctgaaatcaatagacggcagagag


 


ttcatgctactgctgcagaagtacacggccgccgaaacaagcgacgaagaatttgcggatttgagggcc


 


gccataccgcgctattccattgacctggccgagccgggtcaaactaaagtgctttatcgggggatatcg


 


ctgccggagaagactgcggcgcgattactgaatatctcttggggttacgaaagtcgcgdaatagcccat


 


ggtcttatccatggcttgcgggtagttaaggaaggtctgaagtag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 54 as follows:









MNPITHSFSHLGFSNAQSTSALAPGGNKVPNFVSRGRGKGVPLEHFNTADEYRLARQQGGVLKSIDGRE



 


FMLLLQKYTAAETSDEEFADLRAAIPRYSIDLAEPGQTKVLYRGISLPEKTAARLLNISWGYESREIAH


 


GLIHGLRVVKEGLK






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted. Furthermore, the protein of SEQ ID NO: 54 has been shown to be translocated by DC3000 in planta.

A twenty-eighth nucleic acid molecule encodes HopPtoL (ORF29) and has a nucleotide sequence according to SEQ ID NO: 55 as follows:








atgactactctgaccaccagacagatacaactcgcccacgcttggacatccgttcatacaggcgctggc



 


ctggccctggactgggtcgccgatgtggccgaaaaggtcgaggaaatcgccaccaaggccgacgccctc


 


agccgtgacttgcaccgcgcgcgcaacctgtcccgcagccttgggcgggtctcgacgacacccatgggt


 


atcggtttcttcggcttgtctcaggcaggcaagagctacctgatttccgctctggcggcggacgagaaa


 


ggccagttgctgacccggctgggtactcagcaactggacttcatcaagcacgtgaacccggtgggcggc


 


ggtaaggaggccaccggtctggtcacgcggttcacccgcaccgccgcgccaagtctggacccgcacttt


 


ccggtggagctgcgtctgtttcgcgaggtcgagatcgccatcattttggccaacgcctggtttgaggat


 


ttcgatcatcagcgcttgaacagccaagtcaccgatgcgcagatcgatgcccttttgcagcgtttcgag


 


gggcaattggcagccgctccgacacctggcgtcagcagtgacgacgtggtgctgctatgggattacctg


 


gagcaccattacgctaacgccatgcgcccgctgaacgcccgttattggccttgcgtggtcaaactggcg


 


ccgcgcttgtcggcacgcgagcgcgctcaattgttcgagccgctgtggggcggcatcggcaaaatgacc


 


gaaacctatgagcaactggcctcggccctgcaccgcctggggctggcagagacagtttttgcgcccatc


 


agcgcgctggtcaccgagcgcgatgggcaactggtacaaagcaaaagcatcatcaacgtcgacattctc


 


agccgtcttggcggcagcgcggactcggccatcgaggtacgtccggccagtgaaggcactttgcgccct


 


gccgtgtcggtgaatcgggccgaactggcggcgctcaccaacgagttgatttttcgcctggataacgaa


 


ccggccaacgccatcgtcaatagcgtcgatctgctcgacttcccgggctaccgcagccggcagaagctg


 


atgagcatcaacgaggccagcgaagtcgacagcaatggcaccgccaacaatccggtcgccaggctgttg


 


ctgcgcggcaaggtcgcttacttgtttgagcgttacaccaacgagcaggaaatgaacgcgctggtgatg


 


tgcaccagcaccttcaagcagagcgaagtggtgagcgtcggtccggtactcaagagctggatcgacaag


 


acccaaggcaccagcccccagcagcgcgatggtcgggccagcggtctgatctgggcgttgaccatgtgt


 


gacggctttatcggcggcgcgctcaacggcgaggttgtgcagtttcccgaaggttgcgacaacatgctc


 


aaactgaccatgatcgagcgattcggcaacgaagactggatgaaacaatggggcagcacgcctttcaaa


 


aacacctatctggtgcgcaagccgcgcttcaagaccagcttcatcgagttggcggcggacggtgaagaa


 


cgcgcttacaacgactcatcgcactctgcgttacaggcattgcaacaagcgttcagcaacagtgaactg


 


gtcaagcgccatgtggcagaaccgcaggacgcctggcaggcaatgctgacactgaacgacggcggcatg


 


actcgtttcagctcggcgttcagcccgattgccaacatcgacttcaagttacagcgtattgccgagcaa


 


ctggacgagttgatggtgcaattactgccgcgcctggagcagtactacgaagccggtggcgaagacgaa


 


cgggccaggaagaaggttatcgccaacctgattgcccgcccgttcgcgaccacgccgcacggcaaacac


 


gtgcttggcgaactgctcggttacatgtcgttgccggaacagcagttgcgcgacctttacctgaacggt


 


gatttcgccagccctgccagcgacgccactgcaccggtgcaggccgtcggcaagcctgaagtggaatac


 


gacatattcggcgaggccatcgcagccactgccacggtggaaatacccgcggcaccggccgtagcgccg


 


caataccagagccacgaacaccgtttcgcccgagcggccttcgacctgtgggcaacgcacctgcgcaac


 


ctcagccgtcgccagcacctgctggacctgttggagctgcctgccgaggccatcgccctgctggtcaag


 


gaactggtggtctgcgccgagcgcctggacttgccattgcagctcagcaacgcgctgctcaagcgcgcc


 


cagagcggtgtgcgcaaagaaaacctggtgcagcgccaagtgctgaccgcgcaactgctgctcaacgac


 


ttcgccgcctggttcgggcacaccgcccagccggcgggtcagcggccaacgggcctgctgggtgccaaa


 


caaccgctgtttgctttttatcaaaaggaaatgccagggcgcttcccgcacctcgcagcgcaagccgac


 


gaccagagcgtgattttcgccgatgactggatttctggcattgccattcatacccagaaaaacgtcggc


 


caccgcaagggcaaagaaatcactcctgagcagaacgaggccatgggccgcgtcatccaggcgttcaaa


 


gcgagataa






The HopPtoL protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 56 as follows:









MTTLTTRQIQLAHAWTSVHTGAGLALDWVADVAEKVEEIATKADALSRDLHRARNLSRSLGRVSTTPMG



 


IGFFGLSQAGKSYLISALAADEKGQLLTRLGTQQLDFIKHVNPVGGGKEATGLVTRFTRTAAPSLDPHF


 


PVELRLFREVEIAIILANAWFEDFDHQRLNSQVTDAQIDALLQRFEGQLAAAPTPGVSSDDVVLLWDYL


 


EHHYANAMRPLNARYWPCVVKLAPRLSARERAQLFEPLWGGIGKMTETYEQLASALHRLGLAETVFAPI


 


SALVTERDGQLVQSKSIINVDILSRLGGSADSAIEVRPASEGTLRPAVSVNRAELAALTNELIFRLDNE


 


PANAIVNSVDLLDFPGYRSRQKLMSINEASEVDSNGTANNPVARLLLRGKVAYLFERYTNFQEMNALVM


 


CTSTFKQSEVVSVGPVLKSWIDKTQGTSPQQRDGRASGLIWALTMCDGFIGGALNGEVVQFPEGCDNML


 


KLTMIERFGNEDWMKQWGSTPFKNTYLVRKPREKTSFIELAADGEERAYNDSSHSALQALQQAFSNSEL


 


VKRHVAEPQDAWQAMLTLNDGGMTRFSSAFSPIANIDFKLQRIAEQLDELMVQLLPRLEQYYEAGGEDE


 


RARKKVIANLIARPFATTPHGKHVLGELLGYMSLPEQQLRDLYLNGDFASPASDATAPVQAVGKPEVEY


 


DIFGEAIAATATVEIPAAPAVAPQYQSHEHRFARAAFDLWATHLRNLSRRQHLLDLLELPAEAIALLVK


 


ELVVCAERLDLPLQLSNALLKRAQSGVRKENLVQRQVLTAQLLLNDFAAWFGHTAQPAGQRPTGLLGAK


 


QPLFAFYQKEMPGRFPHLAAQADDQSVIFADDWISGIAIHTQKNVGHRKGKEITPEQNEAMGRVIQAFK


 


AR






HopPtoL has been shown to be a protein that is secreted by DC3000. HopPtoL has significant homology, as detected by BLAST search (1e-21), to an SPI-2 regulated SrfC (see Worley et al., Mol. Microbiol. 36:749–761 (2000); GenBank Accession No. AAF74575, each of which is hereby incorporated by reference in its entirety).

A twenty-ninth nucleic acid molecule encodes HopPtoS2 (ORF30) and has a nucleotide sequence according to SEQ ID NO: 57 as follows:








atgaatataaatcgacaactgcctgtatcaggctcggagcgattgttgactcccgacgtgggcgtatct



 


cgccaggcttgttccgaaaggcattattctactggacaggatcggcatgatttttaccgttttgctgcc


 


aggctacatgtggatgcgcagtgttttggtctgtcaatagacgatttgatggataagttttctgacaag


 


cacttcagggctgagcatcctgaatacagggatgtctatccggaggaatgttctgccatttatatgcat


 


accgctcaagactattctagtcacctcgtaaggggggaaataggaacgccgctgtaccgagaggtcaat


 


aattatcttcgacttcaacatgagaattctgggcgagaagctgaaattgataatcacgacgaaaagcta


 


tcgcctcacataaaaatgctttcatctgcgcttaatcgtttaatggatgtcgccgcttttagaggaacg


 


gtttatagaggcattcgcggtgatttagataccattgctcggctctaccatctattcgatacgagcggc


 


cggtacgtagagcccgctttcatgagtacaactcgaataaaggacagtgcccaggtgtttgagccaggc


 


acgccaaacaacatagctttccagataagcctaaaaagaggcgccgacatttcgggatcttcccaagcg


 


ccctcagaggaagaaatcatgctacccatgatgagtgagttcgtcattgaacatgcatccgctctttcc


 


gaaggaaagcatttatttgtattaagtcagatttga






The HopPtoS2 protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 58 as follows:









MNINRQLPVSGSERLLTPDVGVSRQACSERHYSTGQDRHDFYRFAARLHVDAQCFGLSIDDLMDKFSDK



 


HFRAEHPEYRDVYPEECSAIYMHTAQDYSSHLVRGEIGTPLYREVNNYLRLQHENSGREAEIDNHDEKL


 


SPHIKMLSSALNRLMDVAAFRGTVYRGIRGDLDTIARLYHLFDTGGRYVEPAFMSTTRIKDSAQVFEPG


 


TPNNIAFQISLKRGADISGSSQAPSEEEIMLPMMSEFVIEHASALSEGKHLFVLSQI






HopPtoS2 has been shown to be a protein that is secreted by DC3000. HopPtoS2 has significant homology, as detected by BLAST search (1e-5), to Clostridium exoenzyme C3 ADP-ribosyltransferase, (Nolling et al., J. Bacteriol. 183:4823–4838 (2001); GenBank Accession No. NP—346979, each of which is hereby incorporated by reference in its entirety). Further confirming that HopPtoS2 has similarity to ADP-ribosyltransferases, it was determined to possess an ART domain (pfam1129). In addition, HopPtoS2 has 20.5% identity to the HopPtoS1 as determined using EMBOSS software.

A thirtieth nucleic acid molecule encodes HopPtoS3 (ORF31) and has a nucleotide sequence according to SEQ ID NO: 59 as follows:








atgaatatcagtcctgtatcgggtgcccacggtagcagctacccttcagctcaatccacagcatcgacg



 


gcatcgaaaggtccctctggatcctttctcaaacagctcggcggctgtttttcaccctgcctgggtagc


 


agctctactggggccatactttctcccgctcatgagcaggtattgagccacacctattccagcaatatt


 


aaaggaaagttgcgcacgacgcccccaaaaggaccgtcgcccaggttgtctgacacacctatgaagcag


 


gcgctttcttcaatgatcgtacaggagcgaaaacggcttaaaagtcaacccaagtcattggcctcggat


 


atagaacgtccagacagtatgatcaaaaaagcgcttgatgaaaaagacggccacccgtttggcgagcgc


 


ttttcagacgacgaatttcttgcgattcatctctatacgagctgtctttataggccgatcaatcatcat


 


ctgcggtatgccccgaacaatgatgttgcaccggttgtcgaggcactgaaaagtggtttggcaaagctt


 


gctcaagaccctgattatcaagtgtctagccagcttcatagaggcatcaagcaaaagatgagtgatggc


 


gaggtcatgagtcgtttcaaaccgggtaagacctatcgtgatgaagcgttcatgagcacatcaactcat


 


atgcaggtttcagaagagtttacctccgacgttacgttgcacctgcggtcctcatcagctgtcaatata


 


ggccccttttcgaaaaatccatacgaggacgaagcgcttatctcgcccctgacgcctttcaaagtaacc


 


ggtctgcgcaagcaggacgataagtggcacgtcgatttgaacgagatagcagataattcagacgagtga






HopPtoS3 has an amino acid sequence according to SEQ ID NO: 60 as follows:









MNISPVSGAHGSSYRSAQSTASTASKGPSGSFLKQLGGCFSPCLGSSSTGAILSPAHEQVLSHTYSSNI



 


KGKLRTTPPKGPSPRLSDTPMKQALSSMIVQERKRLKSQPKSLASDIERPDSMIKKALDEKDGHPFGER


 


FSDDEFLAIHLYTSCLYRPINHHLRYAPNNDVAPVVEALKSGLAKLAQDPDYQVSSQLHRGIKQKMSDG


 


EVMSRFKPGKTYRDEAFMSTSTHMQVSEEFTSDVTLHLRSSSAVNIGPFSKNPYEDEALISPLTPFKVT


 


GLRKQDDKWHVDLNEIADNSDE






HopPtoS3 has significant homology, as detected by BLAST analysis (5e-3), to chicken ADP-ribosyltransferase (Tsuchiya et al., J. Biol. Chem. 269:27451–27457 (1994); Genbank Accession No. P55807, each of which is hereby incorporated by reference in its entirety). Further confirming that HopPtoS3 is an ADP-ribosyltransferase, it was determined to possess an ART domain (pfam1129). In addition, HopPtoS3 has 71.7% identity to HopPtoS1 as determined using EMBOSS software.

A thirty-first nucleic acid molecule encodes ORF32 and has a nucleotide sequence according to SEQ ID NO: 61 as follows:








atgaatattaacccttccctgggcgctcatggcagcgcctactcgtcgcctcaaagtgatacttcgaag



 


gccactggaaaagcacctgcgccttcttttttcaaacagttgggcggctgtttttcgccgtgccttggt


 


tcccatgcgtcaagcagccaacaactgtccgccagtcatgcgcagacgctcagtcagaattactccagc


 


aacattcaggggacgagccgcacacgccagccgagagcaccctcgccacgcctgtcagatacgcccatg


 


aagcaggcgctttcctcaatgatcgaacgcgagcgtttgcggcttcaaggtctttcgggaggaatgttc


 


tcgggcattgactccgccgatgccatgattggtcgagcgctcacgaagaaggattcaaacccaaaggct


 


gcgcgttttagtgatgatgagtttctcgccgttcacctctacacaacttgcctctacagacctatcaat


 


catcatcttcggtatcaacactag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 62 as follows:









MNINPSLGAHGSAYSSPQSDTSKATGKAPAPSFFKQLGGCFSPCLGSHAS



 


SSQQLSASHAQTLSQNYSSNIQGTSRTRQPRAPSPRLSDTPMKQALSSMI


 


ERERLRLQGLSGGMFSGIDSADAMIGRALTKKDSNPKAARFSDDEFLAVH


 


LYTTCLYRPINHHLRYQH






This protein has significant homology, as detected by BLAST analysis (5e-3), to chicken ADP-ribosyltransferase (Tsuchiya et al., J. Biol. Chem. 269:27451–27457 (1994); Genbank Accession No. P55807, each of which is hereby incorporated by reference in its entirety). Further confirming that protein of SEQ ID NO: 62 is an ADP-ribosyltransferase, it was determined to possess an ART domain (pfam1129). In addition, this protein has 51.3% identity to HopPtoS1 as determined using EMBOSS software.

A thirty-second nucleic acid molecule encodes ORF33 and has a nucleotide sequence according to SEQ ID NO: 63 as follows:








atgagctcgatcacgcacaccaacacgccgcaattggcggtcagcgattcacggggtctgccggtacgc



 


agtgtgcagttctatcgtggcgctgatggtcagcctgttgacgcgagggtgacgcagcactatttcgac


 


aaggccgggcgactgatcgccagtcgcgatccacgtttttccagtcgtttgaaatacggtgtctgtgcg


 


cctgtgaacctgatgcaaatcgtcagcttgtccggggctttgctgttatcgaaaagtgtcgattcaggt


 


tggcgggtgagcctgaacggcgaagcggggcagttagtcgacagctgtgacggacgtgacaacccgcgc


 


cagatcgaatacgacgggctgttgcgccctttggcgatcaacgaatcaggccgaatgaccgagcgcttc


 


acttatggcgggcctgccactgctgagcataaccagtgcaatcaactgattcgccatgacgatacggca


 


ggctcgcgcttgctgcgggactatggactgtcgggtagggcgttgagcgaaaaaaggtacttcctgcag


 


tcgcccgacagcccggactggccacttgccgagcctgatcgtgatgcactgctcgagccggtcggcctg


 


cagacgcgctgggctttcaacgcgcagggcgaggacctggcgcagactgacgcaaacggtaatgtccag


 


cgtttcagtcacggtgtggctgggcaactgcacgctgttgaactgaccctggccaatacggcacagcgg


 


caaacgctggtcagtgcaattcactacgacgcgttcaatcaggccgagcaggagacggcaggaaatggt


 


gtggtcagtcgctatgtgtatgatcaacaggacggtcggctgactgagctcagtgcgctatctgccgac


 


ggctcagtgttgcaaaaactgaactacagctatgacccggcaggtaacgttctactcatcaacgatgcc


 


tcgcaaccagaccggtattgcggcaatcagcgtatcgagccgataaaccgttactgttacgacacgttg


 


tatcagttgatcgaagccacggggcgggaggtcagaaacggggccagccatggtccggcgctacccggt


 


ctgcaacctctgccgacgctcgatccttgccaggtcagcaactacacacagcgttacagctacgacgct


 


gcgggtaacctgctgcaaatgcgccacgaaggcgcgcacaacttcacccgcaacatgcacgttgatccc


 


gacagcaatcgcagcctgcccgacaatgacaggtatgtggatttcgccacgagttttgatgccaacggc


 


aatctgctgcaactcgtgcgtgggcagaccatgagctgggatgtgcgtaatcagttgcggcaaatcact


 


accgtgcaacgtgaagacgcaccgaatgatgaagagcgctatgtatacgacggccagggccagcgctgc


 


cgcaagatcagcaccgcgcaggcatcaggtcgcacactgaccaatgaagttcgctacctgccgggactg


 


gaagttcggaccacggccgatggagaaactcttcacgtcgttacggctcaggcgggtcgcaacagcgtg


 


cgggtgttgcactgggaagccggaaaaccaggcgctattgcgaacgatcaggtgcgttacagcctgggt


 


gatcatctgggctcgagcacgctggagcttgatcagcaaggcggcctgatcagccaggaaagttattac


 


ccctttggcggcacggcctggtgggcggcgcgtagtgcagtggaggccaagtacaaaacagtgcgttat


 


tcgggtaaagagcgcgatgccagcgggctttattattacgggttcaggtattacgcgccgtggttgcag


 


cggtggatcaatcctgacccggcgggggatgtggatgggttgaatctgtacaggatggtcagaaataat


 


ccgcttgtttacgttgatgcgaagggccagcaacctgaacctgttccaaaaactattcaccagatctgg


 


ataggtgaaaacaagaatgccttgagagctcaggttagcaatatcaacagaaccgttgaaatggcttgg


 


gggtataaagtgaagttgcatctggaaacgaggacgccggaagcttattcggaaatcgaaaaggatctg


 


agatccgaagtggttctgcttcctgattcccaggtttttcaaaacttcaaggagaagccgctttatgcg


 


gcctatgaagatttccgaagaaacaatcagaattacgctttcgcggtagacgttttacgtatgcatacc


 


gttcatgatttgggcgggatttattcagatgtcgatgacgtttatgcaggtgcggagactggcggaatg


 


acgcagttgggggataatccgctgtttgcagaacctgatgaggttttgacgctggatcctgttcatgtc


 


ccttgggagccccagaattctgttgaaagttttatggtcaataacagctcatttgccgctcattcaggt


 


gcaggcgtcttacttgacatgatgggggaaggagcgaaacgatatgatgaagccgttgagggcggaagt


 


tatccggatccgacgggcatgaacggtataggtctaagtctgctctggaatcctaacccggcagtaaga


 


gttcgaacgttatcgaatgtagtaggccccggcttgtttacagacacactgcacgcttcggacacagca


 


tacggtgagctttttagtaatctgaaaggcgtcgtctttcaaaaacagccgttcacgtttgccgaccaa


 


atggccaggaagatgccgctgcatcggcatataaaaagcggcgcggcgcaaacctggcgctga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 64 as follows:









MSSITHTNTPQLAVSDSRGLPVRSVQFYRGADGQPVDARVTQHYFDKAGRLIASRDPRFSSRLKYGVCA



 


PVNLMQIVSLSGALLLSKSVDSGWRVSLNGEAGQLVDSCDGRDNPRQIEYDGLLRPLAINESGRMTERF


 


TYGGPATEAHNQCNQLIRHDDTAGSRLLRDYGLSGRALSEKRYFLQSPDSPDWPLAEPDRDALLEPVGL


 


QTRWAFNAQGEDLAQTDANGNVQRFSHGVAGQLHAVELTLANTAQRQTLVSAIHYDAFNQAEQETAGNG


 


VVSRYVYDQQDGRLTELSALSADGSVLQKLNYSYDPAGNVLLINDASQPDRYCGNQRIEPINRYCYDTL


 


YQLIEATGREVRNGASHGPALPGLQPLPTLDPCQVSNYTQRYSYDAAGNLLQMRHEGAHNFTRNMHVDP


 


DSNRSLPDNDRYVDFATSFDANGNLLQLVRGQTMSWDVRNQLRQITTVQREDAPNDEERYVYDGQGQRC


 


RKISTAQASGRTLTNEVRYLPGLEVRTTADGETLHVVTAQAGRNSVRVLHWEAGKPGAIANDQVRYSLG


 


DHLGSSTLELDQQGGLISQESYYPFGGTAWWAARSAVEAKYKTVRYSGKERDASGLYYYGFRYYAPWLQ


 


RWINPDPAGDVDGLNLYRMVRNNPLVYVDAKGQQPEPVPKTIHQIWIGENKNALRAQVSNINRTVEMAW


 


GYKVKLHLETRTPEAYSEIEKDLRSEVVLLPDSQVFQNFKEKPLYAAYEDFRRNNQNYAFAVDVLRMHT


 


VHELGGIYSDVDDVYAGAETGGMTQLGDNPLFAEPDEVLTLDPVHVPWEPQNSVESFMVNNSSFAAHSG


 


AGVLLDMMGEGAKRYDEAVEGGSYPDPTGMNGIGLSLLWNPNPAVRVRTLSNVVGPGLFTDTLHASDTA


 


YGELFSNLKGVVFQKQPFTFADQMARKMPLHRHIKSGAAQTWR






This protein has significant homology, as detected by BLAST analysis (1e-128), to SepC insecticidal toxin (Hurst et al., J. Bacteriol. 182:5127–5138 (2000); Genbank Accession No. NP—065279, each of which is hereby incorporated by reference in its entirety). This protein also has significant homology (2e-128), as detected by BLAST search, to putative insecticidal toxin from Yersinia pestis (Parkhill et al., Nature 413:523–527 (2001); GenBank Accession NC—003143.1, each of which is hereby incorporated by reference in its entirety).

A thirty-third nucleic acid molecule encodes ORF34 and has a nucleotide sequence according to SEQ ID NO: 65 as follows:








atgccgatcaccgcgcagcagttgctgcagatactcccgagcgctggccagaaagccggcgtttttgca



 


cccgtcctgaacacagcgatgagcaagcaccagatcttgacgccgctgcgcatcgcggctttcatcgcc


 


caggtcggtcatgagtccggccaactgcgctacgtccgcgagatttgggggccgactccgcagcagctg


 


ggttatgaaggccgcaaggacctcggcaataccgtggcgggtgatggttcgaagtaccgcgggcgcggc


 


ctgatccagatcaccggccgggccaactatgccgaatgcggcgaggcgctgggcctagacctgatccat


 


cacccggaactgctcgagcagccggagcacgccacaatgtcggcagcgtggtactggagcagccgtggc


 


ctgaactcgctggccgacaaaggggactttcttcaaattacccgaagaatcaacggaggcaccaatgga


 


ctggcggatcggcaggcgctgtacgaccgggcgctgaaggtgctggcgtga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 66 as follows:









MPITAQQLLQILPSAGQKAGVFAPVLNTAMSKHQILTPLRIAAFIAQVGH



 


ESGQLRYVREIWGPTPQQLGYEGRKDLGNTVAGDGSKYRGRGLIQITGRA


 


NYAECGEALGLDLIHHPELLEQPEHATMSAAWYWSSRGLNSLADKGDFLQ


 


ITRRINGGTNGLADRQALYDRALKVLA






This protein has significant homology, as detected by BLAST analysis (3e-36), to a lytic enzyme (Nakayama et al., Mol. Microbiol. 38:213–231 (2000); Genbank Accession No. BAA83137, each of which is hereby incorporated by reference in its entirety).

A thirty-fourth nucleic acid molecule encodes ORF35 and has a nucleotide sequence according to SEQ ID NO: 67 as follows:








atgaatctaacagctttaggttcaaagctgtctcggtatcgcaagcagcttgcgatgagcgaggaagaa



 


gtgtgtgcggtcacccacatcccccttgagcgcctgcagtcagttgaagccggctctcaggcgcctacg


 


ggtgatgaagtgcttatcctggccgatctctaccactgcaacttcaaattcttcatctcgaacgagccg


 


ctcgccccctttgagcagaccgaaatcctgtatcgcaggcacggagctgagttcatcaaggaggatcgt


 


agagccgtccaagaattcctgtacctctgcgaaacagaggacttcctgatgagtgagttgaaggctatg


 


aagctcgaatttccgctgccgcaggcttctgggaattttaagaatgatggaatccgagcggctgaagcc


 


tttcgccttttcaatcagcaccccacaaacgccgtgcctcgggatgtgtatcaggagattcgccaaacc


 


ggagtgcatgtgttccgtagaaagcttggtaactctaacatttcggggcttttcctggctcaccccacg


 


gctgggaagtgcattctggtcaactacagcgaagacgtataccggcagcggtttagcgctgcgcatgaa


 


tttgctcacgctcttttcgatgcgcagggtggccccagtattacctactcccgtacgactaaggctgac


 


ctagtcgaagtgagagcaaacacctttgcctcccggtatctgatgccttcagaaatcctccgacagctg


 


cccaaccctgagcaatggacacaggaaaatacccagtattgggctcatgagttgcgagtcagctgcgtt


 


gccttgggcataggtctgaagtccgagggcttaattagcgagcaagcattccagaggataaagtcgtac


 


cgcgttcctcgtgaactgaagattgacccagaattgccggcccaattgacgacgcaacagcgtgagcga


 


aaggctaagttactggaaaaggggttatctgacagctacgtcgcactgtgcctagacgctcagagccgt


 


ggcatcatcactcaaggtcgattggctgaagccttgcttagtgacttgggaggccttcaagagctgctc


 


agcctttatggaagatcgcgcaatggccattga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 68 as follows:









MNLTALGSKLSRYRKQLAMSEEEVCAVTHIPLERLQSVEAGSQAPTGDEVLILADLYHCNFKFFISNEP



 


LAPFEQTEILYRRHGAEFIKEDRRAVQEFLYLCETEDFLMSELKAMKLEFPLPQASGNFKNDGIRAAEA


 


FRLFNQHPTNAVPRDVYQEIRQTGVHVFRRKLGNSNISGLFLAHPTAGKCILVNYSEDVYRQRFSAAHE


 


FAHALFDAQGGPSITYSRTTKADLVEVRANTFASRYLMPSEILRQLPNPEQWTQENTQYWAHELRVSCV


 


ALGIGLKSEGLISEQAFQRIKSYRVPRELKIDPELPAQLTTQQRERKAKLLEKGLSDSYVALCLDAQSR


 


GIITQGRLAEALLSDLGGLQELLSLYGRSRNGH






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A thirty-fifth nucleic acid molecule encodes ORF36 and has a nucleotide sequence according to SEQ ID NO: 69 as follows:








atgaatatcaaccccttggcttcttcattacagaatcaacagcgcactctcttaggcccgccccccctc



 


aattcatctgctgctctgccgatcaagatccctgtggcgcatgataaagcgcgtgaccctaacgctgaa


 


ttctataccaccgaggaaacgccctggtttgccggctacaaaaagtcggaggcaggacgcgctatttta


 


gagaaaatgtctgagaaggaagcaaaagatatccgaggcgagtatctgggaaactacatgaaagccttt


 


gacgaaaccatatgtcgtatgtacgacaattttcacgatttcaaacagcagcttttttaccttaatacg


 


gagctgtcaaaaaagcatttcggcttcacgctgggctttaatcaggacattcaggtgaccgacccggac


 


gaggtactcaccccggcagagttcacgtacctgaccgagaagctgaacgaacgccaacaactgaaagag


 


gatctgcgtgcgcacgcaaaaattgtgatgacgctgctcgaccattacaccgaaaaattcgataaccgg


 


cacaccctcaatctggagagttacagcaaggtcatcgactacggacagatcttcagccgcaatcatatt


 


ggcaatttcatggacacgattatctaccagatcgagcgcaatgcgccgaagcgtgaggaagaaccaaaa


 


cctctggttgatgtgcacgcttga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 70 as follows:









MNINPLASSLQNQQRTLLGPPPLNSSAALPIKIPVAHDKARDPNAEFYTT



 


EETPWFAGYKKSEAGRAILEKMSEKEAKDIRGEYLGNYMKAFDETICRMY


 


DNFHDFKQQLFYLNTELSKKHFGFTLGFNQDIQVTDPDEVLTPAEFTYLT


 


EKLNERQQLKEDLRAHAKIVMTLLDHYTEKFDNRHTLNLESYSKVIDYGQ


 


IFSRNHIGNFMDTIIYQIERNAPKREEEPKPLVDVHA






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A thirty-sixth nucleic acid molecule encodes ORF37 and has a nucleotide sequence according to SEQ ID NO: 71 as follows:








atgggcctgatcggcgtcaaacagaacaaaccgcaacaggcgcagacctacctgacgcgcctgcaagcg



 


ctgtcgccagcgccctggcaggcggtgcagctggagcaggacattgccctcggccagccgcaaaatcag


 


gcgctgctggatgatgcccgacgcctggccgacgccggtgagcgtgacaaggcgaccggggtgtttcgc


 


cagttgttcaacggccgtttgcctcaaggcactgtcggccgcgagtactacaccaacctgggcttcaac


 


aatgcggactggcccgaggcgcgcaagggttttgaacgcctgatgcggcagaaccctgacgactcgatt


 


ctggcgctgttctttgccaagcacctggcccgccgcgaagacagccgcgccgaaggcatcgccgctctg


 


gcgcgcctgagcactcatccggacatcgccggcgatgccgatcagagctggcgcatggcgctggtctgg


 


atcggcccgcctgcggctgcgcaagtgccactgttcgacgcgtttctcaaggttcatcccgacgatcag


 


gaaatccgcgaccagttgaacaagggtcgccagcagcatgccagcggcgctgcctcaggctggcagcaa


 


gacccgctggtggcgcgcggcttgaaggcgctggaaaaaaatgatcatgtggcggccgaagaagccttt


 


gccgcccgcctgaaaatcaaggcggacgatgccaacgtgcttggcggcctgggcgtggtgcgtcagcag


 


cagaaccggttgcctgaagccgaacaattgctgacccgcgccacgcgccagcagggcggtgcgcgctgg


 


aaaaacgcgctggaaaacgtacagctctggacctcgctgcaagaggcccgtgacctgcaggccaaaggg


 


cagaccggcaaggctcaagcgttgctggctcaggcgcagcggcaaaaccctgacaatatcgacgtgcgt


 


ttgaccctggccgacgtgcaggtgcaggccgggcaactggacgccgcgcaagcgggctatcgtcaggta


 


ctggcgacccagcgcggtaatccgcaggcaatccgcgggctgatcaacgtgctggcccagcgtggtcag


 


gctgatgaagcgttgcgcctgctcgacacattgtcgccaggcgaacaggccaaactgggcgacagcggt


 


cgcttcaaggcgctgcgctccacccaggtggcgcggctggccgagcagcgtggcgatgttcgcgctgcc


 


caggtggccttgaaagacgcggtgaagaacgacccggacaatgtctggacgcgttttgatctggcgcgc


 


ctgtacctcaagaccgacgaagcgcccaaggcccgcgcgctgatcgacgagctgctcaaggctcagccc


 


aacaatatcgatgcgctctacaccagcgcgctgctgtcagtggaaatgggccagtggcaggacgcgcag


 


accacgtttacgcgcatcccggttgatcagcgcacgccggacatgaaagcgcttgctgacgaagtcacc


 


atgaccgtgcagatcaatctggccatcggcatcgcccggcgcggtcagcgccaggaagcgttggcgctg


 


ctcgatcgcttgcaaccggtcgccagcggcagcccggagcgtcaactcacgctggccagcgcttacatc


 


gatgcgggcgagcccgcgcgcggtcgggaaatggcccgtgcggccatcgctcaggcccctttgccgtcg


 


gccgacctgatgctgcaatacgccggtctgctgctcgcagcgggcgatgacgtgcaggtcaatgcgatc


 


ctgcgcaacgtgcagggtcagccgatgagcgtgcagacccgcaaacgttttgatgaccttttgtaccgc


 


taccgcattcgtcaggccgatctgctgcgtgaaggcggtgatctggcgggcgcgtacgacacgctggca


 


cctgctttggcgcagcgcccggacgacattcaggcggtgtcggccttcgcccgcatgtacaccgccaat


 


ggcgacagcgcccgagcgttcgagctgtacaagcctttgttgcagcgccagcccaatgacccgcaagtg


 


ttgctgggcgcagccgatgcggcggtcaaagcgcatgattatggctttgccgaaaaagccctgagccag


 


ttccgcaaactggagcgtaacgacccgcagaccctgacggaggccgcacgtatctaccaaagcatgggg


 


cagaccggcgcggccaccgagttgctgcgcaaggccgtggccatcgaacagagtgaaaaacagcgcgcg


 


atggctgtgcaggctgtgtcgaccagcaccacgtcgtccaacccgtttgcgacgggcggctcacgtagc


 


ctggcggcggcttcggctattccggctccggctcaggtgtcgctcagcggtgggagagcgcttgaaaca


 


aacagtgcgcctgaaatatctgccccgcgtgacaccgcttatcccggccagatcgccgcaccacaaccg


 


ctgtctgccgcacgtacgcaaagtgtgcgcggcaatccgttcatggcagccaccgaccgcgatcaggcc


 


agcagcgcacagcaggcgctcaatcgcattcttgagcagcgcagtggcttcgtcagtcagggcctggcc


 


gtgcgcagcaataacagcgagtcgggtctgagcaaactgaccgtggtcgagaccccgctagaggtcaat


 


ttgcctgccggtgataaccgggtggccgtgcgcgtcacgccggtgtcgctgaatgctggcagcttgaag


 


tcagatgcaggtgcccgttttggcggtggcaccagcggtgctgccggttcgcagagcgacaagggtgtc


 


ggtctggcggtggcgttcgagcgccccgaagaaggcctcaaggccgatatcggcaccacgccgatgggt


 


ttcaaatacaccacggttgccggcggcgcgagtgtcgaccggccgttggctaacaacccggacctgcgc


 


tacggcctcaacgtgtcacggcgtccggtgacggacagcgtgacttcgtttaccggttccacagacgag


 


cgcagcggcctgtcctggggcggcgtcacggccaacggcgggcgcggtcagctcagctatgacgaccag


 


accatcggcggttatggctacggctcgtggcacaaactggttggcaacaacgtgaaatccaacacccga


 


ggcgaagtgggtggcggcgtttactggtacctgcgcaatgccgaggacagcaaactgaccgcaggcctg


 


agcctgatgggcatgagctatgacaatgaccagagctacttcacgtacggccacggtggctatttcagc


 


agcctgatgggcatgagctatgacaatgaccagagctacttcacgtacggccacggtggctatttcagc


 


ccgcagagcttctatgccatcggcgtgccggtgatgtgggcacagcgcaccgagcgtttcagctatcag


 


gtcaagagctcggtcggggtccagcacttcaagcaggacggcgccgaattcttccccgacgacagcacg


 


ctacaggccgcttccgcccagcgctacacagggcaaagcaaaaccggaattggctacaacctgagcgcg


 


gcaggcgagtacaagctcgattccagcctgttcatgggggccagtctgggcctggacaatgcccgggac


 


tatcgccagttcagcggcgcgctttacctgcgttacatgttcgaggacataaccggcccgatggcactg


 


ccggtcagcccttaccgttcaccttattccaactga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 72 as follows:









MGLIGVKQNKPQQAQTYLTRLQALSPAPWQAVQLEQDIALGQPQNQALLDDARRLADAGERDKATGVFR



 


QLFNGRLPQGTVGREYYTNLGFNNADWPEARKGFERLMRQNPDDSILALFFAKHLARREDSRAEGIAAL


 


ARLSTHPDIAGDADQSWRMALVWIGPPAAAQVPLPDAFLKVHPDDQEIRDQLNKGRQQHASGAASGWQQ


 


DPLVARGLKALEKNDHVAAEEAFAARLKIKADDANVLGGLGVVRQQQNRLPEAWQLLTRATRQQGGARW


 


KNALENVQLWTSLQEARDLQAKGQTGKAQALLAQAQRQNPDNIDVRLTLADVQVQAGQLDAAQAGYRQV


 


LATQRGNPQAIRGLINVLAQRGQADEALRLLDTLSPGEQAKLGDSGRFKALRSTQVARLAEQRGDVRAA


 


QVALKDAVKNDPDNVWTRFDLARLYLKTDEAPKARALIDELLKAQPNNIDALYTSALLSVEMGQWQDAQ


 


TTFTRIPVDQRTPDMKALADEVTMTVQINLAIGIARRGQRQEALALLDRLQPVASGSPERQLTLASAYI


 


DAGEPARGREMARAAIAQAPLPSADLMLQYAGLLLAAGDDVQVNAILRNVQGQPMSVQTRKRFDDLLYR


 


YRIRQADLLREGGDLAGAYDTLAPALAQRPDDIQAVSAFARMYTANGDSARAFELYKPLLQRQPNDPQV


 


LLGAADAAVKAHDYGFAEKALSQFRKLERNDPQTLTEAARIYQSMGQTGAATELLRKAVAIEQSEKQRA


 


MAVQAVSTSTTSSNPFATGGSRSLAAASAIPAPAQVSLSGGRALETNSAPEISAPRDTAYPGQIAAPQP


 


LSAARTQSVRGNPFMAATDRDQASSAQQALNRILEQRSGFVSQGLAVRSNNSESGLSKLTVVETPLEVN


 


LPAGDNRVAVRVTPVSLNAGSLKSDAGARFGGGTSGAAGSQSDKGVGLAVAFERPEEGLKADIGTTPMG


 


FKYTTVAGGASVDRPLGNNPDLRYGLNVSRRPVTDSVTSFAGSTDERSGLSWGGVTANGGRGQLSYDDQ


 


TIGGYGYGSWHKLVGNNVKSNTRGEVGGGVYWYLRNAEDSKLTAGLSLMGMSYDNDQSYFTYGHGGYFS


 


PQSFYAIGVPVMWAQRTERFSYQVKSSVGVQHFKQDGAEFFPDDSTLQAASAQRYTGQSKTGIGYNLSA


 


AGEYKLDSSLFMGASLGLDNARDYRQFSGALYLRYMFEDITGPLALPVSPYRSPYSN






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted. This protein also has significant homology (e=0), as detected by BLAST search, to cellulose synthase from Pseudomonas fluorescens (Spiers et al., Genetics 161:33–46 (2002); GenBank Accession P58937, each of which is hereby incorporated by reference in its entirety).

A thirty-seventh nucleic acid molecule encodes ORF38 and has a nucleotide sequence according to SEQ ID NO: 73 as follows:








atgaaactgatacgacagatccgctcgcagggtcgtcagtcgcccttgttcgaggaccttgcccagctc



 


gaggggcgcaagcgtcaatggctggccgagcgcgccgtgcagttcgcactgggcttgcacggccgccgg


 


ccagaggtcgataaccccttcaaaggcaaactgcgtgaagacctgtgctgcatcatgttcgatgacctg


 


tcgctgcacaccctggtcgagcgttacgcggccagtgaagccctgcgacgacacgacagcgagtacttc


 


agcaaactgatcgccacgacacgaaataccgtggaacggcgcatcgtctttcacgggctgctggaacac


 


ttcgacaggctgttgcctatcgaaaagagcatctaccaactcaactaccgcagcgttcaatacgcgcac


 


ctggagcaggaagaagccctgtacggcaaactgataatggaacaacccattagtgcactgctggaagtg


 


cacacgcctgagtggcttcttgagaatctgtcttcgtttgagttttcgattgattga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 74 as follows:









MKLIRQIRSQGRQSPLFEDLAQLEGRKRQWLAERAVQFALGLHGRRPEVD



 


NPFKGKLREDLCCIMFDDLSLHTLVERYAASEALRRHDSEYFSKLIATTR


 


NTVERRIVFHGLLEHFDRLLPIEKSIYQLNYRSVQYAHLEQEEALYGKLI


 


MEQPISALLEVHTPEWLLENLSSFEFSID






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A thirty-eighth nucleic acid molecule encodes ORF39 and has a nucleotide sequence according to SEQ ID NO: 75 as follows:








atgcgactgactactaaaggccgatacgctgtgacagccatgcttgacctggcgttacatgcgcagaac



 


gggccagtgtctctggccgacatctccgagcggcagggcatttccctgtcttatctcgaacagttgttc


 


gccaaactgcgtcgcggcaatctggtttccagtgttcgtggtccgggcggcggttatcagctgtctcgt


 


gacatgaaaggcatccaggtcgcccaagtcgtcgacgcggtcaatgaatcggtcgatgccacgcgttgt


 


caggggctgggtgattgccacgctggcgatacctgcctgacccaccacttgtggtgcgatctgagccag


 


cagattcacgaatttctaagcggtatcagcttggcggatcttgtcactcgccgtgaggtacaagaagtc


 


gctcagcgccaggatatgcgccgtggtcataaccacacgtcgcaactgggtaagatcgaaacgtccgcc


 


gtcgaatga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 76 as follows:









MRLTTKGRYAVTAMLDLALHAQNGPVSLADISERQGISLSYLEQLFAKLR



 


RGNLVSSVRGPGGGYQLSRDMKGIQVAQVVDAVNESVDATRCQGLGDCHA


 


GDTCLTHHLWCDLSQQIHEFLSGISLADLVTRREVQEVAQRQDMRRGHNH


 


TSQLGKIETSAVE






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A thirty-ninth nucleic acid molecule encodes ORF40 and has a nucleotide sequence according to SEQ ID NO: 77 as follows:








atgaataccgtcagaaaacccataacaccacggatgctcagcatgaccgataaaaacggcacccatcga



 


caacgacgtgctgcactgttccccaaaaccccggcgaccgccaccagcctgtgccctttcagagggcct


 


aatatcgccatcgtcccggtgcgctatgcgctggatcgctcgcgctatgacgctgaccccgcgcaactg


 


aagccactgcccaaagacggccaatgggcccacctgccgacgctgaaaactcgcagttacaccttacgc


 


caactgtacgacggctacgtttacgtgttcgacgaaacggccggcacgttgcacgaatacgcagcctca


 


gccagcgacggccatctgagccgcatcgtctggaccgatgcacacatcggtaacgaccagcgaagcggt


 


gccggtgaagggcaaccctttgtgctttacccgcgtgaccaccgcctgcacatcgccttttctcccctg


 


caatggacatggcgaatgtgcgagcacatgcgctcccacgccccaagccgcgcgttgtggatgaaggcg


 


ctggacctggccagctactgcctcaccatggccgaaccggacaccctgccgctggatcgaatcgccgag


 


gccgtggcggatatcgacaaagactgtgttgtggaagatggccgttttgcagattcggcgattcccagt


 


gttcgcccgccatcagaaggtgcagaaccctatccgttatgggcaccgctgggcgccgatgtcttctgg


 


cagggcagcgtctacgatcaggacagctctctggtcattgccctcaatgacccgctcgccgttttcaac


 


gacttgggcatgcagctggcggccgatcaggcggcttttcgggaatggcaaagcgcccacgaacacaag


 


atccagattgcccagaccgtcgccacgctgtgcggtgcagagagcgaagcagagaagctgccagcatcg


 


gtgcgcggtgatgcgctgcgcacgcatcagtacctgagcgaggtcgaagcctactttgaacaatgcatt


 


cttgaagaagcacagatcagcagtagcaacgttcctggagattttctgctgctgccggacatgttcaag


 


agcctggacatgcgcaaatcgatcgaaacacgttatggcagcgcgccgaccgatgagggcgcgcaggcc


 


tggaaagatcgccacaaatggcggcgcgaggtcgatctgagcagtgcgcgtcagtaccttttgcagcac


 


ctgccgaccggagacaaacgcctgcaacaggtgcgtgacacgcaaagcgatttccagcactgggcggca


 


catataggcaccgaaccgctcaagctgttcatcgacaccacacacccgaaaaccctgctgtatttgcag


 


acgatcatgctcaatctgcagatcatctatgcgcattacagcgccgcaaatgcctggctcgccgagcag


 


gaagccaacaccagcagcctgtttggcaccctgcgttatggtttttcgccagcgctcaagcacgccctg


 


catcaggaagccgacgcactgctgaacggcctcggcgacgtcactaatctggccacgcgcatcggtgaa


 


ctcaatggcgtgctcaaccatcagggttttgccgacaagccgtggatgaaggcgctgaaacagcctgtt


 


caagacaccttcaaagccctcggcgaactggccagcggtgccggcaaagccaggtttgaaagtgtatta


 


ctggcatgggtgcccatcgacagccgcatggcccttggcaagcagcagaacatcgttgcgttgcttcgc


 


accctgctgatcggccagatattgctcgactcgacagcacgcgtcgcgatcaatgagcagacagtgacc


 


aagctcaaacagtgggtaagtgagtggcaagtcctcaacaagcaaatcagcgagctggtgcgcagttgg


 


caatacccgaacgcctacaacacgcgccaaagcaccgctcgcaaattgcaggcccataaacacaaactg


 


cgcgttcacgaactgagcatccctgccctgctcgactttcagaacaacgaatacgccaagctattgcag


 


gacgagattcgtcagtacttccagtctggcaaaaccctcgccacggactggctcgcccgcgccaaaggc


 


tggaccgaccgactgggcggcgttgctggcacgatcacctggggcgtggtcatgcttaacctgatcaat


 


accgccttcctctatcgggaccttacccgggacggggatttcagtaccaaggacattggcaaggtgacg


 


tatggattggggtacagcttcaatctgttgatggcggtgtttgtggacgcgccgtggagcatcataagg


 


gacgcaacgccagcgctgatcgatggcaagaatgtggccattctggacaggtccagtgcgtactggaaa


 


gccaagggaaatgcagcgtggggtgatgcgatacgtgggttcagggtttcgatggtggcgatgggtggg


 


tttgggcttgcggcggttacgcttgaattatttgatgttacagatgattttcacgcagctaaaacatca


 


gaagaaacatatggaattggcatcaaggggttttccgtagtggtgatgggattgggtgctgcggcccag


 


ctaatggcaggcatttctcccgctggcgtttttacgattatcgcaatgagtccgtggttcagcgtagcg


 


ctactggcagcaggcttgatttatctttttgctacgatggcccttaattacttcaagcaagacagtgtc


 


ggctggtggctacgcaagtgctgttggtccataacccaagactatcgctatgctgagactgcggaaggt


 


aagcatgacgaagtgcgcgcgctgatggaaataaaattatctccgcaggtccatgtaaaaagcaccgtg


 


aattatgaaaaccgttatcttggcaaaaacgatcactacagcgtagcggtacaaaatggcgcgggggta


 


caagtgcgcttgccgaatcttctacgcgggctgtccgtgcatttcaatatcgttagtagcaagagacca


 


tggggcgtgctgcccgtagaaaaaatagatcagccgatacatgaagcttttctggaccacgggcaattc


 


aggaaagtcgaacagttcgggatgtttaccaacaagcctgctggcaaggcgagtgaagactatacctac


 


ccccgcatgccacctgaaaacgaagacctcatctgggaaacctgggtgccgctcgacaaggacgcaacg


 


tatcttgagttgcaaatctggtacccggccaatcttttaaatcctggcggagacgatagaagctatctg


 


tttcagatggagcttggcacaaaaggcgataccgctattgacggcctggctgcagtggaactcgaggta


 


aaggcatcaagcaggattggcgctctgaccctagaagtcgcagagggcacacctgtatga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 78 as follows:









MNTVRKPITPRMLSMTDKNGTHRQRRAALFPKTPATATSLCPFRGPNIAIVPVRYALDRSRYDADPAQL



 


KPLPKDGQWAHLPTLKTRSYTLRQLYDGYVYVFDETAGTLHEYAASASDGHLSRIVWTDAHIGNDQRSG


 


AGEGQPFVLYPRDHRLHIAFSPLQWTWRMCEHMRSHAPSRALWNKALDLASYCLTMAEPDTLPLDRIAE


 


AVADIDKDCVVEDGRFADSAIPSVRPPSEGAEPYPLWAPLGADVFWQGSVYDQDSSLVIALNDPLAVFN


 


DLGMQLAADQAAFREWQSAHEHKIQIAQTVATLCGAESEAEKLPASVRGDALRTHQYLSEVEAYFEQCI


 


LEEAQISSSNVPGDFLLLPDMFKSLDMRKSIETRYGSAPTDEGAQAWKDRHKWRREVDLSSARQYLLQH


 


LPTGDKRLQQVRDTQSDFQHWAAHIGTEPLKLFIDTTHPKTLLYLQTIMLNLQIIYAQDSAANAWLAEQ


 


EANTSSLFGTLRYGFSPALKHALHQEADALLNGLGDVTNLATRIGELNGVLNHQGFADKPWMKALKQPV


 


QDTFKALGELASGAGKARFESVLLAWVPIDSRMALGKQQNIVALLRTLLIGQILLDSTARVAINEQTVT


 


KLKQWVSEWQVLNKQISELVRSWQYPNAYNTRQSTARKLQAHKHKLRVHELSIPALLDFQNNEYAKLLC


 


DEIRQYFQSGKTLATDWLARAKGWTDRLGGVAGTITWGVVMLNLINTAFLYRDLTRDGDFSTKDIGKVT


 


YGLGYSFNLLMAVFVDAPWSIIRDATPALIDGKNVAILDRSSAYWKAKGNAAWGDAIRGFRVSMVAMGG


 


FGLAAVTLELFDVTDDFHAAKTSEETYGIGIKGFSVVVMGLGAAAQLMAGISPAGVFTIIAMSPWFSVA


 


LLAAGLIYLFATMALNYFKQDSVGWWLRKCCWSITQDYRYAETAEGKHDEVRALMEIKLSPQVHVKSTV


 


NYENRYLGKNDHYSVAVQNGAGVQVRLPNLLRGLSVHFNIVSSKRPWGVLPVEKIDQPIHEAFLDHGQF


 


RKVEQFGMFTNKPAGKASEDYTYPRMPPENEDLIWETWVPLDKDATYLELQIWYPANLLNPGGDDRSYI


 


FQMELGTKGDTAIDGLAAVELEVKASSRIGALTLEVAEGTPV






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A fortieth nucleic acid molecule encodes ORF41 and has a nucleotide sequence according to SEQ ID NO: 79 as follows:








atgtgcctggtggcgagcctgtcggtgctggcaggcatgaccgatgccatcggcttcatggccaccggc



 


gatttcgtctcgttcatgagcggcaacaccacgcgccttgcggtggcgatcagtgatggcgatttgagc


 


gtcacactccgtctggccctggccatctttgcgtttattgccggcaatgcactgggcgttgtcgttgcg


 


cgcctgggcaaccggcgcgccctgcccttactgctggctatcgccacgctgttgtgtgccgctgcggct


 


tggccgttggcgaacaatatgcttgccctgatctgggcgattctggcgatgggcatgctcaacgccgct


 


gtcgagcaggtcaacgggctgccggtgggcctgacctacgtgaccggcgcgctgtcgcgactggggcgc


 


ggtctgggccgctggatgctcggcgaacgccggatggctggcgcattcaactggtcccgtgggccggg


 


atgttcattggcgcagtgatcggcgcgttgcttgaacatcgtctggggctcaatgccttgctggtcagc


 


gccagcctgtcagcgttaatggcgctggtgtcgctgaaaatcccgcatcgctggcaacgtcagtacatg


 


ccgcgctga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 80 as follows:









MCLVASLSVLAGMTDAIGFMATGDFVSFMSGNTTRLAVAISDGDLSVTLR



 


LALAIFAFIAGNALGVVVARLGNRRALPLLLAIATLLCAAAAWPLANNML


 


ALIWAILAMGMLNAAVEQVNGLPVGLTYVTGALSRLGRGLGRWMLGERRD


 


GWRIQLVPWAGMFIGAVIGALLEHRLGLNALLVSASLSALMALVSLKIPH


 


RWQRQYM






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A forty-first nucleic acid molecule encodes ORF42 and has a nucleotide sequence according to SEQ ID NO: 81 as follows:








atgagagggcttggtgttctgagcatgaaccaccagtttcagggcaataccctgttcaaagaaataagc



 


ggtaccagcttttccgcgccctacatcacccatcttgcgggccgtctccttaacgagcacccagaggca


 


tcggcgaacctcttgcgcgctatgctggtgaatcatgcgtcattgtctagcgaggtcgagacgactttc


 


tccgacgacatgaggaagggctacaaagctaataaggcgacccacaaccgtgaaatatcgcgcgatgtg


 


agtggttacggccaagtgaatgaggcagacctgtttcggtcttccgaccattgcgttgtgctgatgtgt


 


gaagagtccattgagaaggactcgtgccagttctacgaactgcctttgcccacttcgtttcttcgcagg


 


gctagaggggcaaggcacctgagcgtcacgctggcttattctcctgccgtcaggacaactcggttggac


 


tatctggcaactcagatcagttatcgcctagtgaaaggttcgtcgcttgaggaagtccaggcctcgttt


 


aactacgacaagcaggacgaaacgaagacccgtggagatgacgctgagcagaatcgagacatcactgct


 


cagttgagaagccgcgggaccgtccagtcctcgcggtggacgttcaagaagcgaaatccagaagaaaaa


 


tggtttgtagttgtgatccgccaggatcgggaatggaatcatcccgacgtgctggatcgagaatcttac


 


gccctggtggtaacagttgctgatcgtgacaacgaacacgcgcagttgtatgccgaaattcaagccaag


 


ctgacgcttcaaaatcaggtgcgtgaagaggcaaggcagcgggctgttctgtaa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 82 as follows:









MRGLGVLSMNHQFQGNTLFKEISGTSFSAPYITHLAGRLLNEHPEASANLLRAMLVNHASLSSEVETTF



 


SDDMRKGYKANKATHNREISRDVSGYGQVNEADLFRSSDHCVVLMCEESIEKDSCQFYELPLPTSFLRR


 


ARGARHLSVTLAYSPAVRTTRLDYLATQISYRLVKGSSLEEVQASFNYDKQDETKTRGDDAEQNRDITA


 


QLRSRGTVQSSRWTFKKRNPEEKWFVVVIRQDREWNHPDVLDRESYALVVTVADRDNEHAQLYAEIQAK


 


LTLQNQVREEARQRAVL






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A forty-second nucleic acid molecule encodes ORF43 and has a nucleotide sequence according to SEQ ID NO: 83 as follows:








atgggcattggcggtttgcttaaacctttggtcgattttttaccgaagttgccgaccttacgcaccaag



 


atttcctcgccttccatcagctacgcgcgtttgcaaagcgatgcgtcccaggtacgcagtaaattggga


 


ttgggtgagcgcagcgtgctgggttatgaagcgctgatcgccgagttcaaggcgtgcggggcggttctg


 


gtgcccgttctttggggacaaaagcagcaacacaagaatgcgttgcacattctattgccggcgtcagat


 


gtcacctttgtcttcgtcaacctggataccaagctggaagacttcaagttttggatggcccacgagtta


 


gcgcatgtctacactcctgagcttgcgggtagtgacgagggggaggattttgcggatgcctttgccggt


 


gccctgctgtttcctgaggcttgcgtgcagctagcgtatgccgaggcggcgcaagcgcctagcgcagct


 


ggggaggtgagtgtccttcagcagcatgcccggcatcaccaaatttcactgaacacggtgttccagcag


 


gcgcagggatatgcggcggaaaacaatctgccatccttacgggtaccggaaaggacaattcacgcggtg


 


cgcaacatctccacgccgcagttggtcagtacgatcctgtttgatccgactccacccaaaccggcgcaa


 


tacattgccgcagcgtcgaatgtgtttcagtctgagttcttcctggcgctgaaacgcatgattcgcgag


 


cacgggacgggcccgtcgtatgttcagcaaatcatggatgtatcactcagtgatgcctccgcgctttac


 


ggcgagctcgcgcgttga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 84 as follows:









MGIGGLLKPLVDFLPKLPTLRTKISSPSISYARLQSDASQVRSKLGLGERSVLGYEALIAEFKACGAVL



 


VPVLWGQKQQHKNALHILLPASDVTFVFVNLDTKLEDFKFWMAHELAHVYTPELAGSDEGEDFADAFAG


 


ALLFPEACVQLAYAEAAQAPSAAGEVSVLQQHARHHQISLNTVFQQAQGYAAENNLPSLRVPERTIHAV


 


RNSSTPQLVSTILFDPTPPKPAQYIAAASNVFQSEFFLALKRMIREHGTGPSYVQQIMDVSLSDASALY


 


GELAR






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A forty-third nucleic acid molecule encodes ORF44 and has a nucleotide sequence according to SEQ ID NO: 85 as follows:








atgaagcagctcgcggcaggcagcaatgtgcatgttcttgaaaatgagtctttccagatagataaggtg



 


cgctttttgggggccacagcttggacagatttcgcaacaggtgaaagcgtgtaccaagcgtcccaggag


 


gcaaggcgaggcatgaatgactttcgcttgatccgtgcaggcgagggttaccgcgcattgagcatcagt


 


gatgtgatcagtcgaaatcatcgaacttacgagtggctcaaggaagagctcgccatggagttcgatggt


 


cagaccattgtcatcactcatcattgcccgttggtcaattactgtggcccagagcagggctcaccgcta


 


atgcctgcttattcaaatgattggccagaactcgttcgtcaggctgatgtgtgggtctttgggcacacg


 


cacagtcatgtcgatgtcatggtggaaggatgccgactcattagtaaccctagaggttatccaggtgag


 


agttgcggctttgccaatgactttgtggtcgatattaactag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 86 as follows:









MKQLAAGSNVHVLENESFQIDKVRFLGATAWTDFATGESVYQASQEARRG



 


MNDFRLIRAGEGYRALSISDVISRNHRTYEQLKEELAMEFDGQTIVITHH


 


CPLVNYCGPEQGSPLMPAYSNDWPELVRQADVWVFGHTHSHVDVMVEGCR


 


LISNPRGYPGESCGFANDFVVDIN.






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A forty-fourth nucleic acid molecule encodes ORF45 and has a nucleotide sequence according to SEQ ID NO: 87 as follows:








atgacgctgacgcagcgtcaggcatggcatcgcgaggcacagcggtttggcgagcaggtggtgaacatg



 


cgcaaagccagcaaggcgcacttcggccaggcggaaaatgacagccgcacctatccggcgcgctttatc


 


gaccagcaactggctcaactgctgaaccggctatccatcgctgcaacggcgcaacagatcaatatttca


 


ctgacctacaggacgggcaccgaagtgctcgaaattcccggcgcgcctgtattgccagaaaccgagacc


 


gagaacgtttcactcaggcaactggtgcatacccaggccctgcgcaccaaggccaaggatgccgtgctt


 


ctacgcgctgtcgacgccgaaggcgtcccccttgcgcacttggacaagcaggccgtaaccgagctgatt


 


gccacgctggaagatcaccgatacctcagtgattaccttgacctgcacctgaaaacctcggcgtatgca


 


cagcagctcaagcggtcagaaaaagccatgttgcaagctcagatgaagatggcgctgctggagatcgag


 


caacaggcttttgcaccagccggtcgcgagtggatcaaggctgtgctggattcgccagccccccaagga


 


cgtcgaaccatggcaggggaaagcattgaagtccgttttttcagcgtcaaccaattcaagatgaccaat


 


gtcatgctgattgctccagccggtaaattcgagaaggggccgctggtgctttgcacgctggatgcttcc


 


gacggtgtggttttccgctggtttaacagcatgtatcacctgaccaccagctttctggaagaggcaccc


 


ttccagcagtatctgattcagcaaataccggtttccaggcgtcttgagacgctgcatgccatgcagtac


 


gaaaaggaagccaagcattggcgtccgccagaagtattcacccaactgacgctgctaccgatcccgtca


 


aggctgctgcgcccagtcgtgtttgtcagccagagcaaagacatttacgaggaaaatcacgagaccaag


 


atcaaccatctgatcaacgaagccaaacggcagatgagcctgtccaccggtacagggcaatcgggtcgg


 


ggcttcgatctgatcgcgagcattgcgattctgtttctgcctggcgcgatcatgatgcctgtctcgctg


 


ggcgctggcctttacaaaacctggagcgctttttcgaaaatcgatgaaaacgacctggaaggtgccgcc


 


gaggagtttctgagcgccctcagctatcttgccattaccttggtcggccatttggcgctggccttgaaa


 


ccggcaggaagcgccgcaaaaacggtgagacgtccgcacctggtacgcagagtcggtcgtgatgggcag


 


gcacagatcggctacctcctgtcgcattcaaaagcgccgcgtttcccagactcgaaattgatcgctgca


 


atggaccccaaacgcttcgtcgccattgaggtagaaggccagacctgcttaataagccggcgggccaac


 


ctgttcggccactcacgcctttatcgggtaaacccgatggatgcaacgcaactggtgcacgagcaggag


 


tttgccttgcgcagcaccaacggcacctggaaaatcgtgggcaaacagatcctgcgcatgagtcagtcc


 


gcaatccgcaatgcccaggctcaactgaccagcctgacaaatctctggccggcgtctctggaggaagca


 


agtagcgccgaacgcttgagcttcgagaccgactacctggcgctggcccagacatccaacgcagaaaac


 


tattccgaaatagtcgcctacgtggaaagcggttcaacagacatcaacccgctgctgcgaagcggcgtg


 


cgcaacgccaccacgcgcagatttttacgtcagttccataaactcaatgcgtgggaaggcactgccttt


 


cgcgccacctatgtgtccagcgacggggtggcatgccttgagcgcgaagtgggttcggtgttcaccgac


 


aacggcgtgcagtctgcatcggtgtcgcgagccaatgcctccagatggagccaggacgggttcgtgagc


 


agcaacgccaatgccgcaaaccacccggtgttcttcatctttgcaccgggagtgcccaagaagaacatg


 


ttcaccggctttcttggcgatcacgtggcaatcccgccaggcacgtgcgtgcaactgggtgcgaccaag


 


cggataaacggacagctgtttgcctggttcgatgcgcccgaacaaatggtcgatcagacctacgatctc


 


tatacaggagaacaggaactctgggtctga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 88 as follows:









MTLTQRQAWHREAQRFGEQVVNMRKASKEHFGQAENDSRTYPARFIDQQLAQLLNRLSIAATAQQINIS



 


LTYRTGTEVLEIPGAPVLPETETENVSLRQLVHTQALRTKAKDAVLLRAVDAEGVPLAHLDKQAVTELI


 


ATLEDHRYLSDYLDLHLKTSAYAQQLKRSEKAMLQAQMKMALLEIEQQAFAPAGREWIKAVLDSPAPQG


 


RRTMAGESIEVRFFSVNQFKMTNVMLIAPAGKFEKGPLVLCTLDASDGVVFRWFNSMYHLTTSFLEEAP


 


FQQYLIQQIPVSRRLETLHAMQYEKEAKHWRPPEVFTQLTLLPIPSRLLRPVVFVSQSKDIYEENHETK


 


INHLINEAKRQMSLSTGTGQSGRGFDLIASIAILFLPGAIMMPVSLGAGLYKTWSAFSKIDENDLEGAA


 


EEFLSALSYLAITLVGHLALALKPAGSAAKTVRRPHLVRRVGRDGQAQIGYLLSHSKAPRFPDSKLIAA


 


MDPKRFVAIEVEGQTCLISRRANLFGHSRLYRVNPMDATQLVHEQEFALRSTNGTWKIVGKQILRMSQS


 


AIRNAQAQLTSLTNLWPASLEEASSAERLSFETDYLALAQTSNAENYSEIVAYVESGSTDINPLLRSGV


 


RNATTRRFLRQFHKLNAWEGTAFRATYVSSDGVACLEREVGSVFTDNGVQSASVSRANASRWSQDGFVS


 


SNANAANHPVFFIFAPGVPKKNMFTGFLGDHVAIPPGTCVQLGATKRINGQLFAWFDAPEQMVDQTYDL


 


YTGEQELWV






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A forty-fifth nucleic acid molecule encodes ORF46 and has a nucleotide sequence according to SEQ ID NO: 89 as follows:








atgactcagctaaaccctgcgggacaaccgcccgcagaaccgacccgaatcgtcaaagctcacattgac



 


ctcatggatcctgccgaaagcgctgactacgaggcgacccgaatggcattgctcgcagcgatgcaaagc


 


ggcaatgccgcgatcaacctcgaacagattcggctcaagcccgacccagcgtccgggttcggcgaatac


 


tgcgctgagaaagctgcgctacctcacccggtccaggccgaaaaccaggaactcccgtttcagatagac


 


agcgatggcagcgtcagtctggcattgatgctgcgctataactacgggttgtcgctgccgcaatcgcct


 


gacgaaacagcgatcaaaaccctgctcaatacgctggcagaacttcgcaccagtcaagaactggggctt


 


attgatcagttcgacatcaaggccatgctgaccatgcaaaatctgcaggatctgaagcgagcctgcatt


 


gagtaccttggcaccgacggtggcacgctgctaggcaagctgggtgctgaaataattgcctcctgccca


 


gagtaccttggcaccgacggtggcacgctgctaggcaagctgggtgctgaaataattgcctcctgccca


 


ctggcagatgtgcagaactccccggtgacggttattgcccggattctcagatcggaaccggcaagggca


 


ttggggcaaacgctgctggcacagcttggtcggcctgaagaagaaacggacgcgtccctgacaacactc


 


gtggaccggattttatggtatgccatcagtagcgatcttcatgatccagaaaaccggaagccaggagaa


 


attgccggctatccattcacccaggccgaaaaccagggacgccgccacgctgacatcctgaacgatatt


 


cacaaccacctgatcaccacgggcaaggctgagtctgtcaacgaagcaataattgcctgcttcatactt


 


gcactcgatgactgcccggaatggctggtcagcagtgttcccgatgatctgccatacggctgtacagag


 


gtgtgggtcaactttcaacatggggtcacacttgcggaagtcatcgagtttggctcgtcacgctggatg


 


aactttgaagacctgatcgagctgccggtgattttcaacaaaaagatggacaccgaagagcagcaagtc


 


gcctatgtcgcaacgcgcatgcccattcttctgacttgggcccaggccaacggttacattcgtacccag


 


agcgacctgccttactccgaacaagagatagaacaggccgccagcgcgtttgaacactccgagaaacaa


 


tcccttgaagctgcgaacgccttgatccggaaagcgccagaacgcaaagccatggctatcagtgccatg


 


aaagaagcgcggaggacgcctgaaatagaaaaaatacttgagcaggaagattactggtttccgcccatc


 


gatctcggcatcaggctggcggtgctacgcaaaaatcacacgcctgtctatcgcgatcaccaaggcacg


 


ctctcaccgtcaaatctgccatacgacccctacggcataaaacacaaggcgtcgtcgttgctggagatc


 


tacatggcaggtgaaaacattgatgactggagactgccggggcgcaacagcaacgagggcctgcttccc


 


atcaaccgtgaaatgcagttgttgtacaaggcgctgccagacatcaatcaaaggttcgagagtgaattt


 


caggcttatctggcagatgcccgtaaggcgtatgcgacgattatcagaaagttgctgactcacctgccg


 


ctcaagcaccgcatggcgatcgaaaatggtgaggtgtcgctacactcactcagattgccgaccaaggac


 


gtgctggcggcgacagagagcgaaaaacatcgggagccgttgcgagggcgcacgggctttgtcatcaaa


 


gctgtctacgagggcaaaaccacgttttacgaggtgtttccgttatcgatgattgtacgctatcgccct


 


gatctggaggcccttctcaagaacggtgtggtcggtatagatttttgggacattctgcctcccacccgt


 


ataccggtagcggtttataacggaatcacaatgccatttgatcagggagcctatttgaacggtcagcta


 


cctgagcctggggcaagcgctgtgatgattgcagaaaccattggtgaacgatttgattcttcaagtgca


 


gaggtcgggcaacaccagcctccgacctcgttttcaaaacgctctactggcattgccgagaccatcaca


 


acatcgcttttctacgtcaacgaagatgcactctttgcacactgcaaaagcctcacgcaggtagaaata


 


gataacggtgccccaggtgcgctcgaagaggtgtccagctttctgatacacctgacgccctggccggaa


 


atcgaaaacattctgtccggagagaaagcgcttatgaggggaggagcaatcggtctggcgctttacatg


 


attccctatgtgggacccgcgggcaagttgctcgcaggcacggcaaaagtcgttacccgcctgggcaaa


 


agcctcataaccagcggtagcaaagtccaggtctcgaaattgctcatcacggccggcaccaccctgaaa


 


gacgccccgctgatcatgatcagacaggcccctgacatgaccagtaaagcaatgactggcgtttcgcaa


 


ttcgtcgtgaaacacgtcacctggaaatttctggcgatacgtataggtattggtttaagccgcaggctt


 


gtagccatcatgagcaggcagcaggcccaggccgcaaagcaagaggccacgtaa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 90 as follows:









MTQLNPAGQPPAEPTRIVKAHIDLMDPAESADYEATRMALLAAMQSGNAAINLEQIRLKPDPASGFGEY



 


CAEKAALPHPVQAENQELPFQIDSDGSVSLALMLRYNYGLSLPQSPDETAIKTLLNTLAELRTSQELGL


 


IDQFDIKAMLTMQNLQDLKRACIEYLGTDGGTLLGKLGAEIIASCPLADVQNSPVTVIARILRSEPARA


 


LGQTLLAQLGRPEEETDASLTTLVDRILWYAISSDLHDPENRKPGEIAGYPFTQAENQGRRHADILNDI


 


HNHLITTGKAESVNEAIIACFILALDDCPEWLVSSVPDDLPYGCTEVWVNFQHGVTLAEVIEFGSSRWM


 


NFEDLIELPVIFNKKMDTEEQQVAYVATRMPILLTWAQANGYIRTQSDLPYSEQEIEQAASAFEHSEKQ


 


SLEAANALIRKAPERKAMAISAMKEARRTPEIEKILEQEDYWFPPIDLGIRLAVLRKNHTPVYRDHQGT


 


LSPSNLPYDPYGIKHKASSLLEIYMAGENIDDWRLPGRNSNEGLLPINREMQLLYKALPDINQRFESEF


 


QAYLADARKAYATIIRKLLTHLPLKHRMAIENGEVSLHSLRLPTKDVLAATESEKHREPLRGRTGFVIK


 


AVYEGKTTFYEVFPLSMIVRYRPDLEALLKNGVVGIDFWDILPPTRIPVAVYNGITMPFDQGAYLNGQL


 


PEPGASAVMIAETIGERFDSSSAEVGQHQPPTSFSKRSTGIAETITTSLFYVNEDALFAHCKSLTQVEI


 


DNGAPGALEEVSSFLIHLTPWPEIENILSGEKALMRGGAIGLALYMIPYVGPAGKLLAGTAKVVTRLGK


 


SLITSGSKVQVSKLLITAGTTLKDAPLIMIRQAPDMTSKAMTGVSQFVVKHVTWKFLAIRIGIGLSRRL


 


VAIMSRQQAQAAKQEAT






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A forty-sixth nucleic acid molecule encodes ORF47 and has a nucleotide sequence according to SEQ ID NO: 91 as follows:








atgtctgttacttcatctgtcctgcgactgtcgcgcctgagcgtgtcgttatcacttttgggcatgctg



 


tcgtctgcactgtttgccggcgcggcattcgccagcgacgagacgcaactgatcgaatccctcaacgcc


 


taccgtggccaggcgcagcgctgtggcgagcaggtgtccatggaactgccgccgctgagcaccgacccg


 


cgtctggtgctgcccgccagtggcaacctgaacctgcaacagtcgctgacccgcgcgtcttatcc9atg


 


gtcaccgtgcaggcgatcagtctgtccggaccgcgagatgcggcgtcggcgttgaaggcggtgcaggag


 


agtttctgccgcgtggtgctggacccgcagttcgtcgatatcggggtcagccgggacgggcgcgactgg


 


cgcatcgtgctggcgcgctcgctggtggcatcacgtctgggtgactggcaagcagaaggtcagaaaatt


 


ctggagatgatcaacaccgcccgtacccaggcgcgtcagtgcggttcgcaatccttcgcggccactaca


 


ccgttgagctggaatcaggtattggggacggccgcacaaggacactcgcaggcaatggccaatcagaac


 


ttctttgaccacaaggggcgcgacggccgcacgccgggtgacagggccgagcttgccggctatctgggc


 


cagcagatcggtgagaatattgccgcaggccaggacactgcccgcaaggtggtggacggctggctggtc


 


agcccgggccactgcgcaaacctgatgacccccggttttcgcgagctgggagccgcctacgcgatggac


 


cccaaaagtgacgcggggatttactggacagccatgttcggcacgcagcaatag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 92 as follows:









MSVTSSVLRLSRLSVSLSLLGMLSSALFAGAAFASDETQLIESLNAYRGQ



 


AQRCGEQVSMELPPLSTDPRLVLPASGNLNLQQSLTRASYPMVTVQAISL


 


SGPRDAASALKAVQESFCRVVLDPQFVDIGVSRDGRDWRIVLARSLVASR


 


LGDWQAFGQKILEMINTARTQARQCGSQSFAATTPLSWNQVLGTAAQGHS


 


QAMANQNFFDHKGRDGRTPGDRAELAGYLGQQIGENIAAGQDTARKVVDG


 


WLVSPGHCANLMTPGFRELGAAYAMDPKSDAGIYWTAMFGTQQ






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A forty-seventh nucleic acid molecule encodes ORF48 and has a nucleotide sequence according to SEQ ID NO: 93 as follows:








atgccgttattaaactggtccagacacatggttcatttaacagccatcggccttatcagcattccggct



 


gcctatgcagcggacaccctgacccgcgacaatggcgcagcggtcggcgacaaccagaactctcagact


 


gcaggcgcccaagggcctgtcctgctgcaagacgtacagctgctgcagaagctgcagcgttttgatcgc


 


gggcgtatcccggagcgtgtggtccacgcacgcggcactggcgtgaaaggcgaattcacagcgtccgcc


 


gacatcagcgacctgagcaaggcgaccgtcttcaaatcgggtgagaagaccccggtattcgtacgtttt


 


tcttccgtggtccacggcaaccactcgccagaaaccctgcgcggcccgcatggcttcgccaccaagttc


 


tacaccgctgatggcaactgggacctggtaggcaacaacttcccgacgttcttcatccgcgacgccatc


 


aagttcccggacatggtgcacgccttcaagcctgacccgcgtaccaacctggacaacgactcgcgccgc


 


ggcggcttcttctcgcatgtaccggaagccacgcgcacgctgaccctgctgtactccaacgaaggcaca


 


ccgaccggctatcgcttcatggacggcaacggcgttcacgcctacaaactggtcaacgccaaaggcgaa


 


gtgcactacgtcaagttccactggaagacgctgcaaggcatcaagaacctcgaccctaaagaagtcgct


 


gaggttcagtccaaggactacagccacctgaccaacgacctggtcggcgccatcaagaagggtgacttc


 


gcgaaatgggacctgtacatccaggtgctgaaacctgaagacctggccaagttcgacttcgacccgctg


 


gacgccaccaaaatctggcctgatgtgccagagaagaaaatcggccagatggtcctgaacaagaacgtc


 


gacaacttcttccaggaaaccgagcaggtcgccatggcacccgccaacctggtccctggtatcgagcct


 


tccgaagaccgtctgctgcaaggtcgagtgttctcctatgccgacacgcaaatgtatcgcctgggtgcc


 


gacagcggcaaaaccaccagcggcgtgaactacgagcctagccgtctggaaccccgtcctgccgatgag


 


aaagcacgttacagcgagctgccaatcagcggcactacccagcaggcgaagatcacgcgtgagcagaac


 


ttcaagcaggcgggtgatctgtatcgctcttacaacgcgaaagagcagaccgacctggtgcagagcttc


 


ggtgaatcgctggccgacactgacaccgaaagcaagaacatcatgctgtcgttcctctacaaggcagac


 


cccacctatggcactcgggtaaccgaagcggccaaaggcgatctggccaaggtcaagtcactggctgcc


 


agcctgaaagactga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 94 as follows:









MPLLNWSRHMVHLTAIGLISIPAAYAADTLTRDNGAAVGDNQNSQTAGAQ



 


GPVLLQDVQLLQKLQRFDRERIPERVVHARGTGVKGEFTASADISDLSKA


 


TVFKSGEKTPVFVRPSSVVHGNHSPETLRDPHGFATKFYTADGNWDLVGN


 


NFPTFFIRDAIKFPDMVHAFKPDPRTNLDNDSRRFDFFSHVPEATRTLTL


 


LYSNEGTPTGYRFMDGNGVHAYKLVNAKGEVHYVKFHWKTLQGIKNLDPK


 


EVAQVQSKDYSHLTNDLVGAIKKGDFPKWDLYIQVLKPEDLAKFDFDPLD


 


ATKIWPDVPEKKIGQMVLNKNVDNFFQETEQVAMAPANLVPGIEPSEDRL


 


LQGRVESYADTQMYRLGANGLSLPVNQPKVAVNNGNQDGAMNSGKTTSGV


 


NYEPSRLEPREADEKARYSELPISGTTQQAKITREQNFKQAGDLYRSYNA


 


KEQTDLVQSFGESLADTDTESKNIMLSFLYKADPTYGTRVTEAAKGDLAK


 


VKSLAASLKD.






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

This protein also has significant homology (e=0), as detected by BLAST search, to catalase isozyme catalytic subunit CatF from Pseudomonas syringae pv. syringae (GenBank Accession AAC61659, which is hereby incorporated by reference in its entirety).

A forty-eighth nucleic acid molecule encodes ORF49 and has a nucleotide sequence according to SEQ ID NO: 95 as follows:








atgggggtttcgagctgcggcaaaagtgccgtcggtgcagaaatcgcccg



 


taacagcggcggtcgcctgatcgaaggcgatgcgttccatccccaggcca


 


acatcgacaagatgagcgccggcacccccctcaccgacgaagaccgtgcc


 


ggctggctgacccgtctgggtgaagaactggccgcagcccttgccaaggg


 


cgaacatccggtgctgacctgttcggcactcaagctcatttatcgtgaac


 


gcctgcgtgcggcggtgccgggcctgggttttgtctttctcgaactgagc


 


aaagagctggccaccgagcgttgcgccaaccggaccgggcatttcatgcc


 


tgcgagtctggtcgatagccagttcgcgaccctggaaccaccgatcggcg


 


agccactgaccctggtggtcgatgccagcaagcctatcgatgtaattggt


 


gaacaagccgcggcatggtggaaaggctctcacgcctga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 96 as follows:









MGVSSCGKSAVGAETARNSGGRLIEGDAFHPQANIDKMSAGTPLTDEDRA



 


GWLTRLGEELAAALAKGEHPVLTCSALKLIYRERLRAAVPGLGFVFLELS


 


KELATERCANRTGHFMPASLVDSQFATLEPPIGEPLTLVVDASKPIDVIG


 


EQAAAWWKGSHA






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted. This protein also has significant homology (1.1e-52), as detected by BLAST search, to gluconokinase from Pseudomonas aeruginosa (Stover et al., Nature 406:959–964 (2000); GenBank Accession AAG05709, each of which is hereby incorporated by reference in its entirety).

A forty-ninth nucleic acid molecule encodes ORF50 and has a nucleotide sequence according to SEQ ID NO: 97 as follows:








atgcgaccggtgtctatgttttccctgcgttccatttgtgctgccgcactgtttgcgctttgcctgtct



 


atcttcccggcgctggccgccgagccgcccacccgcgatgccgtgcagcaaagcctcgacaagattgcc


 


gaccgcaagctgccggatgccgatcagaaggccttgcagcaggtgcttgagcagacgctggcgtttctc


 


aacagcaaagacgacagcgagcaaaagctgaccgcgctcaagcagcagctggctcaagcgccaaaacag


 


acctcggacaaccagcgcgagctggcccggttgaaagaaagcaaagtcgttgccgttgcacagcgctac


 


ggtggcctcgatgtgccgcaactggagcgactgctcagccagcgcagcacccagcaaagtgatctgcaa


 


agcgagcttaacgacgccaacagcctggccatcaccgcgcaaacccggccggagcgggcgcagactgaa


 


atcagcgccaatcagacacgcatccagcagatcaatgccatcctcaagaatggcaaagacaacggcaag


 


accctgagtgccgatcagcgcaatctgctcaatgcggaactggcctcgatcaacgcgctgaacctgctg


 


cgccgtcaggaactggccggcaacagccagttacaggacctgggcaacagccagcacgacttgctgacc


 


gaaaaagtcgcccgccaggagcaggaaattcaggacctgcaaaccctgatcaacgacaagcgccgagcc


 


cagtcgcagaaaaccgtggcggacctgtctctggaagcgcagaaatccggtggcagcagcctcctggcg


 


accgagagcgccgccaacctcaagctgtccgattacctgctgcgcggcaccgaccgtctcaacgagctg


 


acccagcaaaacctcaagaccaagcagcaactggacaacctgacgcagaccgatcaagccctcagcgag


 


cagatcaacgtgctgagcggcagcctgctgctgtccaagattctctacaagcaaaaacagtcgttgccg


 


cacctggaactggacaaaggcctggctgacgaaatcgccaacatccgcctttatcagttcgacatcaat


 


cagcaacgcgagcagatgagcacaccgaccgcttacgtcgaacgactgctcgccacccagcccccggaa


 


aatatcaccccgcaactgcgcaggacgctgcttgatctggccatcacccgcagcgacctgctcgaacgc


 


ctgaaccgcgagctgagcgcgttgctcaacgagtccatcacgctgcaattgaaccagaagcagttgacc


 


agtaccgccgtcggcctgcgctccacgctggacgagcagatgttctggatccccagcaacaagccgctg


 


gatctggagtggttccagaacatctggccgcgcctgcaaaaacaggtcgcgaccctgccctggacgtcc


 


agcctcagcgagctgtcggacggcttgacacaacgcccgctgctgtttctgccattgttactgctgatc


 


ggtgtactgacctggaggcgcaaggcgctttaccagaagctcaaccggctgcacgccgacatcggccac


 


ttcaaacgcgacagtcagtggaaaaccccgttggcgctgctgatcaacgtgctgctggccatgccggtc


 


gcattggggctggcgctgtgcggctacgccttgcaaatcgatgcgcgcgggcaaaacgccaaccttggc


 


gaggccttgctgcagatcgcgctggcctggctagtgttctacaccgcctaccgcgtgctggccccgtcc


 


ggcgttgcgcaactgcactttcgctgggaaccggcgcaggtcgcgttcttgcgcggctgggttcgtcgc


 


ctggggttggtggtgctggcgctggtcgccgtggtggcggtcgccgagcatcaaccggccgcgctggcc


 


gacgacgtgctgggtatcggcgtggtgctgacctgttacgcgctgatgacctggctgctgggccgattg


 


ctgctctccagccctacgcaccacaacgcgtcgctgttccgcaagacgctgggtgtggcgttcacggca


 


ttgccggtcgcgctgtttctggcggtgtgcttcggctactactacaccgcactcaagctcagcgaccgt


 


ctgatcgacacgctgtacctgatgatgatctggctgatggtcgaggccaccttcgttcgtggtctgggc


 


gttgccgcgcggcgactggcctaccagcgtgcgctggccaaacgtcaggctgcgcgagaaaacggtgac


 


agcgacatccccgtcgaagaaccgaaactggacatcgaacaggtcaaccagcagtcgctgcgcctgatt


 


cgtctggccttgctggctggtttcgtcggcgcgttgtacctggtctgggccgagctgatcacggtgttc


 


gcctacctggacaacatcatcctctacgaatacacaagcggcacaggcgccaacatgagcatggtgccg


 


atcagcctgagcgacttcctcggtgccggggtcatcatcgtcattacctttgtgctggcgggcaacctg


 


cccggcttgctcgagtgctggttctgtcacgcatgaacctggtcgcaaggcagcgcctatgcgaccacc


 


acgctgctctcctacaccatcgccggcatcggctttgtgaccacgctgtccacattaggcgtgagctgg


 


gacaagctgcagtggctggtcgcagcgctgtcggtgggcctggggttcggcatgcaggagatcttcgcc


 


aacttcatttccggcatcatgatcctcttcgagcgcccggtacggatcggcgacaccatcaccatcggc


 


gccctgtcgggtacggtcagcaagatccgcatccgcgccacgaccatcaccgacttcgaccgcaaggac


 


attatcgtcccgaacaagaccttcatcaccggccagctcatcaactggtcactgactgacaccgtcacc


 


cgcgtaacgctcaagctgggtgtggattacggctcggacctggacctcgtgcgctccctgctgctgcaa


 


gccgcacgggaaaaccctcgggtgctcaaggagccagagcccattgtctacttcctgaacttcggcgaa


 


agcaccctcgaccacgaactgcgcatgcacgttcgcgacctgggcgaccgcaacccggtactcgacgag


 


atcaaccgcttcatcaaccgcgagttcaagaaacagcacatcaacatctcgttccgccagatggagatc


 


tacctcaaaaacacccagggcctggaatacaaactggtgcccgccgaaccaggcgaaaagcacggcgca


 


ccggctgggcaaaccacgctgcaaccggtaaacaccaaagtagccccggcaaccaaagatgcgccagag


 


ccgccggagttgaggctggactga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 98 as follows:









MRPVSMFSLRSICAAALFALCLSIFPALAAEPPTRDAVQQSLDKIADRKLPDADQKALQQVLEQTLAFL



 


NSKDDSEQKLTALKQQLAQAPKQTSDNQRELARLKESKVVAVAQRYGGLDVPQLEQLLSQRSTQQSDLQ


 


SELNDANSLAITAQTRPERAQTEISANQTRIQQINAILKNGKDNGKTLSADQRNLLNAELASINALNLL


 


RRQELAGNSQLQDLGNSQHDLLTEKVARQEQEIQDLQTLINDKRRAQSQKTVADLSLEAQKSGGSSLLA


 


TESAANLKLSDYLLRGTDRLNELTQQNLKTKQQLDNLTQTDQALSEQINVLSGSLLLSKILYKQKQSLP


 


HLELDKGLADEIANIRLYQFDINQQREQMSTPTAYVERLLATQPPENITPQLRRTLLDLAITRSDLLER


 


LNRELSALLNESITLQLNQKQLTSTAVGLRSTLDEQMFWIPSNKPLDLEWPQNIWPRLQKQVATLPWTS


 


SLSELSDGLTQRPLLFLPLLLLIGVLTWRRKALYQKLNRLHADIGHFKRDSQWKTPLALLINVLLAMPV


 


ALGLALCGYALQIDARGQNANLGEALLQIALAWLVFYTAYRVLAPSGVAQLHFRWEPAQVAFLRGWVRR


 


LGLVVLALVAVVAVAEHQPAALADDVLGIGVVLTCYALMTWLLGRLLLSSPTHHNASLERKTLGVAFTA


 


LPVALFLAVCFGYYYTALKLSDRLIDTLYLMMIWLMVFGATFVRGLGVAARRLAYQRALAKRQGRENGD


 


SDIPVEEPKLDIEQVNQQSLRLIRLALLAGFVGALYLVWAELITVFAYLDNIILYEYTSGTGANMSMVP


 


ISLSDFLGAGVIIVITFVLAGNLPGLLEVLVLSRMNLAQGSAYATTTLLSYTIAGIGFVTTLSTLGVSW


 


DKLQWLVAALSVGLGFGMQEIFANFISGIMILFFRPVRIGDTITIGALSGTVSKIRIRATTITDFDRKD


 


IIVPNKTFITGQLINWSLTDTVTRVTLKLGVDYGSDLDLVRSLLLQAARENPRVLKEPEPIVYFLNFGE


 


STLDHELRMHVRDLGDRNPVLDEINRFINREFKKQHINISFRQMEIYLKNTQGLEYKLVPAEPGEKHGA


 


PAGQTTLQPVNTKVAPATKDAPRPPERLRD






The protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted. This protein also has significant homology (e=0), as detected by BLAST search, to putative potassium efflux system from Yersinia pestis (Parkhill et al., Nature 413:523–527 (2001); GenBank Accession No. NP—406604, each of which is hereby incorporated by reference in its entirety).

A fiftieth nucleic acid molecule encodes ORF51 and has a nucleotide sequence according to SEQ ID NO: 99 as follows:








atgtcaacgttgaatcatacgtctgctgtaaattgccgcgtcagttttgatggtgaccgttgctatgta



 


gacacccccdtccagatcatgccgggtgagcgatgggctgtaaatatcgtacctaacgatttagtcaca


 


atccactacgaggccgccagcaatcacgactaccctttgctgctggccagcataaaaaatctgtttacc


 


gatgagcgttgtgtcgtgctaaagcccggccttacacagcaagctttgaacatgtatttttcagaggtt


 


aacagccttaaacctaacgcgactcatgttcgcttgttgcatcgagcgcagcgtatttttctagaaaac


 


atgatccgtagcgtacagataacctcgcaaggtatcagcgtcactttcgcaaccgccgaattcaaaaat


 


tataactaccagctaaaggtggataaatatacttttgcaaggcttgacaaggggtaccctctctattcg


 


gagctggttgaaaacacctggataacgaaattatccgtagcccataatattctgtattccatctctgtg


 


agcctggaccactcaagcacaccttatacacttttttcaggaaccctcgcggaagacaatatagtccag


 


ccgatacgggcgcttttcaccgacaacaccatgactcaactcacctccttggccgatcagaaaaccgtg


 


gatgccttgtatacgacggtcaatggcaacccggttatcagcatcaaaaaacgcgcagattatcggtct


 


tatctgaacatcgcacagaagttactgcttccaagaacctacaccaaagtagtacggacagtgagcagc


 


ctgtctgtgcattttacgggggaggcgtacaaacaattcaactacaagatgcttgtcaacaatgcttat


 


gcatccgagatcacccgagggaaggcttattactccagcgtgagcaatggggtgtggaccacttccggt


 


acgcatgacagcgacgacaactgcaaagtcacttgtgattacaagggcgcaacctacgtcctgtacgag


 


agtaatgcggcagatagacgcactgaaacctgggcacaagacccgtacgttactcattgcgacccgaga


 


gacctgtaa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 100 as follows:









MSTLNHTSAVNCRVSFDGDRCYVDTPIQIMPGERWAVNIVPNDLVTIHYE



 


AASNHDYPLLLASIKNLFTDERCVVLKPGLTQQALNMYFSEVNSLKPNAT


 


HVRLLHRAQRIFLENMIRSVQITSQGISVTFATAEFKNYNYQLKVDKYTF


 


ARLDKGYPLYSELVENTWITKLSVAHNILYSISVSLDHSSTPYTLFSGTL


 


AEDNIVQPIRALFTDNTMTQLTSLADQKTVDALYTTVNGNPVISIKKRAD


 


YRSYLNIAQKLLLPRTYTKVVRTVSSLSVHFTGEAYKQFNYKMLVNNAYA


 


SEITRGKAYYSSVSNGVWTTSGTHDSDDNCKVTCDYKGATYVLYESNAAD


 


RRTETWAQDPYVTHCDPRDL






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A fifty-first nucleic acid molecule encodes ORF52 and has a nucleotide sequence according to SEQ ID NO: 101 as follows:








atgcgcctgatcgcgcagattctgcccggcctgccggaaaacaccacttacagcgccgccgctgcgtcc



 


aacaccctggcgcgggccatgcccaacgccattcgcaatgcgctgggcaccctggggctggtggctgcg


 


cgcacccagccaagcatctttccgttgccgtcgcgcaacgtcagcggtggcgaaaaagaggacgacctg


 


gagattctgctcaaactcgcggccgccgctgtttcgcgcctgcaaagccaccagttgggcggcctggag


 


cagacccgtaccaatgccgatggcactcaggtgactacatggcaactggaagtgccgatgcgcaacgcc


 


catgacatcgtgccgttgcaggtcaaggtgcagcgcgaagacaagcctgatcaggacgccaccgaagac


 


cgcgacgatatcgagatcaaggaaacccgtgaaaaactctggaaagtcgatctggctttcgacctggag


 


ccgcttggccccatgcaggtgcatgcgcaactgctgcgcggcacgctgtccagccagttatgggccgag


 


cgcccggatagcgcaacactgatcgaacatgaactggggcatttgcgcgagcgcgagcgccgattgccg


 


ctggccgtcggggaactggdgtgcagccatggcgttccgccgcaagggccgcgcaccgccctcgaacaa


 


cgctggatcgacgagaacgcctga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 102 as follows:









MRLIAQILPGLPENTTYSAAAASNTLARAMPNAIRNALGTLGLVAARTQPSIFPLPSRNVSGGEKEDDL



 


EILLKLAAAAVSRLQSHQLGGLEQTRTNADGTQVTTWQLEVPMRNAHDIVPLQVKVQREDKPDQDATED


 


RDDIETKETREKLWKVDLAFDLEPLGPMQVHAQLLRGTLSSQLWAERPDSATLIEHELGHLRERLIACG


 


LAVGEIACSHGVPPQGPRTALEQRWIDENA






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A fifty-second nucleic acid molecule encodes ORF53 and has a nucleotide sequence according to SEQ ID NO: 103 as follows:








atgagtagcgtcgcagcactgatcaccatatcgactggacagacgcagttcgttaaagtcgcgcggacg



 


tcattttctgtgctacgaatccccctcgccggcagatgtcgtgtgtcgtgtccgggatcagttgaccac


 


aagacagagcagaaacccataaaaatagggggaagagacgtgagcctaaatgatcacttgaaaaaagca


 


ttgattctgatccagcgacgagcttgatgaaatcaccgacctttatgtgacgttgcctgcagaggtcga


 


ttcagttgcttgaccatttcactcgaagggaattggaaggaaattgatagcgtctggtctgctcggtta


 


gacgcagcagattcaaagaataatacaaaatgtcacgtccatatcgccaaaaaccaagcatcatcgatc


 


aaaagcaaacaggtttcttggaacagtgatggtagccggcatgataaaaaaacattcgatgtgaacgct






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 104 as follows:









MSSVAALITISTGQTQFVKVARTSFSVLRIPLAGRCRVRDQLTTTIKTEQKPIKIGGRDVSLNDHLKKA



 


LNSDSSDELDEITDLYVTLPAEVFSCLTISLEGNWKEIDSVWSARLDAADSKNNTKCHVHIAKTKHRSS


 


KSKQVSWNSDGSRHDKKTFDVTLGQSRKAQAIARKFLGLGESISLESKDSKQMVERPLLSTATSFSNDG


 


KEVKVEFYVEESTAHLPAWLRW






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A fifty-third nucleic acid molecule encodes ORF54 and has a nucleotide sequence according to SEQ ID NO: 105 as follows:








atgaagccaatccatactgcccgatacaacgcctggaatcagttggagcaggagaccgcccatgactgg



 


ctgggggccaaacccttggccagcagcacccttggctaccgctacgatgactggaaccagcgatgctgc


 


accacgaccgatgacaacgtacagacttatgagtattcagacccgatcggcagcgacgtacataaaggc


 


ccaatccagaaaacctggaaacagagtggcgacccggagggccgcatcagtggccgcagcgaaacctgg


 


ctgaatctgttcggcaaaccggaccggatccggacgctgaccgctggtaaaacgggtcgcagccgcacg


 


cacagcatgagccgcagccggaacctgaccacgactgagcaggaactgagcaggcagacctttctgtac


 


gacgggctgggacgctgcaccgagcagcgcgatgcactccagcaaagcaccctgttcagctacgacadc


 


tggtcacgcatggtctcctccacgcttgcagacggcagcgtcatcaaccggagttatgcgccgcaaagc


 


agcagtgagctggcaacgatgctcgaggtcgtgcaccagaacggcaccaccagaaccgtggcaggtaca


 


cagaaatttgacgggcttgagcgtgtgacgcagaccaaaacaggtgaccgcgtcgaacagttcaactac


 


gacgccggtgagatgcagcccaggtcgcgcacaacagccgggctggacaacatcaactttacctacact


 


cgggcgctcactgatcagattttttccagcacggctccggatgaaacggccaaattcgattatgacaag


 


accagtgcccgcctcatcgaagcgacgaacccgcaaggcacgcgcacttaccgctatgacgtgcacaat


 


caactgacgggagagacttgggacaatctgctgggtcaggcttgggaaacccgacaccaatcatcgctg


 


ctgggtcggccgatcaagcgcaccgatctcaaaaaaggcgaggcggcgggcgcagagacccgttacgac


 


tacgacacgctcggcagaatcaggtttatcaaccagagcaacctgcgcaccacaatcgactatgacgtg


 


ctgggccagctctgcaaggtggccaccgaggacctgcaggccggaactggcgtgatcatcgacatggaa


 


tacgacgaccagggacaggaaattctcagaacccagaccgcaagcaaccaagcggcgttgaccttgact


 


caaacgtgggcagtggacgggcttttgaaaacccgcgacctgcaacaggcgggtagccccctgctgcac


 


gaaacgtttagctacgaccccagaggccgcctgacactggtgaattacctgggtagcagcttgccgaga


 


gacgaactgcaaagggagatgaccagacaaatattcagcttcgacgagctggacaacattacgctatgc


 


cagaccaggtttaccgatggcacctctgagcgagcagctttcaaatacggcagccccggcgacgataag


 


cataaagaccgctgccagcttttgagtattgcctacacgccgcccagaaaaacaccggacccgacattc


 


agttacgacgccaacggtaaccagcttaaagacgagcatggcaacagtctgcattacgatagccagagc


 


cgcctgctgcaggtcgcagaaaccggcggtgcccctatcagccaataccgttatgacggccacaatcaa


 


ctggtcgccaccagggatggcaatgaaagcgagattttgcggttctatgagggtcatcaactgagcagc


 


acggtgcaggaagatcaacgcactcagtacctgcatctcggcgaacagccgctgggccagcagattgtg


 


gacgacgccgagcaaaccctgttgctactgactgacgcaaaccagagcgttatgggtgaatttcaacaa


 


ggccagctgcgcaaggcggtctacagtgcctacggggagcgccacagcgaggaggcgctgctgagcact


 


gccgggtttaacggtgaagtacgcgaagccgccaacggctggtatctgttgggcaatggctaccgggcc


 


tacaaccctctcctgatgcgcttccacagcccggattttctcagccccttcgccgaaggcggcgtcaac


 


ccctacacctactgcctgggcaaccccatcgccctgcgegacccgacaggacatgatgccagcggtcag


 


actggccggttgagacggcccgatgagggggctttgccaatgcaacaaggtggcggagatatcatgggt


 


tgggtgggtgtaggaataggcgttgttttcaccgtattgggcgttgccgctaccatagccacgttagga


 


acagccacaccggttaccggcccggtaactgtcctgggcatttccatgaccgccagcgctgccgcggcc


 


gtttcgacagtctcgaccggtgcgttgatcgtcggtacggcattgacagcggcttcaactacggccaat


 


acagttgccattgtaaataacgatcagacggccggagaagtcggcggctggttgggtattgccgctgtg


 


cccgttggcttggtagggtttggcgcgggggctgtggtggcgagggcagttgcggctgcggctaaagtt


 


gcggctgccaacgctggtacgatcggtgtccgcagcgtcagcagaataggcctcgctgctgctggtgcc


 


cgcagaaccatttccagcgctgccagcagcgctcggcgccaaatcagcaacatgttaggcagaatctta


 


ccccgtgctctaaacaggactgctgctactgcacgccggattccaagcgttacaagtggcggatcagga


 


ccagggccatcattatttacacagaetacctttaacgaategattgggatgacgcagaecaetattttt


 


tcaacgaatgcgagcggaatcccaccggccacgcaggtaactcgaatctag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 106 as follows:









MKPTHTARYNAWNQLEQETAHDWLGAKPLASSTLGYRYDDWNQRCCTTTDDNVQTYEYSDPIGSDVHKG



 


PIQKTWKQSGDPEGRISGRSETWLNLFGKPDRIRTLTAGKTGRSRTHSMSRSRNLTTTEQELSRQTFLY


 


DGLGRCTEQRDALQQSTLFSYDNWSRMVSSTLADGSVTNRSYAPQSSSELATMLEVVHQNGTTRTVAGT


 


QKFDGLERVTQTKTGDRVEQFNYDAGFMQPRSRTTAGLDNINFTYTRALTDQIFSSTAPDETAKFDYDK


 


TSARLIEATNPQGTRTYRYDVHNQLTGETWDNLLGQAWETRHQSSLLGRPIKRTDLKKGEAAGAETRYD


 


YDTLGRIRFINQSNLRTTIDYDVLGQLcKVATEDLQAGTGVIIDMEYDDQGQEILRTQTASNQAALTLT


 


QTWAVDGLLKTRDLQQAGSPLLHETFSYDPRGRLTLVNYLGSSLPRDELQREMTRQIFSFDELDNITLC


 


QTRFTDGTSERKAFKYGSPGDDKHKDRCQLLSIAYTPPRKTPDPTFSYDANGNQLKDEHGNSLHYDSQS


 


RLLQVAETGGAPISQYRYDGHNQLVATRDGNESEILRFYEGHQLSSTVQEDQRTQYLHLGEQPLGQQIV


 


DDAEQTLLLLTDANQSVMGEFQQGQLRKAVYSAYGERHSEEALLSTAGFNGEVREAANGWYLLGNGYRA


 


YNPLLMRFHSPDFLSPFAEGGVNPYTYCLGNPIALRDPTGHDASGQTGRLRRPDEGALPMQQGGGDIMG


 


WVGVGIGVVFTVLGVAATIATLGTATPVTGPVTVLGISMTASAAAAVSTVSTGALIVGTALTAASTTAN


 


TVAIVNNDQTAGEVGGWLGIAAVPVGLVGFGAGAVVARAVAAAAKVAAANAGTIGVRSVSRIGLAAAGA


 


RRTISSAASSARRQISNMLGRILPRALNRTAATARRIPSVTSGGSGPGPSLFTQTTFNESIGMTQTTIF


 


STNASGIPPATQVTRI






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A fifty-fourth nucleic acid molecule encodes ORF55 and has a nucleotide sequence according to SEQ ID NO: 107 as follows:








atgcggtgtgtgaggcgatcaagaaggttctttaagctgcaagctgcaagctgcaagaaaaagcaggac



 


cgctttagcttagctgacgctccactgagtactttccatcgaacgatccgaaaaaccctgcctcgaaag


 


cttgtcagacccttttctgaatcagctatcgaggtagtcatgtccatcgaaccccaacgtcagaaagaa


 


cagccacccggccagcacacgccagcggatcagggcccggatcgcaatgatccggccatcgagccgcag


 


gtttcggacgtagagccggagactgaaaaaggtgacggccagacgcaaggccagacccctgcccccagc


 


caaagccagtcacaaagtcagaatcagagccagcagtccaacggcagcgcttacgtgcctgactatgag


 


ccgcaggaaaaaaaggaagaccagcgcaatcatcagcccactcaaggcactgatgctgatatcgacacc


 


aatgcgggctga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ-ID NO: 108 as follows:









MRCVRRSRRFFKLQAASCKKKQDRFSLADAPLSTFHRTIRKTLPRKLVRPFSESAIEVVMSIEPQRQKE



 


QPPGQHTPADQGPDRNDPAIEPQVSDVEPETEKGDGQTQGQTPAPSQSQSQSQNQSQQSNGSAYVPDYE


 


PQEKKEDQRNHQPTQGTDADIDTNAG






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A fifty-fifth nucleic acid molecule encodes ORF56 and has a nucleotide sequence according to SEQ ID NO: 109 as follows:








atgcccgtcactggtgcaggctttatcaagcgtttgacgcaattgtccctctgcgccggcatggcgctg



 


gtcccggtggccgtacaggcagccgaaagcgatccttgggaaggcatcaaccgttccattttcagcttc


 


aacgatacccttgacgcttatacgctcaagccgctggcaaagggttatcagtacatcgctccgcagttt


 


gtcgaagacggtattcataacttcttcagcaatatcggcgatgtcggcaatctggcgaacaacgtcttg


 


caggccaaacctgaagcggccggtgtagataccgcacgccttatcgtcaacactacgttcggtctgctg


 


ggcttcattgacgtcggcacccgcatgggcctgcaacgcagtgatgaagacttcggccagacactgggc


 


tactggggtgtgccaagcggcccgttcgtggtgattccgctgctgggcccaagcacggtgcgtgacgcc


 


attgccaagtacccggacacctacacctccccgtaccgctatattgatcacgtacccacccgcaacacg


 


gcgttgggcgtcaatctggtcgacacgcgtgccagcctgctgtccgccgagcgcctggtcagtggtgat


 


cgctacaccttcatccgcaacgcttacttgcagaaccgcgaattcaaggtcaaggacgggcaggtcgaa


 


gacgatttttaa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 110 as follows:









MPVTGAGFIKRLTQLSLCAGMALVPVAVQAAESDPWEGINRSIFSFNDTLDAYTLKPLAKGYQYIAPQF



 


VEDGIHNFFSNIGDVGNLANNVLQAKPEAAGVDTARLIVNTTFGLLGFIDVGTRMGLQRSDEDFGQTLG


 


YWGVPSGPFVVIPLLGPSTVRDAIAKYPDTYTSPYRYIDHVPTRNTALGVNLVDTRASLLSAERLVSGD


 


RYTFIRNAYLQNRFFKVKDGQVEDDF






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A fifty-sixth nucleic acid molecule encodes ORF57 and has a nucleotide sequence according to SEQ ID NO: 111 as follows:








atgacactttcaaccctgcgccctaccccgcgccagcagtatgaatcgcccgagtcagccgaggatttc



 


acccagcggctggccgacctgacccgcacgctggccgaaacagccgagcagtacgacatcagcgcgcag


 


ttccctcacgccaacttccgcttgctgcacagccacggactgctcggcctgaccgtgcctgccgaactg


 


ggcggcggcgctgccgacctgtcgcgggcgcagcaggtcatcagcgcagtggccagaggcgagccttcg


 


acagcgctgattctggtcatgcagtacctgcagcattccaggctgcaggacaaccgcaactggccgagc


 


cacctgcgcgaacaggtggccaaagacgccgtgcacgagggcgcgctgatcaacgcgctgcgtgtcgaa


 


cccgacctgggcacacctgcgcgtggcggcttgccgggcaccatcgcccggcgcagcgccgaaggctgg


 


cgcatcagcggcagcaagatctactccaccggcagccatggcctgacctggttcgccgtgtgggcgcgc


 


agcgatgacgaggacccgctggtcggcagttggctggtgcacaaggacacgcccgggatcagcatcgtc


 


gaggactgggaccatctgggcatgcgcgccacctgcagccacgaggtcaggttcgacaacgtgcgagtg


 


ccgctcgaacacgcggtcagcgtcagtccgtggagcgccccgcaatccgagcttgatggtgccggcatg


 


ctgtggatgtcggtgctgctgtcgtcggtctacgatggcatcgctcaatctgcccgcgactggctggtg


 


cactggctggaacagcgcacgccttccaacctgggcgccgcgctgtcgaccctgccgcgctttcaggaa


 


acagtcgggcagatcgacacactgctgttcgccaaccgcagcctgctggagtccgccgcccaagggcac


 


acacccgcacagcatgccgcgcagatcaaatacctggtgaccggcaatgccatccgcgcagtggaactg


 


gccattgaggcctcgggcaatcccgggctttcacgcactaacccgctgcagcgtcattaccgcaacgtg


 


ctatgcggccgggtgcatacgccgcagaacgacgccgtgttgatgggcgtgggcaaagcggtatttgcg


 


gcacgcaagcagagccagtaa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 112 as follows:









MTLSTLRPTPRQQYESPESAEDFTQRLADLTRTLAETAEQYDISAQFPHA



 


NFRLLHSHGLLGLTVPAELGGGAADLSRAQQVISAVARGEPSTALILVMQ


 


YLQHSRLQDNRNWPSHLREQVAKDAVHEGALINALRVEPDLGTPARGGLP


 


GTIARRSAEGWRISGSKIYSTGSHGLTWFAVWARSDDEDPLVGSWLVHKD


 


TPGISIVEDWDHLGMRATCSHEVRFDNVRVPLEHAVSVSPWSAPQSELDG


 


AGMLWMSVLLSSVYDGIAQSARDWLVHWLEQRTPSNLGAALSTLPRFQET


 


VGQIDTLLFANRSLLESAAQGHTPAQHAAQIKYLVTGNAIRAVELAIEAS


 


GNPGLSRTNPLQRHYRNVLCGRVHTPQNDAVLMGVGKAVFAARKQSQ






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A fifty-seventh nucleic acid molecule encodes ORF58 and has a nucleotide sequence according to SEQ ID NO: 113 as follows:








atgaatctcacaacacttcctcttgcgctcagcattgcttgcgctgcggccatcacacctgccttcgcg



 


ggcacaagcgtctctgaggcttcacacaaagtgaatgtgcagcaagttcgtaacgcgacggtaaagatc


 


tcctacggcggcacgacctttctgatcgacccgatgctggccaaaaagggaacctacccagggtttgaa


 


aatacctatcgaagcaatctgcgcaatccactggttgatctgaccgaatcgcccaccgaagtgatcgcc


 


ggtatcgacgcagttatcgtcactcatacgcaccttgaccattgggacgatgctgcacaaaaagtgctg


 


cctaaagacatccctctgttcacccagcatgaaaaagacgcgcagctgattcgctctcaaggtttcaag


 


aacgtacgcgtattgactgatgaagccgaattcggcggcgtcaaaattaccaagaccggtgggcagcat


 


ggcaccgacgaaatgtatgccgtgccagccctcgcgaagcctctgggtgaagcaatgggcgttgtattt


 


caagccccgggctacaagaccctctacctcgctggtgacactgtctggcgtaaagaggtcgatcaggct


 


atcgagaactattgtcccgaagtcatcgtactcaatgccggcaaagcaaaaatgacggggtatgagggg


 


gcgatcatcatgggggaagaggatgtactgcgcgcttcacaggtcgcgaagaacgcgaaaatcgtcgct


 


gtacacatgaatgcaatcaaccatatgtccctgacccgtgagcaattgcgcgcttacgtcaagcagcag


 


ggtatcgaaagtcgtgtagacataccggaagatggcgcttcactggagttctga






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 114 as follows:









MNLTTLPLALSIACAAAITPAFAGTSVSEASHKVNVQQVRNATVKISYG



 


GTTFLIDPMLAKKGTYPGFNTYRSNLRNPLVDLTESPTEVIAGIDAVIVT


 


HTHLDHWDDAAQKVLPKDIPLFTQHEKDAQLIRSQGFKNVRVLTDEAEFG


 


GVKITKTGGQHGTDEMYAVPALAKPLGEANGVVFQAPGYKTLYLAGDTVW


 


RKEVDQAIENYCPEVIVLNAGKAKMTGYEGAIIMGEEDVLRASQVAKNAK


 


IVAVHMNAINHMSLTREQLRAYVKQQGIESRVDIPEDGASLEF






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A fifty-eighth nucleic acid molecule encodes ORF59 and has a nucleotide sequence according to SEQ ID NO: 115 as follows:








atgcatctgttgccgtttgcgcgttaccccttatcacctgcagaaacacctaaacccaaggtgaccatg



 


aaggttggagatttcagggcttacgacaccgctccagcacccggagtgaccactgcgtcctgcggacaa


 


ctggcaatcggcaccaagttagaaatcatcgagaccgccgagaatggcgaacttacttatgccaagggt


 


aagattctatctggcagcgtgaagcagggggcaaccaaaaaacgggtcgagggggcggaggtctggttc


 


gcttatttgaaaaacggcgaaccctacaaaaactcagtccctaagcgcatctggctcgctgacgatgtg


 


cctgagcgagcaagacccaattactggcagggtaaggtcaaagcctcagtagtgaataagttgccgctg


 


tacgatgatcctgccagccctacaaatggccagcctgcaggcgcccggaaggggactctggagctggtc


 


atgaacagcgtcatcgagtttaactcttcggaagtcgtcaacctggcgctggatggcaagctgcatcgg


 


atggccaagtgcacgatgctgagtggcggcctgcggggtcatggtgcggttccccccagcttttgggca


 


tgtgttgaaaatgaccctgctaataaagtattgaaatgggactcggtaacgccgaccagttttgatacg


 


gtcgttatgacgagcaccggagtgaaggcgggcgatccaattggctatcttggacaaaccgaaaatctc


 


accggtgaaaatggcggcgtcagcagcaaataccaggttcacgtcgaaattttcacagccgatgctgag


 


gttaaagacttcctcaagaacaccgcgggtttgaagattgggaagcaatacctgcaccttgcaagcggg


 


gctgtactcaagcaaaaagcgcccgcgaccggcaccacagcactcaagcaagaccatgcggttgacttg


 


gctaaagccacaattgtcaaagaaggcaccgatgactggtatgaggtcagcgtgatcgaggacgatcag


 


cctgtagccggcctgataaaaaaagccactgcgctagtcatcacacagcacgattgggaaaaattgggc


 


tttcagatcgtagaggagaacaacgcagcagccgatggtttcttggacccggatgcaatgccacagttc


 


ttcaaagacctattcgcgaagatcgacaagaaccacgatggtgaggtggagcctgctgaactggctgag


 


gctcttaagaaaccggaaaccagaacccagtgggccaggcttgttgcccatcaccctacggagtggaaa


 


gataaggcaggctcccccaagtggagcaagttggataaactgctggaaacgtcgccgaagatgttgaaa


 


catgaaaaagaacgcattgataaatatgtattttgggatgagttgtcagggaaagctaagatgacctca


 


agtttaatatggcattttcatccggtagaattcatttcaacatttagcgcaaaaaaagtctgcgcttgc


 


aacgccatagttaaggctactcgctgggtttcttccagtaagacgcactatggcccattgcatacgggt


 


gataaagagcttgggagtgcacctcagtgggatgacctggtctcagaaggaaaaataacggaagaggag


 


aaaaaaattattgttgtaatgtctggaaacgaggcaaaaattaacggagtacaaagttatgatagcgaa


 


ataattactgccggcgcgatgcdgaaaacaattaacttgtccggtggcggtgagctgccactacaagtt


 


aagaagtttaaaaatcagcatcccgaggcgtacatcgaatactttgattctcaaggctggaagttggat


 


gagacaggtgattcggcgaaaatgtattatcaagggccggctcgagctagtggcgcaaagctggaagga


 


aaggcgctgaaggataatttaaaaattggttgcagtgaatcgacatttgggaaggtggttgactgtcaa


 


cctgtttcagtgatggcctgcgctatcgcaagtccgttatatatccagatacaaataatggattttata


 


gaaaggttacgtagttctttaacgaagaagcccacaggctataactttactgctgggggatttttcaag


 


acctctctcggaaaagctgtggttttggatcacgatataaatcgacccgggtatgtgaaggatgacttg


 


ggatctgctcttgacactttttttgctcaaaatccaacagtcagccgggatattgatacatggggcgca


 


gcatatagcgttaatgagcgaaaagttttagacctgtatggcgctcgaagaagaatgaccaatgcattg


 


cttcgatacaatcacttgaaggcggagttataa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 116 as follows:









MHLLPFARYPLSPAETPKPKVTMKVGDFRAYDTAPAPGVTTAScGQLAIGTKLEIIETAENGELTYAKG



 


KILSGSVKQGATKKRVEGAEVWFAYLKNGEPYKNSVPKRIWLADDVPERARPNYWQGKVKASVVNKLPL


 


YDDPASPTNGQPAGARKGTLELVMNSVIEFNSSEVVNLALDGKLHRNAKCTMLSGGLRGHGAVPPSFWA


 


CVENDPANKVLKWDSVTPTSFDTVVMTSTGVKAGDPIGYLGQTENLTGENGGVSSKYQVHVEIFTADAE


 


VKDFLKNTAGLKIGKQYLHLASGAVLKQKAPATGTTALKQDHAVDLAKATIVKEGTDDWYEVSVIEDDQ


 


PVAGLIKKATALVITQHDWEKLGFQIVEENNAAADGFLDPDAMPQFFKDLFAKIDKNHDGEVEPAELAE


 


ALKKPETRTQWARLVAHHPTEWKDKAGSPKWSKLDKLLETSPKMLKHEKERIDKYVFWDELSGKAKMTS


 


SLIWHFHPVEFISTFSAKKVCACNATVKATRWVSSSKTHYGPLHTGDKELGSAPQWDDLVSEGKITEEE


 


KKIIVVMSGNEAKINGVQSYDSEIITAGAMQKTINLSGGGELPLQVKKFKNQHPEAYIEYFDSQGWKLD


 


ETGDSAKMYYQGPARASGAKLEGKALKDNLKIGCSESTFGKVVDCQPVSVMACAIASPLYIQIQIMDFI


 


ERLRSSLTKKPTGYNFTAGGFFKTSLGKAVVLDHDINRPGYVKDDLGSALDTFFAQNPTVSRDIDTWGA


 


AYSVNERKVLDLYGARRRMTNALLRYNHLKAEL






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A fifty-ninth nucleic acid molecule encodes ORF60 and has a nucleotide sequence according to SEQ ID NO: 117 as follows:








atgcggccgttgcctgcgttcagtattttgcagtttgatccgttgaaacgttcgggtcctgcgctgacg



 


gtcgaacgtgatacaccggtcgatagcaagcctattaatgacgtgcgttgtcgcttccgtacgtgctac


 


ccgaccgaagttcaggcgctggatctgaccgcgctgaattactcggtgaaaggcggtggttcgttgctc


 


agcctgcgcctggagatgagcgctgaaggtcacttgggtgagcttgaactgagccgcctgcgtctgcac


 


tttgcaggcgagcgctatatcagccagatgctgtacctctgcctgctacgcaatctcgagggtatcgag


 


ctgatccctctggacgctgccggcaagcccatcgacggtgtcaatggcgcgccaatggcgttcaagatg


 


ccgggcgaccgtgtacagccggtagggtttgccgaagaagaggcgttgatcccgtatccgctgaacacg


 


ttccgcggttatcgctacctgcaggagtacttcgcgtttcaggacaagttcctgttcgtcgacatcaac


 


ggtctggatctgctcaacgcactgccagaagagacactcaaacaagtgcgcggccttgagttgcgcttt


 


gatattegeaagageggeatteagegtcttegteeeaeeetggataaegtaaagetgtattgeaegeeg


 


atcgteaaettgtteaageaegaegeettgecgattegeettgatggeaageaggaegagtaeetgetg


 


etgeeegccgaatatggectggaaaeetgtggtgtgtttteggttgaaaeegtgaceggttggaagecg


 


ggaggtettggetateaggattatgtgeegttegaateetttgageaegaeeeeagtttegaegtgeee


 


aaeagcegtcegeattacagcattegeeagegttettetttgeteeatgaaggeetegaeaettatetg


 


agttteggeattegecataeagaagegeaegaaaeeetgtegategagttgatgtgeaeeaateagaae


 


ctgeeacgeaaactcaaaetgggegaaateaaegtggeetgegaagataegecggagtttttgagttte


 


egeaatateaeaeeggetaeeteeagtttegegeeeeegetgaaeegtgaetteetgtggaagttgate


 


agcaatatgtcgeteaattaettgtetetggetgacgteaatgcgetgaaggtgattctggaaaectae


 


gatttgcecegttaetaegaecageaegeggaaaaagteageaagegeetgttgggeggtttgaaateg


 


atcaageateaaeaegtggaeagattgeaeegagggttaeeggtaegeggattgegeaetgagetgaee


 


atcgaeeeggaagggtatateggegaaggcgaeatgtttgtattegettcggtteteaaegagttttte


 


gegetttaegeeagtctcaattegtaecaegagetgcgggtaaaaageaeacagggagaggtgtaeeaa


 


tggaeaeeaegtatgggeetceageeeetgetttaa






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 118 as follows:









MRPLPAFSILQFDPLKRSGPALTVERDTPVDSKPINDVRCRFRTCYPTEV



 


QALDLTALNYSVKGGGSLLSLRLEMSAEGHLGELELSRLRLHFAGERYIS


 


QMLYLCLLRNLEGIELIPLDAAGKPIDGVNGAPMAFKMPGDRVQBVGFAE


 


EEALIPYPLNTFRGYRYLQEYFAFQDKFLFVDINGLDLLNALPEETLKQV


 


RGLELRFDIRKSGIQRLRPTLDNVKLYCTPIVNLFKHDALPIRLDGKQDE


 


YLLLPAEYGLETCGVFSVETVTGWKPGGLGYQDYVPFESFEHDPSFDVPN


 


SRPHYSIRQRSSLLHEGLDTYLSFGIRHTEAHETLSIELMCTNQNLPRKL


 


KLGEINVACEDTPEFLSFRNITPAISSPAPPLNRDFLWKLISNMSLNYLS


 


LADVNALKVILETYDLPRYYDQHAEKVSKRLLGGLKSKHQIIVDRLHRGL


 


PVRGLRTELTIDPEGYIGEGDMPVFASVLNEFFALYASLNSYHELRVKST


 


QGEVYQWTPRMGLQPLL






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A sixtieth nucleic acid molecule encodes ORF61 and has a nucleotide sequence according to SEQ ID NO: 119 as follows:








atggtcaaggttacctcttccggatttactgccaaccctctctctcatcatgcggacagtgtttccccc



 


gcgaacagtccccctcagttaccggagcctgtgcatctggttgatttaagcgagtcgtcccgcaagggc


 


ggcatgcgaaatcggccgcatgccagtttgaacagtcaggtgctcgaactgcaagcggtgccgtcgcaa


 


cgtggaaagcatgttcgtgtcagaagtcatgccgatggcgagagtgtcattaatgcctggctggcaaag


 


cgcccctcggttcaaagcgaaaccagtcttgataacgatggcaaactggtgcgttacacccccgtgaat


 


catgagccgctggcgccgcgcaatgaggcgtttttcacctcggtgccggggatgttgatggccgttttg


 


acggtccaccccgagatggaacatggcatcagcggggacataactgctgatgctgtggctgcccggctt


 


gccgaaccgccaatagggttgctaaccggaatctggcagtcttcccatgatcgagcctatctggagcgt


 


ggcggtgtggtgcataccgccaatatggaagagcgctgggcgccgttgacgctgccaggcatcaatccc


 


cgagagcccctgcgaatggccggtttgcaggccgatggtggagtctatctgcataacggcagccaactg


 


tggcgcttgaccgaaactgccgccgagtccgtgaccaccgaaaaccttcctgaaggtgcggcggtacgc


 


attggcgccggtggcgaggtgcatgggctgcatgaaggcgcgcttcattcgaatggcatttcccgtcca


 


atcgagctttggcggccaaaagctggcgcgccggggcgcgagcagagtccggcgcgccccgttgatttg


 


ctgccgttaccgggtggcaccgctgcactgatccttgatgacaagggacgtatttatcacgctgatctg


 


aaaggcacaggcgctgttgaagcccaccggctgaaattacctgctgactttgcgcagggtaaaggttgg


 


gccgtgaccgccatgggattgtcccgagacgacactgttcatctgatgctgcaggatcagaacgggcgt


 


cgcatgagcttgcagcgagcaccgggcgaggcgctgtttcgtcctgcgtacctgctggatcgcccgttg


 


ctgctgctctataccgaagggctgcatgttccgtcggaggccgcggtgcagtcgcacgttcagcttgat


 


tgtcatgctcaactggggcatatcgatggcgtgctgcattataaagcggctcccgatcagtcatgggaa


 


gggctaaagcagtcgggcggcgaaccgctgacgggtttgactgctctttattccagcccgctgggattt


 


atcgacaggaaaccggttttcgctttagtgggggatgcccggcaggtggtcgagttgaaactggagggg


 


cgtacatcctggttgccgagcgatgccgagcttccgcgtcaccctgcgggcgggcctttggcggtgata


 


ccggatacggtagcgttacgcaccagcccgatcgcgcagtttgacgagcctgtacaggcgctggcggtt


 


cacggtaatcgccgggtcgtcgcgctgacggattcggggcgattaatggctgccgatgcggacacccca


 


gcccgccgacttcccacgttgcagcgccccatcgccatcgccgtagggctcaacgatcagttactggtg


 


ctgcatcatccccatagccagcgcccccagttgaaacggttgagtgcgaaagatgactgggagccggtg


 


ccgataattctgccgggtattgttcacccttcaagtcttcgcgctactcgcacggggcaaatacaagtg


 


cagctgggagaaaactggcatacgttgctgccatcaatgacgtcgcacgataatcagcgcttacctgcc


 


cgcgtaaaacctgaaccagagggggatgaggcgccgtcggcgaatttcctggcgggtagcaacgccctc


 


gccaatcagcagcaagccagtcgtatcagcacaccgcatcatgacgcatcggtggttacgacgctggcg


 


gggacaacagccaacaacccgttgacgatggcgtcgagcctacaggcagtggttgatacgacccgcgct


 


caggtaggcgcgttggcgagagatgtagtgggcgcagcggcgaacagcacgatgcgggcdatggcgcat


 


accttgggtgttgtactgccgccaacgcctcaggagaagcgcctggccagtttccataatgaggcgaaa


 


caggcttatacatcaggaaaaatactgtttgagcatctgccgtcactcgcgcaagtgcgcgtcgcttca


 


gccgtagggccgtcggacggagaaagattcgggctgtcacatcagcaaacgcaacgcttgttgacgctg


 


cgagaggggaagctggaagcgctgttacgcgacttgcgcaagatcggctttcatgaaggggtgatcatg


 


ggcgatatgggcgacagcgacagtgcgcacggtcttgtttcgacgacatcgacaccaacgttccggctg


 


gccgagctatggcgacggcagcattcgcgagtggataaggcgctgtcttccgctggattatccagatcg


 


gaagatatttttccggacttgaacctaagtatcaacgcgttggctggcggcgcggcgctgaatgcggat


 


cgtatgagcgaacgtgaagctgagttgttgagcgttttgtgcgaggtcagcgaaaaaatgatgcgcgct


 


ggcgtacgcttgccggcagatgatggaagcgttgacagcgcccacagccaggcgccatacggcttgaga


 


acagcaggattgattgcaggtctggtggactatgatgcgctgttgagcdgtaccgacgcgcaggcgctg


 


gaaatggcggagcgacttcagcaagatgccaggcttgctgcattgtgcaaactcggtctgtcttcgtgg


 


ggtcaattagcggccttcgatgatgtggtgacgacgtttcgcgaacagatatcgttaccgggctcggca


 


cgccgcacccagttgctcaaaaatcttggcttgccacccgatgccgcgccggacgaaatggcggcgcgc


 


atgtccgacttactcctggatctgttcaaccggagcaccttcttttcgacgcagtcgcgtggtctggaa


 


ctgcgcggttcgttgggatcggctgactggaaacatctcaatgcgttcagcgtcggcgtgactggcgag


 


gcgcttcaagtgctcggcgtagagcgcatcggcgatggcaaggacggcgatgccgggttggtcgcgttt


 


tttgtgcgccacgccaaagcctctgtatctgcgacgtcagggatcggaatcgatttcaagccaggcccc


 


ggcactggcggccgtgttattgattcgcgaccgggtcgctcgatgaactcgacgtggggaggctctacc


 


aacctgggtatttccggcgcgtaccagcatggtcagggcgccgccgtgatcatcgcaccgtcgacgatc


 


tccgatttcgtgcggctgttattcgatgtcaaccatcccgataccacccaaatcctgcgcaccggtgtg


 


aacggtggttcgattggtcttgatctgtttgaaaccaatgtgaatgcctctgtgggggcgaacgtcagc


 


gtatcgccattcagcctgagccagaaatatgggccacagaaaccgacggcagatgcggccgtctctgac


 


ccagacaatcggcgcagcaccgcgtcagggtcgttgtcggtaggcgggacggctcaggctggcAcgcac


 


tgggggcaaatggagttgcacctggatcacgcctgggccgatattatcggtctggaatttcagggccgc


 


acggatttcaatcttgaattcaatagcggcctgaatctgggaggcgcgctgtcttccgcgctgggcgat


 


aacccccaaaagttgataaatgcgtccactggaaacggcaatctgcaactcgccggcatccgcgtcgcg


 


tcaagcgatgtgcagttgccgaccgatgctgtggttgacgacaagcgccgtggccccttcctgtcgacg


 


gccagctataaacgcaccttcgataccgaagttgccaagcctgttacggccggggagtggagccagatg


 


cgccagcgccttgccaaagcctttcctgacaatatcgcagagttgggcgcgctcgattaccccaccagg


 


cccggtgagcgtatcgcgaccatcaaacaggtgattgaccgcatacaaggtgcgaaggcgcgtagcgtg


 


gaagccgtcggtgcaatggacggaaaggcattgcaccgtcagcgtttcgatgccgcgagagaaatgtcg


 


aacgccggcaacagcgtatggcgggcgagttccgaaattgagcgcgcctcgatcgtggagatgctgcat


 


cagttgcgtcagcaggaacaaagcgccgtccagaatcacgcccgagccattcccggcgcgcgtgtggaa


 


ttcaacctgttcggtcgtgaatcgctggaaacggtggtctttcacgccatcggtcatctggggcttggc


 


agcaagctgaacgatctggcggagctgcgtcgcaaggtgccgggtctcgatcaggtcatgctgagtttc


 


cagtcgttgcccaaggtcaatcaggtgcgctacgtttttgagatgcgccctcaggcgaggttcgccatc


 


aatgacgcgctactggcgcgcgagcagcaggcatcggcacgtgcgctcggtttgcagggaccctcggga


 


agtgaattgaattggcgcggcgttctggacaagatcaaaaccacgcctgacctttatcggctggcggcg


 


atcgccgtacataacaccgatgaaaaccccgtgacctcaagaatagggctgecgetgctgaatgtgtcg


 


gccacaggcgcgacatcgcatcagttgttcgaggcggaaatccagttccgatacggtctgtatgacggt


 


ctgcaaggggttgagttgctggaggccggaaacagggcattgcagtcgccgttacgggcattacagcaa


 


tccggtattcaggccctggggcagagaacccaggccggggaggttgcgtatggccccccttcgccgcgc


 


aaagagtcgccgttgcgcaccgcagtggatgctgctgcgctgacaacgagtgacatcgcgcgacaactt


 


gaggttaaagtccagcgcatgaataccgcgcatgagcgtgaggcgaatgctatcagttcgttccagcag


 


gcttatgggatcgcgtccgcgcatctagacaggctgcttttgcgcattcctgaattgccattacctgaa


 


attgatgaccgcgacgtcgatggaggacgtgtgcgcggtacatttgcgtcgctccagcgacatcatcag


 


gcgctggatgacgctataagtgccatgcatcaggccagcgaaaaggtgtacacgatacctggcaagcag


 


gccactcaagagcaagacccggcgctggctcaactgctctctgttgaaaaacgtcggcgttcgctcggg


 


catgccttggaaacactggcgggcagaggggtggaagcgggcacggccacagggcttgaacttaacagg


 


gtctcatcgcaagtgaatgatctggtcgctcgccgggacgcgctgctaaggcagcgtgaaagcggtgtt


 


caggagggcggtctggatagcgaagagctggaaatggaacttcaattgaccacctcagtgctgcagcgg


 


ttgcgcgccgatttgctcggcgagcggcaggcgatggaggctaccgccaaacgcctggatcaggcgagc


 


cgcgctgccctcgaaggtgagcgcagcttcagcgacgccgtgcgtgacagggcgtggggcgaactcgat


 


aacgtgtag






The protein encoded by this nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 120 as follows:









MVKVTSSGFTANPLSHHADSVSPANSPPQLPEPVHLVDLSESSRKGGMRNRPHASLNSQVLELQAVPSQ



 


RGKHVRVRSHADGESVINAWLAKRPSVQSETSLDNDGKLVRYTPVNHEPLAPRNEAFFTSVPGMLMAVL


 


TVHPEMEHGISGDITADAVAARLAEPPIGLLTGIWQSSHDRAYLERGGVVHTANMEERWAPLTLPGINP


 


REPLRMAGLQADGGVYLHNGSQLWRLTETAAESVTTENLPEGAAVRIGAGGEVHGLHEGALHSNGISRP


 


IELWRPKAGAPGREQSPARPVDLLPLPGGTAALILDDKGRIYHADLKGTGAVEAHRLKLPADFAQGKGW


 


AVTAMGLSRDDTVHLMLQDQNGRRMSLQRAPGEALFRPAYLLDRPLLLLYTEGLHVPSEAAVQSHVQLD


 


GHAQLGHIDGVLHYKAAPDQSWERLKQSGGEPLTGLTALYSSPLGFIDRKPVFALVGDARQVVELKLEG


 


RTSWLPSDAELPRHPAGGPLAVIPDTVALRTSPIAQFDEPVQALAVHGNRRVVALTDSGRLMAADADTP


 


ARRLPTLQRPIAIAVGLNDQLLVLHHPHSQRPQLKRLSAKDDWEPVPIILPGIVHPSSLRATRTGQIQV


 


QLGENWHTLLPSMTSHDNQRLPARVKPEPEGDEAPSANFLAGSNALANQQQASRISTPHHDASVVTTLA


 


GTTANNPLTMASSLQAVVDTTRAQVGALARDVVGAAANSTMPAMAHTLGVVLPPTPQEKRLASFHNEAK


 


QAYTSGKILFEHLPSLAQVRVASAVGPSDGERFGLSHQQTQRLLTLREGKLEALLRDLRKIGFHEGVIM


 


GDMGDSDSAHGLVSTTSTPTFRLAELWRRQHSRVDKALSSAGLSRSEDIPPDLNLSINALAGGAALNAD


 


RMSEREAELLSVLCEVSEKMMRAGVRLPADDGSVDSAHSQAPYGLRTAGLIAGLVDYDALLSSTDAQAL


 


EMAERLQQDARLAALCKLGLSSWGQLAAFDDVVTTFREQISLPGSARRTQLLKNLGLPPDAAPDEMAAR


 


MSDLLLDLFNRSTFFSTQSRGLELRGSLGSADWKHLNAFSVGVTGEALQVLGVERIGDGKDGDAGLVAF


 


FVRHAKASVSATSGIGIDFKPGPGTGGRVIDSRPGRSMNSTWGGSTNLGISGAYQHGQGAAVIIAPSTI


 


SDFVRLLFDVNHPDTTQILRTGVNGGSIGLDLFETNVNASVGANVSVSPFSLSQKYGPQKPTADAAVSG


 


PDNRRSTASGSLSVGGTAQAGAHWGQMELHLDHAWADIIGLEFQGRTDPNLEFNSGLNLGGALSSALGD


 


NPQKLINASTGNGNLQLAGIRVASSDVQLPTDAVVDDKRRGPFLSTASYKRTFDTEVAKPVTAGEWSQM


 


RQRLAKAFPDNIAELGALDYPTRPGERIATIKQVIDRIQGAKARSVEAVGAMDGKALHRQRFDAAREMS


 


NAGNSVWRASSEIERASIVEMLHQLRQQEQSAVQNHARAIPGARVEFNLFGRESLETVVFHAIGHLGLG


 


SKLNDLAELRRKVPGLDQVMLSFQSLPKVNQVRYVFEMRPQARFAINDALLAREQQASARALGLQGPSG


 


SELNWRGVLDKIKTTPDLYRLAAIAVHNTDENPVTSRIGLPLLNVSATGATSHQLFEAEIQFRYGLYDG


 


LQGVELLEAGNRALQSPLRALQQSGIQALGQRTQAGEVAYGPPSPRKESPLRTAVDAAALTTSDIARQL


 


EVKVQRMNTAHEREANAISSFQQAYGIASAHLDRLLLRIPELPLPEIDDRDVDGGRVRGTFASLQRHHQ


 


ALDDAISAMHQASEKVYTIPGKQATQEQDPALAQLLSVEKRRRSLGHALETLAGRGVEAGTATGLELNR


 


VSSQVNDLVARRDALLRQRESGVQEGGLDSEELEMELQLTTSVLQRLRADLLGERQAMEATAKRLDQAS


 


RAALEGERSFSDAVRDRAWGELDNV






This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A sixty-first nucleic acid molecule encodes a HrpA-related protein and has a nucleotide sequence according to SEQ ID NO: 121 as follows:








atgaacattacgccgctcacgtcagccgcgggcaagggctcgtccgcacaaggcacagacaaaatttcc



 


attcccaactccacgcgcatgatcaatgccgcttcaatcaagtggttgaataaggtgcgtagcgccatc


 


agtgaccacatccgcaccagcatcgagaaagggaaactgttcgagctcgcctccttgggcagcaacatg


 


ttcggtgtcccggctctttcagcgcgcccctcgacgctccaacctgtgttggcgtttgaggctgacccc


 


aatcacgacctgaaccttgtcagggtctatatgcaggacagcgccggcaagctcactccctgggacccg


 


acgcccaacgcggtcacgacgacgtcgaatccatcagagcctgatgcgcagagcgatacggcttcgtca


 


tcattacctcggcggcctcccgcaggctcggtgctgagtttgctgggcattgcgctggatcacgcgcaa


 


cgccacagtcctcgcgcggacaggtctgccaagggacgacctggccgagaggagaggaacggggcaagg


 


ttcaatgccaagcaaacaaagccgacagaggctgaagcctacggtgatcatcagacacccaatcctgat


 


ttgcacaggcaaaaagagacagctcaacgcgttgctgaaagcatcaacagcatgcgagagcagcaaaat


 


ggaatgcaacgcgccgaagggcttctcagagccaaagaagcgttgcaagctcgggaagccgcgcgcaag


 


cagcttctggacgtgctcgaggccatccaggctggccgtgaagactccaccgacaagaagatcagcgcc


 


actgaaaagaacgccacgggcatcaactaccagtga






The Hrp-A related protein has an amino acid sequence according to SEQ ID NO: 122 as follows:









MNITPLTSAAGKGSSAQGTDKISIPNSTRMINAASIKWLNKVRSAISDHIRTSIEKGKLFELASLGSNM



 


FGVPALSARPSTLQPVLAFEADPNHDLNLVRVYMQDSAGKLTPWDPTPNAVTTTSNPSEPDAQSDTASS


 


SLPRRPPAGSVLSLLGIALDHAQRHSPRADRSAKGRPGREERNGARFNAKQTKPTEAEAYGDHQTPNPD


 


LHRQKETAQRVAESINSMREQQNGMQRAEGLLRAKEALQAREAARKQLLDVLEAIQAGREDSTDKKISA


 


TEKNATGINYQ






The HrpA-related protein, has significant homology, as detected by BLAST analysis (5e-07), to the C-terminal 43 amino acids of HrpA (GenBank Accession AF232004; Alfano et al., Proc. Natl. Acad. Sci. USA 97:4856–4861 (2000), each of which is hereby incorporated by reference in its entirety). Expression of the hrpA-related gene is activated by HrpL, as indicated by miniTn5gus mutagenesis. This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted. HrpA is the Hrp pilus subunit protein (Roine et al., Proc. Natl. Acad. Sci. USA 94:3459–3464 (1997), which is hereby incorporated by reference in its entirety).

A sixty-second nucleic acid encodes ORF01152 and has a nucleotide sequence according to SEQ ID NO: 123 as follows:








atgaccttaagaatcaatactcgttctgctaccccggttgtacctctggaaacaggctctacatcgcag



 


ccgacaccaccgccggtcacggcaagagcgactgagcctccccccgtcgccaatcctgcggcgcctaaa


 


tcagcgccaggtgttcagcaagcacacgggctgaagacgcgcatcgctggcaagctttccgaacgtcag


 


accaatttcagtctcgggattcccggcactggtcgtactctcaaccggcccttgcgcagcgggattccg


 


gaggaaggtgagcaggtatcgaacgaggagagtcatgatccgttgctcaaggaagcgcatgaactgcag


 


cgtatggtggagtcggcgctgacccatctgaaggcggcaccgacgtctctctgggagcgtcccgcccct


 


tcaacggtaaggcgtattaccaccaagatttttccgtggctaaagcctgccccgctgcgcgaagtcgca


 


agcaatggcagcaacgccaagaccaagatcaagatcaactcacagcaaagccctgaaaccatcgcagcg


 


gcggtgaaagagctgagcacccggctcgatcaccagagcaaggtgctcgccacagccacccacgcactg


 


gtcgctgcgcgtgagcatcttgaatcgctcgaacaggccaccccgccctcgtcgaccgaaccactggac


 


catgccagggctcgcgttcaacaagccgactccaccacccgcctggccagtcagcaacttcgtgagctg


 


attcagggtacagacgtgttgcaactgggcgcgctgagtgaagggcaggatcaggttgaacagaaagcc


 


gagttttct






The protein encoded by the nucleic acid molecule has an amino acid sequence according to SEQ ID NO: 124 as follows:









MTLRINTRSATPVVPLETGSTSQPTPPPVTARATEPPPVANPAAPKSAPGVQQAHGLKTRIAGKLSERQ



 


TNFSLGIPGTGRTLNRPLRSGIPEEGEQVSNEESHDPLLKEAHELQRMVESALTHLKAAPTSLWERPAP


 


STVRRITTKIFPWLKPAPLREVASNGSNAKTKIKINSQQSPETIAAAVKELSTRLDHQSKVLATATHAL


 


VAAREHLESLEQATPPSSTEPLDHARARVQQADSTTRLASQQLRELIQGTDVLQLGALSEGQDQVEQKA


 


EFS






Expression of ORF01152 is activated by HrpL, as indicated by miniTn5gus mutagenesis (Fouts et al., Proc. Natl. Acad. Sci. USA 99(4):2275–2280 (2001), which is hereby incorporated by reference in its entirety). This protein possesses N-terminal Hop features (see U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, which is hereby incorporated by reference in its entirety), suggesting that it is likely to be secreted.

A sixty-third nucleic acid molecule encodes HopPtoF (formerly AvrPphFPto ORF2) and has a nucleotide sequence according to SEQ ID NO: 125 as follows:








ataggtaatatttgcggcacctcgggctcacgtcatgtgtatagcccatcccatacacaacgaataact



 


tcagctccctctacatccactcatgttggtggagatacactgacatccattcatcagctttcgcatagt


 


cagagagagcagtttctgaacatgcatgatccaatgagagtaatgggacttgaccatgataccgagctt


 


ttcagaacgacggatagtcgctatataaaaaacgataaactcgcgggcaatccacaatccatggcgagt


 


atccttatgcatgaagaactgcgccccaatcgttttgccagccatacaggtgcccaaccacacgaagca


 


agggcgtacgttccgaaaagaataaaagccaccgatctaggagttccatcactgaacgtaatgactggc


 


tcgctagcgcgagacggaattagagcttatgatcacatgagtgataatcaggtctctgtcaaaatgcga


 


ctgggagattttctcgaaaggggtggcaaggtctatgccgacgcttcgtctgtagctgacgatggggaa


 


acatcacaagctctgattgtcacattgcccaaaggacagaaagtgccggtcgaaagggtctga






HopPtoF has an amino acid sequence according to SEQ ID NO: 126 as follows:









MGNICGTSGSRHVYSPSHTQRITSAPSTSTHVGGDTLTSIHQLSHSQREQ



 


FLNMHDPMRVMGLDHDTELFRTTDSRYIKNDKLAGNPQSMASILMHEELR


 


PNRFASHTGAQPHEARAYVPKRIKATDLGVPSLNVMTGSLARDGIRAYDH


 


MSDNQVSVKMRLGDFLERGGKVYADASSVADDGETSQALIVTLPKGQKVP


 


VERV






Contrary to the previously identified sequence of hopPtoF (see U.S. patent application Ser. No. 10/114,828 to Collmer et al., filed Apr. 2, 2002, which is hereby incorporated by reference in its entirety), hopPtoF possesses a rare ATA start codon, which is believed to be involved in regulating protein synthesis in DC3000. HopPtoF has been shown to be expressed by DC3000 and it has been shown to be translocated in planta, where it is localized to the plant plasma membrane and has a role in virulence. HopPtoF has also been shown to cause a hypersensitive response in Arabidopsis Col-0. The homologous Pseudomonas syringae pv. phaseolicola AvrPpbF effector protein has been shown to play a role in both development of the hypersensitive response and virulence in several plants (Tsiamis et al., “Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease,” EMBO J. 19(13):3204–3214 (2000), which is hereby incorporated by reference in its entirety). Finally, HopPtoF has since been shown to be cytotoxic to eukaryotic cells, specifically cultured mammalian CHO and HEK293 cell lines.

A sixty-fourth nucleic acid molecule encodes IaaLPto and has a nucleic acid sequence according to SEQ ID NO: 208 as follows:








atgactgcctacgatgtagaaaaggaatggagcagaatttccaatactgccgctaaaactcaccagaac



 


aacgattttgaaggtttcacctaccaggacttcagaacccacgtaccgatcatggacaaggaaggcttc


 


gcggcacaaaccgaacgctgccttgagcgcaacgagcgcaactgcctgatcggctttaccagtggcacc


 


agcggcaacctcaaacgctgttattactactacgactgtgaagtcgatgaagacagttcccgctccaac


 


gtcttccgcagcaatggtttcattcaacccggtgatcgctgcgccaacctgttcaccatcaacctgttt


 


tctgccctgaacaacatcaccaccatgatggccggtaactgcggtgcgcatgtggtgtccgtaggcgat


 


atcaccctgctgaccaagagtcacttcgaggcgctcaactcgatcaagctcaacgtactgctcggcgta


 


ccctcgaccatcctgcagttcatcgatgccatgcagcagcacggtgtgcacatcgatatcgaaaaggtc


 


gtcttcaatggcgagggcctgaaaacctttcagaagaaaatcatcagggaagcctttggcgaacaggtc


 


tccatcgtcggcgtatatggcagttccgagggcggcattctgggtttcaccaacagcccttgccacacc


 


gaatacgagtttctttccgacaaatacttcatcgagaaagaaggcgacagcatcctcatcacctcgttg


 


acccgcgagaacttcacaccgctgctccggtatcgcctgggagacaccgcaacgctttcgctgaaaggc


 


gacaagctctatttgactgacatccagcgggaggacatgagcttcaacttcatgggcaacctcattggt


 


ctgggcatcattcaacaagcgatcaaacagacactgggccgcacgctggaaatccaggttcacctgtca


 


gtgactgatgcgcgcaaagaactggtgaccgttttcgttcaggcctcggaagtcaacgaagatgaacgc


 


gccagaatcgaaacagccatcgccgatattccggacatcaacgaggcctatcagaaagaccagggcagc


 


gtgctggttgtgcgcaaggatgccagagactacgccgtctcggagcgaggcaaaatgctctacatcatt


 


gaccgcaggaat






IaaLPto has an amino acid sequence according to SEQ ID NO: 209 as follows:









MTAYDVEKEWSRISNTAAKTHQNNDFEGFTYQDFRTHVPIMDKEGFAAQTERCLERNERNCLIGFTSGT



 


SGNLKRCYYYYDCEVDEDSSRSNVFRSNGFIQPGDRCANLFTINLFSALNNITTMMAGNCGAHVVSVGD


 


ITLLTKSHFEALNSIKLNVLLGVPSTILQFIDAMQQHGVHIDIEKVVFNGEGLKTFQKKIIREAFGEQV


 


SIVGVYGSSEGGILGFTNSPCHTEYEFLSDKYFIEKEGDSILITSLTRENFTPLLRYRLGDTATLSLKG


 


DKLYLTDIQREDMSFNFMGNLIGLGIIQQAIKQTLGRTLEIQVHLSVTDARKELVTVFVQASEVNEDER


 


ARIETAIADIPDINEAYQKDQGSVLVVRKDARDYAVSERGKMLYIIDRRN






IaaLPto has significant homology, as detected by BLAST analysis (0), to IAA-lysine synthetase (GenBank accession M35373; Roberto et al., Proc. Natl. Acad. Sci. USA 87: 5797–5801 (1990), each of which is hereby incorporated by reference in its entirety).

Fragments of the above-identified proteins or polypeptides as well as fragments of full length proteins 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., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), and Ausubel et al. (ed.), Current Protocols in Molecular Biology, John Wiley & Sons (New York, N.Y.) (1999 and preceding editions), each of which is hereby incorporated by reference in its entirety. 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, H. A., et al., “Recent Advances in the Polymerase Chain Reaction,” Science 252:1643–51 (1991), which is hereby incorporated by reference. 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.
Other DNA molecules encoding other effector proteins or polypeptides can also be identified by determining whether such DNA molecules hybridize under stringent conditions to a nucleic acid molecule as identified above. An example of suitable stringency conditions is when hybridization is carried out for about 8 to about 20 hours at a temperature of about 37° C. using a hybridization medium that includes 0.9× sodium citrate (“SSC”) buffer, followed by washing for about 5 minutes to about 1 hour 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 increasing 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. up to and including about 65° C. (inclusive of all temperature in such range) for about 8 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 for about 5 minutes to about 1 hour, at about 42° C. up to and including about 65° C. (inclusive of all temperatures in such range) in a 0.2×SSC buffer. Such hybridizing nucleic acid molecules preferably hybridize over substantially over their entire length. Moreover, such hybridizing nucleic acid molecules does not include previously reported nucleic acid molecules that encode effector proteins.
The delivery of effector proteins or polypeptides can be achieved in several ways: (1) as a stable 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, which is hereby incorporated by reference in its entirety, 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 F. W. Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology vol. 185 (1990), which is hereby incorporated by reference in its entirety), and any derivatives thereof. 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., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety.
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. Furthermore, 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, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference in its entirety.
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., “A Cloned Erwinia chrysanthemi Hrp (Type III Protein Secretion) System Functions in Escherichia coli to Deliver Pseudomonas syringae Avr Signals to Plant Cells and Secrete Avr Proteins in Culture,” Microbiol. 95:10206–10211 (1998), which is hereby incorporated by reference in its entirety) 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., Proc. Natl. Acad. Sci. USA 80:4803–4807 (1983), which is hereby incorporated by reference in its entirety) and the cauliflower mosaic virus 35S promoter (O'Dell et al., “Identification of DNA Sequences Required for Activity of the Cauliflower Mosaic Virus 35S Promoter,” Nature, 313(6005):810–812 (1985), which is hereby incorporated by reference in its entirety). 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., each of which is hereby incorporated by reference in its entirety.
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., Proc. Nat'l. Acad. Sci. USA, 80:4803–4807 (1983), which is hereby incorporated by reference in its entirety) and the cauliflower mosaic virus 3′ regulatory region (Odell, et al., Nature, 313(6005):810–812 (1985), which is hereby incorporated by reference in its entirety).
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., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), which is hereby incorporated by reference in its entirety.
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., each of which is hereby incorporated by reference in its entirety. 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., Proc. Natl. Acad. Sci. USA, 79:1859–63 (1982), which is hereby incorporated by reference in its entirety.
The DNA molecule may also be introduced into the plant cells by electroporation. Fromm, et al., Proc. Natl. Acad. Sci. USA, 82:5824 (1985), which is hereby incorporated by reference in its entirety. 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, Science, 237:1176–83 (1987), which is hereby incorporated by reference in its entirety.
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., Proc. Natl. Acad. Sci. USA, 80:4803–4807 (1983), which is hereby incorporated by reference in its entirety). 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., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad.
Press, Orlando, Vol. I, 1984, and Vol. III (1986), each of which is hereby incorporated by reference in their entirety.
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. 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 whose product 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. By disease resistance, it is believed that the effector proteins of the present invention can impart to plants resistance against bacterial, viral, and/or fungal diseases.
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.
Alternative to transgenic expression is topical application of the effector proteins to plants. 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 can be carried out using an effector-TAT protein, 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: 127) by standard rDNA techniques to the N-terminus of the effector protein, and the resulting tagged protein is taken up into animal 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., “Protein transduction: unrestricted delivery into all cells?” Trends Cell Biol. 10:290–295 (2000), which is hereby incorporated by reference in its entirety). Other PTDs are known and can be used for this purpose (Prochiantz, “Messenger proteins: homeoproteins, TAT and others,” Curr. Opin. Cell Biol. 12:400–406 (2000), which is hereby incorporated by reference in its entirety). See PCT Application Publication No. WO 01/19393 to Collmer et al., which is hereby incorporated by reference in its entirety.
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 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. 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 if 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.
Thus, a further aspect of the present invention relates more generally to a method of modifying a metabolic pathway in a cell by introducing into the cell an effector protein or polypeptide of the present invention which interacts with a native cellular protein involved in a metabolic pathway of the cell. As a result of introducing the protein or polypeptide into the cell, the protein or polypeptide modifies the metabolic pathway through its interaction with the native cellular protein. By way of example, it is believed that HopPtoD2 is a tyrosine phosphatase that interacts with MAPK.
Yet another 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 protein which is cytotoxic and causes cell death. 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.
Another aspect of the present invention relates to a method of inhibiting programmed cell death which is carried out by introducing into a eukaryotic cell susceptible to programmed cell death, a protein that is a hypersensitive response suppressor, where the introduction thereof is performed under conditions effective to inhibit programmed cell death of the eukaryotic cell. By inhibiting programmed cell death, it is intended that such inhibition includes both delaying the occurrence of programmed cell death as well as preventing programmed cell death. 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. By way of example, hypersensitive response suppressor proteins include, without limitation, AvrPphEPto, AvrPpiB1Pto, AvrPtoB, HopPtoD1, HopPtoE, HopPtoF (previously designated AvrPphFPto ORF2), and HopPtoK.
Because programmed cell death (including apoptosis) is involved in the pathogenesis of a variety of diseases, the HR suppressor proteins of the present invention can be used in the regulation thereof and, thus, as therapeutic agents in the intervention of a wide array of disease processes or maladies (see Rudin & Thompson, Ann. Rev. Med. 48:267–81 (1997), which is hereby incorporated by reference in its entirety).
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), each of which is hereby incorporated by reference in their entirety). 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., J. Mol. Biol. 13:238–252 (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., each of which is hereby incorporated by reference in their entirety.
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, which is hereby incorporated by reference in its entirety.
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., which is hereby incorporated by reference in its entirety. 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, Biotechniques 6:616–627 (1988) and Rosenfeld et al., Science 252:431–434 (1991), WO 93/07283, WO 93/06223, and WO 93/07282, each of which is hereby incorporated by reference in their entirety. 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., Science 258:1485–1488 (1992); Walsh et al., Proc. Nat'l. Acad. Sci. 89:7257–7261 (1992); Walsh et al., J. Clin Invest. 94:1440–1448 (1994); Flotte et al., J. Biol. Chem. 268:3781–3790 (1993); Ponnazhagan et al., J. Exp. Med. 179:733–738 (1994); Miller et al., Proc. Nat'l Acad. Sci. 91:10183–10187 (1994); Einerhand et al., Gene Ther. 2:336–343 (1995); Luo et al., Exp. Hematol. 23:1261–1267 (1995); and Zhou et al., Gene Ther. 3:223–229 (1996), each of which is hereby incorporated by reference in their entirety. In vivo use of these vehicles is described in Flotte et al., Proc. Nat'l Acad. Sci. 90:10613–10617 (1993); and Kaplitt et al., Nature Genet. 8:148–153 (1994), each of which is hereby incorporated by reference in their entirety. 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.; and U.S. Pat. No. 5,885,808 to Spooner et al.; and U.S. Pat. No. 5,871,727 to Curiel, each of which is hereby incorporated by reference in their entirety).
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., which is hereby incorporated by reference in its entirety.
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, thereby causing 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 or administering to a patient, a cytotoxic Pseudomonas protein under conditions effective to inhibit cancer cell division, thereby treating the cancer 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 targeted DNA delivery system to the patient such that the cancer cells are targeted and the effector protein is expressed therein. A number of targeted delivery systems are known in the art and can be employed herewith.
EXAMPLES
The following Examples are intended to be illustrative and in no way are intended to limit the scope of the present invention.
Materials & Methods for Example 1–2
Strains and Media: Escherichia coli strain DH5α was used for cloning experiments, and P. s. tomato DC3000 or derivatives and P. s. phaseolicola 3121 were used for secretion or translocation assays, respectively. Routine culture conditions for bacteria are similar to those described (van Dijk et al., J. Bacteriol. 181:4790–4797 (1999), which is hereby incorporated by reference in its entirety). Antibiotics were used at the following concentrations: 100 μg/ml ampicillin, 20 μg/ml chloramphenicol, 10 μg/ml gentamicin, 100 μg/ml rifampicin, 10 μg/ml kanamycin, and 20 μg/ml tetracycline.

Secretion Assays: All of the secretion assays used P. s. tomato DC3000 strains carrying a pML123 derivative containing a PCR-cloned ORF (encoding a candidate Hrp-secreted protein) fused to nucleotide sequences that encoded either the hemagglutinin or FLAG epitopes along with their native ribosome binding sites (Labes et al., Gene 89:37–46 (1990), which is hereby incorporated by reference in its entirety). Details about the primers and the constructs are provided below.

    HopPtoE: The hopPtoE gene was cloned using forward primer
    
        (agtaggatccatagaaaaataccataggggtgca, SEQ ID No: 128) containing a BamHI site and reverse primer
        (agtatctagatcacttgtcatcgtcgtccttgtagtcgtcaatcacatgcgcttg, SEQ ID No: 129) containing an XbaI site and FLAG epitope codons. The hopPtoC gene was cloned into plasmid vector pLN162.
    
    
    HopPtoG: The hopPtoG gene was cloned using forward primer
    
        (atgcggatcccgtatgaccttgtaaaat, SEQ ID No: 130) containing a BamHI site and reverse primer
        (atgctctagatcaagcgtaatctggaacatcgtatgggtagccgttgtaaaactgctt, SEQ ID No: 131) containing an XbaI site and HA epitope codons. The hopPtoG gene was cloned into plasmid vector pLN131.
    
    
    HopPtoH: The hopPtoH gene was cloned using forward primer
    
        (agtcggatccgataatcctggatgatccattg, SEQ ID No: 132) containing a BamHI site and reverse primer
        (agtcctcgagtcacttgtcatcgtcgtccttgtagtcttgatgtgccctgtactt, SEQ ID No: 133) containing an XhoI site and FLAG epitope codons. The hopPtoH gene was cloned into plasmid vector pLN150.
    
    
    HopPtoI: The hopPtoI gene was cloned using forward primer
    
        (agtaaagcttacgggcaggtattgcaag, SEQ ID No: 134) containing a BamHI site and reverse primer
        (agtatctagatcacttgtcatcgtcgtccttgtagtcttttttgggcagccagcg, SEQ ID No: 135) containing an XbaI site and FLAG epitope codons. The hopPtoC gene was cloned into plasmid vector pLN165.
    
    
    HopPtoL: The hopPtoL gene was cloned using forward primer
    
        (agtaggatcctgcctccaactattggct, SEQ ID No: 136) containing a BamHI site and reverse primer
        (agtatctagatcacttgtcatcgtcgtccttgtagtctctcgctttgaacgcctg, SEQ ID No: 137) containing an XbaI site and FLAG epitope codons. The hopPtoL gene was cloned into plasmid vector pLN224.
    
    
    HopPtoS1: The hopPtoS1 gene was cloned using forward primer
    
        (ataggatcccgagaacggcgcggacgtg, SEQ ID No: 138) containing a BamHI site and reverse primer
        (atatctagatcatttatcatcatcatctttataatcctcgtcagagctctctgc, SEQ ID No: 139) containing an XbaI site and FLAG epitope codons. The hopPtoC gene was cloned into plasmid vector pLN142.
    
    
    HopPtoS2: The hopPtoS2 gene was cloned using forward primer
    
        (gatggatccacgcacataacaacggtg, SEQ ID No: 140) containing a BamHI site and reverse primer
        (atatctagatcatttatcatcatcatctttataatcaatctgacttaatac, SEQ ID No: 141) containing an XbaI site and FLAG epitope codons. The hopPtoC gene was cloned into plasmid vector pLN223.

Constructs carrying different epitope-tagged ORFs were electroporated into DC3000 and a DC3000 hrcC mutant and grown in Hrp-inducing conditions (Yuan & He, J. Bacteriol. 178:6399–6402 (1996), which is hereby incorporated by reference in its entirety). Additionally, all of the DC3000 strains also carried pCPP2318, a construct that contains blaM lacking signal peptide sequences (Charkowski et al., J. Bacteriol. 179:3866–3874 (1997), which is hereby incorporated by reference in its entirety). DC3000 cultures were separated into cell-bound and supernatant fractions as described (van Dijk et al., J. Bacteriol. 181:4790–4797 (1999), which is hereby incorporated by reference in its entirety). Proteins were separated with SDS/PAGE by standard procedures (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab Press, Plainview, N.Y. (1989), which is hereby incorporated by reference in its entirety), transferred to polyvinylidene difluoride membranes, and immunoblotted by using anti-FLAG (Sigma), anti-hemagglutinin (Roche Molecular Biochemicals), or anti-β-lactamase (5 Prime→3 Prime) as primary antibodies. Primary antibodies were recognized by goat anti-rabbit IgG-alkaline phosphatase conjugate (Sigma), which were visualized by chemiluminescence by using a Western-Light chemiluminescence detection system (Tropix, Bedford, Mass.) and X-Omat x-ray film.

Plant Materials and Translocation Assays: Arabidopsis thaliana accession Columbia (Col-0) and rps2-201 (Kunkel et al., Plant Cell 5:865–875 (1993), which is hereby incorporated by reference in its entirety) mutant plants were grown in a growth chamber with 12 h of light at 24° C. (22° C. at night) and 70% relative humidity. Details about the primers and constructs described below.

    
    
    AvrRpt2: The avrRpt2 gene was cloned using forward primer
    
        (attggtacctctagaggatccaaccttcaatctgaa, SEQ ID No: 142) containing KpnI, XbaI, and BamHI sites and reverse primer
        (atgtcgacttagcggtagagcattgcg, SEQ ID No: 143) containing an SalI site. The avrRpt2 gene was cloned into plasmid vector pÑavrRpt2.

HopPtoG-AvrRpt2: The chimeric gene was cloned using forward primer

        (gcgaattcgttagttgattttgtctagcg, SEQ ID NO: 144) containing an EcoRI site, and reverse primer (gaggatccgccgttgtaaaactgcttaga, SEQ ID NO: 145) containing a BamHI site. The chimeric gene was cloned into plasmid vector phopPtoGÑavrRpt2.
    
    
    The partial avrRpt2 gene with the N-terminal 40 codons deleted was amplified by using standard PCR procedures and cloned into pMOD (Epicentre Technologies, Madison, Wis.). After confirmation by sequence analysis, it was cloned into the KpnI and SalI sites of the broad-host-plasmid pLK, resulting in pΔavrRpt2. DNA fragments spanning 200 bp upstream of the Hrp boxes and the complete ORF for hopPtoG was cloned into pΔavrRpt2 to produce phopPtoG-ΔavrRpt2. The construct was introduced in P. s. phaseolicola 3121 by electroporation. Bacterial strains in 10 mM MgCl2 at a cell density of 108 colony-forming units/ml were infiltrated into A. thaliana Col-0 and Col-0 rps2-201 plants with a needleless syringe.

Identification of Putative Effector Protein ORFs: Several approaches were employed for the identification of putative effector proteins, including the use of a Hidden Markov Model to analyze regions upstream of ORFs for hrp-related promoters (Fouts et al., Proc. Natl. Acad. Sci. USA 99(4):2275–2280 (2001), which is hereby incorporated by reference in its entirety), a miniTn5gus transposon-based assay which identifies HrpL-activated insertions, via insertions downstream of Hrp boxes (Fouts et al., Proc. Natl. Acad. Sci. USA 99(4):2275–2280 (2001), which is hereby incorporated by reference in its entirety), and computer search for candidate Hrp-secreted proteins based on an algorithm that identifies compliance or non-compliance with export signal rules of known effector proteins (N-terminal 50 amino acids) (Petnicki-Ocwieja et al., Proc. Natl. Acad. Sci. USA 99:7652–7657 (2002); U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, each of which is hereby incorporated by reference in its entirety).



Materials & Methods for Example 3–8
Bacterial strains, plasmids, and media: Escherichia coli strains DH5α and DB3.1. (Invitrogen) were used for general cloning and Gateway technology manipulations, respectively. P. s. pv. tomato DC3000 and P. fluorescens strains were grown in King's B (KB) broth at 30° C. (King et al., J. Lab. Med. 22:301–307 (1954), which is hereby incorporated by reference in its entirety). E. coli and Agrobacterium tumefaciens C58C1 were grown in LB broth at 37° C. or 30° C., respectively. Unless otherwise noted, constructs used were made by PCR and Table 1 below includes a list of nucleotide primer sequences that were used. The pHIR11 derivative, pLN18, which lacks shcA and hopPsyA was generated as described previously (van Dijk et al., Mol. Microbiol. 44:1469–1481 (2002), which is hereby incorporated by reference in its entirety). Briefly, 2 kb regions upstream and downstream of shcA and hopPsyA were PCR cloned into pBluescript-II KS on either side of an nptII antibiotic marker. When transformed into the E. coli strain C2110 (Kahn and Hanawalt, J. Mol. Biol. 128:501–525 (1979), which is hereby incorporated by reference in its entirety) containing pHIR11, this construct recombined into pHIR11 because ColE1 plasmids, such as pBluescript-II KS, cannot replicate in this polA mutant at 42° C. When this strain was grown at 30° C., the ColE1 replicon replicated, forcing it to recombine out of pHIR11. pHIR11 derivatives that lacked shcA and hopPsyA were identified with PCR. Antibiotics were used at the following concentrations (μg/ml): rifampicin, 100; ampicillin, 100; gentamicin, 10; kanamycin, 50; tetracycline, 20; nalidixic acid, 20; and spectinomycin 50.







TABLE 1







Additional information on plasmid constructions











Gene
Primer Nucleotide Sequences (5′→3′) and Other
SEQ ID
Parent
Plasmid


Name
Relevant Features
NO:
Plasmid
Construct














shcA and
P21: gtaaaacgacggccagt
146
pHIR11
pLN18


hopPsyA
P23: atgagaattcgcatctccatgcatctt (Eco RI)
147



P227: cggactcgagctcagggcgcgaaactga (Xho I)
148



P228: gtatggtaccccgacctggcaaccgcag (Kpn I)
149


avrPto
P792: agtcctcgagactaaagagggtatacgaatgggaaatata
150
pBBR1
pLN526



(Xho I)

MCS2



P793: agtcgatatctcattgccagttacggtacgggc
151



(Eco RV)


hopPtoT
P582: gatggatccaagtaaccggtctgcaca (Bam HI)
152
pML123
pLN256



P583: atatctagatcatttatcatcatcatctttatatgacttttgagccgcc
153



tg (Xba I)


mouse
P0942: ggcctcgagatggacgggtccggggagcagctt
154
pTA7002
pLN555


α-Bax
(Xho I)



P0943: ggcactagttcagcccatcttcttccagatggtg (Spe I)
155


avrPphEPto
P683: cacctatttaattcgttgagaaacaatgaaaata
156
Gateway
pCPP5057



P684: gacatctcgtctcgccaagcc
157
entry


avrPpiB1Pto
P685: caccaagcaacgtctggaggcaacaatgca
158
Gateway
pCPP5052



P686: gtcgcctaggaaattatttagttcccatga
159
entry


avrPtoB
P693: caccaagatcggagaggatcagaatatggcg
160
Gateway
pLN323



P694: ggggactattctaaaagcatacttggc
161
entry


hopPsyA
P787: caccttagcgtaaggagctaacaatgaaccc
162
Gateway
pLN458



P788: gtttcgcgccctgagcgc
163
entry


hopPtoE
P695: cacccataggggtgcaataacaatgaataga
164
Gateway
pLN324



P696: gtcaatcacatgcgcttggcc
165
entry


hopPtoF
P900:
166
Gateway
pCPP5070



aaaaagcaggcttcgaaggagatagaaccatgtatagcccatcc

entry



P901: agaaagctgggtaacagaccctttcgac
167


hopPtoG
P0904: cacccacataggatatgtaaacaatgcaaataaagaac
168
Gateway
pLN520



P0905: gccgttgtaaaactgcttagaggc
169
entry


hopPtoK
P940: caccacaaagaggttttcaaacaatgaatc
170
Gateway
pCPP5100



P941: gcagtagagcgtgtcgcgac
171
entry


avrPphEPto
Gateway recombination

pMLl123
pCPP5068





pPZP212
pLN535


avrPpiB1Pto
Gateway recombination

pMLl123
pCPP5063





pPZP212
pLN503


avrPtoB
Gateway recombination

pMLl123
pLN347





pPZP212
pLN502


hopPsyA
Gateway recombination

pPZP212
pLN474


hopPtoE
Gateway recombination

pPZP212
pLN524


hopPtoF
Gateway recombination

pMLl123
pCPP5070





pPZP212
pLN525


hopPtoG
Gateway recombination

pPZP212
pLN530


hopPtoK
Gateway recombination

pML123
pCPP5100


avrPPhEPto
P166: atacataacgctggccta
172
pKnockout-
pLN15



P167: cggatccatgacaatcgt
173


avrPpiB1Pto
P168: gcaaatcctttaagctct
174
pKnockout-
pLN16



P169: tgtttcgctaagccactg
175


avrPtoB
P304: tcgcgccaaaccagggag
176
pKnockout-
pLN42



P305: tcccacattctgcaacgc
177


hopPsyAPto
P188: aaccccattcagtcacgc
178
pKnockout-
pLN23



P189: tttgccatgcgtgattgc
179


hopPtoD1
P160: cctctacgatctattcaa
180
pKnockout-
pLN4



P161: ggcaatgctcgcggcctg
181


hopPtoE
P913: tccggtagctcgtcagcg
182
pKnockout-
pLN543



P914: gtggatgaccacatagttatg
183


hopPtoF
P179: agcccatcccatacacaa
184
pKnockout-
pLN7



P180: cactttctgtcctttggg
185


hopPtoG
P256: tattcagcttcaagaatg
186
pKnockout-
pLN29



P257: acccgcatagacctgtctg
187


hopPtoH
P194: atcactccgtctcgatatc
188
pKnockout-
pLN27



P195: tgccctgtacttcatgcg
189


hopPtoJ
P173: ctatgtatttcaaaacac
190
pKnockout-
pLN8



P174: atcaccctctgtaattccc
191


hopPtoK
P171: cgcatttcaaccagctca
192
pKnockout-
pLN9



P172: cagcaccggaagcccttc
193


hopPtoS1
P190: ggtaatatttgtggtacttc
194
pKnockout-
pLN41



P191: cagatgtaacgtgacatc
195


hopPtoT
P192: acagtcagcaatcactcg
196
pKnockout-
pLN25



P193: tacactccatacactgctg
197


avrPphEPto
P854: ttgaattcatgaaaatacataacgctgg (Eco RI)
198
pGilda
pLN508



P855: ttctcgagtcagacatctcgtctcgc (Xho I)
199


avrPpiB1Pto
P860: ttggatccgtatgcacgcaaatcctttaagctc
200
pGilda
pLN507



(Bam HI)



P861: ttctcgagtcagtcgcctaggaaattatttagttcc
201



(Xho I)


hopPtoE
P858: ttgaattcatgaatagagtttccggtagctc (Eco RI)
202
pGilda
pLN504



P859: ttctcgagtcagtcaatcacatgcgcttgg (Xho I)
203


hopPtoF
P856: ttgaattcatgggtaatatttgcggcacctc (Eco RI)
204
pGilda
pLN505



P857: ttctcgagtcagaccctttcgaccgg (Xho I)
205


hopPtoG
P862: ttgaattcatgcaaataaagaacagtcatctc (Eco RI)
206
pGilda
pLN506



P863: ttctcgagtcagccgttgtaaaactgcttagag (Xho I)
207









Hypersensitive Response Assays: The broad-host-range vector pML123 was used to express effector genes in Pseudomonas strains (Labes et al., Gene 89:37–46 (1990), which is hereby incorporated by reference in its entirety). The pML123 constructs containing hopPtoC, hopPtoD1, hopPtoD2, and hopPtoJ are described in U.S. patent application Ser. No. 10/114,828 to Collmer et al., filed Apr. 2, 2002, which is hereby incorporated by reference in its entirety; and pML123 constructs containing hopPtoE, hopPtoG, hopPtoH, hopPtoI, hopPtoL, hopPtoS1, and hopPtoS2 are described above. A pML123 construct containing hopPtoB was similarly prepared. pML123 constructs containing hopPtoF, hopPtoK, hopPtoT, avrPtoB, avrPphEPto, avrPpiB1Pto, and avrPto are detailed in Table 1. P. fluorescens(pHIR11) carrying pML123 constructs with effector genes or vector controls with an OD600 of 0.2 (ca. 108 cells/ml) in 5 mM MES (pH 5.6) and infiltrated into Nicotiana tabacum cv. Xanthi, N. benthamiana, or A. thaliana Ws-0 leaves. For bacterial mixing experiments involving two different P. fluorescens strains, P. fluorescens(pLN18) and a pML123 effector construct were infiltrated 2 h before P. fluorescens(pHIR11). The plants were scored for the production of an HR after 24 h. DC3000 strains were tested for their ability to elicit an HR on Nicotiana tabacum cv. xanthi by infiltrating strains with an OD600 of 0.2 along with 10-fold serially diluted samples with a needleless syringe.

Type III secretion assays, SDS-PAGE, and immunoblot analysis: DC3000 and DC3000 hrcC mutant (Yuan and He, J. Bacteriol. 178:6399–6402 (1996), which is hereby incorporated by reference in its entirety) carrying the plasmids pLN162, pLN526, pCPP2318, which encode for HopPtoE-FLAG, AvrPto, and β-lactamase, respectively, were grown in type III-inducing minimal medium (Huynh et al., Science 245:1374–1377 (1989), which is hereby incorporated by reference in its entirety). Cells were adjusted to an initial OD600 of 0.3 and grown for 6 h and separated into cell-bound and supernatant fractions by centrifugation at 4° C. Protein samples from bacterial cultures were prepared similarly as described (van Dijk et al., J. Bacteriol. 181:4790–4797 (1999), which is hereby incorporated by reference in its entirety). Cell and supernatant fractions were analyzed by SDS-PAGE (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), which is hereby incorporated by reference in its entirety), transferred to polyvinylidene difluoride membranes, and immunobloted using anti-AvrPto, -β-lactamase, or -FLAG as primary antibodies. Generation of anti-AvrPto antibodies has been described (van Dijk et al., J. Bacteriol. 181:4790–4797 (1999), which is hereby incorporated by reference in its entirety). The anti-β-lactamase antibodies were purchased from Chemicon International and the anti-FLAG antibodies were purchased from Sigma Chemical Co. Primary antibodies were recognized by goat anti-immunoglobulin G-alkaline phosphatase conjugate (Sigma Chemical Co.), and visualized by chemiluminescence using a chemilumincescence detection system and X-Omat X-ray film.

Agrobacterium-mediated transient assays: The avr gene hopPsyA was recombined into a derivative of pPZP212 (Hajdukiewicz et al., Plant Mol. Biol. 25:989–994 (1994), which is hereby incorporated by reference in its entirety), pLN462, which was modified to be a Gateway Destination vector, resulting in pLN474. The bax gene was PCR-cloned into pTA7002, creating pLN531, and expression of bax was induced with dexamethasome as previously described (Aoyama and Chua, Plant Journal 11:605–612 (1997), which is hereby incorporated by reference in its entirety). The effector genes carried on Gateway entry vectors avrPphEPto, avrPpiB1Pto, avrPtoB, hopPtoE, hopPtoF, and hopPtoG were recombined into pLN462 (which fused each gene to a hemagglutinin epitope) creating constructs pLN535, pLN503, pLN502, pLN524, pLN525, and pLN530, respectively. Agrobacterium-mediated transient expression experiments were done by infiltrating A. tumefaciens C58C1 (van Larebeke et al., Nature 252:169–170 (1974), which is hereby incorporated by reference in its entirety) harboring the disabled Ti plasmid pMP90 (Koncz and Schell, Mol. Gen. Genet. 204:383–396 (1986), which is hereby incorporated by reference in its entirety) at an OD600 of 0.4 into N. benthamiana and N. tabacum cv. xanthi plants using a needleless syringe as described (van den Ackerveken et al., Cell 87:1307–1316 (1996), which is hereby incorporated by reference in its entirety). For co-expression experiments, Agrobacterium strains carrying pPZP212 binary plasmids with different effector genes were infiltrated 4 h prior to infiltration of strains expressing either Bax or HopPsyA. Evidence of production of effectors from transient assays was acquired by harvesting 1 cm diameter leaf disks from infiltrated zones, grinding leaf tissue with a mortar and pestle in the presence of liquid nitrogen, and resuspending plant material in 50 μl of 1× SDS-PAGE tracking buffer. SDS-PAGE and immunoblot analysis were performed as described above using high affinity anti-hemagglutinin antibodies (Roche).

Construction of DC3000 effector mutants: In-frame internal fragments of the effector genes were PCR cloned into XcmI digested pKnockout-. (Windgassen et al., FEMS Microbiol. Lett. 193:201–205 (2000), which is hereby incorporated by reference in its entirety) using the primer sets listed in Table 1 above. The resulting constructs were conjugated separately into DC3000 by triparental mating using spectinomycin as selection for the plasmid marker. The following effector mutants were confirmed with primers that flanked each coding region: hopPtoD1, UNL104; hopPtoC, UNL106; hopPtoE, UNL139; hopPtoK, UNL107; hopPtoJ, UNL108; hopPtoF, UNL109; avrPhEPto, UNL113; avrPpiB1Pto, UNL114; hopPtoH, UNL118; hopPtoT, UNL122; hopPtoG, UNL124; hopPtoS1, UNL126; and avrPtoB, UNL127.

Yeast viability assays: To determine whether type III effector-encoding plasmids rescued yeast from Bax-induced lethality, the effector genes avrPphEPto, avrPpiB1Pto, hopPtoG, hopPtoF, and hopPtoE were PCR-cloned into the yeast expression vector pGilda (Clontech, Palo Alto, Calif.) resulting in constructs pLN508, pLN507, pLN506, pLN505, and pLN504, respectively. Table 1 above contains information for the nucleotide primers used to make these constructs. S. cerevisiae EGY48 strain containing pJG4-5-Bax (kindly provided by J. C. Reed, Burnham Institute, La Jolla, Calif.) and various pGilda plasmids containing effector genes were grown in SC-U-L/glucose media overnight. The chicken Bcl-xL cloned in pGilda was kindly provided by C. Thompson (University of Chicago, Chicago, Ill.), which acted as a positive control for PCD suppression in these experiments. The yeast cultures were then serial 10-fold diluted into SC medium, and 5 μl of each dilution was dropped onto SC-U-L/Galactose or SC-UL/Glucose plates. Cells were incubated at 30° C. for 5 days, and photographed. For oxidative stress experiments, EGY48 strains containing pGilda effector constructs were grown in SC-U media overnight and treated as described in Abramovitch et al. (Abramovitch et al., EMBO 22:60–69 (2003), which is hereby incorporated by reference in its entirety).

Example 1
Demonstration of Pseudomonas syringae pv. tomato DC3000 Protein Secretion
From the hidden Markov model analysis, 28 candidate effector ORFs were identified that were not homologs of known Avr proteins/Hops or of any proteins unlikely to be secreted, and whose low G+C % content and association with mobile genetic elements suggested horizontal acquisition. Several of the predicted proteins shared amino acid identity with proteins likely to be effectors. For example, HopPtoS1 (ORF5) yields several ADP-ribosyltransferases in BLASTP searches (highest BLAST E value 1e-5), including a type III-secreted ADP-ribosyltransferase from Pseudomonas aeruginosa (Yahr et al., Mol. Microbiol. 22:991–1003 (1996)), and HopPtoH (ORF2) is homologous to an ORF adjacent to the avrPpiC2 avr gene of P. s. pisi (Arnold et al., Microbiology 147:1171–1182 (2001), which is hereby incorporated by reference in its entirety) (see Table 2 below).
To test whether these proteins travel the Hrp pathway, the ORFs were cloned into a broad-host-range vector fused to either the hemagglutinin or FLAG epitope. DC3000 wild-type and Hrp mutant cultures carrying these constructs were separated into supernatant and cell fractions and analyzed with SDS/PAGE and immunoblots. Five of the eight proteins tested were secreted via the DC3000 Hrp system (FIG. 1A) and consequently were designated as HopPtoE, HopPtoG, HopPtoH, HopPtoI, and HopPtoS1, respectively. Although three ORFs (ORF6, ORF 7, and ORF8) were not detectably secreted in culture, they may still be effectors because AvrB similarly is not secreted in culture although translocated in planta (van Dijk et al., J. Bacteriol. 181:4790–4797 (1999); Gopalan et al., Plant Cell 8:1095–1105 (1996), each of which is hereby incorporated by reference in its entirety).







TABLE 2







ORFs with 5′ Hrp Promoter Sequences and Encoding Proteins


Demonstrated to be Secreted by the P. syringae Hrp System












Initial
New
Size
%
Homolog
GenBank


designation
designation
(bp)
G + C
(BLASTP E value)
Accession 















ORF1
HopPtoI
1,899
48.9
None
NA


ORF2†
HopPtoH
657
47.2
ORF3 from P. s. pisi
CAC 16702






avrPpiC2 locus (le-114)


ORF3
HopPtoE
636
50.7
None
NA


ORF4
HopPtoG
1,482
43.7
Hypothetical protein from
NP_521884






R. solanacearum (le-137)


ORF5‡
HopPtoS1
852
46.5
Chicken ADP-
P55807






ribosyltransferase (1e-5)





 Each of the listed Genbank Accessions is hereby incorporated by reference in its entirety.


†ORF2: homolog described in Arnold et al., Microbiology 147:1171-1182 (2001), which is hereby incorporated by reference in its entirety.


‡ORF5: homolog described in Tsuchiya et al., J. Biol. Chem. 269:27451-27457 (1994), which is hereby incorporated by reference in its entirety. Determined to possess an ART domain (pfam1129), further confirming its similarity to ADP-ribosyltransferases.






To determine whether the export signal-based search had identified any novel Hrp-secreted proteins, secretion assays were also performed on ORF29 and ORF30, both of which seemed to be particularly promising candidates. The products encoded by ORF29 and ORF30 share similarity with a putative type III effector from S. enterica, SrfC, and ADP-ribosyltransferases, respectively. Both ORFs were PCR-cloned into a broad-host-range vector fused to the FLAG epitope, and each construct was introduced into DC3000 wild-type and Hrp mutant strains. The epitope-tagged ORF29 and ORF30 proteins were secreted by DC3000 in a Hrp-dependent manner without leakage of a cytoplasmic marker protein (FIG. 1B), and consequently they were designated as HopPtoL and HopPtoS2, respectively (see Table 3 below).







TABLE 3







Selected ORFs Encoding Candidate Effector Proteins That Were


Identified by the Genomewide Search Based on Export-Signal Patterns




















Homolog




New
Size
%
Hrp
Mobile
(BLASTP E
GenBank


Designation
Designation
(bp)
G + C′
promoter 
DNA†
value)
Accession‡

















ORF29 
HopPtoL
270
61.0
n
n
SPI-2 regulated
AAF74575




0



SrfC (1e-21)


ORF30Ø,¶
HopPtoS2
795
46.5
y
n
Clostridium
NP_346979








exoenzyme C3








ADP ribosyl-








Tansferase (1e-5)


ORF31 ,¶
NA
897
49.8
y
y
Chicken ADP
P55807








ribosyl-








transferase (5e-3)


ORF32 ,¶
NA
507
54.2
y
y
Chicken ADP
P55807








ribosyl-








transferase (5e-3)


ORF33Θ
NA
282
55.2
n
y
SepC insecticidal
NP_065279




3



toxin (1e-128)


ORF34*
NA
534
63.5
y
n
Lytic enzyme
BAA83137








(3e-36)





 Indicates that the ORF is within 10 kb of a HrpL-responsive Hrp promoter identified in Fouts


†Indicates that a transposon, plasmid, or a phage-related sequence is within 10 kb.


‡Each of the listed Genbank Accessions is hereby incorporated by reference in its entirety.


¶ORF was determined to possess an ART domain (pfam1129), further confirming its similarity to ADP-ribosyltransferases.


 Homolog identified in Worley et al., Mol. Microbiol. 36:749-761 (2000), which is hereby incorporated by reference in its entirety.


ØHomolog identified in Nolling et al., J. Bacteriol. 183:4823-4838 (2001), which is hereby incorporated by reference in its entirety.


 Homolog identified in Tsuchiya et al., J. Biol. Chem. 269:27451-27457 (1994), which is hereby incorporated by reference in its entirety.


ΘHomolog identified in Hurst et al., J. Bacteriol. 182:5127-5138 (2000), which is hereby incorporated by reference in its entirety.


*Homolog identified in Nakayama et al., Mol. Microbiol. 38:213-231 (2000), which is hereby incorporated by reference in its entirety.






Interestingly, the export signal-based search found a putative effector, SrfC, that is predicted to travel the type III pathway encoded by SPI2 of S. enterica (Worley et al., Mol. Microbiol. 36:749–761 (2000), which is hereby incorporated by reference in its entirety). A further indicator of the efficacy of the search was the finding of three additional ADP-ribosyltransferases, ORF 30, 31, and 32, all with significant amino acid sequence identity to HopPtoS 1 (Table 3).
Example 2
AvrRpt2 Translocation Assay Indicates that at Least One of the Additional Hops Is Translocated into Plant Cells.
HopPtoG was selected to test for translocation into plant cells because it shared no similarities with any sequences in the databases and was shown to be secreted (FIG. 1B). P. s. phaseolicola carrying a plasmid expressing hopPtoG-Δ avrRpt2 elicited an RPS2-dependent hypersensitive response in A. thaliana Col-0 (FIG. 1C), indicating that targeting information in HopPtoG directed translocation of the AvrRpt2 fusion protein into plant cells. Thus, HopPtoG appears to be a Hrp-injected effector protein.
Discussion of Examples 1–2
One demonstration of the selectivity of the export signal rules is that only the chicken ADP-ribosyltransferase NRT2CHK shows major violations of the rules even though this protein is more similar to HopPtoS1 and S2 than either of the type III-secreted ADP-ribosyltransferases from P. aeruginosa, ExoS and ExoT (see Petnicki-Ocwieja et al., Proc. Natl. Acad. Sci. USA 99:7652–7657 (2002); U.S. patent application Ser. No. 10/341,180 to Collmer et al., filed Jan. 13, 2003, each of which is hereby incorporated by reference in its entirety).
HopPtoS1 and HopPtoS2 share sequence similarity with ADP-ribosyltransferases, proteins that have long been implicated in bacterial pathogenesis in animals through the modification of host signal transduction pathways (Finlay & Falkow, Microbiol. Mol. Biol. Rev. 61:136–169 (1997), which is hereby incorporated by reference in its entirety), but until now have not been implicated in the bacterial pathogenesis of plants. The DC3000 genomic studies described in Fouts et al. (Proc. Natl. Acad. Sci. USA 99:2275–2280 (2002), which is hereby incorporated by reference in its entirety) clearly show that several of the effectors in DC3000 are redundant. By using the pattern-based export prediction, three ADP-ribosyltransferase genes (in addition to hopPtoS1) that have N-termini putative export signals were identified in the genome of DC3000. One of these, ORF32, appears to be truncated. The other two, HopPtoS2 and ORF31, are full-length genes based on sequence alignments. HopPtoS2 is secreted by the Hrp system (FIG. 1B) and ORF31 shares high amino acid sequence identity with the Hrp-secreted HopPtoS1. Interestingly, HopPtoS1 contains putative myristoylation and palmitoylation sites at its N terminus, whereas the other two do not, indicating that HopPtoS1 may be localized to the plasma membrane. Thus, there appear to be at least three Hrp-secreted ADP-ribosyltransferases and these may localize to different regions of the plant cell. The existence of these proteins in Pseudomonas syringae is particularly noteworthy given that ADP-ribosyltransferase genes have not been identified in the bacterial plant pathogen genomes that have been published thus far (Simpson et al., Nature 406:151–159 (2000); Wood et al., Science 294:2317–2323 (2001); Goodner et al., Science 294:2323–2328 (2001); Salanoubat et al., Nature 415:497–502 (2002), each of which is hereby incorporated by reference in its entirety). Significantly, the genomewide search for export signals yielded a homolog of the S. enterica candidate effector SrfC, further adding to the growing list of effectors shared between plant and animal pathogens. It is also noteworthy that one of the ORFs found by the genomewide search (ORF48) is a homolog of a bacterial catalase (BLASTP 1e-126), and another (ORF49) is a glucokinase homolog (BLASTP 3e-42). These putative effectors likely have a role in oxidative stress and regulation of sugar metabolism, respectively.
Example 3
HopPtoE Suppresses the Hypersensitive Response in Tobacco and a DC3000 hopPtoE Mutant Possesses an Enhanced Hypersensitive Response Phenotype
In the course of experiments with confirmed DC3000 type III effectors, the effector HopPtoD2 was observed capable of suppressing the HR elicited by P. s. phaseolicola on Nicotiana benthamiana plants. These results prompted the screening other effector proteins for HR suppressor activity (Collmer et al., Trends Microbiol. 10:462–470 (2002)). To do this, the pHIR11 system was used, allowing nonpathogens such as E. coli and P. fluorescens to elicit the HR and secrete effectors in culture via the TTSS. This tool allowed for testing whether individual effectors were capable of suppressing the HopPsyA-dependent HR as depicted in FIG. 2A. P. fluorescens(pHIR11) strains carrying a number of different effector constructs were infiltrated into tobacco (N. tabacum cv. xanthi). Interestingly, HopPtoD2, the effector that suppressed an HR elicited by P. phaseolicola, did not suppress the HopPsyA-dependent HR (FIG. 4A). The first identified effector to suppress or block the HR elicited by P. fluorescens(pHIR11) was HopPtoE (FIG. 2B). To detect a potential phenotype consistent with HopPtoE acting as an HR suppressor, a DC3000 mutant defective in HopPtoE was constructed. Both DC3000 and the hopPtoE mutant, UNL139, elicited an HR in tobacco when infiltrated into leaf panels at high inoculum (FIG. 2C).
Based on this result, it is likely that effectors have functionally redundant roles, which may partially mask a phenotype. Therefore, a more sensitive HR assay was performed, where 10-fold serially diluted bacterial strains were infiltrated into tobacco leaf panels to detect any subtle difference in the 5 ability of different strains of bacteria to elicit an HR. When UNL139 was tested in this assay, it was more effective than DC3000 at HR elicitation at lower cell density (FIG. 2C). Interestingly, when hopPtoE was provided in trans to UNL139, the mutant strain was less effective at HR elicitation than DC3000 (FIG. 2C). Thus, the enhanced HR phenotype of the hopPtoE mutant was complemented by hopPtoE. These observations are consistent with HopPtoE acting as an HR suppressor and suggest that HopPtoE contributes incrementally to the ability of the pathogen to suppress the HR.
Example 4
HopPtoE Does Not Block the DC3000 Type III Secretion System
One possible explanation for the observed phenotypes was that HopPtoE was blocking the type III secretion of other type III substrates, including Avr proteins. There is actually a precedent for type III substrates, such as HrpZ and HrpW, to block the type III secretion of proteins from P. syringae (Alfano et al., Mol. Microbiol. 19:715–728 (1996); Charkowski et al., J. Bacteriol. 180:5211–5217 (1998), each of which is hereby incorporated by reference in its entirety) and it was crucial to consider this alternative. To test this, DC3000 and a DC3000 hrcC mutant defective in the TTSS, both carrying plasmids that contained avrPto and hopPtoE, were grown in a medium that induced type III secretion. These cultures were separated into supernatant and cell fractions, and analyzed them by SDS-PAGE and immunoblots with either anti-FLAG or -AvrPto antibodies. Both AvrPto and HopPtoE were secreted in culture via the TTSS (FIG. 3B), indicating that, at least in culture, over-expression of hopPtoE did not block type III secretion. It was next determined whether expression of hopPtoE in DC3000 altered its HR-eliciting ability. Because DC3000 contains a native copy of hopPtoE in its genome, these experiments actually tested whether over-expression of HopPtoE altered the HR phenotype. DC3000 strains, with and without plasmid-encoded hopPtoE, were infiltrated into tobacco at high inoculum levels (108 cells/ml). After 24 hours, DC3000 elicited an HR on tobacco, whereas DC3000 with a plasmid containing hopPtoE did not (FIG. 3B), indicating that over-expression of HopPtoE suppressed the HR. However, after approximately 3 h, DC3000 with hopPtoE in trans also elicited an HR. Thus, hopPtoE in trans in DC3000 only delayed the ability of this pathogen to elicit an HR. When these experiments were repeated in N. benthamiana, the HR delay was greater than 24 h, indicating that the suppression ability of HopPtoE depended to a certain extent on the test plant. To eliminate the possibility that HopPtoE affected the ability of P. fluorescens(pHIR11) to deliver the Avr protein HopPsyA into plant cells, a different bacterial strain was used to deliver HopPsyA (as compared to the strain used to deliver HopPtoE) into plant cells. To accomplish this, a pHIR11 derivative, pLN18, was constructed so as to lack hopPsyA and shcA, a gene that encodes a chaperone for HopPsyA (van Dijk et al., Mol. Microbiol. 44:1469–148 (2002), which is hereby incorporated by reference in its entirety). P. fluorescens(pLN18) does not elicit an HR on tobacco because it lacks HopPsyA (FIG. 3C), while maintaining the ability to secrete proteins via its functional TTSS.
In planta mixed-inoculum experiments were performed by first infiltrating into tobacco P. fluorescens(pLN18) with hopPtoE contained in a broad-host-range plasmid and, after 2 h, P. fluorescens(pHIR11). P. fluorescens(pHIR11) was infiltrated at an OD600 sufficient to cause HR elicitation. FIG. 3C shows that P. fluorescens(pLN18) retained the ability to suppress the pHIR11-dependent HR. This indicates that the HR suppression activity does not occur in the bacterial cell.
Example 5
pHIR11 Assays Identify Seven Effectors Capable of Suppressing the HopPsyA-Dependent Hypersensitive Response
Nineteen confirmed effector genes were cloned into a broad-host-range plasmid and tested to determine whether the encoded effectors were able to suppress the HR elicited by P. fluorescens(pHIR11) (see FIG. 4A for a list of the effectors tested). Each candidate suppressor gene was expressed in P. fluorescens(pHIR11) and these strains were infiltrated into tobacco and Arabidopsis thaliana ecotype Ws-0, two plants that produce an HR in response to pHIR11-containing bacteria.
Surprisingly, seven of the nineteen effectors tested were able to suppress the pHIR11-dependent HR on both A. thaliana and tobacco (FIGS. 4B–C). In planta mixed-inoculum experiments similar to those describe in FIG. 3C demonstrated that all of the identified suppressors were able to inhibit the pHIR11-dependent HR. These results indicate that the site of suppressor activity was outside of the bacteria. Two of the identified suppressors, HopPtoF and AvrPtoB, were homologs of AvrPphF and VirPphA, respectively, two Avr proteins able to “block” the HR produced by P. s. phaseolicola (Jackson et al., Proc. Natl. Acad. Sci. USA 96:10875–10880 (1999); Tsiamis et al., EMBO J. 19:3204–3214 (2000), each of which is hereby incorporated by reference in its entirety). The VirPphA homolog, AvrPtoB, was recently reported to suppress the HR elicited by AvrPto (Abramovitch et al., EMBO J. 22:60–69 (2003), which is hereby incorporated by reference in its entirety). Thus, these findings demonstrate that AvrPtoB and HopPtoF are HR suppressors. The other HR suppressors identified were AvrPphEPto, AvrPpiB1Pto, HopPtoD1, and HopPtoK. The HR suppression observed for HopPtoD1 and HopPtoK was not complete (i.e., the HR was present, although much reduced).
Example 6
Agrobacterium Transient Assays that Co-Deliver HopPsyA and Individual Hypersensitive Response Suppressors Confirm that Each Effector Alone Suppresses the HopPsyA-Dependent Hypersensitive Response Inside Plant Cells
To determine if the HR suppression is due solely to the suppressor proteins, both HopPsyA and individual HR suppressor effectors were transiently co-delivered using Agrobacterium-mediated transient assays (agroinfitrations) (van den Ackerveken et al., Cell 87:1307–1316 (1996), which is hereby incorporated by reference in its entirety). In each case, the effector suppressed the HopPsyA-dependent HR (FIG. 5A). Protein expression was confirmed with immunoblots that showed the agroinfiltrations produced both HopPsyA and the specific suppressor tested (FIG. 5B). These data complement the bacteria-delivered suppressor data shown above, because agroinfiltrations demonstrate that the suppressor activity is dependent only on the suppressor and that the suppressor acts within plant cells, whereas the experiments where P. fluorescens(pHIR11) deliver each suppressor resemble what happens in nature and protein levels are closer to the levels that the pathogen “inject” into plant cells.
Example 7
DC3000 Suppressor Mutants Display an Enhanced Ability to Elicit a Hypersensitive Response on Nonhost Plants, Consistent with Loss of Hypersensitive Response Suppression Activity in the Pathogen
Based on the above findings, it was recognized that a pathogen may encode multiple HR suppressors, each contributing, perhaps incrementally, to the suppression of the HR and/or plant defenses. To analyze these proteins in more detail, mutants defective in each gene corresponding to the effectors listed in FIG. 4A were made. The ability of DC3000 and the suppressor mutants to induce defense responses on non-host plants were tested, similar to the experiments described in FIG. 2C. Tobacco leaves were infiltrated with different dilutions of DC3000 or each mutant, and then their ability to elicit an HR was analyzed. Interestingly, all the mutants were more effective at eliciting an HR at lower concentrations, generally producing an HR at 10-fold higher dilution than wild type DC3000 (FIG. 6). As an example, UNL105 caused a confluent HR at a titer of 106 cells/ml, whereas DC3000 only produced a spotty HR or no HR at this titer. It is important to note that DC3000 produced a typical HR at dilutions of 106 cells/ml or higher. This enhanced HR phenotype produced by each suppressor mutant resulted from the absence of the effector, because when each was supplied in trans the HR-eliciting ability returned to a DC3000-like HR (FIG. 6).
Although HopPtoG was not identified as an HR suppressor in the assays with P. fluorescens(pHIR11), the hopPtoG mutant UNL124 caused an enhanced HR phenotype. Moreover, additional assays shown below suggest that HopPtoG does function as a suppressor. Thus, these findings demonstrate that the phenotype of potential suppressor mutants on non-host plants is consistent with and complements HR suppression data. Therefore, the HR titration assays should be useful in the identification of other HR suppressors in bacterial plant pathogens.
Example 8
Pseudomonas syringae Hypersensitive Response Suppressors Inhibit Programmed Cell Death Induced by the Pro-apoptotic Protein Bax in both Plants and Yeast
The pro-apoptotic mouse protein Bax has been shown to induce PCD in plants that resembles the HR (Kawai-Yamada et al., Proc. Natl. Acad. Sci. USA 98:12295–12300 (2001); Lacomme and Santa Cruz, Proc. Natl. Acad. Sci. USA 96:7956–7961 (1999), each of which is hereby incorporated by reference in its entirety). Bax is a member of the Bcl-2 family of pro-apoptotic proteins and is thought to initiate programmed cell death (“PCD”) by localizing to the mitochondria and causing the release of pro-apoptotic factors, including cytochrome c (Jurgensmeier et al., Proc. Natl. Acad. Sci. USA 95:4997–5002 (1998), which is hereby incorporated by reference in its entirety). Recently, Abramovitch et al. (EMBO J. 22:60–69 (2003), which is hereby incorporated by reference in its entirety) reported that AvrPtoB suppresses Bax-induced PCD in plants. The suppressors identified in Examples 3–7 were tested in their ability to suppress Bax-induced PCD in plants. With the exception of AvrPpiB1Pto, all of them suppressed Bax-induced cell death in plants (FIG. 7A). Interestingly, AvrPphE, HopPtoG, HopPtoF, and HopPtoE also suppressed Bax-induced PCD in yeast (FIG. 7B). The fact that AvrPtoB was unable to suppress Bax-induced PCD in yeast highlights a difference between the activity of these suppressors. Moreover, AvrPphE, HopPtoG, HopPtoF, and HopPtoE were unable to suppress the PCD initiated in yeast by H2O2, while AvrPtoB did suppress PCD in this assay (Abramovitch et al., EMBO J. 22:60–69 (2003), which is hereby incorporated by reference in its entirety). This further demonstrates that the PCD suppressors display different PCD suppressing characteristics.
Discussion of Examples 3–8
Effectors delivered by the Hrp TTSS appear central to P. syringae pathogenesis, but the anti-host functions of these proteins remain obscure. The above Examples identify a founding inventory of Hrp effectors from P. s. tomato DC3000 using several novel bioassays and provide evidence that many of these proteins appear to suppress one or more broadly conserved eukaryote PCD pathways. To understand these results, it is useful to consider the collection of effectors that were assayed, the utility and limitations of the bioassays, and the role of plant cell death in disease and defense. The nineteen effectors considered here were recently identified in DC3000 on the basis of their homology with known effectors and/or their ability to be secreted and/or translocated by the Hrp TTSS. In general, P. syringae TTSS effectors identified on the basis of avirulence phenotypes are designated Avrs, whereas those identified through secretion assays are designated Hops. However, the working assumption is that all of the “Avrs” are injected into plant cells by the Hrp system and many of the “Hops” will confer avirulence phenotypes to bacteria if tested in hosts that happen to carry a corresponding R gene; that is, effectors, Avrs, and Hops are synonymous terms.
It must be noted that the effector described herein do not represent the entire inventory of DC3000 effectors. Effectors encoded within the Hrp pathogenicity island are described elswhere (Alfano et al., Proc. Natl. Acad. Sci. USA 97:4856–4861 (2000), which is hereby incorporated by reference in its entirety. Additional candidate effectors in DC3000 have also been identified (Collmer et al., Trends Microbiol. 10:462–470 (2002), which is hereby incorporated by reference in its entirety).
However, the set of effectors analyzed here suggests that many DC3000 effectors have HR suppression activity. The bioassays used herein were designed to efficiently detect HR suppressor activity, determine whether suppressor action occurs in plant cells rather than in bacteria, and determine if the test effectors could also suppress PCD in other plants and the model eukaryote, yeast. The primary screen, based on suppression of the HR elicited by P. fluorescens(pHIR11) in tobacco, proved to be simple and effective (FIG. 2). Although there is the formal possibility that suppressors identified with this bioassay could be merely interfering with the delivery of HopPsyA, multiple lines of evidence indicate that they act after delivery into plant cells. For example, HopPtoE overexpression had no effect on the secretion of AvrPto (FIG. 3A), a representative type III substrate, and HopPtoE suppressed HopPsyA-dependent HR elicitation when delivered by a functional TTSS in a different strain (FIG. 3C) or when transiently expressed in plant cells following inoculation with A. tumefaciens (FIG. 5A). It is also noteworthy that the suppressors identified herein functioned when delivered via the TTSS, a natural route that is thought to yield relatively low levels of effectors within plant cells. Agrobacterium-mediated transient expression, in contrast, can produce far higher levels of effectors within plant cells potentially leading to artifactual responses. It should also be noted that HopPtoD1 and HopPtoK appear to exhibit limited suppressor activity.
In an attempt to identify plant targets or sites of action of the suppressors in plants, HopPtoE, AvrPphEPto, AvrPiB1Pto, AvrPtoB, HopPtoF, and HopPtoG were subjected to cursory bioinformatic analyses. BLASTP and PSI-BLAST searches (Altschul et al., Nucleic Acids Res. 25: 3389–3402 (1997), which is hereby incorporated by reference in its entirety) did not identify any proteins (other than clear Avr homologs) that shared significant similarity with any of the suppressors. However, 3D-PSSM analyses, a method that uses protein fold recognition to identify proteins with similar folding patterns (Kelley et al., J. Mol. Biol. 299:499–520 (2000), which is hereby incorporated by reference in its entirety), indicated that AvrPtoB had similarity to heme-dependent peroxidases (above 90% certainty; PSSM E value 0.0895). Moreover, when AvrPtoB was aligned with a lignin peroxidase, a representative heme-dependent peroxidase (Welinder, Eur. J. Biochem. 151:497–504 (1985), which is hereby incorporated by reference in its entirety), the amino acids within the peroxidase active site were present similarly spaced in AvrPtoB. This result is intriguing because of the clear involvement of reactive oxygen species (ROS) in plant defense (Mittler, Trends Plant Sci. 7:405–410 (2002), which is hereby incorporated by reference in its entirety) and the potential of peroxidases to modulate ROS. Moreover, there have been reports of peroxidases rescuing Bax-induced cell death in yeasts (Kampranis et al., J. Biol. Chem. 275:29207–29216 (2000); Moon et al., Biochem. Biophys. Res. Commun. 290:457–462 (2002), each of which is hereby incorporated by reference in its entirety) and transgenic antisense tobacco plants with reduced amounts of ascorbate peroxidase were “hyperresponsive” to P. syringae (Mittler et al., Proc. Natl. Acad. Sci. USA 96:14165–14170 (1999), which is hereby incorporated by reference in its entirety), producing a phenotype reminiscent of the enhanced HR phenotypes produced by the suppressor mutants reported here. However, Abramovitch et al. (EMBO J. 22:60–69 (2003), which is hereby incorporated by reference in its entirety) reported that AvrPtoB possessed predicted structural features similar to domains within Bcl-2 family members, an equally intriguing finding due to the involvement of these proteins in PCD regulation. Yeast has emerged as model for studying PCD and has proven particularly useful for the analysis of cell death inducers and suppressors obtained from multicellular eukaryotes with more complex PCD pathways (Madeo et al., Curr. Genet. 41:208–216 (2002), which is hereby incorporated by reference in its entirety). An example of the utility of the yeast system to plant PCD research is found in the induction of yeast PCD by the plant defense protein osmotin (Narasimhan et al., Mol. Cell 8:921–930 (2001), which is hereby incorporated by reference in its entirety). A particularly fruitful use of the yeast system involves heterologous expression of the mammalian Bax protein, which induces PCD in yeast. Yeast expressing Bax can be screened, as done here, for heterologously expressed genes that block Bax-induced PCD. This system has been used to identify the Arabidopsis ethylene-responsive element binding protein (AtEBP) as a suppressor of PCD (Pan et al., FEBS Lett. 508:375–378 (2001), which is hereby incorporated by reference in its entirety) and its relevance to plant biology is further indicated by observations that Bax expression in tobacco can induce an apparent HR and that Bcl-2 (an anti-aptototic gene of the Bax/Bcl-2 family) expression in tobacco strongly alters plant-pathogen interactions (Dickman et al., Proc. Natl. Acad. Sci. USA 98:6957–6962 (2001); Lacomme and Santa Cruz, Proc. Natl. Acad. Sci. USA 96:7956–7961 (1999), each of which is hereby incorporated by reference in its entirety).
Four of the five effectors tested (AvrPphEPto, HopPtoG, HopPtoF, and HopPtoE) suppress Bax-induced yeast PCD, indicating that the targets are likely to be broadly conserved and not unique to plants. Interestingly, AvrPpiB1Pto and AvrPtoB failed to do so, even though both suppressed the HR elicited by P. fluorescens(pHIR11) in both tobacco and Arabidopsis, and DC3000 avrPtoB and avrPpiB1Pto mutants produced enhanced HRs. It is also puzzling that HopPtoG failed to suppress the HR elicited by P. fluorescens(pHIR11) although a DC3000 hopPtoG mutant had enhanced HR activity and HopPtoG suppressed Bax-induced yeast PCD. Moreover, it is also noteworthy that HopPtoD2, an effector that was recently identified to suppresses an HR elicited by avirulent P. syrinage strains did not suppress the HR elicited by P. fluorescens(pHIR11). These exceptions suggest that multiple bioassays will be required to identify all of the DC3000 effectors with some ability to suppress PCD.
While the suppressors described here were identified due to their ability to suppress PCD, it is possible they suppress other more general plant defenses as well. Indeed, HopPtoD2 has been found to be an active protein tyrosine phosphatase that appears to modulate a mitogen-activated protein kinase (MAPK) pathway in tobacco. An analogous MAPK pathway in Arabidopsis is part of the plant innate immune system activated in response to bacterial flagellin (Asal et al., Nature 415:977–983 (2002); Felix et al., Plant J. 18:265–276 (1999), each of which is hereby incorporated by reference in its entirety). The innate immune systems of mammals, insects, and plants have the capacity to recognize common markings on microorganisms, such as flagellin or LPS (Boller, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:189–214 (1995); Medzhitov and Janeway, Trends Microbiol. 8:452–456 (2000), each of which is hereby incorporated by reference in its entirety). These common components have been referred to as pathogen-associated molecular patterns (PAMPs) and they are not known to elicit the HR in plants. Thus, the assays used herein would not detect the activity of suppressors that specifically targeted PAMP-induced defense pathways unless the target was at convergence points of PCD pathways and PAMP-induced innate immunity pathways. Future research will determine whether these PCD suppressors specifically target Avr-induced PCD pathways or also suppress other plant defenses generally grouped into a broad category of defenses typically referred to as non-host resistance (Heath, Curr. Opin. Plant Biol. 3:315–319 (2000), which is hereby incorporated by reference in its entirety).
A general model of suppressor function must also reconcile several behaviors of bacterium-plant interactions that involve multiple effectors. Expression in P. syringae of a heterologous effector typically results in HR elicitation in test plants that carry a corresponding R gene despite the presence of resident suppressor effectors. For example, DC3000 heterologously expressing avrRpt2 or avrRps4 elicits the HR in Arabidopsis plants carrying the corresponding R genes (Hinsch and Staskawicz, Mol. Plant—Microbe Interact. 9:55–61 (1996); Kunkel et al., Plant Cell 5:865–875 (1993), which is hereby incorporated by reference in its entirety). On the other hand, suppressors can block HR elicitation by resident effectors, as revealed by the original discovery of suppressors like VirPphA and effectors with masked avirulence activity in P. s. phaseolicola (Jackson et al., Proc. Natl. Acad. Sci. USA 96:10875–10880 (1999), which is hereby incorporated by reference in its entirety) and by the observations presented here that several effectors can block HR elicitation by HopPsyA in the heterologous P. fluorescens(pHIR11) system. Perhaps the simplest explanation is that there is a hierarchy in the delivery of effectors by wild-type strains. Such a hierarchy in delivery has been proposed to explain the deployment of effectors with conflicting activities, such as the Salmonella SopE and SptP proteins, in animal pathogens (Cornelis and van Gujsegem, Annu. Rev. Microbiol. 54:734–774 (2000); Galán and Zhou, Proc. Natl. Acad. Sci. USA 97:8754–8761 (2000), each of which is hereby incorporated by reference in its entirety). The global identification of a set of suppressors in P. s. tomato DC3000 should facilitate systematic investigation of the underlying functions of TTSS effectors in P. syringae pathogenesis.
A final aspect of PCD and pathogenesis is that the ability to elicit host cell death appears to be a general characteristic of TTSS-dependent pathogens like P. syringae despite the fact that these bacteria typically rely upon living host cells as sites of multiplication (Alfano and Collmer, J. Bacteriol. 179:5655–5662 (1997); Knodler and Finlay, Microbes Infect. 3:1321–1326 (2001), each of which is hereby incorporated by reference in its entirety). This is particularly puzzling with P. syringae because late-stage infections with most strains produce necrotic lesions, but the symptomless growth of P. s. syringae gacS mutants suggests that such cell killing maybe gratuitous (Willis et al., Mol. Plant—Microbe Interact. 3:149–156 (1990), which is hereby incorporated by reference in its entirety). Similarly puzzling are recent observations suggesting that plants compromised in PCD pathways are unexpectedly more resistant to P. syringae (Lincoln et al., Proc. Natl. Acad. Sci. USA 99:15217–15221 (2002); Stone et al., Plant Cell 12:1811–1822 (2000); Richael et al., Physiol. Mol. Plant Pathol. 59:213–221 (2001), each of which is hereby incorporated by reference in its entirety). Thus, rapid and delayed host cell death are associated with defense and disease, respectively, and pathogen manipulation of cell death pathways may be a central process in pathogenesis.
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. A method of modifying a cell death pathway in a cell comprising:
introducing into a cell (i) a protein or polypeptide comprising the amino acid sequence of SEQ ID NO: 58 or (ii) a protein or polypeptide encoded by a nucleic acid molecule that hybridizes to the complement of SEQ ID NO: 57 under hybridization conditions comprising 1M Na+ at 65° C. followed by wash conditions comprising 0.2× sodium citrate at 65° C. wherein the protein or polypeptide has ADP-ribosyl transferase activity.

2. The method according to claim 1, wherein said introducing is carried out with the protein or polypeptide comprising the amino acid sequence of SEQ ID NO: 58.

3. The method according to claim 1, wherein said introducing is carried out by transformation of the cell with a transgene encoding the protein or polypeptide.

4. The method according to claim 1, wherein said introducing is carried out by delivery of the protein or polypeptide into the cell.

5. The method according to claim 1, wherein the cell death pathway is suppressed.