WO1998054214A2 - Hypersensitive response elicitor fragments and uses thereof - Google Patents

Hypersensitive response elicitor fragments and uses thereof Download PDF

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Publication number
WO1998054214A2
WO1998054214A2 PCT/US1998/010874 US9810874W WO9854214A2 WO 1998054214 A2 WO1998054214 A2 WO 1998054214A2 US 9810874 W US9810874 W US 9810874W WO 9854214 A2 WO9854214 A2 WO 9854214A2
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fragment
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ala
hypersensitive response
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PCT/US1998/010874
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French (fr)
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WO1998054214A9 (en
WO1998054214A3 (en
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Ronald J. Laby
Zhong-Min Wei
Steven V. Beer
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Cornell Research Foundation, Inc.
Eden Bioscience Corporation
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Priority to BR9809699-0A priority Critical patent/BR9809699A/en
Priority to EP98924950A priority patent/EP0996729A2/en
Priority to AU77004/98A priority patent/AU750732B2/en
Priority to JP50090299A priority patent/JP2002501388A/en
Priority to NZ501138A priority patent/NZ501138A/en
Priority to CA002289905A priority patent/CA2289905A1/en
Application filed by Cornell Research Foundation, Inc., Eden Bioscience Corporation filed Critical Cornell Research Foundation, Inc.
Priority to PL98337094A priority patent/PL337094A1/en
Priority to KR19997011216A priority patent/KR20010013226A/en
Publication of WO1998054214A2 publication Critical patent/WO1998054214A2/en
Publication of WO1998054214A3 publication Critical patent/WO1998054214A3/en
Publication of WO1998054214A9 publication Critical patent/WO1998054214A9/en
Priority to FI992545A priority patent/FI19992545A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/27—Erwinia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to fragments of a hypersensitive response elicitor which fragments elicit a hypersensitive response and uses thereof.
  • Interactions between bacterial pathogens and their plant hosts generally fall into two categories: (1) compatible (pathogen-host), leading to intercellular bacterial growth, symptom development, and disease development in the host plant; and (2) incompatible (pathogen-nonhost), resulting in the hypersensitive response, a particular type of incompatible interaction occurring, without progressive disease symptoms.
  • compatible pathogen-host
  • incompatible pathogen-nonhost
  • the hypersensitive response is a rapid, localized necrosis that is associated with the active defense of plants against many pathogens (Kiraly, Z., "Defenses Triggered by the Invader: Hypersensitivity,” pages 201-224 in: Plant Disease: An Advanced Treatise, Vol. 5, J.G. Horsfall and E.B. Cowling, ed. Academic Press New York (1980); Klement, Z., "Hypersensitivity,” pages 149-177 in: Phvtopatho genie Prokarvotes, Vol. 2, M.S. Mount and G.H. Lacy, ed. Academic Press, New York (1982)).
  • the hypersensitive response elicited by bacteria is readily observed as a tissue collapse if high concentrations (> 10 7 cells/ml) of a limited host-range pathogen like Pseudomonas syringae or Erwinia amylovora are infiltrated into the leaves of nonhost plants (necrosis occurs only in isolated plant cells at lower levels of inoculum)
  • a limited host-range pathogen like Pseudomonas syringae or Erwinia amylovora
  • hrp Lodgren, P.B., et al., "Gene Cluster of Pseudomonas syringae pv. 'phaseolicola' Controls Pathogenicity of Bean Plants and Hypersensitivity on Nonhost Plants," J. Bacteriol. 168:512-22 (1986); Willis, D.K., et al., "hrp Genes of Phytopathogenic Bacteria," Mol. Plant-Microbe Interact. 4:132-138 (1991)). Consequently, the hypersensitive response may hold clues to both the nature of plant defense and the basis for bacterial pathogenicity.
  • hrp genes are widespread in gram-negative plant pathogens, where they are clustered, conserved, and in some cases interchangeable (Willis, D.K., et al., "hrp Genes of Phytopathogenic Bacteria," Mol. Plant-Microbe Interact. 4:132-138 (1991); Bonas, U., "hrp Genes of Phytopathogenic Bacteria," pages 79-98 in: Current Topics in Microbiology and Immunology: Bacterial Pathogenesis of Plants and Animals - Molecular and Cellular Mechanisms, J.L. Dangl, ed. Springer- Verlag, Berlin (1994)).
  • hrp genes encode components of a protein secretion pathway similar to one used by Yersinia, Shigella, and Salmonella spp. to secrete proteins essential in animal diseases (Van Gijsegem, et al., "Evolutionary Conservation of Pathogenicity
  • Syringae HarpinPss a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants," Cell 73:1255-1266 (1993), Wei, Z.-H., et al., "HrpI of Erwinia amylovora Functions in Secretion of Harpin and is a Member of a New Protein Family," J. Bacteriol. 175:7958-7967 (1993); Arlat, M. et al.
  • the P. solanacearum GMI1000 PopAl protein has similar physical properties and also elicits the hypersensitive response in leaves of tobacco, which is not a host of that strain (Arlat, et al. "PopAl, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum," EMBO J. 13:543-53 (1994)).
  • P. solanacearum popA mutants still elicit the hypersensitive response in tobacco and incite disease in tomato.
  • the role of these glycine-rich hypersensitive response elicitors can vary widely among gram-negative plant pathogens.
  • the present invention seeks to identify fragments of hypersensitive response elicitor proteins or polypeptides, which fragments elicit a hypersensitive response, and uses of such fragments.
  • the present invention is directed to an isolated fragment of an Erwinia hypersensitive response elicitor protein or polypeptide where the fragment elicits a hypersensitive response in plants. Also disclosed are isolated DNA molecules which encode such fragments.
  • the fragments of hypersensitive response elicitors can be used to impart disease resistance to plants, to enhance plant growth, and/or to control insects. This involves applying the fragments in a non-infectious form to plants or plant seeds under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.
  • transgenic plants or plant seeds can be utilized.
  • a transgenic plant seed transformed with the DNA molecule encoding such a fragment can be provided and planted in soil. A plant is then propagated under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.
  • Figure 1 shows a deletion and proteolysis analysis for the Erwinia amylovora hypersensitive response elicitor (i.e. harpin).
  • A is the name of the harpin fragment.
  • B is the length of the fragment in amino acid residues.
  • C indicates whether detectable protein is produced.
  • D states whether there is hypersensitive response (i.e., HR) eliciting activity.
  • the solid line indicates that there are additional amino acids which are not harpin encoded, while the dashed line indicates the portion of the harpin that is deleted.
  • the numbers above the fragments in the box represent the amino acid residue present at the end of a given fragment; residue #1 is the N-terminus, and residue #403 is the C-terminus.
  • FIG. 2 is a Western blot illustrating specific secretion of harpin Ea , but not harpin Ea C31.
  • Lane A Ea273(pGPl-2) CFEP;
  • lane B Ea273(pGPl-2)(pCPPl 104) CFEP;
  • lane C E. coli DH5 ⁇ (pCPPl 107) CFEP harpin size standard;
  • lane D BioRad low range molecular weight markers;
  • lane E Ea273(pGPl-2) supernatant;
  • lane F Ea273(pGPl 2)(pCPPl 104) supernatant.
  • the blot was probed with an anti-harpin £a polyclonal antibody.
  • Figure 3 is an HR assay on tobacco leaf infiltrated as follows: (1) A, harpin Ea
  • FIG. 4 shows the digestion of harpin with endoproteinase Glu-C.
  • Lane A is harpin;
  • Lane B is harpin + endoproteinase Glu-C;
  • Lane C is BioRad low range molecular weight markers.
  • Figure 5 A shows the proteolysis of harpin.
  • Coomassie blue stained polyacrylamide gel was loaded as follows: A, BioRad low range molecular weight markers; B, IF-apple; C, IF-raspberry; D, IF-tobacco; E, harpinEa; F, harpin Ea + IF- apple; G, harpin Ea + IF-raspberry; H, harpii Ea + IF-tobacco.
  • Figure 5B shows a Coomassie Blue stained polyacrylamide gel loaded as follows: A, IF-tobacco; B, IF-tobacco + harpin Ea ; C, harpin E a; D, BioRad low range molecular weight markers; E, IF-tobacco + harpinEa + PMSF. HR-eliciting activity of the sample following proteolysis is denoted below the gel.
  • Figure 5C depicts whether proteolytic activity is present in IF from all plants tested. Intercellular fluid harvested from several plants was analyzed by PAGE in a gel containing 0.1% copolymerized gelatin. After washing to remove SDS and incubation to allow proteolysis of gelatin, the gels were stained to demonstrate the presence of gelatinolytic activity.
  • A IF-apple; B, IF-tobacco; C, IF-cotoneaster; D, BioRad mw; E, endoproteinase Glu-C; and F, ground leaf extract-tobacco.
  • Figure 6 shows the refractionation of elicitor-active peptides following proteolysis of harpin E a by tobacco IF. Absorbance was measured at 210 nm. Peak 1 contains peptides P91 and P95; peak 2 contains peptides P65 and P69.
  • Figure 7 shows the predicted proteolytic cleavage sites within harpin of several tested proteinases, and the effect of these cleavages on activity of active harpin fragments. Residues potentially important for HR-eliciting activity, based on the loss of activity following further cleavage, are indicated by upward-pointing arrows at bottom.
  • Figure 8 shows the similarities near N-termini among harpins of Erwinia spp. Underlined residues are present (identical or similar) in at least four out of the five proteins examined. Nine out of the first 26 residues are conserved in this manner.
  • Figures 9 A-B show Kyte-Doolittle hydropathy plots of bacterial HR-eliciting proteins.
  • Ea E. amylovora EA321; Est, E. stewartii DC283; Ech, E. chrysanthemi AC4150; Ecc, E. cartovora subsp. carotovora; Rs, R, solanacearum; Pss, P. syringae pv. syringae.
  • Figure 10 shows truncated proteins of the hypersensitive response elicitor protein or polypeptide.
  • Figure 11 shows a list of synthesized oligonucleotide primers for construction of truncated harpin proteins.
  • N represents the N-terminus (5' region), and C represents the C-terminus (3' region).
  • the primers correspond to the indicated sequence identification numbers for the present application: Nl (SEQ. ID. No. 1), N76 (SEQ. ID. No. 2), N99 (SEQ. ID. No. 3), N105 (SEQ. ID. No. 4), Nl 10 (SEQ. ID. No. 5), N137 (SEQ. ID. No. 6), N150 (SEQ. ID. No. 7), N169 (SEQ. ID. No. 8), N210 (SEQ. ID. No. 9), N267 (SEQ. ID. No.
  • N343 SEQ. ID. No. 11
  • C75 SEQ. ID. No. 12
  • C104 SEQ. ID. No. 13
  • C168 SEQ. ID. No. 14
  • C180 SEQ. ID. No. 15
  • C204 SEQ. ID. No. 16
  • C209 SEQ. ID. No. 17
  • C266 SEQ. ID. No. 18
  • C342 SEQ. ID. No. 19
  • C403 SEQ. ID. No. 20.
  • the present invention is directed to isolated fragments of a hypersensitive response elicitor protein or polypeptide where the fragments elicit a hypersensitive response in plants. Also disclosed are DNA molecules encoding such fragments as well as expression systems, host cells, and plants containing such molecules. Uses of the fragments themselves and the DNA molecules encoding them are disclosed.
  • hypersensitive response elicitor polypeptides or proteins are derived from hypersensitive response elicitor polypeptides or proteins of a wide variety of fungal and bacterial pathogens. Such polypeptides or proteins are able to elicit local necrosis in plant tissue contacted by the elicitor.
  • Suitable bacterial sources of polypeptide or protein elicitors include Erwinia, Pseudomonas, and Xanthamonas species (e.g., the following bacteria: Erwinia amylovora, Erwinia chrysanthemi, Erwinia stewartii, Erwinia carotovora, Pseudomonas syringae, Pseudomonas solancearum, Xanthomonas campestris, and mixtures thereof).
  • Phytophthora An example of a fungal source of a hypersensitive response elicitor protein or polypeptide is Phytophthora.
  • Suitable species of Phytophthora include Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamomi, Phytophthora capsici, Phytophthora megasperma, and Phytophthora citrophthora.
  • the hypersensitive response elicitor polypeptide or protein from Erwinia chrysanthemi has an amino acid sequence corresponding to SEQ. ID. No. 21 as follows:
  • This hypersensitive response elicitor polypeptide or protein has a molecular weight of 34 kDa, is heat stable, has a glycine content of greater than 16%, and contains substantially no cysteine.
  • the Erwinia chrysanthemi hypersensitive response elicitor polypeptide or protein is encoded by a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 22 as follows:
  • GAGCAGCACC ATCGATAAGT TGACCTCCGC GCTGACTTCG ATGATGTTTG GCGGCGCGCT 780
  • the hypersensitive response elicitor polypeptide or protein derived from Erwinia amylovora has an amino acid sequence corresponding to SEQ. ID. No.23 as follows:
  • This hypersensitive response elicitor polypeptide or protein has a molecular weight of about 39 kDa, has a pi of approximately 4.3, and is heat stable at 100°C for at least 10 minutes.
  • This hypersensitive response elicitor polypeptide or protein has substantially no cysteine.
  • the hypersensitive response elicitor polypeptide or protein derived from Erwinia amylovora is more fully described in Wei, Z.-M., R. J. Laby, C. H. Zumoff, D. W. Bauer, S.-Y. He, A. Collmer, and S. V.
  • CTCCTTGGCA ACGGGGGACT GGGAGGTGGT CAGGGCGGTA ATGCTGGCAC GGGTCTTGAC 780
  • Pseudomonas syringae has an amino acid sequence corresponding to SEQ. ID. No. 25 as follows: Met Gin Ser Leu Ser Leu Asn Ser Ser Ser Leu Gin Thr Pro Ala Met 1 5 10 15
  • This hypersensitive response elicitor polypeptide or protein has a molecular weight of
  • Pseudomonas syringae is found in He, S. Y., H. C. Huang, and A. Collmer, "Pseudomonas syringae pv. syringae Harpinp ss : a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants," Cell 73:1255-1266 (1993), which is hereby incorporated by reference.
  • the DNA molecule encoding the hypersensitive response elicitor from Pseudomonas syringae has a nucleotide sequence corresponding to SEQ. ID. No. 26 as follows:
  • ATGCAGAGTC TCAGTCTTAA CAGCAGCTCG CTGCAAACCC CGGCAATGGC CCTTGTCCTG 60
  • the hypersensitive response elicitor polypeptide or protein derived from Pseudomonas solanacearum has an amino acid sequence corresponding to SEQ. ID. No. 27 as follows:
  • the hypersensitive response elicitor polypeptide or protein from Xanthomonas campestris pv. glycines has an amino acid sequence corresponding to SEQ. ID. No. 29 as follows:
  • This sequence is an amino terminal sequence having only 26 residues from the hypersensitive response elicitor polypeptide or protein of Xanthomonas campestris pv. glycines. It matches with fimbrial subunit proteins determined in other
  • the hypersensitive response elicitor polypeptide or protein from Xanthomonas campestris pv. pelargonii is heat stable, protease sensitive, and has a molecular weight of 20 kDa. It includes an amino acid sequence corresponding to SEQ. ID. No.
  • the hypersensitive response elicitor protein or polypeptide of Erwinia stewartii is set forth in Ahmad et al., "Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize," 8th Int'l. Cong. Molec. Plant-Microbe Interact., July 14-19, 1996 and Ahmad, et al., "Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize," Ann. Mtg. Am. Phvtopath. Soc, July 27-31 , 1996, which are hereby incorporated by reference.
  • Hypersensitive response elicitor proteins or polypeptides from Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamoni, Phytophthora capsici, Phytophthora megasperma, and Phytophora citrophthora are described in Kaman, et al., "Extracellular Protein Elicitors from Phytophthora: Most Specificity and Induction of Resistance to Bacterial and Fungal Phytopathogens," Molec.
  • elicitors can be identified by growing fungi or bacteria that elicit a hypersensitive response under which genes encoding an elicitor are expressed.
  • Cell-free preparations from culture supernatants can be tested for elicitor activity (i.e. local necrosis) by using them to infiltrate appropriate plant tissues.
  • Fragments of the above hypersensitive response elicitor polypeptides or proteins as well as fragments of full length elicitors from other pathogens are encompassed by the method of the present invention.
  • Suitable fragments can be produced by several means.
  • subclones of the gene encoding a known elicitor protein are produced by conventional molecular genetic manipulation by subcloning gene fragments. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or peptide that can be tested for elicitor activity according to the procedure described below.
  • fragments of an elicitor protein can be produced by digestion of a full-length elicitor protein with proteolytic enzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin.
  • fragments of the elicitor protein gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These then would be cloned into an appropriate vector for expression of a truncated peptide or protein. Chemical synthesis can also be used to make suitable fragments. Such a synthesis is carried out using known amino acid sequences for the elicitor being produced.
  • fragments can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE).
  • An example of suitable fragments of an Erwinia hypersensitive response elicitor which fragments elicit a hypersensitive response are fragments of the Erwinia amylovora hypersensitive response elicitor.
  • Suitable fragments include a C-terminal fragment of the amino acid sequence of SEQ. ID. No. 23, an N-terminal fragment of the amino acid sequence of SEQ. ID. No. 23, or an internal fragment of the amino acid sequence of SEQ. ID. No. 23.
  • the N-terminal fragment of the amino acid sequence of SEQ. ID. No. 23 can span the following amino acids of SEQ. ID. No. 23: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1 and 372.
  • the internal fragment of the amino acid sequence of SEQ. ID. No. 23 can span the following amino acids of SEQ. ID. No. 23: 76 and 209, 105 and 209, 99 and 209, 137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180, and 105 and 180.
  • Other suitable fragments can be identified in accordance with the present invention.
  • Variants may be made 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.
  • 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 fragment of the present invention is preferably produced in purified form (preferably at least about 60%, more preferably 80%, pure) by conventional techniques.
  • the fragment of the present invention is produced but not secreted into the growth medium of recombinant host cells.
  • the protein or polypeptide of the present invention is secreted into growth medium.
  • the host cell e.g., E. coli
  • the homogenate is centrifuged to remove bacterial debris.
  • the supernatant is then subjected to heat treatment and the fragment is separated by centrifugation.
  • the supernatant fraction containing the fragment is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the fragment.
  • the protein fraction may be further purified by ion exchange or HPLC.
  • the DNA molecule encoding the fragment of the hypersensitive response elicitor polypeptide or protein 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.
  • Recombinant genes may also be introduced into viruses, such as vaccina virus.
  • Recombinant viruses can be generated by transection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see
  • 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, New York (1989), which is hereby inco ⁇ orated by reference.
  • host-vector systems may be utilized to express the protein- encoding sequence(s).
  • 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.
  • mRNA messenger RNA
  • eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promotors are not recognized and do not function in eucaryotic cells.
  • SD Shine- Dalgarno
  • 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.
  • Promotors vary in their "strength" (i.e. their ability to promote transcription). For the pu ⁇ oses of expressing a cloned gene, it is desirable to use strong promotors 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 promotors may be used. For instance, when cloning in E.
  • promotors such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the P R and P L promotors of coliphage lambda and others, including but not limited, to / cUV5, omp , bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • trp-lacUV5 (tac) promotor or other E. coli promotors 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 promotor unless specifically induced.
  • the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
  • the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside).
  • IPTG isopropylthio-beta-D-galactoside
  • trp isopropylthio-beta-D-galactoside
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic 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 promotor, may also contain any combination of various "strong" transcription and/or translation initiation signals.
  • 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.
  • ATG initiation codon
  • 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 TV gene of coliphage lambda, or from the E. coli tryptophan ⁇ , D, C, B or A genes.
  • any SD-ATG combination produced by recombinant DNA or other techniques involving inco ⁇ oration of synthetic nucleotides may be used.
  • Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
  • the present invention further relates to methods of imparting disease resistance to plants, enhancing plant growth, and/or effecting insect control for plants.
  • These methods involve applying the fragment of a hypersensitive response elicitor polypeptide or protein, which fragment itself elicits a hypersensitive response; in a non-infectious form to all or part of a plant or a plant seed under conditions effective for the fragment to impart disease resistance, enhance growth, and/or control insects.
  • these fragments of a hypersensitive response elicitor protein or polypeptide can be applied to plants such that seeds recovered from such plants themselves are able to impart disease resistance in plants, to enhance plant growth, and/or to effect insect control.
  • transgenic plants or plant seeds can be utilized.
  • a transgenic plant seed transformed with a DNA molecule encoding a fragment of a hypersensitive response elicitor polypeptide or protein which fragment elicits a hypersensitive response can be provided and planted in soil.
  • a plant is then propagated from the planted seed under conditions effective to permit that DNA molecule to impart disease resistance to plants, to enhance plant growth, and/or to control insects.
  • the embodiment of the present invention where the hypersensitive response elicitor polypeptide or protein is applied to the plant or plant seed can be carried out in a number of ways, including: 1) application of an isolated fragment or 2) application of bacteria which do not cause disease and are transformed with a genes encoding the fragment.
  • the fragment can be applied to plants or plant seeds by applying bacteria containing the DNA molecule encoding the fragment of the hypersensitive response elicitor polypeptide or protein which fragment elicits a hypersensitive response.
  • bacteria must be capable of secreting or exporting the fragment so that the fragment can contact plant or plant seeds cells.
  • the fragment is produced by the bacteria inplanta or on seeds or just prior to introduction of the bacteria to the plants or plant seeds.
  • the methods of the present invention can be utilized to treat a wide variety of plants or their seeds to impart disease resistance, enhance growth, and/or control insects.
  • Suitable plants include dicots and monocots. More particularly, useful crop plants can include: alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.
  • suitable ornamental plants are: Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
  • absolute immunity against infection may not be conferred, but the severity of the disease is reduced and symptom development is delayed. Lesion number, lesion size, and extent of sporulation of fungal pathogens are all decreased.
  • This method of imparting disease resistance has the potential for treating previously untreatable diseases, treating diseases systemically which might not be treated separately due to cost, and avoiding the use of infectious agents or environmentally harmful materials.
  • the method of imparting pathogen resistance to plants in accordance with the present invention is useful in imparting resistance to a wide variety of pathogens including viruses, bacteria, and fungi.
  • Resistance, ter alia, to the following viruses can be achieved by the method of the present invention: Tobacco mosaic virus and Tomato mosaic virus.
  • Resistance, mter alia, to the following bacteria can also be imparted to plants in accordance with present invention: Pseudomonas solancearum, Pseudomonas syringae pv. tabaci, and Xanthomonas campestris pv. pelargonii.
  • Plants can be made resistant, inter alia, to the following fungi by use of the method of the present invention: Fusarium oxysporum and Phytophthora infestans.
  • plant growth enhancement or promotion can be achieved. This can occur as early as when plant growth begins from seeds or later in the life of a plant.
  • plant growth according to the present invention encompasses greater yield, increased quantity of seeds produced, increased percentage of seeds germinated, increased plant size, greater biomass, more and bigger fruit, earlier fruit coloration, and earlier fruit and plant maturation.
  • the present invention provides significant economic benefit to growers. For example, early germination and early maturation permit crops to be grown in areas where short growing seasons would otherwise preclude their growth in that locale. Increased percentage of seed germination results in improved crop stands and more efficient seed use. Greater yield, increased size, and enhanced biomass production allow greater revenue generation from a given plot of land.
  • insect control encompasses preventing insects from contacting plants to which the hypersensitive response elicitor has been applied, preventing direct insect damage to plants by feeding injury, causing insects to depart from such plants, killing insects proximate to such plants, interfering with insect larval feeding on such plants, preventing insects from colonizing host plants, preventing colonizing insects from releasing phytotoxins, etc.
  • the present invention also prevents subsequent disease damage to plants resulting from insect infection.
  • the present invention is effective against a wide variety of insects.
  • European corn borer is a major pest of corn (dent and sweet corn) but also feeds on over 200 plant species including green, wax, and lima beans and edible soybeans, peppers, potato, and tomato plus many weed species.
  • Additional insect larval feeding pests which damage a wide ' variety of vegetable crops include the following: beet armyworm, cabbage looper, corn ear worm, fall armyworm, diamondback moth, cabbage root maggot, onion maggot, seed corn maggot, pickleworm (melonworm), pepper maggot, tomato pinworm, and maggots.
  • the method of the present invention involving application of the fragment of a hypersensitive response elicitor polypeptide or protein, which fragment elicits a hypersensitive response can be carried out through a variety of procedures when all or part of the plant is treated, including leaves, stems, roots, etc. This may (but need not) involve infiltration of the hypersensitive response elicitor polypeptide or -protein into the plant.
  • Suitable application methods include high or low pressure spraying, injection, and leaf abrasion proximate to when elicitor application takes place.
  • the fragment of the hypersensitive response elicitor protein or polypeptide in accordance with present invention, can be applied by low or high pressure spraying, coating, immersion, or injection. Other suitable application procedures can be envisioned by those skilled in the art provided they are able to effect contact of the fragment with cells of the plant or plant seed.
  • the seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants.
  • the plants may be treated with one or more applications of the fragment of the hypersensitive response elicitor protein or polypeptide or whole elicitors to impart disease resistance to plants, to enhance plant growth, and/or to control insects on the plants.
  • the fragment of the hypersensitive response elicitor polypeptide or protein in accordance with the present invention, can be applied to plants or plant seeds alone or in a mixture with other materials. Alternatively, the fragment can be applied separately to plants with other materials being applied at different times.
  • a composition suitable for treating plants or plant seeds in accordance with the application embodiment of the present invention contains a fragment of a hypersensitive response elicitor polypeptide or protein which fragment elicits a hypersensitive response in a carrier. Suitable carriers include water, aqueous solutions, slurries, or dry powders. In this embodiment, the composition contains greater than 500 nM of the fragment.
  • this composition may contain additional additives including fertilizer, insecticide, fungicide, nematacide, and mixtures thereof.
  • Suitable fertilizers include (NH ) NO .
  • An example of a suitable insecticide is Malathion.
  • Useful fungicides include Captan.
  • Other suitable additives include buffering agents, wetting agents, coating agents, and abrading agents. These materials can be used to facilitate the process of the present invention.
  • the hypersensitive response eliciting fragment can be applied to plant seeds with other conventional seed formulation and treatment materials, including clays and polysaccharides.
  • transgenic plants and transgenic seeds a hypersensitive response eliciting fragment need not be applied topically to the plants or seeds. Instead, transgenic plants transformed with a DNA molecule encoding such a fragment are produced according to procedures well known in the art.
  • the vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA.
  • the genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incorporated by reference.
  • particle bombardment also known as biolistic transformation
  • 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.
  • the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA.
  • 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
  • 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 inco ⁇ orated by reference. In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. 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 A. rhizogenes previously transformed with the gene. 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.
  • assaying for the presence of opines can be used to identify transformed tissue.
  • Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of J. tumefaciens or the Ri plasmid of J.
  • the Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome, j. Schell, Science, 237:1176-83 (1987), which is hereby inco ⁇ orated by reference. After transformation, the transformed plant cells must be regenerated.
  • 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.
  • the expression cassette After the expression cassette is stably inco ⁇ orated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure with the presence of the gene encoding the hypersensitive response eliciting fragment resulting in disease resistance, enhanced plant growth, and/or control of insects on the plant.
  • transgenic seeds or propagules are recovered from the transgenic plants.
  • the seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
  • the transgenic plants are propagated from the planted transgenic seeds under conditions effective to impart disease resistance to plants, to enhance plant growth, and/or to control insects. While not wishing to be bound by theory, such disease resistance, growth enhancement, and/or insect control may be RNA mediated or may result from expression of the polypeptide or protein fragment.
  • transgenic plants and plant seeds When transgenic plants and plant seeds are used in accordance with the present invention, they additionally can be treated with the same materials as are used to treat the plants and seeds to which a hypersensitive response eliciting fragment is applied. These other materials, including hypersensitive response eliciting fragments, can be applied to the transgenic plants and plant seeds by the above-noted procedures, including high or low pressure spraying, injection, coating, and immersion. Similarly, after plants have been propagated from the transgenic plant seeds, the plants may be treated with one or more applications of the hypersensitive response eliciting fragment to impart disease resistance, enhance growth, and/or control insects. Such plants may also be treated with conventional plant treatment agents (e.g., insecticides, fertilizers, etc.).
  • conventional plant treatment agents e.g., insecticides, fertilizers, etc.
  • Example 2 Molecular biology techniques. Several approaches were employed to obtain truncated or otherwise altered versions of both E. amylovora ha ⁇ ins. These techniques included: (i) subcloning of restriction fragments containing portions of the gene encoding the hypersensitive response elicitor protein or polypeptide from Erwinia amylovora (i.e. hrpN) into expression vectors, by standard techniques (Sambrook, et al., Molecular Cloning: a Laboratory Manual 2 nd ed. ed.
  • pCPP1084 DNA was prepared using a Qiagen midiprep column (Qiagen, Chatsworth, CA) and digested with sst / followed by EcoRI. Subsequently, the digested DNA was subjected to exonuclease III digestion, ligation, and transformation into E. coli BL21(D ⁇ 3). Deletion sizes were estimated by agarose gel electrophoresis. Ha ⁇ in fragments were named with respect to the portion of ha ⁇ in deleted (e.g., ha ⁇ inEa C82 lacks the C-terminal 82 amino acid residues of full-length ha ⁇ inEa). Example 3 - Protein expression.
  • T7 RNA polymerase-dependent systems were used for expression from T7 promoters. These systems utilized either strain E. coli BL21(DE3) (Studier, et al, "Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-Level Expression of Cloned Gene," J. Mol Biol. 189:113-130 (1986), which is hereby inco ⁇ orated by reference), or plasmid pGPl-2 (Tabor, et al, "A Bacteriophage T7 DNA Polymerase/Promoter System for Controlled Exclusive Expression of Specific Genes," Proc. Natl Acad. Sci, USA 82:1074-1078 (1985), which is hereby inco ⁇ orated by reference) in E.
  • coli DH5 ⁇ Expression of hrpN from the T7 promoter was induced by addition of IPTG to a final concentration of 0.4 mM.
  • E. amylovora Ea321 i.e. ha ⁇ in E a
  • Ea273 pGPl-2 was introduced by transformation with a 42°C heat shock for 10 minutes, or by electroporation (Biorad Gene PulserTM).
  • Hypersensitive response (i.e. HR)-eliciting activity was screened in tobacco cv. Xanthi leaves by inplanta lysis (He, et al, "Pseudomonas syringae pv.
  • syringae ha ⁇ inp ss a Protein That is Secreted via the H ⁇ Pathway and Elicits the Hypersensitive Response in Plants," Cell 73:1255-1266 (1993), which is hereby inco ⁇ orated by reference) or by preparation of boiled and unboiled “CFEPs" (Wei, et al, "Ha ⁇ in, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora," Science 257:85-88 (1992), which is hereby incorporated by reference).
  • Example 4 In vitro proteolysis of harpin.
  • In vitro proteolysis of ha ⁇ inEa with Staphylococcus V8 proteinase (also termed endoproteinase Glu-C), trypsin, pepsin, and papain was performed as recommended (Scopes, et al, Protein Purification: Principles and Practice. 2 nd ed. Springer-Verlag. New York (1987), which is hereby incorporated by reference), for 2- 16 hrs. at 20-37°.
  • Endoproteinase Glu-C digestion was performed either in 50 mM ammonium bicarbonate, pH 7.8 (in which cleavage occurs only after glutamic acid), or in 50 mM potassium phosphate, pH 7.8 (in which cleavage after both glutamic acid and aspartic acid occurs).
  • Example 5 Plant-derived proteinases.
  • Intercellular fluids were obtained from tobacco, tomato, apple, raspberry, and Laceaster, as described (Hammond-Kosack, et al, "Preparation and Analysis of Intercellular Fluid," p. 15-21. In S.J. Gurr, M.J. McPherson, and D.J. Bowles (ed.), Molecular Plant Pathology A Practical Approach. 2 nd ed., The Practical Approach Series, IRL Publishers, Oxford (1992), which is hereby inco ⁇ orated by reference), by vacuum infiltration of intercellular spaces with high-purity water. Proteolytic digestion of PAGE-purified ha ⁇ in Ea was performed for 2-16 hrs. at 20-37°C, pH, by mixing equal volumes of IF with ha ⁇ inEa.
  • a total leaf extract was obtained by grinding tobacco leaf panels with mortar and pestle in 5 mM potassium phosphate. The extract was centrifuged and filtered, and the clarified ground leaf extract used identically as was the IF. Proteinase inhibitors were employed as follows: Pepstatin A (final concentration l ⁇ M), E-64 (l ⁇ M), Aprotinin (2 ⁇ g/ml), o-phenanthroline (ImM), and p-mercuribenzoate (PCMB) (Sigma, St. Louis, MO).
  • Peptide fragments of ha ⁇ in obtained following digestion with tobacco IF were fractionated by reverse-phase HPLC on a Vydac C 18 column using a 2-60% acetonitrile gradient in 0.1 % trifiuoroacetic acid. Fractions were lyophilized, resuspended in 5 mM potassium phosphate and infiltrated into tobacco leaf panels. The fraction with greatest HR-eliciting activity was refractionated as above with a 35- 70%) acetonitrile gradient, and the purity of each fraction was assayed via gas chromatography-mass spectroscopy (GC-MS) and by N-terminal protein sequencing at the Cornell Biotechnology Program Core Facility.
  • GC-MS gas chromatography-mass spectroscopy
  • Example 7 Proteinase activity-stained gels. Proteinase activity of IF was assayed in activity- stained polyacrylamide gels
  • Ha ⁇ in C31 contains the N-terminal 372 amino acids of harpin, but lacks the C-terminal 31 residues, which are replaced by 47 residues encoded by the vector, resulting in a protein slightly larger than the wild type ha ⁇ in Ea -
  • the C31 protein retains HR-eliciting activity and is stable and easily expressed and detected by western analysis or PAGE but it is no longer secreted into the culture supernatant as is the wild type protein ( Figure 2).
  • the presence of ha ⁇ in Ea C31 does not interfere with secretion of the wild type ha ⁇ in, which is found in both the CFEP and the culture supernatant. However, ha ⁇ inEa C31 is found only in the CFEP.
  • Example 10 Effect of proteolysis on harpin ⁇ a 's HR eliciting activity
  • purified full length protein was proteolyzed in vitro by several proteinases, including endoproteinase Glu-C, trypsin, pepsin, and papain (e.g., Figures 3 and 4). Harpin solutions digested with trypsin or with papain lost all activity. In contrast, following digestion with endoproteinase Glu-C, HR-eliciting activity was retained. No peptides larger than 6 kD were evident by PAGE following trypsin digestion. Endoproteinase Glu-C digestion yielded an approximately 20 kD fragment, larger than expected if all cleavage sites were cut, indicating that digestion was not complete ( Figure 4).
  • Example 11 Apoplastic fluids (IF) contain harpin-degrading proteolytic activity
  • IF intercellular fluids
  • IF intercellular fluids
  • Each IF tested possessed proteinase activity(s), as indicated by the presence of multiple activity-stained bands in polyacrylamide gels containing co-polymerized gelatin ( Figures 5A to 5C), as well as by the disappearance of detectable ha ⁇ in Ea (Schagger, et al, "Tricine-Sodium Dodecyl Sulfate Gel
  • the length of time required for leaf collapse when either intact ha ⁇ in or ha ⁇ in predigested with tobacco IF was infiltrated into tobacco leaf panels was compared. Both preparations elicited the HR in a similar time frame (12-18 hours, depending on the experiment).
  • Peptides resulting from digestion by apoplastic plant proteinase(s) were fractionated by reverse phase HPLC (Vydac C18 column), and tested for activity. Following treatment of intact ha ⁇ inEa with tobacco IF, three fractions contained some HR-eliciting activity on tobacco. Two of the three demonstrated weak activity, and little protein was present. They were not further characterized. Fraction 19, which contained the strongest activity as well as the most protein, was refractionated using a more shallow elution gradient ( Figure 6). Refractionation, N-terminal protein sequencing, and molecular weight analysis by mass spectroscopy indicated that four largely overlapping peptides were present.
  • Peak 19-1 contained peptides P91 and P95, corresponding to ha ⁇ in ⁇ a residues 110-200 and 110-204; peak 19-2 contained peptides P64 and P68, corresponding to ha ⁇ in Ea residues 137-200 and 137-204.
  • 19-1 and 19-2 each possessed HR-eliciting activity.
  • the smallest peptide thus confirmed to retain activity consisted of residues 137-204.
  • the two peptides in each peak were not separable under the conditions used.
  • These active fragments are distinct from the smallest active N-terminal fragment (harpin Ea C305), and indicate that more than one portion of ha ⁇ in ⁇ a displays activity inplanta. Further digestion with trypsin abolished the HR-eliciting activity of 19-2.
  • Example 13 E. amylovora harpin's similarity with other proteins.
  • ha ⁇ in Ea The predicted protein sequences of proteinaceous HR elicitors from several other bacterial plant pathogens, and of other proteins known to be, or thought to be, secreted by a type III secretion pathway were also compared with that of ha ⁇ in Ea .
  • ha ⁇ in Ea was compared with elicitors from E. amylovora Ea246 (i.e. ha ⁇ in Ear ), Erwinia chrysanthemi EC 16 (ha ⁇ in Ec ⁇ (Bauer, et al, "Erwinia chrysanthemi ha ⁇ in Ec ⁇ -,: An Elicitor of the Hypersensitive Response That Contributes to Soft-Rot Pathogenesis," Mol.
  • hydropathy profile of each Erwinia harpin is generally similar to that of the others, yet distinct from that reported for harpinp ss (Alfano, et al, "Analysis of the Role of the Pseudomonas Syringae HrpZ harpin in Elicitation of the Hypersensitive Response to Tobacco Using Functionally Nonpolar hrpZ Deletion Mutations, Truncated H ⁇ Z Fragments, and hrmA Mutations," Mol. Microbiol
  • Ha ⁇ inE CC possesses a strikingly hydrophobic region around residues 54-143 (Mukherjee, et al, Presented at the 8 th International Congress Molecular Plant-Microbe Interactions, Knoxville, TN (1996), which is hereby incorporated by reference). This portion of the protein is also the most hydrophobic region of ha ⁇ inEa and ha ⁇ inEs- The rest of each protein is predominantly hydrophilic.
  • Truncated proteins and fragments of harpin obtained following proteolytic digestion of the full length protein indicate several su ⁇ rising aspects of ha ⁇ in Ea HR- eliciting activity. These harpin fragments demonstrate that HR-eliciting activity resides in distinct regions of the protein, and that relatively small fragments of the protein, as little as 68 residues and possibly less, are sufficient for this activity.
  • Fragments of other plant pathogen-derived elicitor proteins also retain biological activity, including Avr9 from Caldosporium fulvum (Van den Ackervecken, et al, "The AVR9 Race-Specific Elicitor of Cladosporium fulvum is Processed by Endogenous and Plant Proteases," PI Physiol 103:91-96 (1993), which is hereby inco ⁇ orated by reference), Pep-13 of Phytophthora megasperma (N ⁇ rnburger, et al, "High Affinity Binding of a Fungal Oligopeptide Elicitor to the Parsley Plasma
  • ha ⁇ h Ea P64 and P68 demonstrate distinct hydrophobicity during reverse-phase HPLC ( Figure 6), and they correspond to a hydrophobic peak in a Kyte- Doolittle plot ( Figure 9).
  • SMEP a metalloproteinase (Huangpu, et al, "Purification and Developmental Analysis of an Extracellular Proteinase From Young Leaves of Soybean," Plant Phvsiol 108:969-974 (1995); McGeehan, et al, "Sequencing and Characterization of the Soybean Leaf Metalloproteinase," Plant Phvsiol. 99:1179-1183 (1992), which are hereby inco ⁇ orated by reference) sensitive to EDTA, is thought to cleave at G/L and G/I.
  • P91 and P95 thus may represent partial digestion products of a SMEP-like proteinase in the tobacco apoplast.
  • the apoplastic activities degrade ha ⁇ in without destroying its HR-eliciting ability, in contrast to intracellular proteolytic activities present in a ground leaf- extract, which abolish activity. This raises a number of interesting questions, e.g., does the plant use these ha ⁇ in fragments as elicitor-signals?
  • the timing of the HR was examined when full length harpin and ha ⁇ in predigested by tobacco intercellular fluid were each infiltrated into tobacco leaves. The HR elicited by each preparation occurred 12-18 hours after infiltration.
  • PGIP of Phaseolus vulgaris has been suggested to modulate the level of elicitor-active oligogalacturonides present during the plant-parasite interaction in bean (Desiderio, et al, "Polygalacturonase, PGIP, and Oligogalacturonides in Cell-Cell Communication,” Biochem. Sci. Trans. 22:394-397 (1994), which is hereby inco ⁇ orated by reference).
  • HR-eliciting activity by ha ⁇ in fragments contributes to the ability of plants to recognize the presence of a pathogen.
  • transgenic host and non-host plants engineered for apoplastic expression of a ha ⁇ in activity-degrading proteinase, would exhibit reduced or increased sensitivity to E. amylovora, compared to non-engineered plants.
  • infestans (Pieterse, et al, "Structure and Genomic Organization of the ip ⁇ B and ipiO Gene Clusters of Phytophthora infestans," Gene, 138:67-77 (1994), which is hereby inco ⁇ orated by reference) produces a glycine-rich pathogenicity-associated family of proteins of unknown function. Because the primary amino acid sequence of each elicitor protein or peptide fragment shows no obvious similarity to that of the others, it is unclear whether they interact with the same target on or in the plant cell, plasma membrane, or cell wall. In that regard, it might be of interest to test whether any one of these molecules inhibits the action of other(s).
  • Escherichia coli stains used in the following examples include DH5 ⁇ and BL21(DE3) purchased from Gibco BRL and Stratagene, respectively.
  • the pET28(b) vector was purchased from Novagen.
  • Eco DH5 ⁇ /2139 contained the complete hrpN gene.
  • the 2139 construct was produced by D. Bauer at the Cornell University.
  • the h ⁇ N gene was cleaved from the 2139 plasmid by restriction enzyme digestion with Hindlll, then purified from an agarose gel to serve as the DNA template for PCR synthesis of truncated h ⁇ N clones. These clones were subsequently inserted into the (His) 6 vector pET28(b) which contained a Kan r gene for selection of transformants.
  • Restriction enzymes were obtained from Boehringer Mannheim or Gibco BRL. T4 DNA ligase, Calf Intestinal Alkaline Phosphatase (CIAP), and PCR SupermixTM were obtained from Gibco BRL.
  • the QIAprep Spin Miniprep Kit, the Qiagen Plasmid Mini Kit, and the QIAquick PCR Purification Kit were purchased from Qiagen.
  • the PCR primers were synthesized by Lofstrand Labs Limited (Gaithersburg, MD).
  • the oligopeptides were synthesized by Bio-Synthesis, Inc. (Lewisville, TX). All DNA manipulations such as plasmid isolation, restriction enzyme digestion, DNA ligation, and PCR were performed according to standard techniques (molecular cloning) or protocols provided by the manufacturer.
  • pET28(b) vector DNA 5 ⁇ g were digested with 15 units of Nde I and 20 units of Hind III at 37°C for 3 hours followed with CIAP treatment to reduce the background resulting from incomplete single enzyme digestion.
  • Digested vector DNA was purified with the QIAquick PCR purification kit and directly used for ligation. Ligation was carried out at 14-16°C for 5-12 hours in a 15 ⁇ l mixture containing ca. 200 ng of digested pET28(b), 30 ng of targeted PCR fragment, and 1 unit T4 DNA ligase.
  • ligation solution 5 - 7.5 ⁇ l of ligation solution were added to 100 ⁇ l of DH5 ⁇ competent cells in a 15 ml falcon tube and incubated on ice for 30 min. After a heat shock at 42°C for 45 seconds, 0.9 ml SOC solution or 0.45 ml LB media were added to each tube and incubated at 37°C for 1 hour. 20, 100, and 200 ⁇ l of transformed cells were placed onto LB agar with 30 ⁇ g/ml of kanamycin and incubated at 37°C overnight. Single colonies were transferred to 3 ml LB-media and incubated overnight at 37°C. Plasmid DNA was prepared from 2 ml of culture with the QIAprep Miniprep kit.
  • the DNA from the transformed cells was analyzed by restriction enzyme digestion or partial sequencing to verify the success of the transformations. Plasmids with the desired DNA sequence were transferred into the BL21 strain using the standard chemical transformation method as indicated above. A clone containing the full length ha ⁇ in protein in the pet28(b) vector was generated as a positive control, and a clone with only the pET28(b) vector was generated as a negative control.
  • Example 17 Expression of harpin truncated proteins Escherichia coli BL21(DE3) strains containing the h ⁇ N clones were grown in
  • Luria broth medium g/L Difco Yeast extract, 10 g/L Difco Tryptone, 5g/L NaCI, and 1 mM NaOH
  • the bacteria were then inoculated into 100 volumes of the same medium and grown at 37°C to an OD 6 0 of 0.6-0.8.
  • the bacteria were then inoculated into 250 volumes of the same medium and grown at 37°C to an OD 620 of ca. 0.3 or 0.6-0.8.
  • One milli molar IPTG was then added and the cultures grown at 19°C overnight (ca. 18 hours). Not all of the clones were successfully expressed using this strategy.
  • Escherichia coli BL21(DE3) strains containing the h ⁇ N subclones were grown in Luria broth medium (5g/L Difco Yeast extract, 10 g/L Difco Tryptone, 5g/L NaCI, and 1 mM NaOH) containing 30 ⁇ g/ml of kanamycin at 37°C overnight. The bacteria were then inoculated into 100 volumes of the same medium and grown at 37°C to an OD 620 of 0.6-0.8.
  • the bacteria were then inoculated into 250 volumes of the growth medium and grown at 37°C to a specific induction OD 62 o- One milli molar IPTG was then added and the cultures grown at an optimal temperature for protein expression, and harvested at a particular time for recovery of the highest level of protein.
  • a 50 ml culture of a h ⁇ N clone was grown as above to induce expression of the truncated protein.
  • 1.5 ml of the cell suspension were centrifuged at 14,000 ⁇ m for 5 minutes, re-suspended in urea lysis buffer (8 M urea, 0J M Na 2 HPO 4 , and 0.01 M Tris ⁇ pH 8.0), incubated at room temperature for 10 minutes, then centrifuged again at 14,000 ⁇ m for 10 minutes, and the supernatant saved.
  • Example 20 Large scale native purification of harpin truncated proteins for comprehensive biological activity assays
  • the column was centrifuged at 1100 ⁇ m for one minute to remove any residual wash buffer and then the protein was eluted from the resin with 4 ml of imidazole elution buffer (1 M imidazole, 0.5 M NaCI, and 20 mM Tris) by incubating the column with the elution buffer for ten minutes at room temperature and then centrifuging the column at 1100 ⁇ m for one minute.
  • the eluate was run on a 4-20%>, a 16%), or a 10-20%> Tris-Glycine pre-cast gel depending upon the size of the truncated protein to verify the expression.
  • the concentration of the proteins was determined by comparison of the protein bands with a standard protein in the Mark 12 molecular weight marker.
  • Example 21 Large scale urea purification of harpin truncated proteins for comprehensive biological activity assay
  • the procedure was the same as the large scale native purification except that urea lysis buffer, washing buffer, and elution buffer were used, and the cells were not sonicated as in the native purification.
  • the protein was renatured by dialyzing against lower and lower concentrations of urea over an eight hour period, then dialyzing overnight against 10 mM Tris/20 mM NaCI The renaturing process caused the N-terminal proteins to precipitate.
  • the precipitated 1-168 protein was solubilized by the addition of 100 mM Tris-HCl at pH 10.4 then heating the protein at 30°C for ca. one hour. The concentration of the protein was determined by comparison of the protein bands with a standard protein in the Mark 12 molecular weight marker.
  • fragment proteins All of the cloned fragment proteins were expressed to a certain degree except for three small fragments (amino acids 169-209, 150-209, and 150-180). The fragments were expressed at varying levels. Fragments 210-403 and 267-403 were expressed very well, yielding a high concentration of protein from a small scale purification, resulting in a substantial protein band on SDS gel electrophoresis. Other fragments (such as a.a. 1-168 and 1-104) produced much less protein, resulting in faint protein bands upon electrophoresis. It was difficult to determine whether fragment 343-403, the smallest C-terminal protein, was expressed, as there were several background proteins apparent on the gel, in addition to the suspected 343-403 protein.
  • the positive and negative control proteins consisting of the full length harpin protein and only background proteins, respectively, were tested for expression and HR activity as well.
  • the large scale expression and purification of the fragment proteins was done to determine the level of expression and titer of the HR activity (Table 4).
  • the full length positive control protein elicited HR down to only 5-7 ⁇ g/ml.
  • This false HR was likely due an affinity of the imidazole used in the purification process to bind to one or several of the background proteins, thereby not completely dialyzing out. Imidazole at a concentration of ca. 60 mM did elicit a false HR response.
  • the other potential HR domain is thought to be located in the N-terminus of the protein from a.a. 1-104 (possibly a.a. 1-75) (SEQ. ID. No. 23). It was difficult to confirm or narrow down the N-terminus HR domain due to the difficulties encountered in purifying these fragment proteins.
  • the internal domain proteins elicited an HR response between 5 and 10 ⁇ g/ml of protein like the positive control, and the N-terminus domain proteins elicited an HR response between 1 and 3 ⁇ g/ml, lower than the positive control.
  • Example 24 Biological Activity of HR Inducing Fragments
  • the two N-terminal ha ⁇ in fragments spanning nucleotides 1-104 and nucleotides 1-168 of the nucleic acid of SEQ. ID. No. 24 were effective at inducing resistance of tobacco against TMV, in a similar manner as the full length ha ⁇ in protein.
  • the internal fragments spanning nucleotides 76-209 and nucleotides 105-209 of the nucleic acid of SEQ. ID. No. 24 were also effective at inducing TMV resistance.
  • these same four fragments conferred plant growth enhancement ("PGE") in tomato increasing the height of the plants from 4-19%> taller than the buffer control plants.
  • PGE plant growth enhancement
  • the full length ha ⁇ in protein induced growth enhancement of 6% greater than the buffer.
  • the negative control did not induce TMV resistance or growth enhancement.
  • Gly Asn Ala lie Gly Met Gly Val Gly Gin Asn Ala Ala Leu Ser Ala 195 . 200 205
  • Gin Tyr Pro Glu lie Phe Gly Lys Pro Glu Tyr Gin Lys Asp Gly Trp 245 250 255
  • MOLECULE TYPE DNA (genomic)
  • GAGCAGCACC ATCGATAAGT TGACCTCCGC GCTGACTTCG ATGATGTTTG GCGGCGCGCT 780
  • MOLECULE TYPE DNA (genomic)
  • CTCCTTGGCA ACGGGGGACT GGGAGGTGGT CAGGGCGGTA ATGCTGGCAC GGGTCTTGAC 780 GGTTCGTCGC TGGGCGGCAA AGGGCTGCAA AACCTGAGCG GGCCGGTGGA CTACCAGCAG 840
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 26:
  • ATGCAGAGTC TCAGTCTTAA CAGCAGCTCG CTGCAAACCC CGGCAATGGC CCTTGTCCTG 60
  • MOLECULE TYPE DNA (genomic)

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Abstract

The present invention is directed to isolated fragments of an Erwinia hypersensitive response elicitor protein or polypeptide which fragments elicit a hypersensitive response in plants. Also disclosed are isolated DNA molecules which encode the Erwinia hypersensitive response eliciting fragment. Isolated fragments of hypersensitive response elicitor proteins or polypeptides, which elicit a hypersensitive response, and the isolated DNA molecules that encode them can be used to impart disease resistance to plants, to enhance plant growth, and/or to control insects on plants. This can be achieved by applying the hypersensitive response eliciting fragments in a non-infectious form to plants or plant seeds under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds. Alternatively, transgenic plants or plant seeds transformed with a DNA molecule encoding a hypersensitive response eliciting fragment can be provided and the transgenic plants or plants resulting from the transgenic plant seeds are grown under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.

Description

HYPERSENSITIVE RESPONSE ELICITOR FRAGMENTS ELICITING A HYPERSENSITIVE RESPONSE AND USES THEREOF
This application claims benefit of U.S. Patent Application Serial No. 60/048,109.
FIELD OF THE INVENTION
The present invention relates to fragments of a hypersensitive response elicitor which fragments elicit a hypersensitive response and uses thereof.
BACKGROUND OF THE INVENTION
Interactions between bacterial pathogens and their plant hosts generally fall into two categories: (1) compatible (pathogen-host), leading to intercellular bacterial growth, symptom development, and disease development in the host plant; and (2) incompatible (pathogen-nonhost), resulting in the hypersensitive response, a particular type of incompatible interaction occurring, without progressive disease symptoms. During compatible interactions on host plants, bacterial populations increase dramatically and progressive symptoms occur. During incompatible interactions, bacterial populations do not increase, and progressive symptoms do not occur.
The hypersensitive response is a rapid, localized necrosis that is associated with the active defense of plants against many pathogens (Kiraly, Z., "Defenses Triggered by the Invader: Hypersensitivity," pages 201-224 in: Plant Disease: An Advanced Treatise, Vol. 5, J.G. Horsfall and E.B. Cowling, ed. Academic Press New York (1980); Klement, Z., "Hypersensitivity," pages 149-177 in: Phvtopatho genie Prokarvotes, Vol. 2, M.S. Mount and G.H. Lacy, ed. Academic Press, New York (1982)). The hypersensitive response elicited by bacteria is readily observed as a tissue collapse if high concentrations (> 107 cells/ml) of a limited host-range pathogen like Pseudomonas syringae or Erwinia amylovora are infiltrated into the leaves of nonhost plants (necrosis occurs only in isolated plant cells at lower levels of inoculum) (Klement, Z., "Rapid Detection of Pathogenicity of Phytopathogenic Pseudomonads," Nature 199:299-300; Klement, et al., "Hypersensitive Reaction Induced by Phytopathogenic Bacteria in the Tobacco Leaf," Phytopathology 54:474- 477 (1963); Turner, et al., "The Quantitative Relation Between Plant and Bacterial Cells Involved in the Hypersensitive Reaction," Phytopathology 64:885-890 (1974); Klement, Z., "Hypersensitivity," pages 149-177 in Phytopathogenic Prokaryotes. Vol. 2., M.S. Mount and G.H. Lacy, ed. Academic Press, New York (1982)). The capacities to elicit the hypersensitive response in a nonhost and be pathogenic in a host appear linked. As noted by Klement, Z., "Hypersensitivity," pages 149-177 in Phytopathogenic Prokaryotes. Vol. 2., M.S. Mount and G.H. Lacy, ed. Academic Press, New York, these pathogens also cause physiologically similar, albeit delayed, necroses in their interactions with compatible hosts. Furthermore, the ability to produce the hypersensitive response or pathogenesis is dependent on a common set of genes, denoted hrp (Lindgren, P.B., et al., "Gene Cluster of Pseudomonas syringae pv. 'phaseolicola' Controls Pathogenicity of Bean Plants and Hypersensitivity on Nonhost Plants," J. Bacteriol. 168:512-22 (1986); Willis, D.K., et al., "hrp Genes of Phytopathogenic Bacteria," Mol. Plant-Microbe Interact. 4:132-138 (1991)). Consequently, the hypersensitive response may hold clues to both the nature of plant defense and the basis for bacterial pathogenicity.
The hrp genes are widespread in gram-negative plant pathogens, where they are clustered, conserved, and in some cases interchangeable (Willis, D.K., et al., "hrp Genes of Phytopathogenic Bacteria," Mol. Plant-Microbe Interact. 4:132-138 (1991); Bonas, U., "hrp Genes of Phytopathogenic Bacteria," pages 79-98 in: Current Topics in Microbiology and Immunology: Bacterial Pathogenesis of Plants and Animals - Molecular and Cellular Mechanisms, J.L. Dangl, ed. Springer- Verlag, Berlin (1994)). Several hrp genes encode components of a protein secretion pathway similar to one used by Yersinia, Shigella, and Salmonella spp. to secrete proteins essential in animal diseases (Van Gijsegem, et al., "Evolutionary Conservation of Pathogenicity
Determinants Among Plant and Animal Pathogenic Bacteria," Trends Microbiol. 1 :175-180 (1993)). In E. amylovora, P. syringae, and P. solanacearum, hrp genes have been shown to control the production and secretion of glycine-rich, protein elicitors of the hypersensitive response (He, S.Y., et al. "Pseudomonas Syringae pv. Syringae HarpinPss: a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants," Cell 73:1255-1266 (1993), Wei, Z.-H., et al., "HrpI of Erwinia amylovora Functions in Secretion of Harpin and is a Member of a New Protein Family," J. Bacteriol. 175:7958-7967 (1993); Arlat, M. et al. "PopAl, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum," EMBO 13:543-553 (1994)). The first of these proteins was discovered in E. amylovora Ea321, a bacterium that causes fire blight of rosaceous plants, and was designated harpin (Wei, Z.-M., et al, "Harpin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora," Science 257:85-88 (1992)). Mutations in the encoding hrpN gene revealed that harpin is required for E. amylovora to elicit a hypersensitive response in nonhost tobacco leaves and incite disease symptoms in highly susceptible pear fruit. The P. solanacearum GMI1000 PopAl protein has similar physical properties and also elicits the hypersensitive response in leaves of tobacco, which is not a host of that strain (Arlat, et al. "PopAl, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum," EMBO J. 13:543-53 (1994)). However, P. solanacearum popA mutants still elicit the hypersensitive response in tobacco and incite disease in tomato. Thus, the role of these glycine-rich hypersensitive response elicitors can vary widely among gram-negative plant pathogens.
Other plant pathogenic hypersensitive response elicitors have been isolated, cloned, and sequenced. These include: Erwinia chrysanthemi (Bauer, et. al., "Erwinia chrysanthemi HarpinEdl: Soft-Rot Pathogenesis," MPMI 8(4): 484-91 (1995)); Erwinia carotovora (Cui, et. al., "The RsmA" Mutants of Erwinia carotovora subsp. carotovora Strain Ecc71 Overexpress hrpN_ιcc and Elicit a Hypersensitive Reaction-like Response in Tobacco Leaves," MPMI 9(7): 565-73 (1966)); Erwinia stewartii (Ahmad, et. al., "Harpin is not Necessary for the Pathogenicity of Erwinia stewartii on Maize," 8th Int'L Cong. Molec. Plant-Microb. Inter. July 14-19, 1996 and Ahmad, et. al., "Harpin is not Necessary for the Pathogenicity of Erwinia stewartii on Maize," Ann. Mtg. Am. Phytopath. Soc. July 27-31 , 1996); and Pseudomonas syringae pv. syringae (WO 94/26782 to Cornell Research Foundation, Inc.). The present invention seeks to identify fragments of hypersensitive response elicitor proteins or polypeptides, which fragments elicit a hypersensitive response, and uses of such fragments. SUMMARY OF THE INVENTION
The present invention is directed to an isolated fragment of an Erwinia hypersensitive response elicitor protein or polypeptide where the fragment elicits a hypersensitive response in plants. Also disclosed are isolated DNA molecules which encode such fragments.
The fragments of hypersensitive response elicitors can be used to impart disease resistance to plants, to enhance plant growth, and/or to control insects. This involves applying the fragments in a non-infectious form to plants or plant seeds under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.
As an alternative to applying the fragments to plants or plant seeds in order to impart disease resistance, to enhance plant growth, and/or to control insects on plants, transgenic plants or plant seeds can be utilized. When utilizing transgenic plants, this involves providing a transgenic plant transformed with a DNA molecule encoding a fragment of a hypersensitive response elicitor protein or polypeptide which fragments elicit a hypersensitive response in plants and growing the plant under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects in the plants or plants grown from the plant seeds. Alternatively, a transgenic plant seed transformed with the DNA molecule encoding such a fragment can be provided and planted in soil. A plant is then propagated under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a deletion and proteolysis analysis for the Erwinia amylovora hypersensitive response elicitor (i.e. harpin). A is the name of the harpin fragment. B is the length of the fragment in amino acid residues. C indicates whether detectable protein is produced. D states whether there is hypersensitive response (i.e., HR) eliciting activity. The solid line indicates that there are additional amino acids which are not harpin encoded, while the dashed line indicates the portion of the harpin that is deleted. The numbers above the fragments in the box represent the amino acid residue present at the end of a given fragment; residue #1 is the N-terminus, and residue #403 is the C-terminus.
Figure 2 is a Western blot illustrating specific secretion of harpinEa, but not harpinEaC31. Lane A, Ea273(pGPl-2) CFEP; lane B, Ea273(pGPl-2)(pCPPl 104) CFEP; lane C, E. coli DH5α (pCPPl 107) CFEP harpin size standard; lane D, BioRad low range molecular weight markers; lane E, Ea273(pGPl-2) supernatant; lane F, Ea273(pGPl 2)(pCPPl 104) supernatant. The blot was probed with an anti-harpin£a polyclonal antibody. Figure 3 is an HR assay on tobacco leaf infiltrated as follows: (1) A, harpinEa
+ raspberry IF; (2) B, harpinEa + apple IF; (3) C, harpinEa + tobacco IF; (4) D, harpinEa + endoproteinase Glu-C; (5) E, harpinEa + trypsin; (6) F, harpinEa; (7) G, tobacco IF; (8) H, endoproteinase Glu-C; (9) I, trypsin; and (10) J, harpinEa- IF refers to intracelluar fluids. Figure 4 shows the digestion of harpin with endoproteinase Glu-C. Lane A is harpin; Lane B is harpin + endoproteinase Glu-C; Lane C is BioRad low range molecular weight markers.
Figure 5 A shows the proteolysis of harpin. Coomassie blue stained polyacrylamide gel was loaded as follows: A, BioRad low range molecular weight markers; B, IF-apple; C, IF-raspberry; D, IF-tobacco; E, harpinEa; F, harpinEa + IF- apple; G, harpinEa + IF-raspberry; H, harpii Ea + IF-tobacco.
Figure 5B shows a Coomassie Blue stained polyacrylamide gel loaded as follows: A, IF-tobacco; B, IF-tobacco + harpinEa; C, harpinEa; D, BioRad low range molecular weight markers; E, IF-tobacco + harpinEa + PMSF. HR-eliciting activity of the sample following proteolysis is denoted below the gel.
Figure 5C depicts whether proteolytic activity is present in IF from all plants tested. Intercellular fluid harvested from several plants was analyzed by PAGE in a gel containing 0.1% copolymerized gelatin. After washing to remove SDS and incubation to allow proteolysis of gelatin, the gels were stained to demonstrate the presence of gelatinolytic activity. A, IF-apple; B, IF-tobacco; C, IF-cotoneaster; D, BioRad mw; E, endoproteinase Glu-C; and F, ground leaf extract-tobacco. Figure 6 shows the refractionation of elicitor-active peptides following proteolysis of harpinEa by tobacco IF. Absorbance was measured at 210 nm. Peak 1 contains peptides P91 and P95; peak 2 contains peptides P65 and P69.
Figure 7 shows the predicted proteolytic cleavage sites within harpin of several tested proteinases, and the effect of these cleavages on activity of active harpin fragments. Residues potentially important for HR-eliciting activity, based on the loss of activity following further cleavage, are indicated by upward-pointing arrows at bottom.
Figure 8 shows the similarities near N-termini among harpins of Erwinia spp. Underlined residues are present (identical or similar) in at least four out of the five proteins examined. Nine out of the first 26 residues are conserved in this manner.
Figures 9 A-B show Kyte-Doolittle hydropathy plots of bacterial HR-eliciting proteins. Ea, E. amylovora EA321; Est, E. stewartii DC283; Ech, E. chrysanthemi AC4150; Ecc, E. cartovora subsp. carotovora; Rs, R, solanacearum; Pss, P. syringae pv. syringae.
Figure 10 shows truncated proteins of the hypersensitive response elicitor protein or polypeptide.
Figure 11 shows a list of synthesized oligonucleotide primers for construction of truncated harpin proteins. N represents the N-terminus (5' region), and C represents the C-terminus (3' region). The primers correspond to the indicated sequence identification numbers for the present application: Nl (SEQ. ID. No. 1), N76 (SEQ. ID. No. 2), N99 (SEQ. ID. No. 3), N105 (SEQ. ID. No. 4), Nl 10 (SEQ. ID. No. 5), N137 (SEQ. ID. No. 6), N150 (SEQ. ID. No. 7), N169 (SEQ. ID. No. 8), N210 (SEQ. ID. No. 9), N267 (SEQ. ID. No. 10), N343 (SEQ. ID. No. 11), C75 (SEQ. ID. No. 12), C104 (SEQ. ID. No. 13), C168 (SEQ. ID. No. 14), C180 (SEQ. ID. No. 15), C204 (SEQ. ID. No. 16), C209 (SEQ. ID. No. 17), C266 (SEQ. ID. No. 18), C342 (SEQ. ID. No. 19), and C403 (SEQ. ID. No. 20).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to isolated fragments of a hypersensitive response elicitor protein or polypeptide where the fragments elicit a hypersensitive response in plants. Also disclosed are DNA molecules encoding such fragments as well as expression systems, host cells, and plants containing such molecules. Uses of the fragments themselves and the DNA molecules encoding them are disclosed.
The fragments of hypersensitive response elicitor polypeptides or proteins according to the present invention are derived from hypersensitive response elicitor polypeptides or proteins of a wide variety of fungal and bacterial pathogens. Such polypeptides or proteins are able to elicit local necrosis in plant tissue contacted by the elicitor. Examples of suitable bacterial sources of polypeptide or protein elicitors include Erwinia, Pseudomonas, and Xanthamonas species (e.g., the following bacteria: Erwinia amylovora, Erwinia chrysanthemi, Erwinia stewartii, Erwinia carotovora, Pseudomonas syringae, Pseudomonas solancearum, Xanthomonas campestris, and mixtures thereof).
An example of a fungal source of a hypersensitive response elicitor protein or polypeptide is Phytophthora. Suitable species of Phytophthora include Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamomi, Phytophthora capsici, Phytophthora megasperma, and Phytophthora citrophthora.
The hypersensitive response elicitor polypeptide or protein from Erwinia chrysanthemi has an amino acid sequence corresponding to SEQ. ID. No. 21 as follows:
Met Gin He Thr He Lys Ala His He Gly Gly Asp Leu Gly Val Ser 1 5 10 15
Gly Leu Gly Ala Gin Gly Leu Lys Gly Leu Asn Ser Ala Ala Ser Ser 20 25 30
Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Thr He Asp Lys Leu Thr 35 • 40 45
Ser Ala Leu Thr Ser Met Met Phe Gly Gly Ala Leu Ala Gin Gly Leu 50 55 60
Gly Ala Ser Ser Lys Gly Leu Gly Met Ser Asn Gin Leu Gly Gin Ser 65 70 75 80 Phe Gly Asn Gly Ala Gin Gly Ala Ser Asn Leu Leu Ser Val Pro Lys
85 90 95
Ser Gly Gly Asp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu Asp Asp 100 105 110
Leu Leu Gly His Asp Thr Val Thr Lys Leu Thr Asn Gin Ser Asn Gin 115 120 125 Leu Ala Asn Ser Met Leu Asn Ala Ser Gin Met Thr Gin Gly Asn Met 130 • 135 140
Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser Ser He Leu Gly 145 150 155 160 Asn Gly Leu Gly Gin Ser Met Ser Gly Phe Ser Gin Pro Ser Leu Gly
165 170 175
Ala Gly Gly Leu Gin Gly Leu Ser Gly Ala Gly Ala Phe Asn Gin Leu 180 185 190
Gly Asn Ala He Gly Met Gly Val Gly Gin Asn Ala Ala Leu Ser Ala 195 200 205
Leu Ser Asn Val Ser Thr His Val Asp Gly Asn Asn Arg His Phe Val 210 215 220
Asp Lys Glu Asp Arg Gly Met Ala Lys Glu He Gly Gin Phe Met Asp 225 230 235 240 Gin Tyr Pro Glu He Phe Gly Lys Pro Glu Tyr Gin Lys Asp Gly Trp
245 250 255
Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser Lys 260 265 270
Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met Asp Lys Phe Arg Gin 275 280 285
Ala Met Gly Met He Lys Ser Ala Val Ala Gly Asp Thr Gly Asn Thr 290 295 300
Asn Leu Asn Leu Arg Gly Ala Gly Gly Ala Ser Leu Gly He Asp Ala 305 310 315 320 Ala Val Val Gly Asp Lys He Ala Asn Met Ser Leu Gly Lys Leu Ala
325 330 335
Asn Ala
This hypersensitive response elicitor polypeptide or protein has a molecular weight of 34 kDa, is heat stable, has a glycine content of greater than 16%, and contains substantially no cysteine. The Erwinia chrysanthemi hypersensitive response elicitor polypeptide or protein is encoded by a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 22 as follows:
CGATTTTACC CGGGTGAACG TGCTATGACC GACAGCATCA CGGTATTCGA CACCGTTACG 60 GCGTTTATGG CCGCGATGAA CCGGCATCAG GCGGCGCGCT GGTCGCCGCA ATCCGGCGTC 120 GATCTGGTAT TTCAGTTTGG GGACACCGGG CGTGAACTCA TGATGCAGAT TCAGCCGGGG 180
CAGCAATATC CCGGCATGTT GCGCACGCTG CTCGCTCGTC GTTATCAGCA GGCGGCAGAG 240
TGCGATGGCT GCCATCTGTG CCTGAACGGC AGCGATGTAT TGATCCTCTG GTGGCCGCTG 300
CCGTCGGATC CCGGCAGTTA TCCGCAGGTG ATCGAACGTT TGTTTGAACT GGCGGGAATG 360 ACGTTGCCGT CGCTATCCAT AGCACCGACG GCGCGTCCGC AGACAGGGAA CGGACGCGCC 420
CGATCATTAA GATAAAGGCG GCTTTTTTTA TTGCAAAACG GTAACGGTGA GGAACCGTTT 480
CACCGTCGGC GTCACTCAGT AACAAGTATC CATCATGATG CCTACATCGG GATCGGCGTG 540
GGCATCCGTT GCAGATACTT TTGCGAACAC CTGACATGAA TGAGGAAACG AAATTATGCA 600
AATTACGATC AAAGCGCACA TCGGCGGTGA TTTGGGCGTC TCCGGTCTGG GGCTGGGTGC 660 TCAGGGACTG AAAGGACTGA ATTCCGCGGC TTCATCGCTG GGTTCCAGCG TGGATAAACT 720
GAGCAGCACC ATCGATAAGT TGACCTCCGC GCTGACTTCG ATGATGTTTG GCGGCGCGCT 780
GGCGCAGGGG CTGGGCGCCA GCTCGAAGGG GCTGGGGATG AGCAATCAAC TGGGCCAGTC 840
TTTCGGCAAT GGCGCGCAGG GTGCGAGCAA CCTGCTATCC GTACCGAAAT CCGGCGGCGA 900
TGCGTTGTCA AAAATGTTTG ATAAAGCGCT GGACGATCTG CTGGGTCATG ACACCGTGAC 960 CAAGCTGACT AACCAGAGCA ACCAACTGGC TAATTCAATG CTGAACGCCA GCCAGATGAC 1020
CCAGGGTAAT ATGAATGCGT TCGGCAGCGG TGTGAACAAC GCACTGTCGT CCATTCTCGG 1080
CAACGGTCTC GGCCAGTCGA TGAGTGGCTT CTCTCAGCCT TCTCTGGGGG CAGGCGGCTT 1140
GCAGGGCCTG AGCGGCGCGG GTGCATTCAA CCAGTTGGGT AATGCCATCG GCATGGGCGT 1200
GGGGCAGAAT GCTGCGCTGA GTGCGTTGAG TAACGTCAGC ACCCACGTAG ACGGTAACAA 1260 CCGCCACTTT GTAGATAAAG AAGATCGCGG CATGGCGAAA GAGATCGGCC AGTTTATGGA 1320
TCAGTATCCG GAAATATTCG GTAAACCGGA ATACCAGAAA GATGGCTGGA GTTCGCCGAA 1380
GACGGACGAC AAATCCTGGG CTAAAGCGCT GAGTAAACCG GATGATGACG GTATGACCGG 1440
CGCCAGCATG GACAAATTCC GTCAGGCGAT GGGTATGATC AAAAGCGCGG TGGCGGGTGA 1500
TACCGGCAAT ACCAACCTGA ACCTGCGTGG CGCGGGCGGT GCATCGCTGG GTATCGATGC 1560 GGCTGTCGTC GGCGATAAAA TAGCCAACAT GTCGCTGGGT AAGCTGGCCA ACGCCTGATA 1620
ATCTGTGCTG GCCTGATAAA GCGGAAACGA AAAAAGAGAC GGGGAAGCCT GTCTCTTTTC 1680
TTATTATGCG GTTTATGCGG TTACCTGGAC CGGTTAATCA TCGTCATCGA TCTGGTACAA 1740
ACGCACATTT TCCCGTTCAT TCGCGTCGTT ACGCGCCACA ATCGCGATGG CATCTTCCTC 1800
GTCGCTCAGA TTGCGCGGCT GATGGGGAAC GCCGGGTGGA ATATAGAGAA ACTCGCCGGC 1860 CAGATGGAGA CACGTCTGCG ATAAATCTGT GCCGTAACGT GTTTCTATCC GCCCCTTTAG 1920
CAGATAGATT GCGGTTTCGT AATCAACATG GTAATGCGGT TCCGCCTGTG CGCCGGCCGG 1980
GATCACCACA ATATTCATAG AAAGCTGTCT TGCACCTACC GTATCGCGGG AGATACCGAC 2040
AAAATAGGGC AGTTTTTGCG TGGTATCCGT GGGGTGTTCC GGCCTGACAA TCTTGAGTTG 2100 GTTCGTCATC ATCTTTCTCC ATCTGGGCGA CCTGATCGGT T 2141
The hypersensitive response elicitor polypeptide or protein derived from Erwinia amylovora has an amino acid sequence corresponding to SEQ. ID. No.23 as follows:
Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gin He Ser 1 5 10 15
He Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg Gin 20 25 30
Asn Ala Gly Leu Gly Gly Asn Ser Ala Leu Gly Leu Gly Gly Gly Asn 35 40 45
Gin Asn Asp Thr Val Asn Gin Leu Ala Gly Leu Leu Thr Gly Met Met 50 55 60 Met Met Met Ser Met Met Gly Gly Gly Gly Leu Met Gly Gly Gly Leu 65 70 75 80
Gly Gly Gly Leu Gly Asn Gly Leu Gly Gly Ser Gly Gly Leu Gly Glu 85 90 95
Gly Leu Ser Asn Ala Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr 100 105 110
Leu Gly Ser Lys Gly Gly Asn Asn Thr Thr Ser Thr Thr Asn Ser Pro 115 120 125
Leu Asp Gin Ala Leu Gly He Asn Ser Thr Ser Gin Asn Asp Asp Ser 130 135 140 Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Ser Asp Pro Met Gin Gin 145 150 155 160
Leu Leu Lys Met Phe Ser Glu He Met Gin Ser Leu Phe Gly Asp Gly 165 170 175
Gin Asp Gly Thr Gin Gly Ser Ser Ser Gly Gly Lys Gin Pro Thr Glu 180 185 190
Gly Glu Gin Asn Ala Tyr Lys Lys Gly Val Thr Asp Ala Leu Ser Gly 195 200 205 Leu Met Gly Asn Gly Leu Ser Gin Leu Leu Gly Asn Gly Gly Leu Gly 210 215 220
Gly Gly Gin Gly Gly Asn Ala Gly Thr Gly Leu Asp Gly Ser Ser Leu 225 230 235 240 Gly Gly Lys Gly Leu Gin Asn Leu Ser Gly Pro Val Asp Tyr Gin Gin
245 250 255
Leu Gly Asn Ala Val Gly Thr Gly He Gly Met Lys Ala Gly He Gin 260 265 270
Ala Leu Asn Asp He Gly Thr His Arg His Ser Ser Thr Arg Ser Phe 275 280 285
Val Asn Lys Gly Asp Arg Ala Met Ala Lys Glu He Gly Gin Phe Met 290 295 300
Asp Gin Tyr Pro Glu Val Phe Gly Lys Pro Gin Tyr Gin Lys Gly Pro 305 310 315 320 Gly Gin Glu Val Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser
325 330 335
Lys Pro Asp Asp Asp Gly Met Thr Pro Ala Ser Met Glu Gin Phe Asn 340 345 350
Lys Ala Lys Gly Met He Lys Arg Pro Met Ala Gly Asp Thr Gly Asn 355 360 365
Gly Asn Leu Gin Ala Arg Gly Ala Gly Gly Ser Ser Leu Gly He Asp 370 375 380
Ala Met Met Ala Gly Asp Ala He Asn Asn Met Ala Leu Gly Lys Leu 385 390 395 400 Gly Ala Ala
This hypersensitive response elicitor polypeptide or protein has a molecular weight of about 39 kDa, has a pi of approximately 4.3, and is heat stable at 100°C for at least 10 minutes. This hypersensitive response elicitor polypeptide or protein has substantially no cysteine. The hypersensitive response elicitor polypeptide or protein derived from Erwinia amylovora is more fully described in Wei, Z.-M., R. J. Laby, C. H. Zumoff, D. W. Bauer, S.-Y. He, A. Collmer, and S. V. Beer, "Harpin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora," Science 257:85-88 (1992), which is hereby incorporated by reference. The DNA molecule encoding this polypeptide or protein has a nucleotide sequence corresponding to SEQ. ID. No. 24 as follows:
AAGCTTCGGC ATGGCACGTT TGACCGTTGG GTCGGCAGGG TACGTTTGAA TTATTCATAA 60 GAGGAATACG TTATGAGTCT GAATACAAGT GGGCTGGGAG CGTCAACGAT GCAAATTTCT 120
ATCGGCGGTG CGGGCGGAAA TAACGGGTTG CTGGGTACCA GTCGCCAGAA TGCTGGGTTG 180
GGTGGCAATT CTGCACTGGG GCTGGGCGGC GGTAATCAAA ATGATACCGT CAATCAGCTG 240
GCTGGCTTAC TCACCGGCAT GATGATGATG ATGAGCATGA TGGGCGGTGG TGGGCTGATG 300
GGCGGTGGCT TAGGCGGTGG CTTAGGTAAT GGCTTGGGTG GCTCAGGTGG CCTGGGCGAA 360 GGACTGTCGA ACGCGCTGAA CGATATGTTA GGCGGTTCGC TGAACACGCT GGGCTCGAAA 420
GGCGGCAACA ATACCACTTC AACAACAAAT TCCCCGCTGG ACCAGGCGCT GGGTATTAAC 480
TCAACGTCCC AAAACGACGA TTCCACCTCC GGCACAGATT CCACCTCAGA CTCCAGCGAC 540
CCGATGCAGC AGCTGCTGAA GATGTTCAGC GAGATAATGC AAAGCCTGTT TGGTGATGGG 600
CAAGATGGCA CCCAGGGCAG TTCCTCTGGG GGCAAGCAGC CGACCGAAGG CGAGCAGAAC 660 GCCTATAAAA AAGGAGTCAC TGATGCGCTG TCGGGCCTGA TGGGTAATGG TCTGAGCCAG 720
CTCCTTGGCA ACGGGGGACT GGGAGGTGGT CAGGGCGGTA ATGCTGGCAC GGGTCTTGAC 780
GGTTCGTCGC TGGGCGGCAA AGGGCTGCAA AACCTGAGCG GGCCGGTGGA CTACCAGCAG 840
TTAGGTAACG CCGTGGGTAC CGGTATCGGT ATGAAAGCGG GCATTCAGGC GCTGAATGAT 900
ATCGGTACGC ACAGGCACAG TTCAACCCGT TCTTTCGTCA ATAAAGGCGA TCGGGCGATG 960 GCGAAGGAAA TCGGTCAGTT CATGGACCAG TATCCTGAGG TGTTTGGCAA GCCGCAGTAC 1020
CAGAAAGGCC CGGGTCAGGA GGTGAAAACC GATGACAAAT CATGGGCAAA AGCACTGAGC 1080
AAGCCAGATG ACGACGGAAT GACACCAGCC AGTATGGAGC AGTTCAACAA AGCCAAGGGC 1140
ATGATCAAAA GGCCCATGGC GGGTGATACC GGCAACGGCA ACCTGCAGGC ACGCGGTGCC 1200
GGTGGTTCTT CGCTGGGTAT TGATGCCATG ATGGCCGGTG ATGCCATTAA CAATATGGCA 1260 CTTGGCAAGC TGGGCGCGGC TTAAGCTT 1288
The hypersensitive response elicitor polypeptide or protein derived from
Pseudomonas syringae has an amino acid sequence corresponding to SEQ. ID. No. 25 as follows: Met Gin Ser Leu Ser Leu Asn Ser Ser Ser Leu Gin Thr Pro Ala Met 1 5 10 15
Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser 20 25 30 Ser Lys Ala Leu Gin Glu Val Val Val Lys Leu Ala Glu Glu Leu Met 35 40 45
Arg Asn Gly Gin Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu Ala 50 55 60
Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly He Glu Asp Val 65 70 75 80
He Ala Ala Leu Asp Lys Leu He His Glu Lys Leu Gly Asp Asn Phe 85 90 95
Gly Ala Ser Ala Asp Ser Ala Ser Gly Thr Gly Gin Gin Asp Leu Met 100 105 110 Thr Gin Val Leu Asn Gly Leu Ala Lys Ser Met Leu Asp Asp Leu Leu
115 120 125
Thr Lys Gin Asp Gly Gly Thr Ser Phe Ser Glu Asp Asp Met Pro Met 130 135 140
Leu Asn Lys He Ala Gin Phe Met Asp Asp Asn Pro Ala Gin Phe Pro 145 150 155 160
Lys Pro Asp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn Phe 165 170 175
Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp He He 180 185 190 Gly Gin Gin Leu Gly Asn Gin Gin Ser Asp Ala Gly Ser Leu Ala Gly
195 200 205
Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn Asn Ser Ser 210 215 220
Val Met Gly Asp Pro Leu He Asp Ala Asn Thr Gly Pro Gly Asp Ser 225 230 235 240
Gly Asn Thr Arg Gly Glu Ala Gly Gin Leu He Gly Glu Leu He Asp 245 250 255
Arg Gly Leu Gin Ser Val Leu Ala Gly Gly Gly Leu Gly Thr Pro Val 260 265 270 Asn Thr Pro Gin Thr Gly Thr Ser Ala Asn Gly Gly Gin Ser Ala Gin
275 280 285 Asp Leu Asp Gin Leu Leu Gly Gly Leu Leu Leu Lys Gly Leu Glu Ala 290 295 300
Thr Leu Lys Asp Ala Gly Gin Thr Gly Thr Asp Val Gin Ser Ser Ala 305 310 315 320 Ala Gin He Ala Thr Leu Leu Val Ser Thr Leu Leu Gin Gly Thr Arg
325 330 335
Asn Gin Ala Ala Ala 340
This hypersensitive response elicitor polypeptide or protein has a molecular weight of
34-35 kDa. It is rich in glycine (about 13.5%) and lacks cysteine and tyrosine.
Further information about the hypersensitive response elicitor derived from
Pseudomonas syringae is found in He, S. Y., H. C. Huang, and A. Collmer, "Pseudomonas syringae pv. syringae Harpinpss: a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants," Cell 73:1255-1266 (1993), which is hereby incorporated by reference. The DNA molecule encoding the hypersensitive response elicitor from Pseudomonas syringae has a nucleotide sequence corresponding to SEQ. ID. No. 26 as follows:
ATGCAGAGTC TCAGTCTTAA CAGCAGCTCG CTGCAAACCC CGGCAATGGC CCTTGTCCTG 60
GTACGTCCTG AAGCCGAGAC GACTGGCAGT ACGTCGAGCA AGGCGCTTCA GGAAGTTGTC 120
GTGAAGCTGG CCGAGGAACT GATGCGCAAT GGTCAACTCG ACGACAGCTC GCCATTGGGA 180
AAACTGTTGG CCAAGTCGAT GGCCGCAGAT GGCAAGGCGG GCGGCGGTAT TGAGGATGTC 240 ATCGCTGCGC TGGACAAGCT GATCCATGAA AAGCTCGGTG ACAACTTCGG CGCGTCTGCG 300
GACAGCGCCT CGGGTACCGG ACAGCAGGAC CTGATGACTC AGGTGCTCAA TGGCCTGGCC 360
AAGTCGATGC TCGATGATCT TCTGACCAAG CAGGATGGCG GGACAAGCTT CTCCGAAGAC 420
GATATGCCGA TGCTGAACAA GATCGCGCAG TTCATGGATG ACAATCCCGC ACAGTTTCCC 480
AAGCCGGACT CGGGCTCCTG GGTGAACGAA CTCAAGGAAG ACAACTTCCT TGATGGCGAC 540 GAAACGGCTG CGTTCCGTTC GGCACTCGAC ATCATTGGCC AGCAACTGGG TAATCAGCAG 600
AGTGACGCTG GCAGTCTGGC AGGGACGGGT GGAGGTCTGG GCACTCCGAG CAGTTTTTCC 660
AACAACTCGT CCGTGATGGG TGATCCGCTG ATCGACGCCA ATACCGGTCC CGGTGACAGC 720
GGCAATACCC GTGGTGAAGC GGGGCAACTG ATCGGCGAGC TTATCGACCG TGGCCTGCAA 780
TCGGTATTGG CCGGTGGTGG ACTGGGCACA CCCGTAAACA CCCCGCAGAC CGGTACGTCG 840 GCGAATGGCG GACAGTCCGC TCAGGATCTT GATCAGTTGC TGGGCGGCTT GCTGCTCAAG 900
GGCCTGGAGG CAACGCTCAA GGATGCCGGG CAAACAGGCA CCGACGTGCA GTCGAGCGCT 960
GCGCAAATCG CCACCTTGCT GGTCAGTACG CTGCTGCAAG GCACCCGCAA TCAGGCTGCA 1020
GCCTGA 1026
The hypersensitive response elicitor polypeptide or protein derived from Pseudomonas solanacearum has an amino acid sequence corresponding to SEQ. ID. No. 27 as follows:
Met Ser Val Gly Asn He Gin Ser Pro Ser Asn Leu Pro Gly Leu Gin
1 5 10 15
Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gin Gin Ser Gly Gin Ser 20 25 30
Val Gin Asp Leu He Lys Gin Val Glu Lys Asp He Leu Asn He He 35 40 45
Ala Ala Leu Val Gin Lys Ala Ala Gin Ser Ala Gly Gly Asn Thr Gly 50 55 60
Asn Thr Gly Asn Ala Pro Ala Lys Asp Gly Asn Ala Asn Ala Gly Ala 65 70 75 80 Asn Asp Pro Ser Lys Asn Asp Pro Ser Lys Ser Gin Ala Pro Gin Ser
85 90 95
Ala Asn Lys Thr Gly Asn Val Asp Asp Ala Asn Asn Gin Asp Pro Met 100 105 110
Gin Ala Leu Met Gin Leu Leu Glu Asp Leu Val Lys Leu Leu Lys Ala 115 120 125
Ala Leu His Met Gin Gin Pro Gly Gly Asn Asp Lys Gly Asn Gly Val 130 135 140
Gly Gly Ala Asn Gly Ala Lys Gly Ala Gly Gly Gin Gly Gly Leu Ala 145 150 155 160 Glu Ala Leu Gin Glu He Glu Gin He Leu Ala Gin Leu Gly Gly Gly
165 170 175
Gly Ala Gly Ala Gly Gly Ala Gly Gly Gly Val Gly Gly Ala Gly Gly 180 185 190
Ala Asp Gly Gly Ser Gly Ala Gly Gly Ala Gly Gly Ala Asn Gly Ala 195 200 205 Asp Gly Gly Asn Gly Val Asn Gly Asn Gin Ala Asn Gly Pro Gin Asn 210 215 220
Ala Gly Asp Val Asn Gly Ala Asn Gly Ala Asp Asp Gly Ser Glu Asp 225 230 235 240 Gin Gly Gly Leu Thr Gly Val Leu Gin Lys Leu Met Lys He Leu Asn
245 250 255
Ala Leu Val Gin Met Met Gin Gin Gly Gly Leu Gly Gly Gly Asn Gin 260 265 270
Ala Gin Gly Gly Ser Lys Gly Ala Gly Asn Ala Ser Pro Ala Ser Gly 275 280 285
Ala Asn Pro Gly Ala Asn Gin Pro Gly Ser Ala Asp Asp Gin Ser Ser 290 295 300
Gly Gin Asn Asn Leu Gin Ser Gin He Met Asp Val Val Lys Glu Val 305 310 315 320 Val Gin He Leu Gin Gin Met Leu Ala Ala Gin Asn Gly Gly Ser Gin
325 330 335
Gin Ser Thr Ser Thr Gin Pro Met 340
It is encoded by a DNA molecule having a nucleotide sequence corresponding SEQ. ID. No. 28 as follows:
ATGTCAGTCG GAAACATCCA GAGCCCGTCG AACCTCCCGG GTCTGCAGAA CCTGAACCTC 60
AACACCAACA CCAACAGCCA GCAATCGGGC CAGTCCGTGC AAGACCTGAT CAAGCAGGTC 120
GAGAAGGACA TCCTCAACAT CATCGCAGCC CTCGTGCAGA AGGCCGCACA GTCGGCGGGC 180
GGCAACACCG GTAACACCGG CAACGCGCCG GCGAAGGACG GCAATGCCAA CGCGGGCGCC 240 AACGACCCGA GCAAGAACGA CCCGAGCAAG AGCCAGGCTC CGCAGTCGGC CAACAAGACC 300
GGCAACGTCG ACGACGCCAA CAACCAGGAT CCGATGCAAG CGCTGATGCA GCTGCTGGAA 360
GACCTGGTGA AGCTGCTGAA GGCGGCCCTG CACATGCAGC AGCCCGGCGG CAATGACAAG 420
GGCAACGGCG TGGGCGGTGC CAACGGCGCC AAGGGTGCCG GCGGCCAGGG CGGCCTGGCC 480
GAAGCGCTGC AGGAGATCGA GCAGATCCTC GCCCAGCTCG GCGGCGGCGG TGCTGGCGCC 540 GGCGGCGCGG GTGGCGGTGT CGGCGGTGCT GGTGGCGCGG ATGGCGGCTC CGGTGCGGGT 600
GGCGCAGGCG GTGCGAACGG CGCCGACGGC GGCAATGGCG TGAACGGCAA CCAGGCGAAC 660
GGCCCGCAGA ACGCAGGCGA TGTCAACGGT GCCAACGGCG CGGATGACGG CAGCGAAGAC 720
CAGGGCGGCC TCACCGGCGT GCTGCAAAAG CTGATGAAGA TCCTGAACGC GCTGGTGCAG 780 ATGATGCAGC AAGGCGGCCT CGGCGGCGGC AACCAGGCGC AGGGCGGCTC GAAGGGTGCC 840
GGCAACGCCT CGCCGGCTTC CGGCGCGAAC CCGGGCGCGA ACCAGCCCGG TTCGGCGGAT 900
GATCAATCGT CCGGCCAGAA CAATCTGCAA TCCCAGATCA TGGATGTGGT GAAGGAGGTC 960
GTCCAGATCC TGCAGCAGAT GCTGGCGGCG CAGAACGGCG GCAGCCAGCA GTCCACCTCG 1020 ACGCAGCCGA TGTAA 1035
Further information regarding the hypersensitive response elicitor polypeptide or protein derived from Pseudomonas solanacearum is set forth in Arlat, M., F. Van Gijsegem, J. C. Huet, J. C. Pemollet, and C. A. Boucher, "PopAl, a Protein which Induces a Hypersensitive-like Response in Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum," EMBO J. 13:543-533 (1994), which is hereby incorporated by reference.
The hypersensitive response elicitor polypeptide or protein from Xanthomonas campestris pv. glycines has an amino acid sequence corresponding to SEQ. ID. No. 29 as follows:
Thr Leu He Glu Leu Met He Val Val Ala He He Ala He Leu Ala 1 5 10 15
Ala He Ala Leu Pro Ala Tyr Gin Asp Tyr 20 25
This sequence is an amino terminal sequence having only 26 residues from the hypersensitive response elicitor polypeptide or protein of Xanthomonas campestris pv. glycines. It matches with fimbrial subunit proteins determined in other
Xanthomonas campestris pathovars.
The hypersensitive response elicitor polypeptide or protein from Xanthomonas campestris pv. pelargonii is heat stable, protease sensitive, and has a molecular weight of 20 kDa. It includes an amino acid sequence corresponding to SEQ. ID. No.
30 as follows:
Ser Ser Gin Gin Ser Pro Ser Ala Gly Ser Glu Gin Gin Leu Asp Gin 1 5 10 15 Leu Leu Ala Met 20 Isolation of Erwinia carotovora hypersensitive response elictor protein or polypeptide is described in Cui et al., "The RsmA Mutants of Erwinia carotovora subsp. carotovora Strain Ecc71 Overexpress hrp NECC and Elicit a Hypersensitive Reaction-like Response in Tobacco Leaves," MPMI, 9(7):565-73 (1996), which is hereby incorporated by reference. The hypersensitive response elicitor protein or polypeptide of Erwinia stewartii is set forth in Ahmad et al., "Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize," 8th Int'l. Cong. Molec. Plant-Microbe Interact., July 14-19, 1996 and Ahmad, et al., "Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize," Ann. Mtg. Am. Phvtopath. Soc, July 27-31 , 1996, which are hereby incorporated by reference.
Hypersensitive response elicitor proteins or polypeptides from Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamoni, Phytophthora capsici, Phytophthora megasperma, and Phytophora citrophthora are described in Kaman, et al., "Extracellular Protein Elicitors from Phytophthora: Most Specificity and Induction of Resistance to Bacterial and Fungal Phytopathogens," Molec. Plant- Microbe Interact., 6(1): 15-25 (1993), Ricci et al., "Structure and Activity of Proteins from Pathogenic Fungi Phytophthora Eliciting Necrosis and Acquired Resistance in Tobacco," Eur. J. Biochem., 183:555-63 (1989), Ricci et al., "Differential Production of Parasiticein, and Elicitor of Necrosis and Resistance in Tobacco, by Isolates of Phytophthora parasitica," Plant Path. 41 :298-307 (1992), Baillreul et al, "A New Elicitor of the Hypersensitive Response in Tobacco: A Fungal Glycoprotein Elicits Cell Death, Expression of Defence Genes, Production of Salicylic Acid, and Induction of Systemic Acquired Resistance," Plant J., 8(4):551-60 (1995), and Bonnet et al., "Acquired Resistance Triggered by Elicitors in Tobacco and Other Plants," Eur. J. Plant Path., 102:181-92 (1996), which are hereby incorporated by reference. The above elicitors are exemplary. Other elicitors can be identified by growing fungi or bacteria that elicit a hypersensitive response under which genes encoding an elicitor are expressed. Cell-free preparations from culture supernatants can be tested for elicitor activity (i.e. local necrosis) by using them to infiltrate appropriate plant tissues. Fragments of the above hypersensitive response elicitor polypeptides or proteins as well as fragments of full length elicitors from other pathogens are encompassed by the method of the present invention.
Suitable fragments can be produced by several means. In the first, subclones of the gene encoding a known elicitor protein are produced by conventional molecular genetic manipulation by subcloning gene fragments. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or peptide that can be tested for elicitor activity according to the procedure described below. As an alternative, fragments of an elicitor protein can be produced by digestion of a full-length elicitor protein with proteolytic enzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin. Different proteolytic enzymes are likely to cleave elicitor proteins at different sites based on the amino acid sequence of the elicitor protein. Some of the fragments that result from proteolysis may be active elicitors of resistance. In another approach, based on knowledge of the primary structure of the protein, fragments of the elicitor protein gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These then would be cloned into an appropriate vector for expression of a truncated peptide or protein. Chemical synthesis can also be used to make suitable fragments. Such a synthesis is carried out using known amino acid sequences for the elicitor being produced. Alternatively, subjecting a full length elicitor to high temperatures and pressures will produce fragments. These fragments can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE). An example of suitable fragments of an Erwinia hypersensitive response elicitor which fragments elicit a hypersensitive response are fragments of the Erwinia amylovora hypersensitive response elicitor. Suitable fragments include a C-terminal fragment of the amino acid sequence of SEQ. ID. No. 23, an N-terminal fragment of the amino acid sequence of SEQ. ID. No. 23, or an internal fragment of the amino acid sequence of SEQ. ID. No. 23. The C-terminal fragment of the amino acid sequence of SEQ. ID. No. 23 can span amino acids 105 and 403 of SEQ. ID. No. 23. The N-terminal fragment of the amino acid sequence of SEQ. ID. No. 23 can span the following amino acids of SEQ. ID. No. 23: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1 and 372. The internal fragment of the amino acid sequence of SEQ. ID. No. 23 can span the following amino acids of SEQ. ID. No. 23: 76 and 209, 105 and 209, 99 and 209, 137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180, and 105 and 180. Other suitable fragments can be identified in accordance with the present invention.
Variants may be made 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 fragment of the present invention is preferably produced in purified form (preferably at least about 60%, more preferably 80%, pure) by conventional techniques. Typically, the fragment of the present invention is produced but not secreted into the growth medium of recombinant host cells. Alternatively, the protein or polypeptide of the present invention is secreted into growth medium. In the case of unsecreted protein, to isolate the protein fragment, 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 heat treatment and the fragment is separated by centrifugation. The supernatant fraction containing the fragment is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the fragment. If necessary, the protein fraction may be further purified by ion exchange or HPLC.
The DNA molecule encoding the fragment of the hypersensitive response elicitor polypeptide or protein 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. Patent No. 4,237,224 to Cohen and Boyer, which is hereby incoφorated by reference, 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 procaryotic organisms and eucaryotic cells grown in tissue culture.
Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transection of plasmids into cells infected with virus.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see
"Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incoφorated 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 incoφorated by reference), 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, New York (1989), which is hereby incoφorated by reference.
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 promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promotors are not recognized and do not function in eucaryotic cells.
Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes 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.
Promotors vary in their "strength" (i.e. their ability to promote transcription). For the puφoses of expressing a cloned gene, it is desirable to use strong promotors 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 promotors may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promotors such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promotors of coliphage lambda and others, including but not limited, to / cUV5, omp , bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promotors 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 promotor 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 procaryotic 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 promotor, 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 TV gene of coliphage lambda, or from the E. coli tryptophan Ε, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incoφoration of synthetic nucleotides may be used.
Once the isolated DNA molecule encoding the fragment of a hypersensitive response elicitor polypeptide or protein has been cloned into an expression system, it is ready to be incoφorated into a host cell. Such incoφoration 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.
The present invention further relates to methods of imparting disease resistance to plants, enhancing plant growth, and/or effecting insect control for plants. These methods involve applying the fragment of a hypersensitive response elicitor polypeptide or protein, which fragment itself elicits a hypersensitive response; in a non-infectious form to all or part of a plant or a plant seed under conditions effective for the fragment to impart disease resistance, enhance growth, and/or control insects. Alternatively, these fragments of a hypersensitive response elicitor protein or polypeptide can be applied to plants such that seeds recovered from such plants themselves are able to impart disease resistance in plants, to enhance plant growth, and/or to effect insect control.
As an alternative to applying a fragment of a hypersensitive response elicitor polypeptide or protein to plants or plant seeds in order to impart disease resistance in plants, to effect plant growth, and/or to control insects on the plants or plants grown from the seeds, transgenic plants or plant seeds can be utilized. When utilizing transgenic plants, this involves providing a transgenic plant transformed with a DNA molecule encoding a fragment of a hypersensitive response elicitor polypeptide or protein, which fragment elicits a hypersensitive response, and growing the plant under conditions effective to permit that DNA molecule to impart disease resistance to plants, to enhance plant growth, and/or to control insects. Alternatively, a transgenic plant seed transformed with a DNA molecule encoding a fragment of a hypersensitive response elicitor polypeptide or protein which fragment elicits a hypersensitive response can be provided and planted in soil. A plant is then propagated from the planted seed under conditions effective to permit that DNA molecule to impart disease resistance to plants, to enhance plant growth, and/or to control insects.
The embodiment of the present invention where the hypersensitive response elicitor polypeptide or protein is applied to the plant or plant seed can be carried out in a number of ways, including: 1) application of an isolated fragment or 2) application of bacteria which do not cause disease and are transformed with a genes encoding the fragment. In the latter embodiment, the fragment can be applied to plants or plant seeds by applying bacteria containing the DNA molecule encoding the fragment of the hypersensitive response elicitor polypeptide or protein which fragment elicits a hypersensitive response. Such bacteria must be capable of secreting or exporting the fragment so that the fragment can contact plant or plant seeds cells. In these embodiments, the fragment is produced by the bacteria inplanta or on seeds or just prior to introduction of the bacteria to the plants or plant seeds. The methods of the present invention can be utilized to treat a wide variety of plants or their seeds to impart disease resistance, enhance growth, and/or control insects. Suitable plants include dicots and monocots. More particularly, useful crop plants can include: alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane. Examples of suitable ornamental plants are: Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia. With regard to the use of the fragments of the hypersensitive response elicitor protein or polypeptide of the present invention in imparting disease resistance, absolute immunity against infection may not be conferred, but the severity of the disease is reduced and symptom development is delayed. Lesion number, lesion size, and extent of sporulation of fungal pathogens are all decreased. This method of imparting disease resistance has the potential for treating previously untreatable diseases, treating diseases systemically which might not be treated separately due to cost, and avoiding the use of infectious agents or environmentally harmful materials. The method of imparting pathogen resistance to plants in accordance with the present invention is useful in imparting resistance to a wide variety of pathogens including viruses, bacteria, and fungi. Resistance, ter alia, to the following viruses can be achieved by the method of the present invention: Tobacco mosaic virus and Tomato mosaic virus. Resistance, mter alia, to the following bacteria can also be imparted to plants in accordance with present invention: Pseudomonas solancearum, Pseudomonas syringae pv. tabaci, and Xanthomonas campestris pv. pelargonii.
Plants can be made resistant, inter alia, to the following fungi by use of the method of the present invention: Fusarium oxysporum and Phytophthora infestans.
With regard to the use of the fragments of the hypersensitive response elicitor protein or polypeptide of the present invention to enhance plant growth, various forms of plant growth enhancement or promotion can be achieved. This can occur as early as when plant growth begins from seeds or later in the life of a plant. For example, plant growth according to the present invention encompasses greater yield, increased quantity of seeds produced, increased percentage of seeds germinated, increased plant size, greater biomass, more and bigger fruit, earlier fruit coloration, and earlier fruit and plant maturation. As a result, the present invention provides significant economic benefit to growers. For example, early germination and early maturation permit crops to be grown in areas where short growing seasons would otherwise preclude their growth in that locale. Increased percentage of seed germination results in improved crop stands and more efficient seed use. Greater yield, increased size, and enhanced biomass production allow greater revenue generation from a given plot of land.
Another aspect of the present invention is directed to effecting any form of insect control for plants. For example, insect control according to the present invention encompasses preventing insects from contacting plants to which the hypersensitive response elicitor has been applied, preventing direct insect damage to plants by feeding injury, causing insects to depart from such plants, killing insects proximate to such plants, interfering with insect larval feeding on such plants, preventing insects from colonizing host plants, preventing colonizing insects from releasing phytotoxins, etc. The present invention also prevents subsequent disease damage to plants resulting from insect infection.
The present invention is effective against a wide variety of insects. European corn borer is a major pest of corn (dent and sweet corn) but also feeds on over 200 plant species including green, wax, and lima beans and edible soybeans, peppers, potato, and tomato plus many weed species. Additional insect larval feeding pests which damage a wide' variety of vegetable crops include the following: beet armyworm, cabbage looper, corn ear worm, fall armyworm, diamondback moth, cabbage root maggot, onion maggot, seed corn maggot, pickleworm (melonworm), pepper maggot, tomato pinworm, and maggots. Collectively, this group of insect pests represents the most economically important group of pests for vegetable production worldwide.
The method of the present invention involving application of the fragment of a hypersensitive response elicitor polypeptide or protein, which fragment elicits a hypersensitive response, can be carried out through a variety of procedures when all or part of the plant is treated, including leaves, stems, roots, etc. This may (but need not) involve infiltration of the hypersensitive response elicitor polypeptide or -protein into the plant. Suitable application methods include high or low pressure spraying, injection, and leaf abrasion proximate to when elicitor application takes place. When treating plant seeds or propagules (e.g., cuttings), in accordance with the application embodiment of the present invention, the fragment of the hypersensitive response elicitor protein or polypeptide, in accordance with present invention, can be applied by low or high pressure spraying, coating, immersion, or injection. Other suitable application procedures can be envisioned by those skilled in the art provided they are able to effect contact of the fragment with cells of the plant or plant seed. Once treated with the fragment of the hypersensitive response elicitor of the present invention, the seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants. After plants have been propagated from seeds treated in accordance with the present invention, the plants may be treated with one or more applications of the fragment of the hypersensitive response elicitor protein or polypeptide or whole elicitors to impart disease resistance to plants, to enhance plant growth, and/or to control insects on the plants.
The fragment of the hypersensitive response elicitor polypeptide or protein, in accordance with the present invention, can be applied to plants or plant seeds alone or in a mixture with other materials. Alternatively, the fragment can be applied separately to plants with other materials being applied at different times. A composition suitable for treating plants or plant seeds in accordance with the application embodiment of the present invention contains a fragment of a hypersensitive response elicitor polypeptide or protein which fragment elicits a hypersensitive response in a carrier. Suitable carriers include water, aqueous solutions, slurries, or dry powders. In this embodiment, the composition contains greater than 500 nM of the fragment.
Although not required, this composition may contain additional additives including fertilizer, insecticide, fungicide, nematacide, and mixtures thereof. Suitable fertilizers include (NH ) NO . An example of a suitable insecticide is Malathion. Useful fungicides include Captan. Other suitable additives include buffering agents, wetting agents, coating agents, and abrading agents. These materials can be used to facilitate the process of the present invention. In addition, the hypersensitive response eliciting fragment can be applied to plant seeds with other conventional seed formulation and treatment materials, including clays and polysaccharides.
In the alternative embodiment of the present invention involving the use of transgenic plants and transgenic seeds, a hypersensitive response eliciting fragment need not be applied topically to the plants or seeds. Instead, transgenic plants transformed with a DNA molecule encoding such a fragment are produced according to procedures well known in the art.
The vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics, 202:179-85 (1985), which is hereby incoφorated by reference. The genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incorporated by reference.
Another approach to transforming plant cells with a gene which imparts resistance to pathogens 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. Patent Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which are hereby incoφorated by reference. 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.
Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies. Fraley, et al., Proc. Natl. Acad. Sci. USA. 79:1859-63 (1982), which is hereby incorporated by reference.
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 incoφorated by reference. In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. 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 A. rhizogenes previously transformed with the gene. 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 can be introduced into appropriate plant cells, by means of the Ti plasmid of J. tumefaciens or the Ri plasmid of J. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome, j. Schell, Science, 237:1176-83 (1987), which is hereby incoφorated by reference. After transformation, the transformed plant cells must be regenerated.
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. Ill (1986), which are hereby incoφorated by reference. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes.
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 expression cassette is stably incoφorated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure with the presence of the gene encoding the hypersensitive response eliciting fragment resulting in disease resistance, enhanced plant growth, and/or control of insects on the plant.
Alternatively, transgenic seeds or propagules (e.g., cuttings) are recovered from the transgenic plants. The seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants. The transgenic plants are propagated from the planted transgenic seeds under conditions effective to impart disease resistance to plants, to enhance plant growth, and/or to control insects. While not wishing to be bound by theory, such disease resistance, growth enhancement, and/or insect control may be RNA mediated or may result from expression of the polypeptide or protein fragment.
When transgenic plants and plant seeds are used in accordance with the present invention, they additionally can be treated with the same materials as are used to treat the plants and seeds to which a hypersensitive response eliciting fragment is applied. These other materials, including hypersensitive response eliciting fragments, can be applied to the transgenic plants and plant seeds by the above-noted procedures, including high or low pressure spraying, injection, coating, and immersion. Similarly, after plants have been propagated from the transgenic plant seeds, the plants may be treated with one or more applications of the hypersensitive response eliciting fragment to impart disease resistance, enhance growth, and/or control insects. Such plants may also be treated with conventional plant treatment agents (e.g., insecticides, fertilizers, etc.).
EXAMPLES Example 1 - Strains and plasmids used
The strains and plasmids used are set forth in Table 1 below
Table 1
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Example 2 - Molecular biology techniques. Several approaches were employed to obtain truncated or otherwise altered versions of both E. amylovora haφins. These techniques included: (i) subcloning of restriction fragments containing portions of the gene encoding the hypersensitive response elicitor protein or polypeptide from Erwinia amylovora (i.e. hrpN) into expression vectors, by standard techniques (Sambrook, et al., Molecular Cloning: a Laboratory Manual 2nd ed. ed. Cold Spring Harbor, Laboratory," Cold Spring Harbor, NY (1989), which is hereby incoφorated by reference); (ii) insertion of an Ω- fragment (Fellay, et al, "Interposon Mutagenesis of Soil and Water Bacteria a Family of DNA Fragments Designed for in vitro Insertional Mutagenesis of Gram-Negative Bacteria," Gene 52:147-154 (1987), which is hereby incoφorated by reference) into hrpN; (iii) site-specific mutagenesis approaches (Innis, et al, PCR Protocols. A
Guide to Methods and Applications. Academic Press San Diego, CA (1990); Kunkel, et al, "Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection," Proc. Nat. Acad. Sci. USA 82:488-492 (1985), which are hereby incoφorated by reference); and (iv) creation of nested deletions (Erase-a-Base™ kit; Promega, Madison, WI). C-terminal deletion analysis of the hypersensitive response elicitor protein or polypeptide from Erwinia amylovora (i.e. haφhiEa) in pCPP1084 could not be performed because of the location of restriction enzyme cleavage sites in pCPP1084. For N-terminal deletions, pCPP1084 DNA was prepared using a Qiagen midiprep column (Qiagen, Chatsworth, CA) and digested with sst / followed by EcoRI. Subsequently, the digested DNA was subjected to exonuclease III digestion, ligation, and transformation into E. coli BL21(DΕ3). Deletion sizes were estimated by agarose gel electrophoresis. Haφin fragments were named with respect to the portion of haφin deleted (e.g., haφinEa C82 lacks the C-terminal 82 amino acid residues of full-length haφinEa). Example 3 - Protein expression.
For expression from T7 promoters, T7 RNA polymerase-dependent systems were used. These systems utilized either strain E. coli BL21(DE3) (Studier, et al, "Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-Level Expression of Cloned Gene," J. Mol Biol. 189:113-130 (1986), which is hereby incoφorated by reference), or plasmid pGPl-2 (Tabor, et al, "A Bacteriophage T7 DNA Polymerase/Promoter System for Controlled Exclusive Expression of Specific Genes," Proc. Natl Acad. Sci, USA 82:1074-1078 (1985), which is hereby incoφorated by reference) in E. coli DH5α. Expression of hrpN from the T7 promoter was induced by addition of IPTG to a final concentration of 0.4 mM. For expression in E. amylovora Ea321 (i.e. haφinEa) or Ea273, pGPl-2 was introduced by transformation with a 42°C heat shock for 10 minutes, or by electroporation (Biorad Gene Pulser™). Hypersensitive response (i.e. HR)-eliciting activity was screened in tobacco cv. Xanthi leaves by inplanta lysis (He, et al, "Pseudomonas syringae pv. syringae haφinpss: a Protein That is Secreted via the Hφ Pathway and Elicits the Hypersensitive Response in Plants," Cell 73:1255-1266 (1993), which is hereby incoφorated by reference) or by preparation of boiled and unboiled "CFEPs" (Wei, et al, "Haφin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora," Science 257:85-88 (1992), which is hereby incorporated by reference).
Example 4 - In vitro proteolysis of harpin. In vitro proteolysis of haφinEa with Staphylococcus V8 proteinase (also termed endoproteinase Glu-C), trypsin, pepsin, and papain was performed as recommended (Scopes, et al, Protein Purification: Principles and Practice. 2nd ed. Springer-Verlag. New York (1987), which is hereby incorporated by reference), for 2- 16 hrs. at 20-37°. Endoproteinase Glu-C digestion was performed either in 50 mM ammonium bicarbonate, pH 7.8 (in which cleavage occurs only after glutamic acid), or in 50 mM potassium phosphate, pH 7.8 (in which cleavage after both glutamic acid and aspartic acid occurs). Example 5 - Plant-derived proteinases.
Intercellular fluids (IF) were obtained from tobacco, tomato, apple, raspberry, and cotoneaster, as described (Hammond-Kosack, et al, "Preparation and Analysis of Intercellular Fluid," p. 15-21. In S.J. Gurr, M.J. McPherson, and D.J. Bowles (ed.), Molecular Plant Pathology A Practical Approach. 2nd ed., The Practical Approach Series, IRL Publishers, Oxford (1992), which is hereby incoφorated by reference), by vacuum infiltration of intercellular spaces with high-purity water. Proteolytic digestion of PAGE-purified haφinEa was performed for 2-16 hrs. at 20-37°C, pH, by mixing equal volumes of IF with haφinEa. A total leaf extract was obtained by grinding tobacco leaf panels with mortar and pestle in 5 mM potassium phosphate. The extract was centrifuged and filtered, and the clarified ground leaf extract used identically as was the IF. Proteinase inhibitors were employed as follows: Pepstatin A (final concentration lμM), E-64 (lμM), Aprotinin (2μg/ml), o-phenanthroline (ImM), and p-mercuribenzoate (PCMB) (Sigma, St. Louis, MO).
Example 6 - Peptide purification.
Peptide fragments of haφin obtained following digestion with tobacco IF were fractionated by reverse-phase HPLC on a Vydac C 18 column using a 2-60% acetonitrile gradient in 0.1 % trifiuoroacetic acid. Fractions were lyophilized, resuspended in 5 mM potassium phosphate and infiltrated into tobacco leaf panels. The fraction with greatest HR-eliciting activity was refractionated as above with a 35- 70%) acetonitrile gradient, and the purity of each fraction was assayed via gas chromatography-mass spectroscopy (GC-MS) and by N-terminal protein sequencing at the Cornell Biotechnology Program Core Facility.
Example 7 - Proteinase activity-stained gels. Proteinase activity of IF was assayed in activity- stained polyacrylamide gels
(Laemmli, "Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4," Nature 227:680-685 (1970), which is hereby incorporated by reference) copolymerized with 0.1 % gelatin (Heussen, et al, "Electrophoretic Analysis of Plasminogen Activators in Polyacrylamide Gels Containing Sodium Dodecyl Sulfate and Copolymerized Substrates," Anal Biochem. 102:196-202 (1980), which is hereby incoφorated by reference). After electrophoresis, each gel was rinsed extensively to remove SDS and allow refolding of proteinases in the gel. Following additional incubation to allow proteolysis to occur, the gels were stained with 0.1%) Amido Black in 30%> methanol/10%> acetic acid. Each gel stained darkly (due to the presence of copolymerized gelatin) except where proteinases had digested the gelatin, resulting in colorless bands representing the sites of proteinase activity.
Example 8 - Truncated harpins retain HR-eliciting activity.
The stability and the HR-eliciting activity of proteins encoded by various DNA constructs is shown in Figure 1. Many DNA constructs encoding portions of haφinEa or ha inEar did not yield detectable protein products following induction of expression in the T7 promoter-polymerase system (Tabor, et al, "A Bacteriophage T7 DNA Polymerase/Promoter System for Controlled Exclusive Expression of Specific Genes," Proc. Natl Acad. Sci. USA 82:1074-1078 (1985), which is hereby incoφorated by reference) and analysis of cell extracts by PAGE, possibly due to instability of the encoded proteins. No DNA constructs (e.g., those obtained via Erase-a-Base™ protocol) yielded detectable protein products displaying N-terminal deletions relative to the full-length protein. No stable but inactive proteins were identified. Several constructs encoding proteins truncated at their C-terminus and often including additional vector-encoded amino acids yielded detectable products (e.g. haφin a C82). In contrast, a construct encoding the same 321 N-terminal amino acid residues of haφinEa, but yielding a protein truncated by the presence of an Ω- fragment (ha inEa C82 Ω) was unstable (i.e. no product was detected). A construct encoding a ha inEa fragment with a large internal deletion (haφinEa 1175) was also successfully used to express protein. These various truncated proteins were tested for HR-eliciting activity. A 98 residue N-terminal haφinEa fragment (haφiiiEa C305) was the smallest bacterially-produced peptide that displayed HR-eliciting activity. Example 9 - Secretion of harpinEa with an altered C-terminus.
The effect of alteration at the haφin C-terminus on its secretion was examined. Haφin C31 contains the N-terminal 372 amino acids of harpin, but lacks the C-terminal 31 residues, which are replaced by 47 residues encoded by the vector, resulting in a protein slightly larger than the wild type haφinEa- The C31 protein retains HR-eliciting activity and is stable and easily expressed and detected by western analysis or PAGE but it is no longer secreted into the culture supernatant as is the wild type protein (Figure 2). The presence of haφinEa C31 does not interfere with secretion of the wild type haφin, which is found in both the CFEP and the culture supernatant. However, haφinEa C31 is found only in the CFEP.
Example 10 - Effect of proteolysis on harpinεa's HR eliciting activity In order to generate additional harpinEa fragments, purified full length protein was proteolyzed in vitro by several proteinases, including endoproteinase Glu-C, trypsin, pepsin, and papain (e.g., Figures 3 and 4). Harpin solutions digested with trypsin or with papain lost all activity. In contrast, following digestion with endoproteinase Glu-C, HR-eliciting activity was retained. No peptides larger than 6 kD were evident by PAGE following trypsin digestion. Endoproteinase Glu-C digestion yielded an approximately 20 kD fragment, larger than expected if all cleavage sites were cut, indicating that digestion was not complete (Figure 4).
Example 11 - Apoplastic fluids (IF) contain harpin-degrading proteolytic activity
Apoplastic fluids (intercellular fluids; IF) from tobacco and other plants were also employed to proteolyze haφin. Each IF tested possessed proteinase activity(s), as indicated by the presence of multiple activity-stained bands in polyacrylamide gels containing co-polymerized gelatin (Figures 5A to 5C), as well as by the disappearance of detectable haφinEa (Schagger, et al, "Tricine-Sodium Dodecyl Sulfate Gel
Electrophoresis for the Separation of Proteins in the Range From 1 to 100 kDa," Anal Biochem. 166:368-379 (1987), which is hereby incorporated by reference) following overnight digestion of purified haφinEa with IF. Proteinase activity was substantially greater at 37°C than at 20°C, and activity was higher at pH 8.5 than at pH 7. Several inhibitors were employed in order to define the proteolytic activity(s) of the IF. No single proteinase inhibitor which was employed prevented degradation of haφinEa- However, a mixture of the inhibitors Pepstatin A (lμM), E-64 (lμM), Aprotinin (2 μg/ml), and o-phenanthroline (ImM), targeted at acid proteinases, cysteine proteinases, serine proteinases, and metalloproteinases, respectively, partially inhibited proteolysis.
HaφinEa degraded by proteolytic activities present in the plant apoplast retained HR-eliciting activity (Figure 3). In contrast, haφinEa proteolyzed by a clarified extract produced by grinding tobacco leaf tissue with mortar and pestle lost HR-eliciting activity. In order to study whether apoplastic degradation of haφin was a prerequisite to its HR-eliciting activity, the length of time required for leaf collapse when either intact haφin or haφin predigested with tobacco IF was infiltrated into tobacco leaf panels was compared. Both preparations elicited the HR in a similar time frame (12-18 hours, depending on the experiment).
Example 12 - Characterization of HR-eliciting peptide fragments
Peptides resulting from digestion by apoplastic plant proteinase(s) were fractionated by reverse phase HPLC (Vydac C18 column), and tested for activity. Following treatment of intact haφinEa with tobacco IF, three fractions contained some HR-eliciting activity on tobacco. Two of the three demonstrated weak activity, and little protein was present. They were not further characterized. Fraction 19, which contained the strongest activity as well as the most protein, was refractionated using a more shallow elution gradient (Figure 6). Refractionation, N-terminal protein sequencing, and molecular weight analysis by mass spectroscopy indicated that four largely overlapping peptides were present. Peak 19-1 contained peptides P91 and P95, corresponding to haφinεa residues 110-200 and 110-204; peak 19-2 contained peptides P64 and P68, corresponding to haφinEa residues 137-200 and 137-204. 19-1 and 19-2 each possessed HR-eliciting activity. The smallest peptide thus confirmed to retain activity consisted of residues 137-204. The two peptides in each peak were not separable under the conditions used. These active fragments are distinct from the smallest active N-terminal fragment (harpinEaC305), and indicate that more than one portion of haφinεa displays activity inplanta. Further digestion with trypsin abolished the HR-eliciting activity of 19-2. This proteinase cleaves P64 and P68 as shown in Figure 7. Further digestion with endoproteinase Glu-C in ammonium bicarbonate buffer abolished the HR-eliciting activity of 19-1. Endoproteinase Glu-C is predicted to cleave P91 and P95 as shown in Figure 7. Loss of elicitor-activity followed further digestion of these peptides with endoproteinase Glu-C or trypsin.
Example 13 - E. amylovora harpin's similarity with other proteins.
The predicted protein sequences of proteinaceous HR elicitors from several other bacterial plant pathogens, and of other proteins known to be, or thought to be, secreted by a type III secretion pathway were also compared with that of haφinEa. When haφinEa was compared with elicitors from E. amylovora Ea246 (i.e. haφinEar), Erwinia chrysanthemi EC 16 (haφinEcι (Bauer, et al, "Erwinia chrysanthemi haφinEcι-,: An Elicitor of the Hypersensitive Response That Contributes to Soft-Rot Pathogenesis," Mol. Plant-Microbe Interact 8:484-491 (1995), which is hereby incoφorated by reference), Erwinia carotovora subsp. carotovora (haφinεcc) (Mukherjee, et al, Presented at the 8th International Congress Molecular Plant- Microbe Interactions, Knoxville, TN (1996), which is hereby incoφorated by reference), Erwinia stewartii (Haφinεs) (Frederick, et al, "The wts Water-Soaking Genes of Erwinia stewartii are Related to hrp genes," Presented at the Seventh International Symposium on Molecular Plant-Microbe Interactions, Edinburgh, Scotland (1994), which is hereby incoφorated by reference), Ralstonia (Pseudomonas) solanacearum (PopA) (Arlat, et al, "PopAl, a Protein Which Induces a Hypersensitivity-Like Response on Specific Petunia Genotypes, is Secreted via the Hφ Pathway of Pseudomonas solanacearum," EMBO J. 13:543-553 (1994), which is hereby incoφorated by reference), Pseudomonas syringae 61 (haφinpss) (He, et al, "Pseudomonas syringae pv. syringae haφinpss: a Protein That is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants," Cell 73:1255-1266 (1993), which is hereby incorporated by reference), Pseudomonas syringae pv. tomato (haφinpst) (Preston, et al, "The HrpZ Proteins of Pseudomonas syringae pvs. syringae, glycinea, and tomato Are Encoded By An Operon Containing Yersiniaysc Homologs and Elicit the Hypersensitive Response in Tomato But Not Soybean," Mol Plant-Microbe Interact 8:717-732 (1995), which is hereby incoφorated by reference), the Erwinia-derw' ed haφins contained significant regions of similarity at the C-terminus. In addition, all the elicitors are glycine-rich, secreted, and heat-stable. Limited similarity between haφinpss and haφinπa had been reported previously (He, et al, "Pseudomonas syringae pv. syringae haφinpss: a Protein That is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants," Cell 73:1255-1266 (1993), which is hereby incoφorated by reference), (Laby, et al, Presented at the Seventh International Symposium on Molecular Plant-Microbe Interactions, Edinburgh, Scotland (1994), which is hereby incoφorated by reference). A limited region of similarity between haφinEa and other haφins frm Erwinia spp. was also evident at the extreme N-terminus of each protein, where 9 out of the first 26 residues are conserved (Figure 8). Kyte-Doolittle hydropathy plots for each of the haφins produced by different Erwinia spp. are illustrated in Figure 9. Each of the Erwinia haφins examined displays a generally similar hydrophobicity profile along the full length of the protein. This profile is distinct from the profile demonstrated by PopAl and by haφinpss, and does not possess the symmetry evident in the profile of those two proteins. The hydropathy profile of each Erwinia harpin is generally similar to that of the others, yet distinct from that reported for harpinpss (Alfano, et al, "Analysis of the Role of the Pseudomonas Syringae HrpZ harpin in Elicitation of the Hypersensitive Response to Tobacco Using Functionally Nonpolar hrpZ Deletion Mutations, Truncated HφZ Fragments, and hrmA Mutations," Mol. Microbiol
19:715-728 (1996), which is hereby incorporated by reference). HaφinECC possesses a strikingly hydrophobic region around residues 54-143 (Mukherjee, et al, Presented at the 8th International Congress Molecular Plant-Microbe Interactions, Knoxville, TN (1996), which is hereby incorporated by reference). This portion of the protein is also the most hydrophobic region of haφinEa and haφinEs- The rest of each protein is predominantly hydrophilic.
Truncated proteins and fragments of harpin obtained following proteolytic digestion of the full length protein indicate several suφrising aspects of haφinEa HR- eliciting activity. These harpin fragments demonstrate that HR-eliciting activity resides in distinct regions of the protein, and that relatively small fragments of the protein, as little as 68 residues and possibly less, are sufficient for this activity. Fragments of other plant pathogen-derived elicitor proteins also retain biological activity, including Avr9 from Caldosporium fulvum (Van den Ackervecken, et al, "The AVR9 Race-Specific Elicitor of Cladosporium fulvum is Processed by Endogenous and Plant Proteases," PI Physiol 103:91-96 (1993), which is hereby incoφorated by reference), Pep-13 of Phytophthora megasperma (Nϋrnburger, et al, "High Affinity Binding of a Fungal Oligopeptide Elicitor to the Parsley Plasma
Membranes Triggers Multiple Defence Responses," Cell, 78:449-460 (1994), which is hereby incoφorated by reference), and haφinpss of P. syringae pv. syringae (Alfano, et al, "Analysis of the Role of the Pseudomonas syringae HrpZ haφin in Elicitation of the Hypersensitive Response in Tobacco Using Functionally Nonpolar hrpZ Deletion Mutations, Truncated HφZ Fragments, and hrmA Mutations," Mol Microbiol 19:715-728 (1996), which is hereby incoφorated by reference).
Expression of truncated haφin fragments and proteolysis of full-length haφins showed that two distinct fragments retain HR-eliciting activity. The primary sequence of each active fragment show no discernable similarity with each other, or with other elicitor-active peptides. However, the sites of cleavage by trypsin and endoproteinase Glu-C suggest portions of each fragment required for activity. It would be of interest to alter specifically the amino acid residues at or near these cleavage sites to determine whether HR-eliciting activity is altered or lost. Additionally, haφh Ea P64 and P68 demonstrate distinct hydrophobicity during reverse-phase HPLC (Figure 6), and they correspond to a hydrophobic peak in a Kyte- Doolittle plot (Figure 9). The role of this putative hydrophobic domain could be tested by mutagenesis, or by synthesis of altered peptides. However, the fact that multiple fragments independently possess HR-eliciting activity complicates analysis of full-length proteins. This finding, that fragments of the protein retain HR-eliciting activity, also allowed (at least) two apoplastic proteinase activities, which are distinct from intracellular plant proteinases, to be defined. Two apoplastic plant proteinases (from soybean) have been studied in some detail. SMEP, a metalloproteinase (Huangpu, et al, "Purification and Developmental Analysis of an Extracellular Proteinase From Young Leaves of Soybean," Plant Phvsiol 108:969-974 (1995); McGeehan, et al, "Sequencing and Characterization of the Soybean Leaf Metalloproteinase," Plant Phvsiol. 99:1179-1183 (1992), which are hereby incoφorated by reference) sensitive to EDTA, is thought to cleave at G/L and G/I. Interestingly, although there are 19 potential SMEP cleavage sites in the intact h φinEa, only one of them is located within fragments P91 and P95, and none are within fragments P64 and P68 (Figure 7). P91 and P95 thus may represent partial digestion products of a SMEP-like proteinase in the tobacco apoplast. The other studies soybean apoplastic proteinase, SLAP, a sulfhydryl proteinase (Huangpu, et al, "Purification and Developmental Analysis of an Extracellular Proteinase From Young Leaves of Soybean," Plant Phvsiol 108:969- 974 (1995), which is hereby incorporated by reference) sensitive to p- chloromercuribenzoic acid (pCMB). Several lines of evidence suggest that multiple proteolytic activities in the IF are degrading haφinEa- PMSF, a serine protease inhibitor, decreases but does not entirely block haφinEa degradation (Figure 5C); no single proteinase inhibitor tested blocks haφin degradation, and the cleavage sites after residues 109, 136, 200, and 204 are dissimilar. Endoproteinase Glu-C does not abolish activity of full-length haφin, but does abolish activity of P91 and P95 (and presumably P64 and P68); trypsin abolishes the activity of P64 and P68 (and presumably P91 and P95). These final digests suggest specific portions of each distinct HR-eliciting peptide which are presumably necessary for its activity, as mentioned previously.
The apoplastic activities degrade haφin without destroying its HR-eliciting ability, in contrast to intracellular proteolytic activities present in a ground leaf- extract, which abolish activity. This raises a number of intriguing questions, e.g., does the plant use these haφin fragments as elicitor-signals? The timing of the HR was examined when full length harpin and haφin predigested by tobacco intercellular fluid were each infiltrated into tobacco leaves. The HR elicited by each preparation occurred 12-18 hours after infiltration. Co-infiltration of proteinase inhibitors into tobacco leaf panels along with haφin also had no effect on haφin' s HR eliciting activity, although limited proteolytic degradation cannot be ruled out in this case, particularly since it seems that at least two, and perhaps more, apoplastic proteinases are present in tobacco. Because predigested harpin elicited the HR no faster than undigested protein, proteolytic digestion seems unlikely to be a rate-limiting step required for HR to occur. The role of these apoplastic proteinases which are able to hydro lyze haφin partially, yet unable to abolish haφin' s HR-eliciting activity on tobacco, remains unclear. Salzer et al, "Rapid Reactions of Spruce Cells to Elicitors Released From the Ectomycorrhizal Fungus Hebeloma crustuliniforme and Inactivation of These Elicitors by Extracellular Spruce Cell Enzymes," Planta 198:118-126 (1996), which is hereby incoφorated by reference, have noted that spruce (Picea abies (L.) Karst.) modulates the level of fungal cell wall elicitors released by the ectomycorrhizal fungus Hebeloma crustuliniforme by inactivating these molecules in the apoplast. They propose that Picea controls the elicitor level as part of its symbiotic interaction with the fungus. Similarly, PGIP of Phaseolus vulgaris has been suggested to modulate the level of elicitor-active oligogalacturonides present during the plant-parasite interaction in bean (Desiderio, et al, "Polygalacturonase, PGIP, and Oligogalacturonides in Cell-Cell Communication," Biochem. Sci. Trans. 22:394-397 (1994), which is hereby incoφorated by reference). Perhaps the retention of HR-eliciting activity by haφin fragments contributes to the ability of plants to recognize the presence of a pathogen. In this regard, it would be interesting to explore whether transgenic host and non-host plants, engineered for apoplastic expression of a haφin activity-degrading proteinase, would exhibit reduced or increased sensitivity to E. amylovora, compared to non-engineered plants.
Despite numerous attempts, only a handful of truncated derivatives of harpinEa and haφinεar were successfully expressed from portions of hrpN. Problems with protein stability were evident in that several truncated haφins were unstable and difficult to purify. Additionally, expression of truncated harpins may be deleterious to bacteria. Truncated haφinEaC31 was, however, stable and easily purified, but not secreted, suggesting that C-terminal sequences are involved in haφin secretion. Unfortunately, the presence of vector-encoded amino acids in this protein complicates this conclusion. All attempts to clone β-galactosidase-haφin fusion proteins have been unsuccessful, as were attempts to clone and express hrpN downstream of the lacL promoter in several commonly used vectors such as pBluescript. Expression of such constructs is evidently deleterious to bacterial strains.
Wei, et al, "Haφin, Elicitor of the Hypersensitive Response Produced By the Plant Pathogen Erwinia amylovora," Science. 257:85-88 (1992), which is hereby incoφorated by reference, reported previously that BLAST searches indicated haφinεa possessed slight similarity to several other glycine-rich proteins, including keratins and glycine-rich cell wall proteins. However, this is thought to be due to the high glycine content of haφinEa, and as such does not suggest a role for haφinEa- Examination of N-terminal sequences from several HR-eliciting proteins produced by phytopathogenic bacteria (Figure 8) yielded some potential similarities. However, the region in question is quite short. The region of putative primary sequence similarity is limited to the first 26 residues at the N-terminus, and its role remains unclear. Suφrisingly, E. carotovora haφinEcc appears more similar to the haφins from E. amylovora and E. stewartii than to that from E. chrysanthemi, to which it is more closely related with respect to its taxonomic position as well as its mechanism of pathogenicity (i.e. soft-rots). In addition, although primary sequence similarity is strongest only in the C-terminal third of each protein, the Erwinia haφins possess broadly similar hydrophobicity profiles along their entire lengths (Figure 9). Based on its hydrophobicity profile, Alfano, et al, "Analysis of the Role of the Pseudomonas syringae HrpZ haφin in Elicitation of the Hypersensitive Response in Tobacco Using Functionally Nonpolar hrpZ Deletion Mutations, Truncated HφZ Fragments, and hrmA Mutations," Mol Microbiol 19:715-728 (1996), which is hereby incoφorated by reference, speculated that haφinPss may have an amphiphilic nature. However, the Erwinia haφins' profiles do not match that of haφinpss.
Recently, a number of other secreted glycine-rich pathogenicity associated proteins, elicitors of the HR or other plant-defense responses, have been described from other plant pathogenic bacteria and fungi (Boiler, "Chemoperception of Microbial Signals in Plant Cells," Ann. Rev. Plant Phvsiol Plant Molec. Biol 46:189- 214 (1996), which is hereby incoφorated by reference), including Phytophthora megasperma (Ballieul, et al, "A New Elicitor of the Hypersensitive Response in Tobacco: a Fungal Glycoprotein Elicits Cell Death, Expression of Defence Genes, Production of Salicylic Acid, and Induction of Systemic Acquired Resistance," Plant Journal 8:551-560 (1995); Nϋrnburger, et al, "High Affinity Binding of a Fungal Oligopeptide Elicitor to the Parsley Plasma Membranes Triggers Multiple Defence Responses," Cell 78:449-460 (1994), which are hereby incoφorated by reference), and Magnaporthe grisea (Sweigard, et al, "Identification, Cloning, and
Characterization of PWL2, a Gene For Host Species Specificity in the Rice Blast Fungus," Plant Cell 7:1221-1233 (1995), which is hereby incoφorated by reference). Proteinaceous HR-elicitors have also now been described from Phynchosporium secalis (Rohe, et al, "The Race-Specific Elicitor, NIP1, From the Barley Pathogen, Rhynchosporium secalis, Determines Avirulence on Host Plants of the Rrsl Resistance Genotype," EMBO Journal 14:4168-4177 (1995) which is hereby incoφorated by reference, while P. infestans (Pieterse, et al, "Structure and Genomic Organization of the ipϊB and ipiO Gene Clusters of Phytophthora infestans," Gene, 138:67-77 (1994), which is hereby incoφorated by reference) produces a glycine-rich pathogenicity-associated family of proteins of unknown function. Because the primary amino acid sequence of each elicitor protein or peptide fragment shows no obvious similarity to that of the others, it is unclear whether they interact with the same target on or in the plant cell, plasma membrane, or cell wall. In that regard, it might be of interest to test whether any one of these molecules inhibits the action of other(s). The increasing availability of peptides such as Pepl3, Avr9, P68, and haφinEa C305 with plant-defense response-eliciting activity (HR and otherwise) should enable precise probing of their targets on or in plant cells, as well as determination of whether their mechanisms of activity are similar, distinct, or overlapping.
Example 14 - Bacterial strains and plasmids
Escherichia coli stains used in the following examples include DH5α and BL21(DE3) purchased from Gibco BRL and Stratagene, respectively. The pET28(b) vector was purchased from Novagen. Eco DH5α/2139 contained the complete hrpN gene. The 2139 construct was produced by D. Bauer at the Cornell University. The hφN gene was cleaved from the 2139 plasmid by restriction enzyme digestion with Hindlll, then purified from an agarose gel to serve as the DNA template for PCR synthesis of truncated hφN clones. These clones were subsequently inserted into the (His)6 vector pET28(b) which contained a Kanr gene for selection of transformants.
Example 15 - DNA Manipulation
Restriction enzymes were obtained from Boehringer Mannheim or Gibco BRL. T4 DNA ligase, Calf Intestinal Alkaline Phosphatase (CIAP), and PCR Supermix™ were obtained from Gibco BRL. The QIAprep Spin Miniprep Kit, the Qiagen Plasmid Mini Kit, and the QIAquick PCR Purification Kit were purchased from Qiagen. The PCR primers were synthesized by Lofstrand Labs Limited (Gaithersburg, MD). The oligopeptides were synthesized by Bio-Synthesis, Inc. (Lewisville, TX). All DNA manipulations such as plasmid isolation, restriction enzyme digestion, DNA ligation, and PCR were performed according to standard techniques (molecular cloning) or protocols provided by the manufacturer.
Example 16 - Fragmentation of hrpN gene
A series of N-terminal and C-terminal truncated hrpN genes and internal fragments were generated via PCR (Fig. 10). The full length hφN gene was used as the DNA template and 3' and 5' primers were designed for each truncated clone (Fig. 11). The 3' primers contained in the Ndel enzyme cutting site which contained the start codon ATG (Methionine) and the 5' primers contained the stop codon TAA and a Hindlll enzyme cutting site for ligation into the pET28(b) vector. PCR was carried out in 0.5 ml tubes in a GeneAmp™ 9600 or 9700. 45 μl of Supermix™ were mixed with 20 pmoles of each pair of DNA primers, 10 ng of full length haφin DNA, and diH O to a final volume of 50 μl. After heating the mixture at 95 °C for 2 min, the PCR was performed for 30 cycles at 94°C for 1 min, 58°C for 1 min and 72°C for 1.5 min. The PCR products were verified on a 6% TBE gel (Novex). Amplified DNA was purified with the QIAquick PCR purification kit, digested with Nde I and Hind III at 37°C for 5 hours, extracted once with phenol:chloroform:isoamylalcohol (25:25:1) and precipitated with ethanol. 5 μg of pET28(b) vector DNA were digested with 15 units of Nde I and 20 units of Hind III at 37°C for 3 hours followed with CIAP treatment to reduce the background resulting from incomplete single enzyme digestion. Digested vector DNA was purified with the QIAquick PCR purification kit and directly used for ligation. Ligation was carried out at 14-16°C for 5-12 hours in a 15 μl mixture containing ca. 200 ng of digested pET28(b), 30 ng of targeted PCR fragment, and 1 unit T4 DNA ligase. 5 - 7.5 μl of ligation solution were added to 100 μl of DH5α competent cells in a 15 ml falcon tube and incubated on ice for 30 min. After a heat shock at 42°C for 45 seconds, 0.9 ml SOC solution or 0.45 ml LB media were added to each tube and incubated at 37°C for 1 hour. 20, 100, and 200 μl of transformed cells were placed onto LB agar with 30 μg/ml of kanamycin and incubated at 37°C overnight. Single colonies were transferred to 3 ml LB-media and incubated overnight at 37°C. Plasmid DNA was prepared from 2 ml of culture with the QIAprep Miniprep kit. The DNA from the transformed cells was analyzed by restriction enzyme digestion or partial sequencing to verify the success of the transformations. Plasmids with the desired DNA sequence were transferred into the BL21 strain using the standard chemical transformation method as indicated above. A clone containing the full length haφin protein in the pet28(b) vector was generated as a positive control, and a clone with only the pET28(b) vector was generated as a negative control.
Example 17 - Expression of harpin truncated proteins Escherichia coli BL21(DE3) strains containing the hφN clones were grown in
Luria broth medium (g/L Difco Yeast extract, 10 g/L Difco Tryptone, 5g/L NaCI, and 1 mM NaOH) containing 30 μg/ml of kanamycin at 37°C overnight. The bacteria were then inoculated into 100 volumes of the same medium and grown at 37°C to an OD6 0 of 0.6-0.8. The bacteria were then inoculated into 250 volumes of the same medium and grown at 37°C to an OD620 of ca. 0.3 or 0.6-0.8. One milli molar IPTG was then added and the cultures grown at 19°C overnight (ca. 18 hours). Not all of the clones were successfully expressed using this strategy. Several of the clones had to be grown in Terrific broth (12 g/L Bacto Tryptone, 24 g/L Bacto yeast, 0.4% glycerol, 0.17 M KH2PO4, and 0.72 K2HPO4), and/or grown at 37°C after IPTG induction, and/or harvested earlier than overnight (Table 2).
Table 2: Expression of haφin truncated proteins
Figure imgf000050_0001
General expression method: Escherichia coli BL21(DE3) strains containing the hφN subclones were grown in Luria broth medium (5g/L Difco Yeast extract, 10 g/L Difco Tryptone, 5g/L NaCI, and 1 mM NaOH) containing 30 μg/ml of kanamycin at 37°C overnight. The bacteria were then inoculated into 100 volumes of the same medium and grown at 37°C to an OD620 of 0.6-0.8. The bacteria were then inoculated into 250 volumes of the growth medium and grown at 37°C to a specific induction OD62o- One milli molar IPTG was then added and the cultures grown at an optimal temperature for protein expression, and harvested at a particular time for recovery of the highest level of protein.
Example 18 - Small scale purification of harpin truncated proteins (verification of expression)
A 50 ml culture of a hφN clone was grown as above to induce expression of the truncated protein. Upon harvesting of the culture, 1.5 ml of the cell suspension were centrifuged at 14,000 φm for 5 minutes, re-suspended in urea lysis buffer (8 M urea, 0J M Na2HPO4, and 0.01 M Tris ~ pH 8.0), incubated at room temperature for 10 minutes, then centrifuged again at 14,000 φm for 10 minutes, and the supernatant saved. A 50 μl aliquot of a 50%) slurry of an equilibrated (His)6-binding nickel agarose resin was added to the supernatant and mixed at 4°C for one hour. The nickel agarose was then washed three times with urea washing buffer (8 M urea, 0J M Na2HPO4, and 0.01 M Tris - pH 6.3), centrifuging at 5,000 φm for five minutes between washings. The protein was eluted from the resin with 50 μl of urea elution buffer (8 M urea, 0.1 M Na2HPO4, 0.01 M Tris, and 0.1 M EDTA - pH 6.3). The eluate was run on a 4-20%), a 16%>, or a 10-20%> Tris-Glycine pre-cast gel depending upon the size of the truncated protein to verify the expression.
Example 19 - Induction of HR in tobacco
A 1.5 ml aliquot from the 50 ml cultures grown for small scale purification of the truncated proteins was centrifuged at 14,000 φm for four minutes and re-suspended in an equal volume of 5 mM potassium phosphate buffer, pH 6.8. The cell suspension was sonicated for ca. 30 seconds then diluted 1 :2 and 1 :10 with phosphate buffer. Both dilutions plus the neat cell lysate were infiltrated into the fourth to ninth leaves of 10-15 leaf tobacco plants by making a hole in single leaf panes and infiltrating the bacterial lysate into the intercellular leaf space using a syringe without a needle. The HR response was recorded 24-48 hr post infiltration. Tobacco (Nicotiana tabacum v. Xanthi) seedlings were grown in an environmental chamber at 20-25°C with a photoperiod of 12-h light /l 2-h dark and ca. 40% RH.
Cell lysate was used for the initial HR assays (in order to screen the truncated proteins for HR activity) as the small scale urea purification yielded very little protein which was denatured due to the purification process.
Example 20 - Large scale native purification of harpin truncated proteins for comprehensive biological activity assays
Six 500 ml cultures of a hφN clone were grown as described earlier to induce expression of the truncated protein. Upon harvesting of the culture the cells were centrifuged at 7,000 φm for 5 minutes, re-suspended in imidazole lysis buffer (5 mM imidazole, 0.5 M NaCI, 20 mM Tris) plus Triton X-100 at 0.05% and lysozyme at 0.1 mg/ml, and incubated at 30°C for 15 minutes, sonicated for two minutes, then centrifuged again at 15,000 φm for 20 minutes, and the supernatant was saved. A 4 ml aliquot of a 50%> slurry of an equilibrated (His)6-binding nickel agarose resin was added to the supernatant and mixed at 4°C for ca. four hours. The nickel agarose was then washed three times with imidazole washing buffer (20 mM imidazole, 0.5 M NaCI, and 20 mM Tris), centrifuging at 5,000 φm for five minutes between washings, then placed in a disposable chromatography column. The column was centrifuged at 1100 φm for one minute to remove any residual wash buffer and then the protein was eluted from the resin with 4 ml of imidazole elution buffer (1 M imidazole, 0.5 M NaCI, and 20 mM Tris) by incubating the column with the elution buffer for ten minutes at room temperature and then centrifuging the column at 1100 φm for one minute. The eluate was run on a 4-20%>, a 16%), or a 10-20%> Tris-Glycine pre-cast gel depending upon the size of the truncated protein to verify the expression. The concentration of the proteins was determined by comparison of the protein bands with a standard protein in the Mark 12 molecular weight marker.
Example 21 - Large scale urea purification of harpin truncated proteins for comprehensive biological activity assay
The procedure was the same as the large scale native purification except that urea lysis buffer, washing buffer, and elution buffer were used, and the cells were not sonicated as in the native purification. After purification, the protein was renatured by dialyzing against lower and lower concentrations of urea over an eight hour period, then dialyzing overnight against 10 mM Tris/20 mM NaCI The renaturing process caused the N-terminal proteins to precipitate. The precipitated 1-168 protein was solubilized by the addition of 100 mM Tris-HCl at pH 10.4 then heating the protein at 30°C for ca. one hour. The concentration of the protein was determined by comparison of the protein bands with a standard protein in the Mark 12 molecular weight marker. The 1-75 and 1-104 protein fragments were not successfully solubilized using this strategy so they were sonicated in 100 mM Tris-HCl at pH 10.4 to solubilize as much of the protein as possible and expose the active sites of the protein for the biological activity assays. Example 22 - Expression of harpin truncated proteins
The small scale expression and purification of the fragment proteins was done to screen for expression and HR activity (Table 3).
Table 3 Expression and HR activity of haφin truncated proteins (small scale screening)
Figure imgf000053_0001
All of the cloned fragment proteins were expressed to a certain degree except for three small fragments (amino acids 169-209, 150-209, and 150-180). The fragments were expressed at varying levels. Fragments 210-403 and 267-403 were expressed very well, yielding a high concentration of protein from a small scale purification, resulting in a substantial protein band on SDS gel electrophoresis. Other fragments (such as a.a. 1-168 and 1-104) produced much less protein, resulting in faint protein bands upon electrophoresis. It was difficult to determine whether fragment 343-403, the smallest C-terminal protein, was expressed, as there were several background proteins apparent on the gel, in addition to the suspected 343-403 protein. The positive and negative control proteins, consisting of the full length harpin protein and only background proteins, respectively, were tested for expression and HR activity as well. The large scale expression and purification of the fragment proteins was done to determine the level of expression and titer of the HR activity (Table 4).
Table 4 Expression level and HR titer of haφin truncated proteins (large sale purification)
Figure imgf000054_0001
Not all of the proteins were expressed in large scale due to time constraints. The truncated proteins deemed to be the most important in characterizing haφin were chosen. The positive control (full length haφin) was expressed in a relatively high level at 3.7 mg/ml. All of the C-terminal proteins were expressed at relatively high levels from 2-5 mg/ml, except for fragment 343-403 as discussed earlier. The N-terminal fragments were expressed very well also, however, during the purification process, the protein precipitated and very little was resolubilized. The concentrations in Table 3 reflect only the solubilized protein. The internal fragments were expressed in the range of 2-3.6 mg/ml. It was extremely difficult to determine the concentration of fragment 105-168 (it was suspected that the concentration was much higher than indicated), as the protein bands on the SDS gel were large, but poorly stained. The negative control contained several background proteins as expected, but no obviously induced dominant protein. Example 23 - Induction of HR in tobacco
The full length positive control protein elicited HR down to only 5-7μg/ml. The negative control (pET 28) imidazole purified "protein" - which contained only background proteins - elicited an HR response down to the 1 :2 dilution, which lowered the sensitivity of the assay as the 1 :1 and 1 :2 dilutions could not be used. This false HR was likely due an affinity of the imidazole used in the purification process to bind to one or several of the background proteins, thereby not completely dialyzing out. Imidazole at a concentration of ca. 60 mM did elicit a false HR response.
One definitive domain encompassed a small internal region of the protein from a.a. 137-180 (SEQ. ID. No. 23), a mere 44 a.a, is identified as the smallest HR domain. The other potential HR domain is thought to be located in the N-terminus of the protein from a.a. 1-104 (possibly a.a. 1-75) (SEQ. ID. No. 23). It was difficult to confirm or narrow down the N-terminus HR domain due to the difficulties encountered in purifying these fragment proteins. The N-terminus fragment proteins had to be purified with urea as no protein was recovered when the native purification process was used. Consequently, these proteins precipitated during the renaturing process and were difficult or nearly impossible to get back into solution, thereby making it hard to run the proteins through the HR assay, as only soluble protein is able to elicit HR. Difficulty narrowing the N-terminus HR domain was only compounded by the fact that the negative control elicited false HR at the low dilution levels thereby reducing the sensitivity of the assay.
The internal domain proteins elicited an HR response between 5 and 10 μg/ml of protein like the positive control, and the N-terminus domain proteins elicited an HR response between 1 and 3 μg/ml, lower than the positive control.
Suφrisingly, when the internal HR domain was cleaved between a.a. 168 and 169 (fragments 76-168 and 105-168) (SEQ. ID. No. 23) the fragment lost its HR activity. This suggests that the HR activity of fragment 1-168 (SEQ. ID. No. 23) should not be attributed to the internal HR domain, but rather to some other domain, leading to the assumption that there was likely a second HR domain to be found in the N-terminal region of the protein. However, as discussed earlier it was difficult to confirm this assumption. The haφin C-terminus (a.a. 210-403 (SEQ. ID. No. 23)) did not contain an HR domain. It did not elicit HR at a detectable level using the current HR assay. Even the large C-terminal fragment from a.a. 169-403 (SEQ. ID. No. 23) did not elicit HR even though it contained part of the internal HR domain. As stated above, the protein between a.a. 168 and 169 (SEQ. ID. No. 23) causes a loss of HR activity. Because some of the small cloned proteins with 61 a.a. or less were not expressed, several oligopeptides were synthesized with 30 a.a. to narrow down the functional region of the internal HR domain. The oligopeptides were synthesized within the range of a.a. 121-179 (SEQ. ID. No. 23). However, these oligos did not elicit the HR response. It was not expected that there would be an HR response from oligos 137-166, 121-150, and 137-156 (SEQ. ID. No. 23) as these fragments did not contain the imperative amino acids 168 and 169 (SEQ. ID. No. 23). It was expected that the oligo 150-179 (SEQ. ID. No. 23) would elicit an HR response. It is possible that 30 a.a. is too small for the protein to elicit any activity due to a lack of folding and, therefore, a lack of binding or that during the synthesis of the peptides important amino acids were missed (either in the process, or simply by the choice of which 30 amino acids to synthesize) and, therefore, the fragments would not be able to elicit HR. It is also possible, although unlikely, that these small proteins would have undergone some form of post-translational modification within the E.coli cell that they did not contain when synthesized and, therefore, were not able to elicit an HR response.
Example 24 - Biological Activity of HR Inducing Fragments The two N-terminal haφin fragments spanning nucleotides 1-104 and nucleotides 1-168 of the nucleic acid of SEQ. ID. No. 24 were effective at inducing resistance of tobacco against TMV, in a similar manner as the full length haφin protein. The internal fragments spanning nucleotides 76-209 and nucleotides 105-209 of the nucleic acid of SEQ. ID. No. 24 were also effective at inducing TMV resistance. In addition, these same four fragments conferred plant growth enhancement ("PGE") in tomato increasing the height of the plants from 4-19%> taller than the buffer control plants. The full length haφin protein induced growth enhancement of 6% greater than the buffer. The negative control did not induce TMV resistance or growth enhancement.
Table 5 TMV resistance and PGE activity of HR inducing fragments derived from haφin
Figure imgf000057_0001
Although the invention has been described in detail for the puφose of illustration, it is understood that such detail is solely for that puφose, 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.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Cornell Research Foundation, Inc. and EDEN Bioscience Corporation
(ii) TITLE OF INVENTION: HYPERSENSITIVE RESPONSE ELICITOR
FRAGMENTS ELICITING A HYPERSENSITIVE RESPONSE AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 30
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Nixon, Hargrave, Devans & Doyle LLP
(B) STREET: Clinton Square, P.O. Box 1051
(C) CITY: Rochester
(D) STATE: New York
(E) COUNTRY: U.S.A.
(F) ZIP: 14603
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/048,109
(B) FILING DATE: 30-MAY-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Goldman, Michael L.
(B) REGISTRATION NUMBER: 30,727
(C) REFERENCE/DOCKET NUMBER: 19603/1302
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (716) 263-1304
(B) TELEFAX: (716) 263-1600
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GGGAATTCAT ATGAGTCTGA ATACAAGTGG G 31
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : GGGAATTCAT ATGGGCGGTG GCTTAGGCGG T 31
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE : . cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : GGCATATGTC GAACGCGCTG AACGATATG 29
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
GGGAATTCAT ATGTTAGGCG GTTCGCTGAA C . 31
(2) INFORMATION FOR SEQ ID NO : 5 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : GGCATATGCT GAACACGCTG GGCTCGAAA 29
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : GGCATATGTC AACGTCCCAA AACGACGAT 29
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE : • cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : GGCATATGTC CACCTCAGAC TCCAGCG 27
(2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : GGGAATTCAT ATGCAAAGCC TGTTTGGTGA TGGG 34
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : GGGAATTCAT ATGGGTAATG GTCTGAGCAA G 31
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGGAATTCAT ATGAAAGCGG GCATTCAGGC G 31
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY : ' linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GGGAATTCAT ATGACACCAG CCAGTATGGA GCAG 34 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE : • CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12 : GCAAGCTTAA CAGCCCACCA CCGCCCATCA T 31
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GCAAGCTTAA ATCGTTCAGC GCGTTCGACA G 31
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCAAGCTTAA TATCTCGCTG AACATCTTCA GCAG 34
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GCAAGCTTAA GGTGCCATCT TGCCCATCAC 30
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 'linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GCAAGCTTAA ATCAGTGACT CCTTTTTTAT AGGC 34
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GCAAGCTTAA CAGGCCCGAC AGCGCATCAG T 31
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GCAAGCTTAA ACCGATACCG GTACCCACGG C 31 (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GCAAGCTTAA TCCGTCGTCA TCTGGCTTGC TCAG 34
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GCAAGCTTAA GCCGCGCCCA GCTTG 25
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 338 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Met Gin lie Thr lie Lys Ala His lie Gly Gly Asp Leu Gly Val Ser 1 5 10 15
Gly Leu Gly Ala Gin Gly Leu Lys Gly Leu Asn Ser Ala Ala Ser Ser 20 25 30
Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Thr lie Asp Lys Leu Thr 35 40 45 Ser Ala Leu Thr Ser Met Met Phe Gly Gly Ala Leu Ala Gin Gly Leu 50 55 60
Gly Ala Ser Ser Lys Gly Leu Gly Met Ser Asn Gin Leu Gly Gin Ser 65 70 75 80
Phe Gly Asn Gly Ala Gin Gly Ala Ser Asn Leu Leu Ser Val Pro Lys 85 90 95
Ser Gly Gly Asp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu Asp Asp 100 105 110
Leu Leu Gly His Asp Thr Val Thr Lys Leu Thr Asn Gin Ser Asn Gin 115 120 125
Leu Ala Asn Ser Met Leu Asn Ala Ser Gin Met Thr Gin Gly Asn Met 130 135 140
Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser Ser lie Leu Gly 145 150 155 160
Asn Gly Leu Gly Gin Ser Met Ser Gly Phe Ser Gin Pro Ser Leu Gly 165 170 175
Ala Gly Gly Leu Gin Gly Leu Ser Gly Ala Gly Ala Phe Asn Gin Leu 180 185 190
Gly Asn Ala lie Gly Met Gly Val Gly Gin Asn Ala Ala Leu Ser Ala 195 . 200 205
Leu Ser Asn Val Ser Thr His Val Asp Gly Asn Asn Arg His Phe Val 210 215 220
Asp Lys Glu Asp Arg Gly Met Ala Lys Glu lie Gly Gin Phe Met Asp 225 230 235 240
Gin Tyr Pro Glu lie Phe Gly Lys Pro Glu Tyr Gin Lys Asp Gly Trp 245 250 255
Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser Lys 260 265 270
Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met Asp Lys Phe Arg Gin 275 280 285
Ala Met Gly Met lie Lys Ser Ala Val Ala Gly Asp Thr Gly Asn Thr 290 295 300
Asn Leu Asn Leu Arg Gly Ala Gly Gly Ala Ser Leu Gly lie Asp Ala 305 310 315 320
Ala Val Val Gly Asp Lys lie Ala Asn Met Ser Leu Gly Lys Leu Ala 325 330 335
Asn Ala (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2141 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
CGATTTTACC CGGGTGAACG TGCTATGACC GACAGCATCA CGGTATTCGA CACCGTTACG 60
GCGTTTATGG CCGCGATGAA CCGGCATCAG GCGGCGCGCT GGTCGCCGCA ATCCGGCGTC 120
GATCTGGTAT TTCAGTTTGG GGACACCGGG CGTGAACTCA TGATGCAGAT TCAGCCGGGG 180
CAGCAATATC CCGGCATGTT GCGCACGCTG CTCGCTCGTC GTTATCAGCA GGCGGCAGAG 240
TGCGATGGCT GCCATCTGTG CCTGAACGGC AGCGATGTAT TGATCCTCTG GTGGCCGCTG 300
CCGTCGGATC CCGGCAGTTA TCCGCAGGTG ATCGAACGTT TGTTTGAACT GGCGGGAATG 360
ACGTTGCCGT CGCTATCCAT AGCACCGACG GCGCGTCCGC AGACAGGGAA CGGACGCGCC 420
CGATCATTAA GATAAAGGCG GCTTTTTTTA TTGCAAAACG GTAACGGTGA GGAACCGTTT 480
CACCGTCGGC GTCACTCAGT AACAAGTATC CATCATGATG CCTACATCGG GATCGGCGTG 540
GGCATCCGTT GCAGATACTT TTGCGAACAC CTGACATGAA TGAGGAAACG AAATTATGCA 600
AATTACGATC AAAGCGCACA TCGGCGGTGA TTTGGGCGTC TCCGGTCTGG GGCTGGGTGC 660
TCAGGGACTG AAAGGACTGA ATTCCGCGGC TTCATCGCTG GGTTCCAGCG TGGATAAACT 720
GAGCAGCACC ATCGATAAGT TGACCTCCGC GCTGACTTCG ATGATGTTTG GCGGCGCGCT 780
GGCGCAGGGG CTGGGCGCCA GCTCGAAGGG GCTGGGGATG AGCAATCAAC TGGGCCAGTC 840
TTTCGGCAAT GGCGCGCAGG GTGCGAGCAA CCTGCTATCC GTACCGAAAT CCGGCGGCGA 900
TGCGTTGTCA AAAATGTTTG ATAAAGCGCT GGACGATCTG CTGGGTCATG ACACCGTGAC 960
CAAGCTGACT AACCAGAGCA ACCAACTGGC TAATTCAATG CTGAACGCCA GCCAGATGAC 1020
CCAGGGTAAT ATGAATGCGT TCGGCAGCGG TGTGAACAAC GCACTGTCGT CCATTCTCGG 1080
CAACGGTCTC GGCCAGTCGA TGAGTGGCTT CTCTCAGCCT TCTCTGGGGG CAGGCGGCTT 1140
GCAGGGCCTG AGCGGCGCGG GTGCATTCAA CCAGTTGGGT AATGCCATCG GCATGGGCGT 1200
GGGGCAGAAT GCTGCGCTGA GTGCGTTGAG TAACGTCAGC ACCCACGTAG ACGGTAACAA 1260 CCGCCACTTT GTAGATAAAG AAGATCGCGG CATGGCGAAA GAGATCGGCC AGTTTATGGA 1320
TCAGTATCCG GAAATATTCG GTAAACCGGA ATACCAGAAA GATGGCTGGA GTTCGCCGAA 1380
GACGGACGAC AAATCCTGGG CTAAAGCGCT GAGTAAACCG GATGATGACG GTATGACCGG 1440
CGCCAGCATG GACAAATTCC GTCAGGCGAT GGGTATGATC AAAAGCGCGG TGGCGGGTGA 1500
TACCGGCAAT ACCAACCTGA ACCTGCGTGG CGCGGGCGGT GCATCGCTGG GTATCGATGC 1560
GGCTGTCGTC GGCGATAAAA TAGCCAACAT GTCGCTGGGT AAGCTGGCCA ACGCCTGATA 1620
ATCTGTGCTG GCCTGATAAA GCGGAAACGA AAAAAGAGAC GGGGAAGCCT GTCTCTTTTC 1680
TTATTATGCG GTTTATGCGG TTACCTGGAC CGGTTAATCA TCGTCATCGA TCTGGTACAA 1740
ACGCACATTT TCCCGTTCAT TCGCGTCGTT ACGCGCCACA ATCGCGATGG CATCTTCCTC 1800
GTCGCTCAGA TTGCGCGGCT GATGGGGAAC GCCGGGTGGA ATATAGAGAA ACTCGCCGGC 1860
CAGATGGAGA CACGTCTGCG ATAAATCTGT GCCGTAACGT GTTTCTATCC GCCCCTTTAG 1920
CAGATAGATT GCGGTTTCGT AATCAACATG GTAATGCGGT TCCGCCTGTG CGCCGGCCGG 1980
GATCACCACA ATATTCATAG AAAGCTGTCT TGCACCTACC GTATCGCGGG AGATACCGAC 2040
AAAATAGGGC AGTTTTTGCG TGGTATCCGT GGGGTGTTCC GGCCTGACAA TCTTGAGTTG 2100
GTTCGTCATC ATCTTTCTCC ATCTGGGCGA CCTGATCGGT T 2141
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 403 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gin lie Ser 1 5 10 15 lie Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg Gin 20 25 30
Asn Ala Gly Leu Gly Gly Asn Ser Ala Leu Gly Leu Gly Gly Gly Asn 35 40 45 Gln Asn Asp Thr Val Asn Gin Leu Ala Gly Leu Leu Thr Gly Met Met 50 55 60
Met Met Met Ser Met Met Gly Gly Gly Gly Leu Met Gly Gly Gly Leu 65 70 75 80
Gly Gly Gly Leu Gly Asn Gly Leu Gly Gly Ser Gly Gly Leu Gly Glu 85 90 95
Gly Leu Ser Asn Ala Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr 100 105 110
Leu Gly Ser Lys Gly Gly Asn Asn Thr Thr Ser Thr Thr Asn Ser Pro 115 120 125
Leu Asp Gin Ala Leu Gly lie Asn Ser Thr Ser Gin Asn Asp Asp Ser 130 135 140
Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Ser Asp Pro Met Gin Gin 145 150 155 160
Leu Leu Lys Met Phe Ser Glu lie Met Gin Ser Leu Phe Gly Asp Gly 165 170 175
Gin Asp Gly Thr Gin Gly Ser Ser Ser Gly Gly Lys Gin Pro Thr Glu 180 185 190
Gly Glu Gin Asn Ala Tyr Lys Lys Gly Val Thr Asp Ala Leu Ser Gly 195 200 205
Leu Met Gly Asn Gly Leu Ser Gin Leu Leu Gly Asn Gly Gly Leu Gly 210 215 220
Gly Gly Gin Gly Gly Asn Ala Gly Thr Gly Leu Asp Gly Ser Ser Leu 225 230 235 240
Gly Gly Lys Gly Leu Gin Asn Leu Ser Gly Pro Val Asp Tyr Gin Gin 245 250 255
Leu Gly Asn Ala Val Gly Thr Gly lie Gly Met Lys Ala Gly He Gin 260 265 270
Ala Leu Asn Asp He Gly Thr His Arg His Ser Ser Thr Arg Ser Phe 275 280 285
Val Asn Lys Gly Asp Arg Ala Met Ala Lys Glu He Gly Gin Phe Met 290 295 300
Asp Gin Tyr Pro Glu Val Phe Gly Lys Pro Gin Tyr Gin Lys Gly Pro 305 310 315 320
Gly Gin Glu Val Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser 325 330 335 Lys Pro Asp Asp Asp Gly Met Thr Pro Ala Ser Met Glu Gin Phe Asn 340 345 350
Lys Ala Lys Gly Met He Lys Arg Pro Met Ala Gly Asp Thr Gly Asn 355 360 365
Gly Asn Leu Gin Ala Arg Gly Ala Gly Gly Ser Ser Leu Gly He Asp 370 375 380
Ala Met Met Ala Gly Asp Ala He Asn Asn Met Ala Leu Gly Lys Leu 385 • 390 395 400
Gly Ala Ala
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1288 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
AAGCTTCGGC ATGGCACGTT TGACCGTTGG GTCGGCAGGG TACGTTTGAA TTATTCATAA 60
GAGGAATACG TTATGAGTCT GAATACAAGT GGGCTGGGAG CGTCAACGAT GCAAATTTCT 120
ATCGGCGGTG CGGGCGGAAA TAACGGGTTG CTGGGTACCA GTCGCCAGAA TGCTGGGTTG 180
GGTGGCAATT CTGCACTGGG GCTGGGCGGC GGTAATCAAA ATGATACCGT CAATCAGCTG 240
GCTGGCTTAC TCACCGGCAT GATGATGATG ATGAGCATGA TGGGCGGTGG TGGGCTGATG 300
GGCGGTGGCT TAGGCGGTGG CTTAGGTAAT GGCTTGGGTG GCTCAGGTGG CCTGGGCGAA 360
GGACTGTCGA ACGCGCTGAA CGATATGTTA GGCGGTTCGC TGAACACGCT GGGCTCGAAA 420
GGCGGCAACA ATACCACTTC AACAACAAAT TCCCCGCTGG ACCAGGCGCT GGGTATTAAC 480
TCAACGTCCC AAAACGACGA TTCCACCTCC GGCACAGATT CCACCTCAGA CTCCAGCGAC 540
CCGATGCAGC AGCTGCTGAA GATGTTCAGC GAGATAATGC AAAGCCTGTT TGGTGATGGG 600
CAAGATGGCA CCCAGGGCAG TTCCTCTGGG GGCAAGCAGC CGACCGAAGG CGAGCAGAAC 660
GCCTATAAAA AAGGAGTCAC TGATGCGCTG TCGGGCCTGA TGGGTAATGG TCTGAGCCAG 720
CTCCTTGGCA ACGGGGGACT GGGAGGTGGT CAGGGCGGTA ATGCTGGCAC GGGTCTTGAC 780 GGTTCGTCGC TGGGCGGCAA AGGGCTGCAA AACCTGAGCG GGCCGGTGGA CTACCAGCAG 840
TTAGGTAACG CCGTGGGTAC CGGTATCGGT ATGAAAGCGG GCATTCAGGC GCTGAATGAT 900
ATCGGTACGC ACAGGCACAG TTCAACCCGT TCTTTCGTCA ATAAAGGCGA TCGGGCGATG 960
GCGAAGGAAA TCGGTCAGTT CATGGACCAG TATCCTGAGG TGTTTGGCAA GCCGCAGTAC 1020
CAGAAAGGCC CGGGTCAGGA GGTGAAAACC GATGACAAAT CATGGGCAAA AGCACTGAGC 1080
AAGCCAGATG ACGACGGAAT GACACCAGCC AGTATGGAGC AGTTCAACAA AGCCAAGGGC 1140
ATGATCAAAA GGCCCATGGC GGGTGATACC GGCAACGGCA ACCTGCAGGC ACGCGGTGCC 1200
GGTGGTTCTT CGCTGGGTAT TGATGCCATG ATGGCCGGTG ATGCCATTAA CAATATGGCA 1260
CTTGGCAAGC TGGGCGCGGC TTAAGCTT 1288
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 341 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Met Gin Ser Leu Ser Leu Asn Ser Ser Ser Leu Gin Thr Pro Ala Met 1 5 10 15
Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser 20 25 30
Ser Lys Ala Leu Gin Glu Val Val Val Lys Leu Ala Glu Glu Leu Met 35 40 45
Arg Asn Gly Gin Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu Ala 50 55 60
Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly He Glu Asp Val 65 70 75 80
He Ala Ala Leu Asp Lys Leu He His Glu Lys Leu Gly Asp Asn Phe 85 90 95
Gly Ala Ser Ala Asp Ser Ala Ser Gly Thr Gly Gin Gin Asp Leu Met 100 105 110
Thr Gin Val Leu Asn Gly Leu Ala Lys Ser Met Leu Asp Asp Leu Leu 115 120 125 Thr Lys Gin Asp Gly Gly Thr Ser Phe Ser Glu Asp Asp Met Pro Met 130 135 140
Leu Asn Lys He Ala Gin Phe Met Asp Asp Asn Pro Ala Gin Phe Pro 145 150 155 160
Lys Pro Asp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn Phe 165 170 175
Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp He He 180 185 190
Gly Gin Gin Leu Gly Asn Gin Gin Ser Asp Ala Gly Ser Leu Ala Gly 195 200 205
Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn Asn Ser Ser 210 215 220
Val Met Gly Asp Pro Leu He Asp Ala Asn Thr Gly Pro Gly Asp Ser 225 230 235 240
Gly Asn Thr Arg Gly Glu Ala Gly Gin Leu He Gly Glu Leu He Asp 245 250 255
Arg Gly Leu Gin Ser Val Leu Ala Gly Gly Gly Leu Gly Thr Pro Val 260 265 270
Asn Thr Pro Gin Thr Gly Thr Ser Ala Asn Gly Gly Gin Ser Ala Gin 275 280 285
Asp Leu Asp Gin Leu Leu Gly Gly Leu Leu Leu Lys Gly Leu Glu Ala 290 295 300
Thr Leu Lys Asp Ala Gly Gin Thr Gly Thr Asp Val Gin Ser Ser Ala 305 310 315 320
Ala Gin He Ala Thr Leu Leu Val Ser Thr Leu Leu Gin Gly Thr Arg 325 330 335
Asn Gin Ala Ala Ala 340
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1026 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
ATGCAGAGTC TCAGTCTTAA CAGCAGCTCG CTGCAAACCC CGGCAATGGC CCTTGTCCTG 60
GTACGTCCTG AAGCCGAGAC GACTGGCAGT ACGTCGAGCA AGGCGCTTCA GGAAGTTGTC 120
GTGAAGCTGG CCGAGGAACT GATGCGCAAT GGTCAACTCG ACGACAGCTC GCCATTGGGA 180
AAACTGTTGG CCAAGTCGAT GGCCGCAGAT GGCAAGGCGG GCGGCGGTAT TGAGGATGTC 240
ATCGCTGCGC TGGACAAGCT GATCCATGAA AAGCTCGGTG ACAACTTCGG CGCGTCTGCG 300
GACAGCGCCT CGGGTACCGG ACAGCAGGAC CTGATGACTC AGGTGCTCAA TGGCCTGGCC 360
AAGTCGATGC TCGATGATCT TCTGACCAAG CAGGATGGCG GGACAAGCTT CTCCGAAGAC 420
GATATGCCGA TGCTGAACAA GATCGCGCAG TTCATGGATG ACAATCCCGC ACAGTTTCCC 480
AAGCCGGACT CGGGCTCCTG GGTGAACGAA CTCAAGGAAG ACAACTTCCT TGATGGCGAC 540
GAAACGGCTG CGTTCCGTTC GGCACTCGAC ATCATTGGCC AGCAACTGGG TAATCAGCAG 600
AGTGACGCTG GCAGTCTGGC AGGGACGGGT GGAGGTCTGG GCACTCCGAG CAGTTTTTCC 660
AACAACTCGT CCGTGATGGG TGATCCGCTG ATCGACGCCA ATACCGGTCC CGGTGACAGC 720
GGCAATACCC GTGGTGAAGC GGGGCAACTG ATCGGCGAGC TTATCGACCG TGGCCTGCAA 780
TCGGTATTGG CCGGTGGTGG ACTGGGCACA CCCGTAAACA CCCCGCAGAC CGGTACGTCG 840
GCGAATGGCG GACAGTCCGC TCAGGATCTT GATCAGTTGC TGGGCGGCTT GCTGCTCAAG 900
GGCCTGGAGG CAACGCTCAA GGATGCCGGG CAAACAGGCA CCGACGTGCA GTCGAGCGCT 960
GCGCAAATCG CCACCTTGCT GGTCAGTACG CTGCTGCAAG GCACCCGCAA TCAGGCTGCA 1020 GCCTGA 1026
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 344 amino acids
(B) TYPE: amino acid
( C ) STRANDEDNESS :
(D) TOPOLOGY : linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Met Ser Val Gly Asn He Gin Ser Pro Ser Asn Leu Pro Gly Leu Gin 1 5 10 15 Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gin Gin Ser Gly Gin Ser 20 25 30
Val Gin Asp Leu He Lys Gin Val Glu Lys Asp He Leu Asn He He 35 40 45
Ala Ala Leu Val Gin Lys Ala Ala Gin Ser Ala Gly Gly Asn Thr Gly 50 55 60
Asn Thr Gly Asn Ala Pro Ala ,Lys Asp Gly Asn Ala Asn Ala Gly Ala 65 70 75 80
Asn Asp Pro Ser Lys Asn Asp Pro Ser Lys Ser Gin Ala Pro Gin Ser 85 90 95
Ala Asn Lys Thr Gly Asn Val Asp Asp Ala Asn Asn Gin Asp Pro Met 100 105 110
Gin Ala Leu Met Gin Leu Leu Glu Asp Leu Val Lys Leu Leu Lys Ala 115 120 125
Ala Leu His Met Gin Gin Pro Gly Gly Asn Asp Lys Gly Asn Gly Val 130 135 140
Gly Gly Ala Asn Gly Ala Lys Gly Ala Gly Gly Gin Gly Gly Leu Ala 145 150 155 160
Glu Ala Leu Gin Glu He Glu Gin He Leu Ala Gin Leu Gly Gly Gly 165 170 175
Gly Ala Gly Ala Gly Gly Ala Gly Gly Gly Val Gly Gly Ala Gly Gly 180 185 190
Ala Asp Gly Gly Ser Gly Ala Gly Gly Ala Gly Gly Ala Asn Gly Ala 195 200 205
Asp Gly Gly Asn Gly Val Asn Gly Asn Gin Ala Asn Gly Pro Gin Asn 210 215 220
Ala Gly Asp Val Asn Gly Ala Asn Gly Ala Asp Asp Gly Ser Glu Asp 225 230 235 240
Gin Gly Gly Leu Thr Gly Val Leu Gin Lys Leu Met Lys He Leu Asn 245 250 255
Ala Leu Val Gin Met Met Gin Gin Gly Gly Leu Gly Gly Gly Asn Gin 260 265 270
Ala Gin Gly Gly Ser Lys Gly Ala Gly Asn Ala Ser Pro Ala Ser Gly 275 280 285
Ala Asn Pro Gly Ala Asn Gin Pro Gly Ser Ala Asp Asp Gin Ser Ser 290 295 300 Gly Gin Asn Asn Leu Gin Ser Gin He Met Asp Val Val Lys Glu Val 305 310 315 320
Val Gin He Leu Gin Gin Met Leu Ala Ala Gin Asn Gly Gly Ser Gin 325 330 335
Gin Ser Thr Ser Thr Gin Pro Met 340
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1035 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
ATGTCAGTCG GAAACATCCA GAGCCCGTCG AACCTCCCGG GTCTGCAGAA CCTGAACCTC 60
AACACCAACA CCAACAGCCA GCAATCGGGC CAGTCCGTGC AAGACCTGAT CAAGCAGGTC 120
GAGAAGGACA TCCTCAACAT CATCGCAGCC CTCGTGCAGA AGGCCGCACA GTCGGCGGGC 180
GGCAACACCG GTAACACCGG CAACGCGCCG GCGAAGGACG GCAATGCCAA CGCGGGCGCC 240
AACGACCCGA GCAAGAACGA CCCGAGCAAG AGCCAGGCTC CGCAGTCGGC CAACAAGACC 300
GGCAACGTCG ACGACGCCAA CAACCAGGAT CCGATGCAAG CGCTGATGCA GCTGCTGGAA 360
GACCTGGTGA AGCTGCTGAA GGCGGCCCTG CACATGCAGC AGCCCGGCGG CAATGACAAG 420
GGCAACGGCG TGGGCGGTGC CAACGGCGCC AAGGGTGCCG GCGGCCAGGG CGGCCTGGCC 480
GAAGCGCTGC AGGAGATCGA GCAGATCCTC GCCCAGCTCG GCGGCGGCGG TGCTGGCGCC 540
GGCGGCGCGG GTGGCGGTGT CGGCGGTGCT GGTGGCGCGG ATGGCGGCTC CGGTGCGGGT 600
GGCGCAGGCG GTGCGAACGG CGCCGACGGC GGCAATGGCG TGAACGGCAA CCAGGCGAAC 660
GGCCCGCAGA ACGCAGGCGA TGTCAACGGT GCCAACGGCG CGGATGACGG CAGCGAAGAC 720
CAGGGCGGCC TCACCGGCGT GCTGCAAAAG CTGATGAAGA TCCTGAACGC GCTGGTGCAG 780
ATGATGCAGC AAGGCGGCCT CGGCGGCGGC AACCAGGCGC AGGGCGGCTC GAAGGGTGCC 840
GGCAACGCCT CGCCGGCTTC CGGCGCGAAC CCGGGCGCGA ACCAGCCCGG TTCGGCGGAT 900
GATCAATCGT CCGGCCAGAA CAATCTGCAA TCCCAGATCA TGGATGTGGT GAAGGAGGTC 960 GTCCAGATCC TGCAGCAGAT GCTGGCGGCG CAGAACGGCG GCAGCCAGCA GTCCACCTCG 1020 ACGCAGCCGA TGTAA 1035
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Thr Leu He Glu Leu Met He Val Val Ala He He Ala He Leu Ala 1 5 10 15
Ala He Ala Leu Pro Ala Tyr Gin Asp Tyr 20 25
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Ser Ser Gin Gin Ser Pro Ser Ala Gly Ser Glu Gin Gin Leu Asp Gin 1 5 10 15
Leu Leu Ala Met 20

Claims

WHAT IS CLAIMED:
1. An isolated fragment of an Erwinia hypersensitive response elicitor" protein or polypeptide, wherein said fragment elicits a hypersensitive response in plants.
2. An isolated fragment according to claim 1, wherein the hypersensitive response elicitor protein or polypeptide is derived from an Erwinia amylovora, Erwinia carotovora, Erwinia chrysanthemi, or Erwinia stewartii.
3. An isolated fragment according to claim 2, wherein the hypersensitive response elicitor protein or polypeptide is derived from Erwinia amylovora.
4. An isolated fragment according to claim 3, wherein the fragment is selected from the group consisting of a C-terminal fragment of the amino acid sequence of SEQ. ID. No. 23, an N-terminal fragment of the amino acid sequence of SEQ. ID. No. 23, and an internal fragment of the amino acid sequence of SEQ. ID.
No. 23.
5. An isolated fragment according to claim 4, wherein the fragment is a
C-terminal fragment of the amino acid sequence of SEQ. ID. No. 23 spanning amino acids 105 and 403 of SEQ. ID. No. 23.
6. An isolated fragment according to claim 4, wherein the fragment is an N-terminal fragment of the amino acid sequence of SEQ. ID. No. 23 spanning the following amino acids of SEQ. ID. No. 23: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1 and 372.
7. An isolated fragment according to claim 4, wherein the fragment is an internal fragment of the amino acid sequence of SEQ. ID. No. 23 spanning the following amino acids of SEQ. ID. No. 23: 76 and 209, 105 and 209, 99 and 209, 137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180, and 105 and 180.
8. An isolated DNA molecule encoding a fragment according to claim 1.
9. An isolated DNA molecule according to claim 8, wherein the hypersensitive response elicitor protein or polypeptide is derived from an Erwinia amylovora, Erwinia carotovora, Erwinia chrysanthemi, or Erwinia stewartii.
10. An isolated DNA molecule according to claim 9, wherein the hypersensitive response elicitor protein or polypeptide is derived from Erwinia amylovora.
11. An isolated isolated DNA molecule according to claim 10, wherein the fragment is selected from the group consisting of a C-terminal fragment of the amino acid sequence of SEQ. ID. No. 23, an N-terminal fragment of the amino acid sequence of SEQ. ID. No. 23, and an internal fragment of the amino acid sequence of SEQ. ID. No. 23.
12. An isolated DNA molecule according to claim 10, wherein the fragment is a C-terminal fragment of the amino acid sequence of SEQ. ID. No. 23 spanning amino acids 105 and 403 of SEQ. ID. No. 23.
13. An isolated DNA molecule according to claim 10, wherein the fragment is an N-terminal fragment of the amino acid sequence of SEQ. ID. No. 23 spanning the following amino acids of SEQ. ID. No. 23: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1 and 372.
14. An isolated DNA molecule according to claim 10, wherein the fragment is an internal fragment of the amino acid sequence of SEQ. ID. No. 23 spanning the following amino acids of SEQ. ID. No. 23: 76 and 209, 105 and 209, 99 and 209, 137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180, and 105 and 180.
15. An expression system transformed with a DNA molecule according to claim 8.
16. An expression system according to claim 15, wherein said DNA molecule is in proper sense orientation and correct reading frame.
17. A host cell transformed with a DNA molecule according to claim 8.
18. A host cell according to claim 17, wherein the host cell is selected from the group consisting of a plant cell and a bacterial cell
19. A host cell according to claim 17, wherein the DNA molecule is transformed with an expression system.
20. A transgenic plant transformed with the DNA molecule of claim 8.
21. A transgenic plant according to claim 20, wherein the plant is selected from the group consisting of alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.
22. A transgenic plant according to claim 20, wherein the plant is selected from the group consisting of Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
23. A transgenic plant seed transformed with the DNA molecule of claim 8.
24. A transgenic plant seed according to claim 23, wherein the plant seed is selected from the group consisting of alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.
25. A transgenic plant seed according to claim 23, wherein the plant seed is selected from the group consisting of Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
26. A method of imparting disease resistance to plants comprising: applying a fragment of a hypersensitive response elicitor protein or polypeptide, which fragment elicits a hypersensitive response, in a non-infectious form to a plant or plant seed under conditions impart disease resistance.
27. A method according to claim 26, wherein plants are treated during said applying.
28. A method according to claim 26, wherein plant seeds are treated during said applying, said method further comprising: planting the seeds treated with the fragment of the hypersensitive response elicitor in natural or artificial soil and propagating plants from the seeds planted in the soil.
29. A method of enhancing plant growth comprising: applying a fragment of a hypersensitive response elicitor protein or polypeptide, which fragment elicits a hypersensitive response, in a non-infectious form to a plant or plant seed under conditions effective to enhance plant growth.
30. A method according to claim 29, wherein plants are treated during said applying.
31. A method according to claim 29, wherein plant seeds are treated during said applying, said method further comprising: planting the seeds treated with the fragment of the hypersensitive response elicitor in natural or artificial soil and propagating plants from the seeds planted in the soil.
32. A method of insect control for plants comprising: applying a fragment of a hypersensitive response elicitor protein or polypeptide, which fragment elicits a hypersensitive response, in a non-infectious form to a plant or plant seed under conditions effective to control insects.
33. A method according to claim 32, wherein plants are treated during said applying.
34. A method according to claim 32, wherein plant seeds are treated during said applying, said method further comprising: planting the seeds treated with the fragment of the hypersensitive response elicitor in natural or artificial soil and propagating plants from the seeds planted in the soil.
35. A method of imparting disease resistance to plants comprising: providing a transgenic plant or plant seed transformed with a DNA molecule which encodes a fragment of a hypersensitive response elicitor protein or polypeptide, which fragment elicits a hypersensitive response, and growing the transgenic plant or transgenic plants produced from the transgenic plant seeds under conditions effective to impart disease resistance.
36. A method according to claim 35, wherein a transgenic plant is provided.
37. A method according to claim 35, wherein a transgenic plant seed is provided.
38. A method of enhancing plant growth comprising : providing a transgenic plant or a plant seed transformed with a DNA molecule which encodes a fragment of a hypersensitive response elicitor protein or polypeptide, which fragment elicits a hypersensitive response, and growing the transgenic plant or transgenic plants produced from the transgenic plant seeds under conditions effective to enhance plant growth.
39. A method according to claim 38, wherein a transgenic plant is provided.
40. A method according to claim 38, wherein a transgenic plant seed is provided.
41. A method of insect control for plants comprising: providing a transgenic plant or plant seed transformed with a DNA molecule which encodes a fragment of a hypersensitive response elicitor protein or polypeptide, which fragment elicits a hypersensitive response, and growing the transgenic plant or transgenic plants produced from the transgenic plant seeds under conditions effective to control insects.
42. A method according to claim 41 , wherein a transgenic plant is provided.
43. A method according to claim 41 , wherein a transgenic plant seed is provided.
PCT/US1998/010874 1997-05-30 1998-05-28 Hypersensitive response elicitor fragments and uses thereof WO1998054214A2 (en)

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EP98924950A EP0996729A2 (en) 1997-05-30 1998-05-28 Hypersensitive response elicitor fragments and uses thereof
AU77004/98A AU750732B2 (en) 1997-05-30 1998-05-28 Hypersensitive response elicitor fragments and uses thereof
JP50090299A JP2002501388A (en) 1997-05-30 1998-05-28 Hypersensitive response inducer fragment and its use
NZ501138A NZ501138A (en) 1997-05-30 1998-05-28 Hypersensitive response elicitor protein or polypeptide fragments from Erwinia
CA002289905A CA2289905A1 (en) 1997-05-30 1998-05-28 Hypersensitive response elicitor fragments and uses thereof
BR9809699-0A BR9809699A (en) 1997-05-30 1998-05-28 Isolated fragment of a protein or polypeptide eliciting hypersensitive response to erwinia, isolated DNA molecule, expression system, host cell, transgenic plant, transgenic plant seed, and, processes to confer disease resistance and intensify plant growth and to control plant growth insects for plants
PL98337094A PL337094A1 (en) 1997-05-30 1998-05-28 Fragments causing a hypersensitive reaction and their applications
KR19997011216A KR20010013226A (en) 1997-05-30 1998-05-28 Hypersensitive response elicitor fragments and uses thereof
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AU7700498A (en) 1998-12-30
WO1998054214A9 (en) 1999-04-01
US7132525B2 (en) 2006-11-07
NZ501138A (en) 2002-11-26
JP2002501388A (en) 2002-01-15
PL337094A1 (en) 2000-07-31
BR9809699A (en) 2000-07-11
US20010011380A1 (en) 2001-08-02
EP0996729A2 (en) 2000-05-03
US6583107B2 (en) 2003-06-24
US20030182683A1 (en) 2003-09-25
WO1998054214A3 (en) 1999-03-04
KR20010013226A (en) 2001-02-26
AU750732B2 (en) 2002-07-25
CA2289905A1 (en) 1998-12-03
FI19992545A (en) 2000-01-28
CN1265145A (en) 2000-08-30

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