Classifications

• • 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
  • 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
• • 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
  • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
• • 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
• • 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 active fragments of a hypersensitive response elicitor which fragments do not elicit a hypersensitive response.
• 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: Phytopatho enie Prokaryotes. 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 Determinants Among Plant and Animal Pathogenic Bacteria," Trends Microbiol. 1 :175-180 (1993)).
• E. amylovora, P. syringae, and P. sol ⁇ n ⁇ ce ⁇ rum 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., "Hrpl of Erwinia amylovora Functions in Secretion of Harpin and is a Member of aNew Protein Family," J. Bacteriol. 175:7958-7967 (1993); Arlat, M. et al.
• 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)).
• 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)).
• 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 do not elicit a hypersensitive response but are active when utilized in conjunction with plants.
• the present invention is directed to isolated fragments of an Erwinia hypersensitive response elicitor protein or polypeptide which fragments do not elicit a hypersensitive response in plants but are otherwise active when utilized in conjunction with plants. Also disclosed are isolated DNA molecules which encode such fragments.
• the fragments of hypersensitive response elicitors according to the present invention have the following activity when utilized in conjunction with plants: imparting disease resistance to plants, enhancing plant growth and/or controlling 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 truncated proteins of the hypersensitive response elicitor protein or polypeptide.
• Figure 2 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.
• 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
• C403 SEQ. ID. No. 20
• the present invention is directed to isolated fragments of a hypersensitive response elicitor protein or polypeptide where the fragments do not elicit a hypersensitive response but have other activity 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 Xanthomonas 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 par asitica, 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
• 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 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 CCATTCGG 1080 CAACGGTCTC GGCCAGTCGA TGAGTGGCTT CTCTCAGCCT TCTCTGGGGG CAGGCGGCTT 1140 GCAGGGCCTG AGCG
• 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 pl 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
• CTGAAAAACG TCACCATGGG CGACGACGGG GCGGATGGTA TTCATCTTTA CGGTGATGCC 960
• the isolated DNA molecule of the present invention encodes a hypersensitive response elicitor protein or polypeptide having an amino acid sequence of SEQ. ID. No. 26 as follows:
• This protein or polypeptide is acidic, rich in glycine and serine, and lacks cysteine. It is also heat stable, protease sensitive, and suppressed by inhibitors of plant metabolism.
• the protein or polypeptide of the present invention has a predicted molecular size of ca. 4.5 kDa.
• Erwinia amylovora is disclosed in U.S. Patent Application Serial No. 09/120,663 which is hereby incorporated by reference.
• the protein is encoded by a DNA molecule having a nucleic acid sequence of SEQ. ID. No. 27 as follows: ATGGAATTAA AATCACTGGG AACTGAACAC AAGGCGGCAG TACACACAGC GGCGCACAAC 60
• AAAATGGCTC ACCCGGCTTC AGCCAACGCC GGCGATCGCC TGCAGCATTC ACCGCCGCAC 600
• GGCAGTAAAC CAAATGGTGT CACTGCCCGT GTTTCTGCCG GGCTAAGTGC ATCGGCAAAC 4440
• GCCAGCAATA ACCGCCCAAC CTTCCTCAAC GGGGTCGGCG CGGGTGCTAA CCTGACGGCT 4560
• This DNA molecule is known as the dspE gene for Erwinia amylovora.
• This isolated DNA molecule of the present invention encodes a protein or polypeptide which elicits a plant pathogen's hypersensitive response having an amino acid sequence of SEQ. ID. No. 28 as follows: Met Glu Leu Lys Ser Leu Gly Thr Glu His Lys Ala Ala Val His Thr 1 5 10 15
• Asp Arg Val Glu lie Ala Gin Glu Asp Asp Asp Ser Glu Phe Gin Gin 225 230 235 240
• Ser Gly Lys lie Ser Leu Gly Ser Gly Thr Gin Ser His Asn Lys Thr 405 410 415
• Val Asp Gin Arg Gly Gin Val Ala lie Leu Thr Asp Thr Pro Gly Arg 515 520 525 His Lys Met Ser lie Met Pro Ser Leu Asp Ala Ser Pro Glu Ser His 530 535 540 lie Ser Leu Ser Leu His Phe Ala Asp Ala His Gin Gly Leu Leu His 545 550 555 560
• Trp Asn Leu Thr Asp Ala Leu Val lie Asp Asn Gin Leu Gly Leu His 660 665 670
• This protein or polypeptide is about 198 kDa and has a pl of 8.98.
• the present invention relates to an isolated DNA molecule having a nucleotide sequence of SEQ. ID. No. 29 as follows:
• This isolated DNA molecule of the present invention encodes a hypersensitive response elicitor protein or polypeptide having an amino acid sequence of SEQ. ID. No. 30 as follows:
• This protein or polypeptide is about 16 kDa and has a pi of 4.45.
• the hypersensitive response elicitor polypeptide or protein derived from Pseudomonas syringae has an amino acid sequence corresponding to SEQ. ID. No. 31 as follows:
• 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 Harpin Pss : 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. 32 as follows:
• ATGCAGAGTC TCAGTCTTAA CAGCAGCTCG CTGCAAACCC CGGCAATGGC CCTTGTCCTG 60
• Pseudomonas syringae is disclosed in U.S. Patent Application Serial No. 09/120,817, which is hereby incorporated by reference.
• the protein has a nucleotide sequence of SEQ. ID. No. 33 as follows:
• This DNA molecule is known as the dspE gene for Pseudomonas syringae.
• This isolated DNA molecule of the present invention encodes a protein or polypeptide which elicits a plant pathogen's hypersensitive response having an amino acid sequence of SEQ. ID. No. 34 as follows:
• This protein or polypeptide is about 42.9 kDa.
• the hypersensitive response elicitor polypeptide or protein derived from Pseudomonas solanacearum has an amino acid sequence corresponding to SEQ. ID. No. 35 as follows:
• the hypersensitive response elicitor polypeptide or protein from Xanthomonas campestris pv. glycines has an amino acid sequence corresponding to SEQ. ID. No. 37 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 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. 38 as follows:
• 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. Phytopath. Soc. July 27-31, 1996, which are hereby incorporated by reference.
• 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(l):15-25 (1993), Ricci et al., "Structure and Activity of Proteins from Pathogenic Fungi Phytophthora Eliciting Necrosis and
• elicitors are exemplary.
• Other elicitors can be identified by growing fungi or bacteria that elicit a hypersensitive response under conditions 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 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. 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.
• 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).
• Suitable fragments of a hypersensitive response elicitor which do not elicit a hypersensitive response include 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 the following amino acids of SEQ. ID. No. 23: 169 and 403, 210 and 403, 267 and 403, or 343 and 403.
• the internal fragment of the amino acid sequence of SEQ. ID. No. 23 can span the following amino acids of SEQ. ID. No. 23: 105 and 179, 137 and 166, 121 and 150, or 137 and 156.
• Other suitable fragments can be identified in accordance with the present invention.
• a useful fragment of a hypersensitive response elicitor which fragment does not itself elicit a hypersensitive response is the protein fragment containing amino acids 190 to 294 of the amino acid sequence (SEQ. ID. No. 31) for the Pseudomonas syringae pv. syringae hypersensitive response elicitor. This fragment is useful in imparting disease resistance and enhancing plant growth.
• a useful fragment of a hypersensitive response elicitor is the peptide having an amino acid sequence corresponding to SEQ. ID. No. 39. This peptide is derived from the hypersensitive response eliciting glycoprotein of Phytophthora megasperma and enhances plant growth.
• 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 in isolated form (i.e. separated from its host organism) and more 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. 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.
• Recombinant genes may also be introduced into viruses, such as vaccina virus.
• Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
• Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system 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 incorporated by reference), pQE, pIH821, pGEX, pET series (see F.W.
• 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.
• 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
• Promotors vary in their "strength" (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong 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 promotor, 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 lacOY5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments.
• a hybrid trp-lac ⁇ JY5 (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.
• the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside).
• IPTG 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. For instance, efficient translation in E. coli requires an SD sequence about 7-9 bases 5' to the initiation codon ("ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E.
• any SD- ATG combination produced by recombinant D ⁇ A or other techniques involving incorporation of synthetic nucleotides may be used.
• 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 does not elicit 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.
• 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 does not elicit 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 gene 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 does not elicit a hypersensitive response.
• bacteria must be capable of secreting or exporting the fragment so that the fragment can contact plant or plant seed 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.
• 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, 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.
• 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, inter alia, to the following bacteria can also be imparted to plants in accordance with present invention: Pseudomonas solanacearum, Pseudomonas syringae pv. tabaci, and Xanthamonas 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 army worm, cabbage looper, corn ear worm, fall army worm, 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 does not elicit 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, propagules (e.g., cuttings), etc. This may (but need not) involve infiltration of the fragment 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 does not elicit 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 4 ) 2 NO 3 .
• 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.
• a fragment of a hypersensitive response elicitor need not be applied topically to the plants or seeds.
• 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 incorporated 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.
• 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 incorporated 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 A. tumefaciens or the Ri plasmid of A. rhizogenes.
• the Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science. 237:1176-83 (1987), which is hereby incorporated by reference.
• 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 incorporated 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 fragment of the hypersensitive response elicitor 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 fragment of a hypersensitive response elicitor in accordance with the present invention is applied. These other materials, including a fragment of a hypersensitive response elicitor in accordance with the present invention, 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 fragment of a hypersensitive response elicitor in accordance with the present invention 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.
• Escherichia coli strains used in the following examples include DH5 ⁇ and BL21(DE3) purchased from Gibco BRL (Grand Island, NY.) and Stratagene (La Jolla, CA), respectively.
• the pET28(b) vector was purchased from Novagen (Madison, WI).
• Eco DH5 ⁇ /2139 contained the complete hrpN gene.
• the 2139 construct was produced by D. Bauer at Cornell University.
• the hrpN 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 hrpN 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 (Indianapolis, IN) 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 (Hilden, Germany).
• 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 (Sambrook, et al., Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)) or protocols provided by the manufacturer.
• Example 3 Fragmentation of hrpN Gene
• the full length hrpN gene was used as the DNA template and 3' and 5' primers were designed for each truncated clone (Fig. 2).
• the 3' primers contained an 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 GeneAmpTM 9700 (Perkin-Elmer, Foster City, CA).
• 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 ⁇ g 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 ⁇ l mixture containing ca.
• Plasmid DNA was prepared from 2 ml of culture with the QIAprep Miniprep kit (QIAGEN, Hilden, Germany). 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 harpin 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.
• Escherichia coli BL21(DE3) strains containing the hrpN clones were grown in Luria broth medium (5 g/L Difco Yeast extract, 10 g/L Difco Tryptone, 5 g/L NaCl, 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 6 20 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 6 2o 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.
• a 50 ml culture of a hrpN clone was grown as above to induce expression of the truncated protein.
• 1.5 ml of the cell suspension were centrifuged at 14,000 rpm for 5 minutes, re-suspended in urea lysis buffer (8 M urea, 0.1 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 rpm for 10 minutes, and the supernatant saved.
• Tobacco (Nicotiana tabacum v. Xanthi) seedlings were grown in an environmental chamber at 20-25°C with a photoperiod of 12-h light /12-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.
• the column was centrifuged at 1100 rpm 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 NaCl, 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.
• 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 NaCl. 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 were expressed at varying levels except for three small fragments (amino acids 169-209, 150-209, and 150-180). 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 hypersensitive response elicitor protein and only background proteins, respectively, were tested for expression and HR activity as well.
• the truncated proteins deemed to be the most important in characterizing the hypersensitive response elicitor were chosen for large scale expression.
• the positive control full length hypersensitive response elicitor
• 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.
• 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.
• 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 HR. It was not expected that there would be an HR 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.).
• the C-terminal fragments enhanced the growth of tomato by 9% to 21%.
• the N-terminal fragments enhanced the growth of tomato by 4% to 13%).
• the internal fragments enhanced growth by 9% to 20%>.
• the 76-209 fragment enhanced growth by 18% at a concentration of 60 ⁇ g/ml, but not at the typical 20 ⁇ g/ml. This was attributed to the inaccuracy of the quantification process (Table 4).
• the oligopeptides enhanced growth from 7.4% to 17.3% (Table 5).
• Example 14 Induction of Systemic Acquired Resistance (SAR) All of the hypersensitive response elicitor fragments tested to date appear to have 60% efficacy or greater, except for the oligopeptide 137-156 (Tables 5 and 6). Table 6
• the hypersensitive response activity is separable from the plant growth enhancement activity.
• the C-terminal fragments clearly enhance the growth of tomato by ca. 20% at a concentration of only 20 ⁇ g/ml, but these same fragments were not able to elicit HR in tobacco, even at higher concentrations than 200 ⁇ g/ml.
• the SAR activity also appears to be separable from the HR activity. This finding is highly significant for future work on transgenic applications of the hypersensitive response elicitor technology.
• the fragments that induce PGE and/or SAR but do not elicit HR will be imperative for this technology, as constitutive expression of even low levels of an HR elicitor might kill a plant.
• hrpZ The following segments of hrpZ, the gene encoding the hypersensitive response elicitor from Pseudomonas syringae pv. syringae, were amplified by PCR using Pfu Turbo (Stratagene): Regions coding for amino acids 152-190, aa 152-294, aa 190-294, aa 301-341, and full length HrpZ (aa 1-341). The DNA fragments were cloned into pCAL-n (Stratagene) to create C-terminal fusion proteins to the calmodulin-binding peptide. pCAL-n was chosen, because the fusion protein could be easily and gently purified on calmodulin resin.
• the DNA was transformed into E coli DH5oc, and the correct clones were identified. The clones were then transferred to E. coli BLR D ⁇ 3 for protein expression. The bacteria were grown in Terrific Broth to an OD 620 of 0.8-1.0. Protein expression was then induced with IPTG and the bacteria were incubated for an additional 3 h. All of the HrpZ fragments were able to be expressed this way. Amino acid fragments 152-294 and 190-294 were chosen for further analysis and characterization. It was expected that the fragment 152-294 contained a domain that elicited the HR, while fragment 190-294 contained no domain that elicited the HR.
• the cultures were spun down, and the bacteria resuspended in 40 ml of 10 mM Tris pH 8.0. Twenty ⁇ l of antifoam and 40 ⁇ l of 200 mM PMSF were added, and the bacteria was sonicated to break open the cells. The bacterial debris was removed by centrifugation, and the supernatant was placed in a boiling water bath for 10 min. The precipitate was removed by centrifugation and the supernatant, a crude protein preparation, was retained for tests.
• the fragment preparations were then tested for inducing resistance to TMV and for growth enhancement. Due to the difference in concentration of the HrpZ fragments, the 152-294 preparation was diluted 40-fold and the 190-294 preparation was diluted 8-fold. The results showed that the 190-294 aa fragment reduced the number of TMV lesions by 85% in comparison to buffer controls (Table 8). In contrast, the 152-294 aa fragment reduced the number of TMV lesions by only 55%. As also shown in Table 8, plants treated with the 152-294 aa fragment grew 4.64%o more than buffer treated plants, while plants treated with the 190-294 aa fragment grew 2.62% more than the buffer treated plants.
• Phytophthora megasperma Several years ago, a 42 kDa glycoprotein elicitor was purified from the fungal culture filtrate of Phytophthora megasperma (Parker et al., "An Extracellular Glycoprotein from Phytophthora megasperma f.sp. glycinea Elicits Phytoalexin Synthesis in Cultured Parsley Cells and Protoplasts," Mol. Plant Microbe Interact. 4:19-27 (1991), which is hereby incorporated by reference).
• an oligopeptide of 13 amino acid was identified within the 42 kDa glycoprotein.
• the 13 amino acids peptide appeared to have similar biological activity as that of the full length glycoprotein (42 kDa). It is sufficient to elicit a complex defense response in parsley cells including H+/Ca2+ influxes, K+/C1- effluxes, active oxygen production, SAR gene induction, and phytoalexin compound accumulation (Nurnberger et al., "High Affinity Binding of a Fungal Oligopeptide Elicitor to Parsley Plasma Membranes Triggers Multiple Defense Response," Cell 78:449-460 (1994), which is hereby incorporated by reference).
• the synthesized sequence of the peptide is NH2-Val-Trp-Asn- Gln-Pro-Val-Arg-Gly-Phe-Lys-Val-Tyr-Glu-COOH (SEQ. ID. No. 39).
• the synthesized peptide was resuspended in 10 ml of 5 mM potassium phosphate buffer and, then, diluted to 1 and 100 ng/ml with the same buffer. About 100 tomato seeds (variety, Marglobe) were submerged in 20 ml of peptide solution overnight. The soaked seeds were planted in an 8 inch pot with artificial soil. Seeds soaked in the buffer without the peptide were used as a control.

Landscapes

• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Molecular Biology (AREA)
• Engineering & Computer Science (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Gastroenterology & Hepatology (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Plant Pathology (AREA)
• Cell Biology (AREA)
• Virology (AREA)
• Insects & Arthropods (AREA)
• Pest Control & Pesticides (AREA)
• Peptides Or Proteins (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Pretreatment Of Seeds And Plants (AREA)
• Agricultural Chemicals And Associated Chemicals (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=22293092

Family Applications (1)

Country Status (15)

Families Citing this family (13)

Family Cites Families (2)

• 1999
  • 1999-10-05 WO PCT/US1999/023181 patent/WO2000020452A2/en not_active Application Discontinuation
  • 1999-10-05 TR TR2001/00967T patent/TR200100967T2/xx unknown
  • 1999-10-05 CA CA002344593A patent/CA2344593A1/en not_active Abandoned
  • 1999-10-05 KR KR1020017004332A patent/KR20010080011A/ko not_active Application Discontinuation
  • 1999-10-05 PL PL99348045A patent/PL348045A1/xx unknown
  • 1999-10-05 CN CN99814028A patent/CN1329619A/zh active Pending
  • 1999-10-05 IL IL14232499A patent/IL142324A0/xx unknown
  • 1999-10-05 HU HU0104245A patent/HUP0104245A3/hu unknown
  • 1999-10-05 AU AU65085/99A patent/AU6508599A/en not_active Abandoned
  • 1999-10-05 MX MXPA01003461A patent/MXPA01003461A/es unknown
  • 1999-10-05 BR BR9915345-9A patent/BR9915345A/pt not_active Application Discontinuation
  • 1999-10-05 JP JP2000574563A patent/JP2002526101A/ja active Pending
  • 1999-10-05 EP EP99953057A patent/EP1119582A2/en not_active Withdrawn
• 2001
  • 2001-03-28 ZA ZA200102536A patent/ZA200102536B/en unknown
  • 2001-04-05 NO NO20011729A patent/NO20011729L/no not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events