Patent Grant
6858707
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. 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: Phypahogenic 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 (≧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,” Phytopatholgy 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 Grain-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. “PopA1, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum,” EMBO J. 13:543-553 (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 GMI 1000 PopA1 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. “PopA1, 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 HarpinEch: Soft-Rot Pathogenesis,” MPMI 8(4): 484-91 (1995)); Erwinia carolovora (Cui, et al., “The RsmA− Mutants of Erwinia carotovora subsp. carotovora Strain Ecc71 Overexpress hrpNEcc and Elicit a Hypersensitive Reaction-like Response in Tobacco Leaves,” MPMI 9(7): 565-73 (1996)); 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. Jul. 14-19, 1996 and Ahmad, et. al., “Harpin is not Necessary for the Pathogenicity of Erwinia stewartii on Maize,” Ann. Mtg. Am. Phytopath. Soc. Jul. 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 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.
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 in accordance with the present invention 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.
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.
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 Xanthomonas species (e.g., the following bacteria: Erwinia amylovora, Erwinia chrysanthemi, Erwinia stewartit, 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 Phytophihora parasitica, Phytophthora cryptogea, Phytophthora cinnamomi, Phytophthora capsici, Phytophthora megasperma, and Phytophthora citrophihora.
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:
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. 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:
Another potentially suitable hypersensitive response elicitor from Erwinia amylovora is disclosed in U.S. patent application Ser. No. 09/120,927, which is hereby incorporated by reference. The protein is encoded by a DNA molecule having a nucleic acid sequence of SEQ ID NO: 25 as follows:
See GenBank Accession No. U94513. 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.
Another potentially suitable hypersensitive response elicitor from Erwinia amylovora is disclosed in U.S. patent application Ser. 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:
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:
This protein or polypeptide is about 198 kDa and has a pI of 8.98.
The present invention relates to an isolated DNA molecule having a nucleotide sequence of SEQ ID NO: 29 as follows:
This is known as the dspF gene. 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 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: 32 as follows:
Another potentially suitable hypersensitive response elicitor from Pseudomonas syningae is disclosed in U.S. patent application Ser. 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 43.9 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:
It is encoded by a DNA molecule having a nucleotide sequence corresponding to SEQ ID NO: 36 as follows:
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, “PopA I, 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: 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:
Isolation of Erwinia carotovora hypersensitve response elictor protein or polypeptide is described in Cui et al., “The Rsm Mutants of Erwinia carotovora subsp. carotovora Strain Ecc71 Overexpress hro NEcc and Elicit a Hypersensitive Reason-like Response is Tobacco Leaves, ” MPMI, 9(7):563‥73)1996), which is hereby incorporated by reference. The hypersensitve 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., Jul. 14-19, 1996 and Ahmad, et al., “Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize,” Ann. Mtg. Am. Phytopath. Soc., Jul. 27-31, 1996, which are hereby incorporated by reference.
Hypersensitive response elicitor proteins or polypeptides from Phytophthoraparasitica, Phytophthora cryptogea, Phytophihora cinnamoni, Phytophthora capsici, Phytophthora megasperma, and Phytophora citrophthora are described in Karnan, 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.
Another hypersensitive response elicitor in accordance with the present invention is from Clavibacter michiganensis subsp. sepedonicus which is fully described in U.S. patent application Ser. No. 09/136,625, which is 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 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. 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 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: 150 and 179, 137 and 166, 121 and 150, 76 and 168, 105 and 168, or 137 and 156. Other suitable fragments can be identified in accordance with the present invention.
Another example of 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.
Yet another example of 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. 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 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. 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 centriugation. 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. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated 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 transfection of plasmids into cells infected with virus.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/− or KS +/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see F. W. Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference.
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 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. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promotor, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coil 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 N gene of coliphage lambda, or from the E. coil tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
Once the isolated DNA molecule encoding the fragment of a hypersensitive response elicitor polypeptide or protein has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
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.
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 does not elicit 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 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. 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 does not elicit a hypersensitive response. Such bacteria must be capable of secreting or exporting the fragment so that the fragment can contact plant or plant seed cells. In these embodiments, the fragment is produced by the bacteria in plants 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, 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, Sainipaulia, 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, inter 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. pelargonil. 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 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. 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 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.
Although not required, this composition may contain additional additives including fertilizer, insecticide, fungicide, nematacide, and mixtures thereof. Suitable fertilizers include (NH4)2NO3. An example of a suitable insecticide is Malathion. Useful fungicides include Captan.
Other suitable additives include buffering agents, wetting agents, coating agents, and 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 fragment of a hypersensitive response elicitor 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 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.
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. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which are hereby incorporated 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. From 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. lumefaciens 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.
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. III (1986), which are hereby incorporated 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 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.
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 fragment of the hypersensitive response elicitor 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 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.).
Escherichia coli strains used in the following examples include DH5α and BL21(DE3) purchased from Gibco BRL (Grand Island, N.Y.) and Stratagene (La Jolla, Calif.), respectively. The pET28(b) vector was purchased from Novagen (Madison, Wis.). Eco DH5α/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 HindIII, 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 Kanr gene for selection of transformants.
Restriction enzymes were obtained from Boehringer Mannheim (Indianapolis, Ind.) or Gibco BRL. T4 DNA ligase, Calf Intestinal Alkaline Phosphatase (CIAP), and PCR Supermix198 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 Lofstnd Labs Limited (Gaithersburg, Md.). The oligopeptides were synthesized by Bio-Synthesis, Inc. (Lewisville, Tex.). 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.
A series of N-terminal and C-terminal truncated hrpN genes and internal fragments were generated via PCR (FIG. 1). 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 NdeI enzyme cutting site which contained the start codon ATG (methionine) and the 5′ primers contained the stop codon TAA and a HindIII enzyme cutting site for ligation into the pET28(b) vector. PCR was carried out in 0.5 ml tubes in a GeneAmp™ 9700 (Perkin-Elmer, Foster City, Calif.). 45 μl of Supermix™ (Life Technology, Gaithersburg, Md.) were mixed with 20 pmoles of each pair of DNA primers, 10 ng of full length harpin DNA, and deionized H2O 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, San Diego, Calif.). 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 NdeI 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 (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 fill 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 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 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 1).
A 50 ml culture of a hrpN 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 rpm for 5 minutes, re-suspended in urea lysis buffer (8 M urea, 0.1 M Na2HPO4, 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. 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, 0.1 M Na2HPO4, and 0.01 M Tris—pH 6.3), centrifuging at 5,000 rpm 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.
A 1.5 ml aliquot from the 50 ml cultures grown for small scale purification of the truncated proteins was centrifuged at 14,000 rpm 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/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.
Six 500 ml cultures of a hrpN 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 rpm for 5 minutes, re-suspended in imidazole lysis buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris) plus Triton X-100 at 0.05% and lysozyme at 0.1 mg/ml, incubated at 30° C. for 15 minutes, sonicated for two minutes, centrifuged again at 15,000 rpm 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 NaCl, and 20 mM Tris), centrifuging at 5,000 rpm for five minutes between washings, then placed in a disposable chromatography column. 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 rpm 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. 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 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. 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.
Sixty tomato (Lycopersicon spp. cv. Marglobe) seeds were soaked overnight in 10 and 20 μg/ml of the truncated protein diluted with 5mM potassium phosphate buffer, pH 6.8. The next morning, the sixty seeds were sewn in three pots and 12-15 days later and again 18-20 days later the heights of the 10 tallest tomato plants per pot were measured and compared with the heights of the control plants treated only with phosphate buffer. Analyses were done on the heights to determine if there was a significant difference in the height of the plants treated with the truncated proteins compared with the buffer control, and thereby determine whether the proteins induced growth enhancement.
Three tobacco (Nicotiana tabacum cv. Xanthi) plants with 8-12 leaves (ca. 75 day old plants) were used in the assay. One leaf of the tobacco plants was covered up and the rest of the leaves were sprayed with ca. 50 ml of a 20 μg/ml solution of the truncated proteins diluted with 5 mM potassium phosphate buffer. Five to seven days later two leaves (the unsprayed leaf and the sprayed leaf opposite and just above the unsprayed leaf) were inoculated with 20 μl of a 1.8 μg/ml solution of TMV along with a pinch of diatomaceous earth by rubbing the mixture along the top surface of the leaves. The TMV entered the plants through tiny lesions made by the diatomaceous earth. Ca. 3-4 days post TMV inolucation, the number of TMV lesions was counted on both leaves compared with the number of lesions on the negative control buffer treated leaves. Analyses were done to determine the efficacy of reducing the number of TWV lesions by the protein fragents compared to the buffer control. Percentage of efficacy was calculated as: Reduction in TMV lesions (% efficacy)=100×(1−mean # of lesions on treated leaves/mean # of lesions on buffer control leaves).
The small scale expression and purification of the fragment proteins was done expression and HR activity (Table 2).
All of the cloned fragment proteins were expressed at varying levels except for three small fragment (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 large scale expression and purification of the fragment proteins was done to determine the level of expression and titer of the HR activity (Table 3).
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) was expressed at 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.
The full length positive control protein elicited HR down to only 5-7 μg/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 to 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 encompassing a small internal region of the protein from aa. 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. a1-104 (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.
Surprisingly, 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 hypersensitive response elicitor 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, cleaving the protein between amino acids 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 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: 23) would elicit an HR. 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.
The C-terminal fragments enhanced the growth of tomato by 9% to 2 1%. 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 μg/ml, but not at the typical 20 μg/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).
The data suggests that there is more than one PGE domain, although the C-terminal and internal domains appear to be dominant over the N-terminal domain, as the N-terminal fragments enhanced growth the least amount.
All of the hypersensitive response elicitor fragments tested to date appear to have 60% effilcacy or greater, except for the oligopeptide 137-156 (Tables 5 and 6).
These data suggest that there are multiple SAR domains within the protein.
It is clear that 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 μg/ml, but these same fragments were not able to elicit HR in tobacco, even at higher concentrations than 200 μg/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.
To test whether non-HR eliciting fragments derived from HrpZ, the hypersensitive response elicitor from Pseudomonas syringae pv. syringae, is able to induce disease resistance, several fragment constructs were made and the expressed fragment proteins were tested for HR elicitation and disease resistance induction in tobacco and growth promotion in tomato.
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. coil DH5α, and the correct clones were identified. The clones were then transferred to E. coil BLR DE3 for protein expression. The bacteria were grown in Terrific Broth to an OD620 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 μl of antifoam and 40 μl 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.
Fifteen μl of each supernatant was run on a gel and stained to determine if the protein was present. It was estimated that about five times as much of the 152-294 fragment was present as the 190-294 fragment. Several dilutions of each preparation were infiltrated into tobacco leaves on two plants for HR tests (Table 7). As shown in Table 7, the 152-294 fragment elicited an HR, but the 190-294 fragment did not.
aThe preparations were diluted with MilliQ water.
bThe results are indicated for each of two plants. +, HR; −, no HR.
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% more than buffer treated plants, while plants treated with the 190-294 aa fragment grew 2.62% more than the buffer treated plants.
a+, elicits HR in tobacco leaves; −, no HR in tobacco leaves.
b% reduction in TMV lesions in unsprayed leaf of tobacco.
c% greater height than buffer sprayed plants.
The results of these tests show that amino acids 152-190 appear to be involved in HR elicitation, because their removal eliminated the ability to elicit the HR. Both fragment preparations achieved disease control and growth enhancement. Thus, the ability to elicit the HR is not the determining factor for reduction in TMV infection and growth enhancement.
Parsley leaves develop a typical resistance reaction against the soybean pathogen Phytophthora megasperma comprising hypersensitive cell death, defense related gene activation, and phytoalexin formulation. 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). Then, 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+/Cl− effluxes, active oxygen production, SAR gene induction, and phytoalexin compound accumulation (Numberger 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).
To test if the 13 amino acid peptide derived from the 42 kDa protein also enhanced plant growth, 20 mg of the oligopeptide was synthesized from Biosynthesis Corp. 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. After seedlings emerged and the first two true leaves fully expanded, the height of the tomato seedlings was recorded. The peptide was not able to elicit the HR in tobacco and other tested plants. However, it had a profound effect on plant growth promotion. Table 9 shows that tomato seedlings treated with the peptide increased 12.6% in height, indicating that the fungal peptide derived from the 42 kDa glycroprotein can promote tomato seedling growth. Extended studies showed that the peptide also had similar growth effect in other crops including tobacco. Similar growth promotion effects were achieved by plants sprayed with the peptide solution.
Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.
This application claims benefit of U.S. Provisional Patent Application Ser. No. 60/103,050, filed Oct. 5, 1998.
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