Patent Grant
7041876
The present invention relates to transgenic plants resistant to oomycete infection which contain a heterologous hypersensitive response elicitor under the control of a promoter responsive to infection by an oomycete.
In general, fungal plant diseases can be classified into two types: those caused by soilborne fungi and those caused by airborne fungi. Soilborne fungi cause some of the most widespread and serious plant diseases, such as root and stem rot caused by Fusarium spp. and root rot caused by Phytophthora spp. For example, Phytophthora parasitica var. nicotiana, a soilborne oomycete found in many tobacco growing regions worldwide, causes black shank, a highly destructive root and stem rot disease of many varieties of cultivated tobacco.
Since airborne fungi can be spread long distances by wind, they can cause devastating losses, particularly in crops which are grown over large regions. A number of pathogens have caused widespread epidemics in a variety of crops. Important diseases caused by airborne fungi are stem rust (Puccinia graminis) on wheat, corn smut (Ustilago maydis) on corn, and late blight disease (Phytophthora infestans) on potato and tomato. Plasmopera viticola is an airborne oomycete that causes downy mildew disease on grape vines. The blue mold fungus (Peronospora tabacina) has caused catastrophic losses in tobacco crops, particularly in the United States and Cuba.
Most of these fungal diseases are difficult to combat, and farmers and growers must use a combination of practices, such as sanitary measures, resistant cultivars, and effective fungicide against such diseases. Hundreds of millions of dollars are spent annually for chemical control of plant-pathogenic fungi. As a result, there is today a real need for new, more effective and safe means to control plant-pathogenic fungi, particularly oomycetes which are responsible for major crop loss.
Genetic engineering promises to be an effective strategy for reducing the losses associated with diseases of field crops. Several successful approaches have been reported where the constitutive expression of antimicrobial peptides such as cecropins (Arce et al., “Enhanced Resistance to Bacterial Infection by Erwinia Carotovora Susp. Atroseptica in Transgenic Potato Plants Expressing the Attacin or the Cecropin SB-37 Genes,” Am. J. Potato Res. 76:169–177 (1999)), lysozyme (Nakajima et al., “Fungal and Bacterial Disease Resistance in Transgenic Plants Expressing Human Lysozyme,” Plant Cell Reports 16:674–679 (1997)), and monoclonal antibodies (Tavladoraki et al, “Transgenic Plants Expressing a Functional Single Chain FV Antibody are Specifically Protected from Virus Attack,” Nature 366:468–472 (1993)) effectively protected plants from parasitic organisms. However successful, these approaches have limited application to food production since many of these antimicrobial peptides and plant defense molecules are potentially toxic or allergenic to humans (Franck-Oberaspach et al., “Consequences of Classical and Biotechnological Resistance Breeding for Food Toxicology and Allergenicity,” Plant Breeding 116:1–17 (1997)). Thus, alternative approaches for genetically engineering disease resistance would be more desirable.
Plants posses a highly evolved pathogen surveillance system which allows for recognition of specific pathogen derived molecules known as elicitors. Elicitor recognition results in an incompatible plant-microbe interaction, defined as the rapid activation of plant defense genes, typically resulting in the hypersensitive response and the onset of systemic acquired resistance.
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: Phytopathogenic 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,” Phytopathology 54:474–477 (1963); Turner, et al., “The Quantitative Relation Between Plant and Bacterial Cells Involved in the Hypersensitive Reaction,” Phytopathology 64:885–890 (1974); Klement, Z., “Hypersensitivity,” pages 149–177 in Phytopathogenic Prokarvotes, 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, (1982), these pathogens also cause physiologically similar, albeit delayed, necroses in their interactions with compatible hosts. Furthermore, the ability to produce the hypersensitive response or pathogenesis is dependent on a common set of genes, denoted hrp (Lindgren, P. B., et al., “Gene Cluster of Pseudomonas syringae pv. ‘phaseolicola’ Controls Pathogenicity of Bean Plants and Hypersensitivity on Nonhost Plants,” J. Bacteriol. 168:512–22 (1986); Willis, D. K., et al., “hrp Genes of Phytopathogenic Bacteria,” Mol. Plant-Microbe Interact. 4:132–138 (1991)). Consequently, the hypersensitive response may hold clues to both the nature of plant defense and the basis for bacterial pathogenicity.
The hrp genes are widespread in Gram-negative plant pathogens, where they are clustered, conserved, and in some cases interchangeable (Willis, D. K., et al., “hrp Genes of Phytopathogenic Bacteria,” Mol. Plant-Microbe Interact. 4:132–138 (1991); Bonas, U., “hrp Genes of Phytopathogenic Bacteria,” pages 79–98 in: Current Topics in Microbiology and Immunology: Bacterial Pathogenesis of Plants and Animals—Molecular and Cellular Mechanisms, J. L. Dangl, ed. Springer-Verlag, Berlin (1994)). Several hrp genes encode components of a protein secretion pathway similar to one used by Yersinia, Shigella, and Salmonella spp. to secrete proteins essential in animal diseases (Van Gijsegem, et al., “Evolutionary Conservation of Pathogenicity Determinants Among Plant and Animal Pathogenic Bacteria,” Trends Microbiol. 1:175–180 (1993)). In E. amylovora, P. syringae, and P. solanacearum, hrp genes have been shown to control the production and secretion of glycine-rich, protein elicitors of the hypersensitive response (He, S. Y., et al. “Pseudomonas Syringae pv. Syringae HarpinPSS: a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants,” Cell 73:1255–1266 (1993); Wei, Z. -M., 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 PopA 1 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 carotovora (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 (1966)); Erwinia stewartii (Ahmad, et. al., “Harpin is not Necessary for the Pathogenicity of Erwinia stewartii on Maize,” 8th Int'l. Cong. Molec. Plant-Microb. Inter. 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.).
Because the hypersensitive response results in localized necrosis of plant tissue, it is desirable to limit expression of a heterologous hypersensitive response elicitor to certain tissues in transgenic plants. This approach is discussed generally in PCT publication WO 94/01546 to Beer et al., but no specific transgenic plants are identified and only two suitable fungus-responsive promoters are suggested, e.g., the phenylalanine ammonia lyase and chalcone synthase promoters. No promoters responsive specifically to infection by oomycetes are identified therein.
The present invention is directed to overcoming these and other deficiencies in the art.
The present invention relates to a chimeric gene that includes a first DNA molecule encoding a hypersensitive response elicitor protein or polypeptide, a promoter operably linked 5′ to the first DNA molecule to induce transcription of the first DNA molecule in response to activation of the promoter by an oomycete, and a 3′ regulatory region operably linked to the first DNA molecule. Also disclosed are an expression system that includes a vector in which is inserted a chimeric gene of the present invention and a host cell that includes a chimeric gene of the present invention.
Another aspect of the present invention relates to a transgenic plant resistant to disease resulting from oomycete infection. The transgenic plant includes a chimeric gene of the present invention, wherein the promoter induces transcription of the first DNA molecule in response to infection of the plant by an oomycete. Transgenic seeds and transgenic cultivars obtained from the transgenic plant are also disclosed.
An additional aspect of the present invention relates to a method of making a recombinant plant cell. This is accomplished by transforming a plant cell with a chimeric gene of the present invention under conditions effective to yield transcription of the first DNA molecule in response to oomycete-induced activation of the promoter.
A further aspect of the present invention relates to a method of making a plant resistant to disease resulting from oomycete infection. This is accomplished by transforming a plant cell with a chimeric gene of the present invention under conditions effective to yield transcription of the first DNA molecule in response to oomycete-induced activation of the promoter and regenerating the plant from the transformed plant cell.
The present invention confers oomycete-induced disease resistance to plants transformed with a chimeric gene encoding a hypersensitive response elicitor protein or polypeptide, which is transcribed within a limited population of plant cells in response to infection of the plant by an oomycete. To limit transcription of the chimeric gene within a certain population of plant cells, the chimeric gene includes a promoter that is responsive to infection by an oomycete (i.e., it is activated by the oomycete). The hypersensitive response elicitor protein or polypeptide can cause tissue collapse at the site of infection and/or induce systemic resistance against the oomycete and other pathogens. By using the promoter from the potato gst1 gene, for example, which is activated by infection with oomyceteous fungi, the present invention can control fungal pathogens within crops without harming the transgenic plant and without resorting to use of environmentally damaging chemicals.
One aspect of the present invention relates to a novel DNA construct in the form of a chimeric gene. The chimeric gene includes a first DNA molecule encoding a hypersensitive response elicitor protein or polypeptide, a promoter operably linked 5′ to the first DNA molecule to induce transcription of the first DNA molecule in response to activation of the promoter by an oomycete, and a 3′ regulatory region operably linked to the first DNA molecule. As discussed more fully hereinafter, a chimeric gene of the present invention is particularly useful in preparing a transgenic plant for the purpose of rendering the transgenic plant resistant to disease resulting from infection thereof by an oomycete.
The first DNA molecule can encode any hypersensitive response elicitor protein or polypeptide which is effective in triggering a hypersensitive response (i.e., in a particular host plant selected for transformation). Generally, it is desirable to express hypersensitive response elicitors only in plants which are non-hosts for the source organism of the hypersensitive response elicitor. Suitable hypersensitive elicitor proteins or polypeptides are those derived from a wide variety of bacterial and fungal pathogens, preferably bacterial pathogens.
Exemplary hypersensitive response elicitor proteins and polypeptides from bacterial sources include, without limitation, the hypersensitive response elicitors fromErwinia species (e.g., Erwinia amylovora, Erwinia chrysanthemi, Erwinia stewartii, Erwinia carotovora, etc.), Pseudomonas species (e.g., Pseudomonas syringae, Pseudomonas solanacearum, etc.), and Xanthomonas species (e.g., Xanthomonas campestris). In addition to hypersensitive response elicitors from these Gram-negative bacteria, it is possible to use elicitors from Gram-positive bacteria. One example is the hypersensitive response elicitor from Clavibacter michiganensis subsp. sepedonicus.
Exemplary hypersensitive response elicitor proteins or polypeptides from fungal sources include, without limitation, the hypersensitive response elicitors (i.e., elicitins) from various Phytophora species (e.g., Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamomi, Phytophthora capsici, Phytophthora megasperma, Phytophthora citrophthora, etc.).
Preferably, the first DNA molecule encodes a hypersensitive response elicitor protein or polypeptide of Erwinia chrysanthemi, Erwinia amylovora, Pseudomonas syringae, or Pseudomonas solanacearum.
The hypersensitive response elicitor protein or polypeptide from Erwinia chrysanthemi has an amino acid sequence corresponding to SEQ. ID. No. 1 as follows:
This hypersensitive response elicitor protein or polypeptide has a molecular weight of 34 kDa, is heat stable, has a glycine content of greater than 16%, and contains substantially no cysteine. This Erwinia chrysanthemi hypersensitive response elicitor protein or polypeptide is encoded by a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 2 as follows:
This hypersensitive response elicitor protein or polypeptide 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 protein or polypeptide has substantially no cysteine. The hypersensitive response elicitor protein or polypeptide derived from Erwinia amylovora is more fully described in Wei, Z-M., et al., “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 hypersensitive response elicitor protein or polypeptide has a nucleotide sequence corresponding to SEQ. ID. No. 4 as follows:
The hypersensitive response elicitor protein or polypeptide derived from Pseudomonas syringae has an amino acid sequence corresponding to SEQ. ID. No. 5 as follows:
This hypersensitive response elicitor protein or polypeptide 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., 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), which is hereby incorporated by reference. The DNA molecule encoding this hypersensitive response elicitor from Pseudomonas syringae has a nucleotide sequence corresponding to SEQ. ID. No. 6 as follows:
Another potentially suitable hypersensitive response elicitor from Pseudomonas syringae is disclosed in U.S. patent application Ser. No. 09/120,817, which is hereby incorporated by reference.
The hypersensitive response elicitor protein or polypeptide derived from Pseudomonas solanacearum has an amino acid sequence corresponding to SEQ. ID. No. 7 as follows:
Further information regarding this hypersensitive response elicitor protein or polypeptide derived from Pseudomonas solanacearum is set forth in Arlat, M., et al., “PopA1, 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. It is encoded by a DNA molecule from Pseudomonas solanacearum having a nucleotide sequence corresponding SEQ. ID. No. 8 as follows:
Other embodiments of the present invention include, but are not limited to, use of the nucleotide sequence encoding for the hypersensitive response elicitor protein or polypeptide from Erwinia carotovora and Erwinia stewartii. Isolation of Erwinia carotovora hypersensitive response elicitor protein or polypeptide is described in Cui, et al., “The RsmA Mutants of Erwinia carotovora subsp. carotovora Strain Ecc71 Overexpress hrp NEcc and Elicit a Hypersensitive Reaction-like Response in Tobacco Leaves,” MPMI, 9(7):565–73 (1996), which is hereby incorporated by reference. The hypersensitive response elicitor protein or polypeptide of Erwinia stewartii is set forth in Ahmad, et al., “Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize,” 8th Int'l. Cong. Molec. Plant-Microbe Interact., 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.
The hypersensitive response elicitor proteins or polypeptides from various Phytophora species are described in Kaman, et al., “Extracellular Protein Elicitors from Phytophthora: Most Specificity and Induction of Resistance to Bacterial and Fungal Phytopathogens,” Molec. Plant-Microbe Interact., 6(1):15–25 (1993); Ricci, et al., “Structure and Activity of Proteins from Pathogenic Fungi Phytophthora Eliciting Necrosis and Acquired Resistance in Tobacco,” Eur. J. Biochem., 183:555–63 (1989); Ricci, et al., “Differential Production of Parasiticein, and Elicitor of Necrosis and Resistance in Tobacco, by Isolates of Phytophthora parasitica,” Plant Path. 41:298–307 (1992); Baillreul, et al., “A New Elicitor of the Hypersensitive Response in Tobacco: A Fungal Glycoprotein Elicits Cell Death, Expression of Defense 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 described in U.S. patent application Ser. No. 09/136,625, which is hereby incorporated by reference.
Other elicitors can be readily identified by isolating putative hypersensitive response elicitors and testing them for elicitor activity as described, for example, in Wei, Z-M., et al., “Harpin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora,” Science 257:85–88 (1992), which is hereby incorporated by reference. Cell-free preparations from culture supernatants can be tested for elicitor activity (i.e., local necrosis) by using them to infiltrate appropriate plant tissues. Once identified, DNA molecules encoding a hypersensitive response elicitor can be isolated using standard techniques known to those skilled in the art. The isolated DNA molecule can then be introduced into the chimeric gene for expression in a transgenic plant of the present invention.
The first DNA molecule can also encode fragments of the above hypersensitive response elicitor proteins or polypeptides as well as fragments of full length elicitors from other pathogens.
Suitable fragments can be produced by several means. Subclones of the gene encoding a known elicitor protein can be produced using conventional molecular genetic manipulation for subcloning gene fragments, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), and Ausubel et al. (ed.), Current Protocols in Molecular Biology, John Wiley & Sons (New York, N.Y.) (1999 and preceding editions), which are hereby incorporated by reference. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or polypeptide that can be tested for elicitor activity, e.g., using procedures set forth in Wei, Z-M., et al., Science 257: 85–88 (1992), which is hereby incorporated by reference.
In another approach, based on knowledge of the primary structure of the protein, fragments of the elicitor protein gene may be synthesized using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. Erlich, H. A., et al., “Recent Advances in the Polymerase Chain Reaction,” Science 252:1643–51 (1991), which is hereby incorporated by reference. These can then be cloned into an appropriate vector for expression of a truncated protein or polypeptide from bacterial cells as described above.
An example of suitable fragments of a hypersensitive response elicitor which elicit a hypersensitive response are fragments of the Erwinia amylovora hypersensitive response elicitor protein or polypeptide of SEQ. ID. No. 3. The fragments can be a C-terminal fragment of the amino acid sequence of SEQ. ID. No. 3, an N-terminal fragment of the amino acid sequence of SEQ. ID. No. 3, or an intern al fragment of the amino acid sequence of SEQ. ID. No. 3. The C-terminal fragment of the amino acid sequence of SEQ. ID. No. 3 can span amino acids 105 and 403 of SEQ. ID. No. 3. The N-terminal fragment of the amino acid sequence of SEQ. ID. No. 3 can span the following amino acids of SEQ. ID. No. 3: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1 and 372. The internal fragment of the amino acid sequence of SEQ. ID. No. 3 can span the following amino acids of SEQ. ID. No. 3: 76 and 209, 105 and 209,99 and 209, 137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180, and 105 and 180. DNA molecules encoding these fragments can also be utilized in the chimeric gene of the present invention.
The first DNA molecule also can be a DNA molecule that hybridizes under stringent conditions to the DNA molecule having nucleotide sequence of SEQ. ID. Nos. 2, 4, 6, or 8. An example of suitable stringency conditions is when hybridization is carried out at a temperature of about 37° C. using a hybridization medium that includes 0.9M sodium citrate (“SSC”) buffer, followed by washing with 0.2×SSC buffer at 37° C. Higher stringency can readily be attained by increasing the temperature for either hybridization or washing conditions or increasing the sodium concentration of the hybridization or wash medium. Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase. Wash conditions are typically performed at or below stringency. Exemplary high stringency conditions include carrying out hybridization at a temperature of about 42° C. to about 65° C. for up to about 20 hours in a hybridization medium containing 1 M NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 50 μg/ml E. coli DNA, followed by washing carried out at between about 42° C. to about 65° C. in a 0.2×SSC buffer.
Variants of suitable hypersensitive response elicitor proteins or polypeptides can also be expressed by the first DNA molecule. Variants may be made by, for example, the deletion, addition, or alteration 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 (such as a 6×His tag).
The promoter of the chimeric gene should be selected on the basis of its ability to induce transcription of the first DNA molecule in response to infection of the plant by an oomycete (i.e., the oomycete activates the promoter). According to one embodiment, the promoter preferably includes some or all of the promoter-effective regions of a gst1 gene from potato. The gst1 promoter is activated in response to infection by oomycetes and not by wounding or other environmental perturbations. The gst1 promoter from potato has a nucleic acid sequence corresponding to SEQ. ID. No. 9 as follows:
Effective fragments of SEQ. ID. No. 9 are also encompassed by the present invention. U.S. Pat. Nos. 5,750,874 and 5,723,760 to Strittmayer et al., which are hereby incorporated by reference, define promoter-effective regions of the potato gst1 promoter. Preferably, the gst1 promoter includes a nucleotide sequence corresponding, at a minimum, to nucleotides 295–567 of SEQ. ID. No. 9. The gst1 promoter can also include effective portions containing nucleotides 295–696 of SEQ. ID. No. 9.
The chimeric gene of the present invention also includes an operable 3′ regulatory region, selected from among those which are capable of providing correct transcription termination and polyadenylation of mRNA for expression in plant cells, operably linked to the first DNA molecule which encodes for a hypersensitive response elicitor. A number of 3′ regulatory regions are known to be operable in plants. Exemplary 3′ regulatory regions include, without limitation, the nopaline synthase 3′ regulatory region (Fraley, et al., “Expression of Bacterial Genes in Plant Cells,” Proc. Nat'l Acad. Sci. USA, 80:4803–4807 (1983), which is hereby incorporated by reference) and the cauliflower mosaic virus 3′ regulatory region (Odell, et al., “Identification of DNA Sequences Required for Activity of the Cauliflower Mosaic Virus 35S Promoter,” Nature, 313(6005):810–812 (1985), which is hereby incorporated by reference). Virtually any 3′ regulatory region known to be operable in plants would suffice for proper expression of the coding sequence of the chimeric gene of the present invention.
The first DNA molecule, promoter, and a 3′ regulatory region can be ligated together using well known molecular cloning techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y. (1989), which is hereby incorporated by reference.
The chimeric gene can also include a second DNA molecule encoding a secretion signal. A number of suitable secretion signals are known in the art and other are continually being identified. The secretion signal can be an RNA leader which directs secretion of the subsequently transcribed protein or polypeptide, or the secretion signal can be an amino terminal peptide sequence that is recognized by a host plant secretory pathway. The second DNA molecule can be ligated between the promoter and the first DNA molecule, using known molecular cloning techniques as indicated above.
According to one embodiment, the second DNA molecule encodes a secretion signal derived from Nicotiana tabacum. Specifically, this DNA molecule encodes the secretion signal polypeptide for PR1-b gene of Nicotiana tabacum. This second DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No. 10 as follows:
tctagaccat gggatttttt ctcttttcac aaatgccctc attttttctt gtgtcgacac
The above sequence includes XbaI sites (underlined) at each end to facilitate insertion of the second DNA molecule into the chimeric gene of the present invention. The coding sequence of SEQ. ID. No. 10 starts at base 8. The polypeptide encoded by this nucleic acid molecule has an amino acid sequence corresponding to SEQ. ID. No. 11 as follows:
An alternative second DNA molecule encoding the secretion signal polypeptide for PR1-B gene of Nicotiana tabacum has a nucleotide sequence corresponding to SEQ. ID. No. 12 as follows:
This nucleotide sequence is disclosed in Genbank Accession No. X03465, which is hereby incorporated by reference. The polypeptide encoded by this nucleic acid molecule has an amino acid sequence corresponding to SEQ. ID. No. 13 as follows:
Yet another second DNA molecule encodes the secretion signal for the PR1-a gene of Nicotiana tabacum. This DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No. 14 as follows:
This DNA molecule is disclosed in Genbank Accession No. X06361, which is hereby incorporated by reference. The polypeptide encoded by this nucleic acid molecule has an amino acid sequence corresponding to SEQ. ID. No. 15 as follows:
Still another second DNA molecule encodes the secretion signal for the PR4-a gene of Nicotiana tabacum. This DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No. 16 as follows:
This DNA molecule is disclosed in Genbank Accession No. X58546, which is hereby incorporated by reference. The polypeptide encoded by this nucleic acid molecule has an amino acid sequence corresponding to SEQ. ID. No. 17 as follows:
Each second DNA molecule can be cloned using primers that introduce restriction sites at the 5′ and 3′ ends thereof to facilitate insertion of the second DNA molecule into the chimeric gene of the present invention. SEQ. ID. No. 10 is shown to include such restriction sites (e.g., XbaI).
Further aspects of the present invention include an expression system that includes a vector containing a chimeric gene of the present invention, as well as a host cell which includes a chimeric gene of the present invention. As described more fully hereinafter, the recombinant host cell can be either a bacterial cell (i.e., Agrobacterium) or a plant cell. In the case of recombinant plant cells, it is preferable that the chimeric gene is stably inserted into the genome of the recombinant plant cell.
The chimeric gene can be incorporated into cells using conventional recombinant DNA technology. Generally, this involves inserting the chimeric gene into an expression vector or system to which it is heterologous (i.e., not normally present). As described above, the chimeric gene contains the necessary elements for the transcription and translation in plant cells of the first DNA molecule (i.e., encoding the hypersensitive response elicitor protein or polypeptide) and, if present, the second DNA molecule.
U.S. Pat. No. 4,237,224 issued 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.
Once the chimeric gene of the present invention has been prepared, it is ready to be incorporated into a host cell. 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, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like. Preferably the host cells are either a bacterial cell or a plant cell.
Accordingly, another aspect of the present invention relates to a method of making a recombinant plant cell. Basically, this method is carried out by transforming a plant cell with a chimeric gene of the present invention under conditions effective to yield transcription of the first DNA molecule in response to oomycete-induced activation of the promoter. Preferably, the chimeric gene is stably inserted into the genome of the recombinant plant cell as a result of the transformation.
A related aspect of the present invention concerns a method of making a plant resistant to disease resulting from oomycete infection. Basically, this method is carried out by transforming a plant cell with a chimeric gene of the present invention under conditions effective to yield transcription of the first DNA molecule in response to oomycete-induced activation of the promoter and regenerating a plant from the transformed plant cell.
One approach to transforming plant cells with a chimeric gene of the present invention is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford, et al., 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. Other variations of particle bombardment, now known or hereafter developed, can also be used.
Another method of introducing the chimeric gene is fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies that contain the chimeric gene. Fraley, et al., Proc. Natl. Acad. Sci. USA, 79:1859–63 (1982), which is hereby incorporated by reference.
The chimeric gene 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 chimeric gene. 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 chimeric gene into plant cells is to infect a plant cell with Agrobacterium tumefaciens or Agrobacterium rhizogenes previously transformed with the chimeric 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 such as a chimeric gene of the present invention can be introduced into appropriate plant cells by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. Schell, J., Science, 237:1176–83 (1987), which is hereby incorporated by reference.
Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers.
After transformation, the transformed plant cells can be selected and regenerated.
Preferably, transformed cells are first identified using, e.g., a selection marker simultaneously introduced into the host cells along with the chimeric gene of the present invention. Suitable selection markers include, without limitation, markers coding for antibiotic resistance, such as kanamycin resistance (Fraley, et al., Proc. Natl. Acad. Sci. USA, 80:4803–4807 (1983), which is hereby incorporated by reference). A number of antibiotic-resistance markers are known in the art and other are continually being identified. Any known antibiotic-resistance marker can be used to transform and select transformed host cells in accordance with the present invention. Cells or tissues are grown on a selection media containing an antibiotic, whereby generally only those transformants expressing the antibiotic resistance marker continue to grow.
Once a recombinant plant cell or tissue has been obtained, it is possible to regenerate a full-grown plant therefrom. Thus, another aspect of the present invention relates to a transgenic plant that is resistant to disease resulting from oomycete infection. The transgenic plant includes a chimeric gene of the present invention, wherein the promoter induces transcription of the first DNA molecule in response to infection of the plant by an oomycete. Preferably, the chimeric gene is stably inserted into the genome of the transgenic plant of the present invention.
Plant regeneration from cultured protoplasts is described in Evans, et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. 1, 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 rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.
Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
After the chimeric gene is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing or by preparing cultivars. With respect to sexual crossing, any of a number of standard breeding techniques can be used depending upon the species to be crossed. Cultivars can be propagated in accord with common agricultural procedures known to those in the field.
Resistance against different types of oomycetes may be imparted to transgenic plants according to the present invention. Without being bound by any particular theory, it is believed that a hypersensitive response elicitor protein or polypeptide encoded by the first DNA molecule is transcribed in response to infection of the plant by an oomycete. The exact mechanism by which the promoter is activated to regulate transcription of sequences under its control is not fully understood; however, the first DNA molecule is transcribed and the hypersensitive response elicitor is expressed in a limited population of cells (i.e., those in which transcription has been induced following oomycete infection). Once expressed, it is believed that the hypersensitive response elicitor can either be secreted from the plant cell (assuming the chimeric gene also contains a second DNA molecule encoding an N-terminal secretion signal) or leaked from an oomycete-infected plant cell. Regardless of how the hypersensitive response elicitor is delivered to the intercellular environment, it is believed that the hypersensitive response elicitor protein or polypeptide will initiate a hypersensitive response to cause localized necrosis of oomycete-infected tissues. In addition, systemic acquired resistance may be developed by the transgenic plant following initiation of the hypersensitive response. This may yield broad disease and/or pathogen resistance to the transgenic plants of the present invention.
Oomycetes against which resistance is imparted include, without limitation, species of Plasmopara, Phytophthora, Peronospora, Pseudoperonospora, Bremia, Sclerospora, Aphanomyces, Pythium, and Albugo.
According to one embodiment of the present invention, an oomycete resistant transgenic tobacco plant includes a chimeric gene of the present invention, wherein expression of the encoded hypersensitive response elicitor is responsive to infection of the plant by an oomycete that is a pathogen of tobacco, including, but not limited to, Peronospora tabacina (which causes blue mold) and Phyophthora parasitica (which causes black shank).
The chimeric gene of the present invention can be utilized to impart oomycete resistance for a wide variety of tobacco plants, some of which may possess varying levels of natural resistance against pathogenic oomycetes. The varieties of tobacco plants which can be protected include, without limitation, those referred to as Coker 371 Gold, K 149, K 326, K 346, K 394, K 730, RG 11, RG17, RG22, Speight G-70, Speight G-117, Speight G-126, GL939, NC 55, NC 71, NC 72, NC 95, NC 2326, OX 207, OX 940, RG 81, RG H4, RG H61, Speight 168, SpeightNF3, Speight 172, CU 236, CU 387, CU 368, NC TG91, OX 4142NF, OX 4083, RG 4H2-12, RG 4H2-17, RG 4H2-20, Speight 177, Speight 178, Speight 179, VPI 107, VPI 605, NG TG94, KY 14, KY 8959, KY 907, KY 908, TN 86, TN 90, TN 97, VA 116, VA 509, B 21×KY 10, KY 14×L8, NC 3, NC BH129, DH332, COOP 313, COOP 543, Clay's 403, Clay's 502, HY 402, PF 561, and R 711.
According to another embodiment of the present invention, an oomycete resistant transgenic grape plant includes a chimeric gene of the present invention, wherein expression of the encoded hypersensitive response elicitor is responsive to infection of the plant by an oomycete that is a pathogen of grape, including, but not limited to, Plasmopara viticola (which causes downy mildew), Pythium spp. (which cause root and/or stem rot), and Phytophthora spp. (which cause root and/or stem rot).
The chimeric gene of the present invention can be utilized to impart oomycete resistance for a wide variety of grapevine plants. The chimeric gene is particularly well suited to imparting resistance to Vitis scion or rootstock cultivars. Scion cultivars which can be protected include, without limitation, those commonly referred to as Table or Raisin Grapes, such as Alden, Almeria, Anab-E-Shahi, Autumn Black, Beauty Seedless, Black Cornish, Black Damascus, Black Malvoisie, Black Prince, Blackrose, Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic, Ferdinand de Lesseps, Fiesta, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta, Kourgane, Kishmishi, Loose Perlette, Malaga, Monukka, Muscat of Alexandria, Muscat Flame, Muscat Hamburg, New York Muscat, Niabell, Niagara, Olivette blanche, Ontario, Pierce, Queen, Red Malaga, Ribier, Rish Baba, Romulus, Ruby Seedless, Schuyler, Seneca, Suavis (IP 365), Thompson seedless, and Thomuscat. They also include, without limitation, those used in wine production, such as Aleatico, Alicante Bouschet, Aligote, Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet franc, Cabernet, Sauvignon, Calzin, Carignane, Charbono, Chardonnay, Chasselas dore, Chenin blanc, Clairette blanche, Early Burgundy, Emerald Riesling, Feher Szagos, Fernao Pires, Flora, French Colombard, Fresia, Furmint, Gamay, Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, Green Veltliner, Grenache, Grillo, Helena, Inzolia, Lagrein, Lambrusco de Salamino, Malbec, Malvasia bianca, Mataro, Melon, Merlot, Meunier, Mission, Montua de Pilas, Muscadelle du Bordelais, Muscat blanc, Muscat Ottonel, Muscat Saint-Vallier, Nebbiolo, Nebbiolo fino, Nebbiolo Lampia, Orange Muscat, Palomino, Pedro Ximenes, Petit Bouschet, Petite Sirah, Peverella, Pinot noir, Pinot Saint-George, Primitivo di Gioa, Red Veltliner, Refosco, Rkatsiteli, Royalty, Rubired, Ruby Cabernet, Saint-Emilion, Saint Macaire, Salvador, Sangiovese, Sauvignon blanc, Sauvignon gris, Sauvignon vert, Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant, Shiraz, Souzao, Sultana Crimson, Sylvaner, Tannat, Teroldico, Tinta Madeira, Tinto cao, Touriga, Traminer, Trebbiano Toscano, Trousseau, Valdepenas, Viognier, Walschriesling, White Riesling, and Zinfandel. Rootstock cultivars which can be protected include Couderc 1202, Couderc 1613, Couderc 1616, Couderc 3309, Dog Ridge, Foex 33 EM, Freedom, Ganzin 1 (A×R #1), Harmony, Kober 5BB, LN33, Millardet & de Grasset 41B, Millardet & de Grasset 420A, Millardet & de Grasset 101–14, Oppenheim 4 (SO4), Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A, Vitis rupestris Constantia, Vitis california, and Vitis girdiana.
Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedures. Alternatively, transgenic seeds or propagules (e.g., scion or rootstock cultivars) 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 oomycete resistance to plants.
The following examples are provided to illustrate embodiments of the present invention, but they are by no means intended to limit its scope.
Cloning of gst1 Promoter
The gst1 promoter region from nucleotides (539 to +48) (Martini et al., “Promoter Sequences of a Potato Pathogenesis-related Gene Mediate Transcriptional Activation Selectively upon Fungal Infection,” Mol. Gen. Genet. 236 (2–3):179–86 (1993), which is hereby incorporated by reference), was PCR amplified using DNA from potato cultivar Atlantic, using a forward primer containing a BamHI site (SEQ. ID. No. 18) as follows:
a reverse primer containing an EcoRI site (SEQ. ID. No. 19) as follows:
and PrimeZyme DNA polymerase (Whatman Biometra, Goettingen, Germany). The DNA was ligated into the LITMUS 38 vector (New England Biolabs, Beverly, Mass.) and three clones were sequenced on an ABI 377 sequencer at the Cornell BioResource Center. Each clone had two to three nucleotide changes when compared to the published sequence (Martini et al., “Promoter Sequences of a Potato Pathogenesis-related Gene Mediate Transcriptional Activation Selectively upon Fungal Infection,” Mol. Gen. Genet. 236: (2–3) 179–86 (1993), which is hereby incorporated by reference). The changes were most likely due to mistakes made by the polymerase because the promoter is extremely A-T rich and all but one of the changes were in different places in the three clones. One clone, pCPP1308, with a single change in the cis-acting region identified by Martini et al. (“Promoter Sequences of a Potato Pathogenesis-related Gene Mediate Transcriptional Activation Selectively upon Fungal Infection,” Mol. Gen. Genet. 236: (2–3) 179–86 (1993), which is hereby incorporated by reference) was used as the source of the gst1 promoter in all subsequent constructions.
Plant Transformation Constructs
The gst1:uidA construct was made by ligating the gst1 promoter from pCPP1308 into pBI101 (Clontech Labs, Palo Alto, Calif.). For the gst1:hrpN and gst1:signal sequence:hrpN constructs (described below), the gst1 promoter region was engineered to have a 5′ HindIII site and a 3′ XbaI site by the polymerase chain reaction (PCR) using pCPP1308 as the template. The forward primer had the nucleotide sequence of SEQ. ID. No. 18 and the reverse primer had a nucleotide sequence according to SEQ. ID. No. 20 as follows:
tacgtctaga tatgtgtggt tggtctccct tg 32
For gst1:hrpN constructs, the hrpN gene of Erwinia amylovora (i.e., encoding a hypersensitive response elicitor identified as harpinea) was engineered to have a 5′ XbaI restriction site and a 3′ SstI restriction site by PCR using pCPP1084 (Wei et al., “Harpin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia Amylovora,” Science 257:85–88 (1992), which is hereby incorporated by reference) as the template. The forward primer had a nucleotide sequence corresponding to SEQ. ID. No. 21 as follows:
and the reverse primer had a nucleotide sequence corresponding to SEQ. ID. No. 22 as follows:
For gst1:signal sequence:hrpN, the hrpN gene was engineered to have a BamHI site on each end. The forward primer had a nucleotide sequence corresponding to SEQ. ID. No. 23 as follows:
and the reverse primer had a nucleotide sequence corresponding to SEQ. ID. No. 24 as follows:
The nopaline synthase terminator was extracted from pBI101 by digesting with SstI and EcoRI.
The nucleic acid molecule encoding the PR1-b signal sequence (of SEQ. ID. No. 1) was engineered to have XbaI restriction sites on both ends. The forward primer had a nucleotide sequence corresponding to SEQ. ID. No. 25 as follows:
and the reverse primer had a nucleotide sequence corresponding to SEQ. ID. No. 26 as follows:
The fragment was amplified using pSKG55 as a template (Gopalan et al., “Expression of the Pseudomonas Syringae Avirulence Protein AvrB in Plant Cells Alleviates its Dependence on the Hypersensitive Response and Pathogenicity (Hrp) Secretion System in Elicitating Genotype-Specific Hypersensitive Cell Death.” Plant Cell 8:1095–1105 (1996), which is hereby incorporated by reference).
PrimeZyme DNA polymerase (Whatman Biometra, Goettingen, Germany) was used with a hot start procedure for amplification of all fragments. The amplified fragments were purified, digested with the appropriate enzymes, and ligated into the binary vector pPZP221 (Hajdukiewicz et al., “The Small Versatile pPZP Family of Agrobacterium Binary Vectors for Plant Transformation,” Plant Mol. Bio. 25:989–994 (1994), which is hereby incorporated by reference) or intermediate constructs, to build up the final constructs. The proper construction of pCPP1294 (
The final constructs were transformed into Agrobacterium tumefaciens strain GV3101 (Martin et al., “The GUS Reporter System as a Tool to Study Plant Gene Expression,” in Gallagher, ed., GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression, Academic Press, pp. 23–43 (1992), which is hereby incorporated by reference) by electroporation using a Bio-Rad GenePulser (Bio-Rad Ltd., York, UK).
To evaluate the activity of the gst1 promoter in a plant other than potato, transgenic Arabidopsis were constructed containing the E. coli uidA gene for β-glucuronidase (GUS) under control of the gst1 promoter. Histochemical GUS assays of were performed essentially as described by Martin et al., “The GUS Reporter System as a Tool to Study Plant Gene Expression,” in Gallagher, ed., GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression, Academic Press, pp. 23–43 (1992), which is hereby incorporated by reference. Uninoculated and inoculated whole small Arabidopsis plants were submerged for 30 minutes on ice in six well microtiter plates in a solution of 1.5% freshly prepared paraformaldehyde in 100 mM sodium phosphate buffer, pH 7.2, containing 0.1% Triton X-100. The plants were washed twice for 5 minutes with sodium phosphate buffer pH 7.2. The plants were then submerged in a solution of 2 mM X-gluc (5-bromo-4-chloro-3-indolyl β-D-glucuronide), 50 mM sodium phosphate, pH 7.2, 0.5% Triton X-100. The solution was vacuum infiltrated into the plants and the plants were then incubated for 16 hours in the dark at 37° C. The staining was stopped by rinsing the plants several times in water and the tissue was then cleared by incubating in several changes of 70% ethanol.
Twenty lines were evaluated for GUS expression in uninoculated leaves, leaves inoculated with Peronospora parasitica isolate NOCO, and whole plants using a histochemical staining procedure (Martin et al., “The GUS Reporter System as a Tool to Study Plant Gene Expression,” in Gallagher, ed., GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression, Academic Press, pp 23–43 (1992), which is hereby incorporated by reference). Five lines showed more intense staining of the inoculated areas than the uninoculated areas and two lines showed no visible staining of any plant parts except the inoculated leaves (
To generate transgenic Arabidopsis expressing hrpN in a pathogen-inducible manner, plant transformation vectors, pCPP 1292 for cytoplasmic localization of HrpN in plants, and pCPP 1294 for extracellular localization of HrpN, were constructed. (
Transgenic Arabidopsis lines were inoculated 2 weeks after sowing with a 5×104 conidiospore suspension of P. parasitica isolate NOCO. Flats were covered with a humidity dome and moved to the growth chamber maintained at 18° C., 16 hours light, and 100% humidity. Plants were scored for infection 7 days after inoculation with a disease rating system adapted from Cao et al., “Generation of Broad-Spectrum Disease Resistance by Overexpression of an Essential Regulatory Gene in Systemic Acquired Resistance,” Proc. Natl. Acad. Sci. USA 95:6531–6536 (1998), which is hereby incorporated by reference. A rating of 1, 0 conidiophores present; 2, 0–5 conidiophores present; 3, 6–20 conidiophores on a few leaves; 4, 6–20 conidiophores on all leaves; 5, 20 or more conidiophores present on all leaves. Inoculated leaves were stained with lactophenol-trypan blue (Keogh et al., “Comparison of Histological and Physiological Responses to Phakopsora Pachyrhizi in Resistant and Susceptible Soybean,” Trans. Br. Mycol. Soc. 74:329–333 (1980), which is hereby incorporated by reference) to observe the extent of fungal colonization under the microscope.
Plants were selected that lacked segregation of antibiotic resistance in the T3 generation. Lines containing the gst1:hrpN construct (“GN lines”) lines were tested for resistance to P. parasitica isolate NOCO in an initial screen.
Thirty lines containing the gst1:signal sequence:hrpN construct (“GSSN lines”) were tested for resistance to P. parasitica isolate NOCO in an initial screen. All but one of the lines was free of any signs of the oomycete ten days after inoculation. Ten GSSN lines were chosen for further study and inoculated by spraying with a conidiospore suspension (5×104 spores/ml) of P. parasitica NOCO. Northern analysis revealed that expression of hrpN was induced by P. parasitica 2 days after inoculation with strong induction at 4 days (
RNA was isolated from inoculated plants over a 4 day interval to analyze hrpN gene expression. RNA was isolated from 1 g of plant tissue as described by Carpenter et al., “Preparation of RNA, in Arabidopsis Protocols,” (Martinez-Zapater, J M. and Salinas, J., eds.), Humana Press, Totowata, N.J., pp. 85–89 (1998). Twenty micro-gram samples were separated by formaldehyde-agarose gel electrophoresis and blotted onto Hybond N+ membranes (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). Hybridizations and washing were performed according to Church et al., “Genomic Sequencing,” Proc. Natl. Acad. Sci. USA 81:1991–1995 (1984), which is hereby incorporated by reference, using P32 labeled hrpN DNA as a probe.
The Arabidopsis lines GSSN 8–4 (test), Col-0 WT (wild type, control), and Col-0 EV (empty vector, control) were inoculated by drop inoculation with a conidiospore suspension (5×104 spores/ml) of P. parasitica. Plants were maintained in a growth chamber (16 hours of light, 18° C., 100% humidity) and were scored for infection ten days post inoculation. Nearly all (29 out of 30) 8–4 plants were free of any signs of P. parasitica (
Plants were rated for disease severity based on the number of conidiophores per leaf. Nearly all GSSN 8–4 plants received a disease rating of 1 with only one being scored 3. The majority of the Col-0 WT and Col-0 EV plants were rated 5, the remainder were rated 4 (
This example demonstrates that pathogen inducible expression of the harpinEa hypersensitive response elicitor of Erwinia amylovora in transgenic plants is a potentially useful strategy for engineering plants for disease resistance. Challenge with Peronospora parasitica resulted in accumulation of hrpN mRNA, production of harpinEa protein, and resistance to P. parasitica. Upon challenge by P. parasitica, it is believed that the transgenic plants most likely mount a hypersensitive response at the site of inoculation, conferring resistance. Subsequently the plants may develop systemic resistance.
For the purposes of the present invention, the gst1 promoter was most applicable to the Arabidopsis/P. parasitica pathosystem since it is well documented that transcription from gst1 is activated by other oomycete pathogens (Martini et al., “Promoter Sequences of a Potato Pathogenesis-related Gene Mediate Transcriptional Activation Selectively upon Fungal Infection,” Mol. Gen. Genet. 236: (2–3) 179–86 (1993), which is hereby incorporated by reference). Additionally, it has been reported that gst1 activation is stimulated by ascomycete, viral, and nematode infection and mycorrhization (Strittmatter et al., “Infections with Various Types of Organisms Stimulate Transcription From a Short Promoter Fragment of the Potato gst1 Gene,” Mol. Plant-Microbe Interact. 9:68–73 (1996), which is hereby incorporated by reference). Therefore, it is possible that both gst1:hrpN and gst1:signal sequence:hrpN constructs may also confer resistance against ascomycete, virus, and nematode infection, as well as mycorrhization.
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.
All of the references designated as being incorporated herein by reference are intended to be incorporated in their entirety unless specific portions thereof have been identified with particularity.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/178,565, filed Jan. 26, 2000, which is hereby incorporated by reference in its entirety.
This invention was made in part with support by the U.S. Government under Grant No. 97-34367-3937 from the U.S. Department of Agriculture. The U.S. Government may have certain rights in this invention.
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| 60178565 | Jan 2000 | US |
