EP1187910A1 - Procedes de renforcement de la defense de vegetaux - Google Patents

Procedes de renforcement de la defense de vegetaux

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Publication number
EP1187910A1
EP1187910A1 EP00921952A EP00921952A EP1187910A1 EP 1187910 A1 EP1187910 A1 EP 1187910A1 EP 00921952 A EP00921952 A EP 00921952A EP 00921952 A EP00921952 A EP 00921952A EP 1187910 A1 EP1187910 A1 EP 1187910A1
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European Patent Office
Prior art keywords
nucleic acid
seq
plant
plants
acid molecule
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EP00921952A
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German (de)
English (en)
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Clarence A. Ryan
Martha O. Cardenas
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University of Washington
Washington State University Research Foundation
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University of Washington
Washington State University Research Foundation
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Publication of EP1187910A1 publication Critical patent/EP1187910A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/38—Solanaceae [Potato family], e.g. nightshade, tomato, tobacco or chilli pepper
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)

Definitions

  • This invention relates to methods of enhancing the natural defensive responses of plants against pathogens, viruses and herbivores.
  • PG has been studied in fruit, abscission zones, and pollen for many years, but it had not been previously identified in leaves.
  • the PG enzyme can liberate pectic fragments from the plant cell wall thereby activating plant defense genes.
  • PG-liberated cell wall fragments are signals that activate active oxygen species (AOS) during pathogenesis
  • AOS is a key signaling component for inducing plant defenses against pathogens (Doke, N. et al. (1996), Gene, 179, 45-51; Lamb, C. and Dixon, R.A. (1997), Annu. Rev. Plant Physiol. Mol. Biol., 48, 251-275; Low, P.S. and Merida, J.R. (1996), Physiol. Plant., 96, 533-542; Levine, A. et al., Cell, 79, 583-593; Draper, J. (1997), Trends in Plant Sci., 2, 162-165.).
  • AOS signaling pathway
  • AOS active oxygen species
  • O 2 and H 2 O 2 frequently called the 'oxidative burst' (Lamb, C. and Dixon, R.A. (1997), Annu. Rev. Plant Physiol. Mol. Biol, 48, 251-275; Low, P.S. and Merida, J.R. (1996), Physiol. Plant., 96, 533-542.).
  • This burst occurs in two phases, a very rapid increase followed by a slower, prolonged response.
  • AOS production has been used as one gauge of the ability of the plant to respond successfully to pathogen attacks.
  • the oxidative burst serves to drive crosslinking of cell wall structural elements and to trigger cell death and induction of defense proteins (Levine, A. et al., Cell, 79, 583-593.).
  • AOS is generated by the activation of a NADPH oxidase that is at the cell surface (Lamb, C. and Dixon, R.A. (1997), Annu. Rev. Plant Physiol. Mol. Biol, 48, 251-275) producing O that is spontaneously dismutated to the more stable active oxygen species, H 2 O .
  • SA salicylic acid
  • NADPH oxidase gene has been identified in plants that bears homology to the enzyme involved in the oxidative burst that accompanies damage or infection in animals (Keller, T. et al. (1998), The Plant Cell, 10, 255-266).
  • Systemin is an 18 amino acid polypeptide, isolated from the leaves of tomato plants, that induces the synthesis of numerous wound-inducible proteins, such as wound- inducible proteinase inhibitor proteins.
  • Systemin is synthesized as part of a larger, 200 amino acid precursor protein called prosystemin.
  • prosystemin The present inventors have newly discovered that systemin induces an active oxygen species response, and that plants that overexpress prosystemin have an augmented active oxygen species response.
  • the present invention provides methods of augmenting plant defenses, in particular augmenting the generation of active oxygen species (AOS), against predator attack that include the steps of (a) introducing a nucleic acid molecule that encodes a protein selected from the group consisting of systemin, prosystemin and polygalacturonase into plant cells; (b) regenerating plants from the plant cells treated in accordance with step (a); and (c) screening the regenerated plants to identify regenerated plants having an augmented active oxygen species response.
  • the plants having an augmented AOS response can be further propagated and screened, for example to generate populations of plants (such as genetically true-breeding plant lines) having an augmented AOS response.
  • nucleic acid molecules that encode systemin, prosystemin or polygalacturonase are cDNA molecules or genomic DNA molecules.
  • nucleic acid molecules encoding systemin, prosystemin or polygalacturonase are cDNA molecules isolated from solanaceous plants, such as tomato and potato.
  • nucleic acid molecules, encoding systemin, prosystemin or polygalacturonase proteins, utilized in the practice of the present invention are under the control of a constitutive promoter, such as the CaMN 35S promoter.
  • the regenerated plants are screened for augmented AOS response by utilizing a colorimetric assay for AOS that is based on the oxidation of a solution of 3,3 diaminobenzidine (DAB), as set forth in Thordal-Christensen, H. et al. (1997), Plant, ]., 11, 1187-1194, which publication is incorporated herein by reference.
  • DAB 3,3 diaminobenzidine
  • the methods of the present invention can be utilized, for example, to augment the endogenous defense responses of plants, i.e., to enhance the production of a plant's own chemicals and proteins, such as active oxygen species and polygalacturonase, that are involved in mounting a defense response against pests, pathogens and/or herbivores.
  • Representative examples of plants that can be treated in accordance with the present invention include tomato, potato and bell pepper plants. Detailed Description of the Preferred Embodiment
  • amino acid and “amino acids” refer to all naturally occurring L- -amino acids or their residues.
  • the amino acids are identified by either the single-letter or three-letter designations:
  • nucleotide means a monomeric unit of D ⁇ A or R ⁇ A containing a sugar moiety (pentose), a phosphate and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (1' carbon of pentose) and that combination of base and sugar is called a nucleoside.
  • the base characterizes the nucleotide with the four bases of DNA being adenine ("A"), guanine ("G”), cytosine ("C”) and thymine (“T”).
  • Inosine (“I") is a synthetic base that can be used to substitute for any of the four, naturally-occurring bases (A, C, G or T).
  • the four RNA bases are A,G,C and uracil (“U”).
  • the nucleotide sequences described herein comprise a linear array of nucleotides connected by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.
  • Oligonucleotide refers to short length single or double stranded sequences of deoxyribonucleotides linked via phosphodiester bonds.
  • the oligonucleotides are chemically synthesized by known methods and purified, for example, on polyacrylamide gels.
  • protein useful in the practice of the present invention refers to proteins such as systemin, prosystemin and polygalacturonase, capable of augmenting one or more plant defense responses, preferably the production of polygalacturonase and/or active oxygen species.
  • systemin refers to the 18 amino acid inducer of wound- inducible proteins (such as wound-inducible proteinase inhibitors) purified from tomato plant leaves and disclosed in Pearce, G. et al., Science 253: 895-898 (1991), and to functional homologs of the systemin protein, disclosed in the foregoing Pearce et al publication, that also induce the synthesis of wound-inducible proteins.
  • wound- inducible proteins such as wound-inducible proteinase inhibitors
  • prosystemin refers to the 200 amino acid precursor of systemin disclosed in McGurl, B. et al., Science 255: 1570-1573 (1992), and to functional homologs of the prosystemin protein disclosed in the foregoing McGurl et al publication.
  • polygalacturonase means an enzyme that catalyzes the hydrolysis of glycosidic linkages of polymerized galacturonic acid residues. Polygalacturonase (PG) activity can be measured, for example, as described in Pressey, R., Hort. Sci. 21:490-492 (1986), which publication is incorporated herein by reference.
  • active oxygen response refers to the production in plants of chemically reactive oxygen species, such as O 2 and H 2 O , in response to cellular damage (and/or changes in cells) caused by pests, pathogens and/or herbivores.
  • the active oxygen species can act as signalling molecules or as effector molecules that harm or kill pests, pathogens and/or herbivores that attack plants.
  • predator includes herbivorous, multicellular organisms that consume plants, and microorganisms, including viruses and fungi, that infect plants.
  • the term "augmenting the active oxygen species response of a plant”, and grammatical equivalents thereof, means augmenting (by a quantifiable, statistically significant amount, such as by greater than a two-fold, five-fold or ten-fold increase) the active oxygen species response in plants treated in accordance with the methods of the present invention compared to control plants that were not treated in accordance with the methods of the present invention.
  • An example of a control group of plants are plants of the same variety as the variety that was treated, in accordance with the methods of the present invention, to produce plants having augmented active oxygen species response.
  • both groups are preferably grown under conditions that are substantially similar or identical.
  • alteration refers to protein molecules with some differences in their amino acid sequences as compared to the corresponding, native, i.e., naturally-occurring, proteins. Ordinarily, the variants will possess at least about 70% homology with the corresponding native protein, and preferably, they will be at least about 80% homologous with the corresponding, native protein.
  • the amino acid sequence variants of the proteins (such as systemin, prosystemin and polygalacturonase) useful in the practice of the present invention possess substitutions, deletions, and/or insertions at certain positions. Sequence variants of proteins may be used to attain desired enhanced or reduced enzymatic activity, modified regiochemistry or stereochemistry, or altered substrate utilization or product distribution.
  • Substitutional protein variants are those that have at least one amino acid residue in the native protein sequence removed and a different amino acid inserted in its place at the same position.
  • the substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
  • Substantial changes in the activity of the proteins useful in the practice of the present invention may be obtained by substituting an amino acid with a side chain that is significantly different in charge and/or structure from that of the native amino acid. This type of substitution would be expected to affect the structure of the polypeptide backbone and/or the charge or hydrophobicity of the molecule in the area of the substitution.
  • Moderate changes in the activity of the protein molecules useful in the present invention would be expected by substituting an amino acid with a side chain that is similar in charge and/or structure to that of the native molecule.
  • This type of substitution referred to as a conservative substitution, would not be expected to substantially alter either the structure of the polypeptide backbone or the charge or hydrophobicity of the molecule in the area of the substitution.
  • Insertional protein variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in the native protein molecule. Immediately adjacent to an amino acid means connected to either the -carboxy or ⁇ -amino functional group of the amino acid. The insertion may be one or more amino acids. Ordinarily, the insertion will consist of one or two conservative amino acids.
  • Insertional protein variants also include insertion of an amino acid with a charge and/or structure that is substantially different from the amino acids adjacent to the site of insertion.
  • Deletional variants are those where one or more amino acids in the native protein molecules have been removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the protein molecule.
  • DNA sequence encoding refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the translated polypeptide chain. The DNA sequence thus codes for the amino acid sequence.
  • replicable vector refers to a piece of DNA, usually double-stranded, which may have inserted into it another piece of DNA (the insert DNA) such as, but not limited to, a cDNA molecule.
  • the vector is used to transport the insert DNA into a suitable host cell.
  • the insert DNA may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of or coincidental with the host chromosomal DNA, and several copies of the vector and its inserted DNA may be generated.
  • the terms “replicable expression vector” and “expression vector” refer exclusively to vectors that contain the necessary elements that permit the expression of a polypeptide encoded by the insert DNA. Many molecules of the polypeptide encoded by the insert DNA can thus be rapidly synthesized.
  • transformed host cell refers to the introduction of DNA into a cell.
  • the cell is termed a "host cell”, and it may be a prokaryotic or a eukaryotic cell.
  • Typical prokaryotic host cells include various strains of E. coli.
  • Typical eukaryotic host cells are plant cells, such as tomato cells, yeast cells, insect cells or animal cells.
  • the introduced DNA is usually in the form of a vector containing an inserted piece of DNA.
  • the introduced DNA sequence may be from the same species as the host cell or from a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign DNA and some DNA derived from the host species.
  • the present invention provides methods for augmenting plant defenses, in particular the generation of active oxygen species (AOS), against predator attack.
  • the methods each include the steps of (a) introducing a nucleic acid molecule that encodes a protein selected from the group consisting of systemin, prosystemin and polygalacturonase into plant cells; (b) regenerating plants from the plant cells treated in accordance with step (a); and (c) screening the regenerated plants to identify regenerated plants having an augmented active oxygen species response.
  • the plants having an augmented AOS response can be further cultured and screened to generate populations of plants (such as genetically true-breeding plant lines) having an augmented AOS response.
  • nucleic acid molecules that encode systemin, prosystemin or polygalacturonase are cDNA molecules or genomic DNA molecules.
  • nucleic acid molecules encoding prosystemin are prosystemin cDNA molecules isolated from solanaceous plants (i.e., members of the family Solanaceae), including tomato (Lycopersicon esculentum), potato (Solanum tuberosum), black nightshade (Solanum nigrum) and bell pepper (Capsicum annuum), such as those described in McGurl, B.
  • nucleic acid molecules that encode prosystemin and that are useful in the practice of the present invention, hybridize to the tomato prosystemin cDNA, having the sequence set forth in SEQ ID NO:l, or to the complementary sequence of the sequence set forth in SEQ ID NO:l, under conditions of 2 X SSC, 55°C (preferably 2 X SSC, 62°C), more preferably under conditions of 1 X SSC, 55°C (most preferably 1 X SSC, 62°C).
  • Hybridization can be carried out by utilizing, for example, the technique of hybridizing radiolabelled nucleic acid probes to nucleic acids immobilized on nitrocellulose filters or nylon membranes as set forth, for example, at pages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual (2nd edition), J. Sambrook, E.F. Fritsch and T. Maniatis eds, the cited pages of which are incorporated herein by reference.
  • nucleic acid molecules that are useful in the practice of the present invention encode a prosystemin protein molecule that has at least 70% amino acid sequence identity, more preferably at least 80% amino acid sequence identity, with the tomato prosystemin protein sequence set forth in SEQ ID NO:2.
  • Sequence identity is detemined by aligning the tomato prosystemin protein sequence set forth in SEQ ID NO:2 with another sequence and introducing gaps, if necessary, to achieve the maximum percent identity, and not considering any conservative substitutions as part of the sequence identity.
  • N- or C- terminal extensions nor insertions shall be construed as reducing identity. No weight is given to the number or length of gaps introduced, if necessary, to achieve the maximum percent identity.
  • Amino acid sequence identity can be determined, for example, in the following manner.
  • the tomato prosystemin protein sequence set forth in SEQ ID NO:2 can be used to search a nucleic acid sequence database, such as the Genbank database, using the program TBLASTN version 2.0.9 (Altschul et al., Nucleic Acids Research 25: 3389-3402 (1997)).
  • the program is used in the ungapped mode.
  • Default filtering is used to remove sequence identities due to regions of low complexity as defined in Wootton, J.C. and S. Federhen, Methods in Enzymology 266: 554-571 (1996).
  • nucleic acid molecules encoding systemin and/or prosystemin can be isolated, for example, by the polymerase chain reaction strategy described in Constabel et al., Plant Molecular Biology 36:55-62 (1998), which publication is incorporated herein by reference.
  • Any nucleic acid molecule encoding a functional polygalacturonase protein is useful in the practice of the present invention.
  • nucleic acid molecules encoding a functional polygalacturonase protein useful in the practice of the present invention are disclosed in: Allen, R.L. and Lonsdale, D.M., Plant Mol. Biol. 20:343-345 (1992); Brown S.M. and Crouch M.L., Plant Cell 2:263-274 (1990) and Robert L.S. et al., Plant Mol. Biol. 23:1213-121 & (1993), each of which publications is incorporated herein by reference.
  • nucleic acid molecules encoding polygalacturonase are polygalacturonase cDNA molecules isolated from solanaceous plants, such as tomato and potato.
  • tomato cDNA molecules encoding polygalacturonase include the tomato fruit polygalacturonase cDNA molecule disclosed in Grierson et al., Nucleic Acids Research 14:8595-8601 (1986) and the tomato polygalacturonase cDNA encoding an mRNA expressed during abscission reported in Kalaitzis et al., Plant Molecular Biology 28:647-656 (1995), each of which publications is incorporated herein by reference.
  • DNA molecules encoding polygalacturonase useful in the practice of the present invention include the DNA sequences having the following GenBank Accession Numbers: Q02096, P35336, P05117, P35337, Q05967, P24548, P26216 and X72291, each of which nucleic acid sequences is incorporated herein by reference.
  • nucleic acid molecules encoding polygalacturonase proteins can be isolated by using cloned polygalacturonase cDNAs or genes as hybridization probes utilizing, for example, the technique of hybridizing radiolabelled nucleic acid probes to nucleic acids immobilized on nitrocellulose filters or nylon membranes as set forth, for example, at pages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual (2nd edition), J. Sambrook, E.F. Fritsch and T. Maniatis eds, the cited pages of which are incorporated herein by reference.
  • nucleic acid molecules encoding polygalacturonase proteins can be identified by the presence of one or more conserved amino acid sequence domains in the carboxyl terminal half of the polygalacturonase protein, such as those described at page 341 of Hadfield K.A. and Bennett A.B., Plant Physiology, 117:337-343 (1998), and in Lester et al., Plant Physiol. 105:225-231 (1994), Jenkins E.S. et al., J. Exp. Bot. 47:111-115 (1996), Petersen et al., Plant Mol. Biol, 31:517-527 (1996) and Hadfield et al., Plant Physiol.
  • nucleic acid molecule encoding a functional systemin is useful in the practice of the present invention.
  • the amino acid sequences of representative examples of functional systemin molecules are set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
  • Hybridization can be carried out by utilizing, for example, the technique of hybridizing radiolabelled nucleic acid probes to nucleic acids immobilized on nitrocellulose filters or nylon membranes as set forth, for example, at pages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual (2nd edition), J. Sambrook, E.F. Fritsch and T. Maniatis eds, the cited pages of which are incorporated herein by reference.
  • nucleic acid molecules, encoding systemin, prosystemin or polygalacturonase proteins, utilized in the practice of the present invention are under the control of a constitutive promoter, such as the CaMV 35S promoter.
  • Routine genetic manipulation techniques can be utilized to add a transport sequence to the amino terminal end of proteins useful in the practice of the present invention.
  • transport sequences well known in the art are disclosed, for example, in the following publications, the cited portions of which are incorporated by reference herein: von Heijne et al., Eur. J. Biochem., 180:535-545, 1989; Stryer, Biochemistry, W.H. Freeman and Company, New York, NY, p. 769 [1988].
  • Transport sequences may be employed to direct proteins useful in the practice of the present invention to a variety of cellular or extracellular locations.
  • Sequence variants, produced by deletions, substitutions, mutations and/or insertions, of the proteins useful in the practice of the present invention can also be used in the methods of the present invention.
  • the amino acid sequence variants of the proteins useful in the practice of the present invention may be constructed by mutating the DNA sequences that encode the wild-type proteins, such as by using techniques commonly referred to as site-directed mutagenesis.
  • Nucleic acid molecules encoding the proteins useful in the practice of the present invention can be mutated by a variety of PCR techniques well known to one of ordinary skill in the art. (See, for example, the following publications, the cited portions of which are incorporated by reference herein: "PCR Strategies", M.A. Innis, D.H. Gelfand and J.J.
  • the two primer system utilized in the Transformer Site-Directed Mutagenesis kit from Clontech may be employed for introducing site-directed mutants into nucleic acid molecules encoding proteins useful in the practice of the present invention.
  • two primers are simultaneously annealed to the plasmid; one of these primers contains the desired, site-directed mutation, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site.
  • Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli.
  • Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli.
  • This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids.
  • the tight linkage of the two mutations and the subsequent linearization of unmutated plasmids results in high mutation efficiency and allows minimal screening. Following synthesis of the initial restriction site primer, this method requires the use of only one new primer type per mutation site.
  • a set of "designed degenerate" oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously.
  • Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be fully sequenced or restricted and analyzed by electrophoresis on Mutation Detection Enhancement gel (J.T. Baker) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control).
  • the two primer system utilized in the QuikChangeTM Site-Directed Mutagenesis kit from Stratagene may be employed for introducing site-directed mutants into nucleic acid molecules encoding proteins useful in the practice of the present invention.
  • Double-stranded plasmid DNA containing the insert bearing the target mutation site, is denatured and mixed with two oligonucleotides complementary to each of the strands of the plasmid DNA at the target mutation site.
  • the annealed oligonucleotide primers are extended using Pfu DNA polymerase, thereby generating a mutated plasmid containing staggered nicks.
  • the unmutated, parental DNA template is digested with restriction enzyme Dpnl which cleaves methylated or hemimethylated DNA, but which does not cleave unmethylated DNA.
  • the parental, template DNA is almost always methylated or hemimethylated since most strains of E. coli, from which the template DNA is obtained, contain the required methylase activity.
  • the remaining, annealed vector DNA incorporating the desired mutation(s) is transformed into E. coli.
  • a non-conservative substitution e.g., Ala for Cys, His or Glu
  • the properties of the mutagenized protein are then examined with particular attention to the kinetic parameters of K m and k cat as sensitive indicators of altered function, from which changes in binding and/or catalysis per se may be deduced by comparison to the native enzyme. If the residue is by this means demonstrated to be important by activity impairment, or knockout, then conservative substitutions can be made, such as Asp for Glu to alter side chain length, Ser for Cys, or Arg for His.
  • hydrophobic segments it is largely size that is usefully altered, although aromatics can also be substituted for alkyl side chains. Changes in the normal product distribution can indicate which step(s) of the reaction sequence have been altered by the mutation. Modification of the hydrophobic pocket can be employed to change binding conformations for substrates and result in altered regiochemistry and/or stereochemistry.
  • site directed mutagenesis techniques may also be employed with nucleic acid molecules encoding proteins useful in the practice of the present invention.
  • restriction endonuclease digestion of DNA followed by ligation may be used to generate deletion variants of proteins useful in the practice of the present invention, as described in Section 15.3 of Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ⁇ d., Cold Spring Harbor Laboratory Press, New York, NY (1989), incorporated herein by reference.
  • a similar strategy may be used to construct insertion variants, as described in section 15.3 of Sambrook et al., supra.
  • Oligonucleotide-directed mutagenesis may also be employed for preparing substitution variants of proteins useful in the practice of the present invention. It may also be used to conveniently prepare the deletion and insertion variants of proteins useful in the practice of the present invention.
  • This technique is well known in the art as described by Adelman et al. (DNA 2:183 [1983]); Sambrook et al., supra; "Current Protocols in Molecular Biology", 1991, Wiley (NY), F.T. Ausubel, R. Brent, R.E. Scientific, D.D. Moore, J.D. Seidman, J.A. Smith and K. Struhl, eds., incorporated herein by reference.
  • oligonucleotides of at least 25 nucleotides in length are used to insert, delete or substitute two or more nucleotides in the nucleic acid molecules encoding proteins useful in the practice of the present invention.
  • An optimal oligonucleotide will have 12 to 15 perfectly matched nucleotides on either side of the nucleotides coding for the mutation.
  • the oligonucleotide is annealed to the single-stranded DNA template molecule under suitable hybridization conditions.
  • a DNA polymerizing enzyme usually the Klenow fragment of E. coli DNA polymerase I, is then added.
  • This enzyme uses the oligonucleotide as a primer to complete the synthesis of the mutation-bearing strand of DNA.
  • a heteroduplex molecule is formed such that one strand of DNA encodes the wild-type protein inserted in the vector, and the second strand of DNA encodes the mutated form of the protein inserted into the same vector.
  • This heteroduplex molecule is then transformed into a suitable host cell.
  • Mutants with more than one amino acid substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than ten amino acids, for example) it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted.
  • the oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.
  • An alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: wild- type protein DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated.
  • the second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations.
  • the oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis.
  • This resultant DNA can be used as a template in a third round of mutagenesis, and so on.
  • plants are genetically transformed with one or more nucleic acids encoding one or more proteins capable of augmenting one or more plant defense responses, such as the production of polygalacturonase and/or active oxygen species.
  • Transgenic plants can be obtained, for example, by transferring plasmids that encode a protein useful in the practice of the present invention and a selectable marker gene, e.g., the kan gene encoding resistance to kanamycin, into Agrobacterium tumifaciens containing a helper Ti plasmid as described in Hoeckema et al., Nature, 303:179-181 (1983) and culturing the Agrobacterium cells with leaf slices, or other tissues or cells, of the plant to be transformed as described by An et al., Plant Physiology, 81:301-305 (1986).
  • Transformation of cultured plant host cells is normally accomplished through Agrobacterium tumifaciens.
  • other methods for introducing DNA into cells such as Polybrene (Kawai and Nishizawa, Mol. Cell. Biol, 4:1172 [1984]), protoplast fusion (Schaffner, Proc. Natl. Acad. Sci. USA, 77:2163 [1980]), electroporation (Neumann et al., EMBO J., 1:841 [1982]), and direct microinjection into nuclei (Capecchi, Cell, 22:479 [1980]) may also be used.
  • Transformed plant calli may be selected through the selectable marker by growing the cells on a medium containing, e.g., kanamycin, and appropriate amounts of phytohormone such as naphthalene acetic acid and benzyladenine for callus and shoot induction. The plant cells may then be regenerated and the resulting plants transferred to soil using techniques well known to those skilled in the art.
  • a representative protocol for the genetic transformation of potato is set forth in Kumar, A., et al., Plant J. 9: 821-829 (1996), and a representative protocol for the genetic transformation of tomato is set forth in Filatti, J.J., et al., Bio/Technology 5: 726-730 (1987), each of which publications are incorporated herein by reference.
  • a gene encoding a protein useful in the practice of the present invention can be incorporated into a plant along with a promoter, such as a constitutive promoter (such as the Cauliflower Mosaic Virus 35S promoter), or a tissue-specific promoter, or an inducible promoter.
  • a promoter such as a constitutive promoter (such as the Cauliflower Mosaic Virus 35S promoter), or a tissue-specific promoter, or an inducible promoter.
  • a responsive promoter system that can be used in the practice of this invention is the glutathione-S-transferase (GST) system in maize. GSTs are a family of enzymes that can detoxify a number of hydrophobic electrophilic compounds that often are used as pre-emergent herbicides (Weigand et al., Plant Molecular Biology, 7:235-243 [1986]).
  • the GSTs are directly involved in causing this enhanced herbicide tolerance. This action is primarily mediated through a specific 1.1 kb mRNA transcription product.
  • maize has a naturally occurring quiescent gene already present that can respond to external stimuli and that can be induced to produce a gene product. This gene has previously been identified and cloned.
  • the promoter is removed from the GST responsive gene and attached to a nucleic acid encoding a protein useful in the practice of the present invention that previously has had its native promoter removed.
  • This engineered gene is the combination of a promoter that responds to an external chemical stimulus and a gene responsible for successful production of a protein useful in the practice of the present invention.
  • Representative examples include electroporation-facilitated DNA uptake by protoplasts in which an electrical pulse transiently permeabilizes cell membranes, permitting the uptake of a variety of biological molecules, including recombinant DNA (Rhodes et al., Science, 240(4849):204-207 [1988]); treatment of protoplasts with polyethylene glycol (Lyznik et al., Plant Molecular Biology, 13:151-161 [1989]); and bombardment of cells with DNA-laden microprojectiles which are propelled by explosive force or compressed gas to penetrate the cell wall (Klein et al., Plant Physiol.
  • Transformation of gymnosperm species can be achieved, for example, by employing the methods set forth in Han et al, Plant Science, 95:187-196 (1994), incorporated by reference herein.
  • a method that has been applied to Rye plants is to directly inject plasmid DNA, including a selectable marker gene, into developing floral tillers (de la Pena et al., Nature 325:274-276 (1987)).
  • plant viruses can be used as vectors to transfer genes to plant cells.
  • the aforementioned publications disclosing plant transformation techniques are incorporated herein by reference, and minor variations make these technologies applicable to a broad range of plant species.
  • DNA from a plasmid is genetically engineered such that it contains not only the gene of interest, but also selectable and screenable marker genes.
  • a selectable marker gene is used to select only those cells that have integrated copies of the plasmid (the construction is such that the gene of interest and the selectable and screenable genes are transferred as a unit).
  • the screenable gene provides another check for the successful culturing of only those cells carrying the genes of interest.
  • a commonly used selectable marker gene is neomycin phosphotransferase II (NPT II). This gene conveys resistance to kanamycin, a compound that can be added directly to the growth media on which the cells grow.
  • Plant cells are normally susceptible to kanamycin and, as a result, die.
  • the presence of the NPT II gene overcomes the effects of the kanamycin and each cell with this gene remains viable.
  • Another selectable marker gene which can be employed in the practice of this invention is the gene which confers resistance to the herbicide glufosinate (Basta).
  • a screenable gene commonly used is the ⁇ -glucuronidase gene (GUS). The presence of this gene is characterized using a histochemical reaction in which a sample of putatively transformed cells is treated with a GUS assay solution. After an appropriate incubation, the cells containing the GUS gene turn blue.
  • the plasmid containing one or more of these genes is introduced into either plant protoplasts or callus cells by any of the previously mentioned techniques. If the marker gene is a selectable gene, only those cells that have incorporated the DNA package survive under selection with the appropriate phytotoxic agent. Once the appropriate cells are identified and propagated, plants are regenerated. Progeny from the transformed plants must be tested to ensure that the DNA package has been successfully integrated into the plant genome.
  • Prokaryotes may also be used as host cells for the initial cloning steps of proteins useful in the practice of the present invention. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated.
  • Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli X1776 (ATCC No. 31,537), and E. coli B; however many other strains of E.
  • coli such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts.
  • Prokaryotic host cells or other host cells with rigid cell walls are preferably transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells.
  • any plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell may also be used to clone, express and/or manipulate nucleic acid molecules encoding proteins useful in the practice of the present invention.
  • the vector usually has a replication site, marker genes that provide phenotypic selection in transformed cells, one or more promoters, and a polylinker region containing several restriction sites for insertion of foreign DNA.
  • Plasmids typically used for transformation of E. coli include pBR322, pUC18, pUC19, pUCI18, pUC119, and Bluescript M13, all of which are described in sections 1.12-1.20 of Sambrook et al., supra. However, many other suitable vectors are available as well. These vectors contain genes coding for ampicillin and/or tetracycline resistance which enables cells transformed with these vectors to grow in the presence of these antibiotics.
  • proteins normally secreted from the cell contain an endogenous secretion signal sequence as part of the amino acid sequence.
  • proteins normally found in the cytoplasm can be targeted for secretion by linking a signal sequence to the protein. This is readily accomplished by ligating DNA encoding a signal sequence to the 5' end of the DNA encoding the protein and then expressing this fusion protein in an appropriate host cell.
  • the DNA encoding the signal sequence may be obtained as a restriction fragment from any gene encoding a protein with a signal sequence.
  • prokaryotic, yeast, and eukaryotic signal sequences may be used herein, depending on the type of host cell utilized to practice the invention.
  • the DNA and amino acid sequence encoding the signal sequence portion of several eukaryotic genes including, for example, human growth hormone, proinsulin, and proalbumin are known (see Stryer, Biochemistry W.H. Freeman and Company, New York, NY, p. 769 [1988]), and can be used as signal sequences in appropriate eukaryotic host cells.
  • Yeast signal sequences as for example acid phosphatase (Arima et al., Nuc. Acids Res., 11:1657 [1983]), -factor, alkaline phosphatase and invertase may be used to direct secretion from yeast host cells.
  • Prokaryotic signal sequences from genes encoding, for example, LamB or OmpF (Wong et al., Gene, 68:193 [1988]), MalE, PhoA, or beta-lactamase, as well as other genes, may be used to target proteins from prokaryotic cells into the culture medium.
  • Trafficking sequences from plants, animals and microbes can be employed in the practice of the invention to direct proteins useful in the practice of the present invention to the cytoplasm, endoplasmic reticulum, mitochondria or other cellular components, or to target the protein for export to the medium. These considerations apply to the overexpression of proteins useful in the practice of the present invention, and to direction of expression within cells or intact organisms to permit gene product function in any desired location.
  • suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes and the DNA encoding proteins useful in the practice of the present invention are prepared using standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well known in the art (see, for example, Sambrook et al., supra).
  • variants of proteins useful in the practice of the present invention are preferably produced by means of mutation(s) that are generated using the method of site-specific mutagenesis.
  • This method requires the synthesis and use of specific oligonucleotides that encode both the sequence of the desired mutation and a sufficient number of adjacent nucleotides to allow the oligonucleotide to stably hybridize to the DNA template.
  • "Digestion,” “cutting,” or “cleaving” of DNA refers to catalytic cleavage of the
  • restriction endonucleases DNA with an enzyme that acts only at particular locations in the DNA. These enzymes are called restriction endonucleases, and the site along the DNA sequence where each enzyme cleaves is called a restriction site.
  • restriction enzymes used in this invention are commercially available and are used according to the instructions supplied by the manufacturers. (See also sections 1.60-1.61 and sections 3.38-3.39 of Sambrook et al., supra.)
  • DAB 3,3 diaminobenzidine
  • Diphenylene iodonium chloride (DPI, Sigma), an inhibitor of NADPH oxidase, was applied at a final concentration of 250 ⁇ M to excised plants previously incubated in
  • polygalacturonase and protein assays were conducted in the following manner. At specified times after wounding, extracts from leaves were prepared for PG assays as follows. 20 g of leaves were homogenized in a Sorvall Omnimizer (Sorvall, Du Pont Company, Wilmington, Delaware) in 60 ml of ice- cold 0.1 M sodium citrate buffer, pH 6.0, containing 1 M NaCl, 4 mM ascorbic acid, 5 mM dithiothreitol (DTT), 2% polyvinylpyrrolidone (PNP), 0.1% bovine serum albumin (BSA).
  • Sorvall Omnimizer Sorvall Omnimizer
  • PNP polyvinylpyrrolidone
  • BSA bovine serum albumin
  • the extracts were filtered through four layers of cheesecloth and incubated at 4°C for three hours, and centrifuged at 10,000g for 20 minutes at 4°C. Proteins were precipitated at 4°C by the slow addition of solid ammonium sulfate to a final concentration of 80% and stirred for one hour at 4°C. The precipitates were recovered by centrifugation at 10,000g for 20 minutes at 4°C, the supernatant was discarded, the pellet was dissolved in 20 ml of 1 M ⁇ aCl, and the solution was dialyzed against 1 M ⁇ aCl at 4°C for 24 hours. Aliquots equivalent to 0.1 g fresh weight were assayed for polygalacturonase (PG) activity. Protein was analyzed with a BCA protein assay kit (Pierce).
  • a wound-inducible polygalacturonase enzyme in tomato leaves may promote the generation of AOS
  • tomato plants were assayed for the production of AOS in leaves in response to wounding.
  • a colorimetric assay for AOS was used that is based on the oxidation of a solution of 3,3 diaminobenzidine (DAB) (as described in Thordal-Christensen, H. et al. (1997), Plant, J., 11, 1187-1194, wbich publication is incorporated herein by reference), that is supplied to excised plants or leaves at a 1 mg/ml concentration for about eight hours.
  • DAB 3,3 diaminobenzidine
  • the assay reactant for AOS, DAB turns deep brown in the presence of active oxygen species, and both the intensity of the DAB coloration and its localization can be qualitatively assessed.
  • the foregoing AOS assay disclosed in Thordal-Christensen, H. et al. (1997) was used to detect AOS in all of the subsequent examples.
  • the development of the DAB-H 2 O 2 reaction product in the tomato leaves in response to wounding can be seen as early as one hour, with the color deepening for about four to six hours, then declining. The color was initially visible at the wound site, deepened in tissue surrounding the wound site, and then appeared in the major veins throughout the plants. Unwounded leaves exhibit only low levels of PG or AOS.
  • Unwounded plants show a low, constitutive level of PG and AOS, but it increases substantially in response to wounding.
  • Example 2 Induction of AOS in Tomato Plant Leaves by Systemin and Oligogalacturonic Acid Systemin, a signal for inducing systemic wound response proteins (including PG), and the enzymatic products of the action of PG on plant cell walls, oligogalacturonic acid (OGA), were both assayed for their abilities to activate AOS production by supplying systemin and OGA to young tomato plants through their cut stems.
  • Systemin and oligogalacturonic acid were supplied to excised tomato plants through their cut stems by the feeding procedure described in Ryan, C.A., Anal. Biochem. 19: 434-440 (1967), which publication is incorporated herein by reference.
  • both systemin and the OGA fragments generated a strong AOS response in the plant leaves).
  • AOS production was particularly evident in the vasculature of plants induced with systemin.
  • DPI diphenylene iodonium chloride
  • the octadecanoid pathway is compromised because of a single mutation.
  • the defense genes of these plants are not activated in response to wounding or systemin because of the defective gene, but the plants do synthesize defense proteins in response to methyl jasmonate, a derivative of jasmonic acid (JA), the downstream product of the octadecanoid pathway (Howe, G. et al. 1996., 77 ⁇ e Plant Cell, 8, 2067-2077) that activates defense genes in vivo, including PG. Because of the mutation, the plants are compromised in their ability to defend themselves against Manduca sexta larvae, an insect predator of tomato plants.
  • JA jasmonic acid
  • the accumulation of wound-induced AOS in the leaves of various plant species in response to wounding was measured.
  • the plant leaves were wounded with a hemostat across the main vein and the assay described in Thordal-Christensen, H. et al. (1997), Plant, J., ⁇ , 1187-1194 was used to measure the AOS levels in the wounded leaves four hours after wounding.
  • All of the monocot species assayed (corn, barley, wheat and rice), which are major agricultural crops worldwide, exhibited a strong PG activity in the leaves of unwounded plants, but exhibited no AOS. When wounded, they all showed an increase in PG activity and exhibited a strong generation of AOS, suggesting that the constitutive PG did not generate AOS, but the wound-inducible PG did.
  • AOS has been shown to be a lethal antibiotic agent that can directly react with pathogens to kill them outright (Doke, N. et al. (1996), Gene, 179, 45-51). It is also possible that AOS can interact with the digestive system of herbivores to make the tissues unpalatable.
  • a second role may be to activate genes for defense, as has been hypothesized for AOS in response to pathogen attacks (Doke, N. et al. (1996), Gene, 119, 45-51; Lamb, C. and Dixon, R.A. (1997), Annu. Rev. Plant Physiol. Mol.
  • the response is systemic and maximizes at about four to six hours in both wounded and unwounded leaves, and then declines.
  • the timing of the response corresponds with an increase in wound-inducible polygalacturonase (PG) mRNA and enzyme activity previously reported, suggesting that oligogalacturonide (OGA) fragments produced by PG are triggering the active oxygen species response.
  • PG wound-inducible polygalacturonase
  • OOA oligogalacturonide
  • Systemin, oligogalacturonide fragments, chitosan and methyl jasmonate all induce the accumulation of active oxygen species in leaves.
  • Tomato plants transformed with an antisense prosystemin gene produce neither PG activity nor active oxygen species in leaves in response to wounding, implicating systemin as a primary wound signal.
  • prosystemin antisense plants do produce both PG activity and active oxygen species when supplied with systemin, OGA, chitosan or MJ.
  • a mutant tomato line (defl) compromised in the octadecanoid pathway does not exhibit PG activity or active oxygen species in response to wounding, systemin, OGA or chitosan, but does respond to MJ, indicating that the generation of active oxygen species requires a functional octadecanoid signaling pathway.

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Abstract

La présente invention se rapporte à des procédés visant à améliorer les défenses de végétaux, et notamment à accroître la génération d'espèces à oxygène actif (AOS) face à une attaque de prédateurs. Un tel procédé consiste (a) à introduire dans les cellules végétales une molécule d'acide nucléique qui code une protéine sélectionnée du groupe constitué par une systémine, une prosystémine et une polygalacturonase; (b) à régénérer des végétaux à partir des cellules végétales traitées conformément à l'étape (a); et (c) à détecter parmi les végétaux régénérés ceux qui présentent une réponse accrue de génération d'espèces à oxygène actif.
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