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WO1999050423A2 - Genes de la voie de l'acide salicylique et leur utilisation pour induire la resistance chez les vegetaux - Google Patents

Genes de la voie de l'acide salicylique et leur utilisation pour induire la resistance chez les vegetaux Download PDF

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Publication number
WO1999050423A2
WO1999050423A2 PCT/EP1999/002176 EP9902176W WO9950423A2 WO 1999050423 A2 WO1999050423 A2 WO 1999050423A2 EP 9902176 W EP9902176 W EP 9902176W WO 9950423 A2 WO9950423 A2 WO 9950423A2
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Prior art keywords
plants
isochorismate
gene
ics
gene coding
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PCT/EP1999/002176
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WO1999050423A3 (fr
Inventor
Hubertus Josephus Maria Linthorst
Robert Verpoorte
Maria Catharina Verberne
Paulo R. H. Moreno
Leonardus Johannes Petronella Van Tegelen
George Joseph Wullems
Anton Felix Croes
Maarten Hendrik Stuiver
Jerôme Hubertina Henricus Victor CUSTERS
Lambertus Henricus Simons
Leo Sjoerd Melchers
John Ferdinand Bol
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Universiteit Leiden
Mogen International NV
Radboud Universiteit Nijmegen
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Universiteit Leiden
Mogen International NV
Radboud Universiteit Nijmegen
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Priority to BR9909303-0A priority Critical patent/BR9909303A/pt
Priority to AU36025/99A priority patent/AU746787B2/en
Priority to MXPA00009573A priority patent/MXPA00009573A/es
Priority to CA002333433A priority patent/CA2333433A1/fr
Priority to EP99917919A priority patent/EP1066389A2/fr
Priority to JP2000541311A priority patent/JP2003513608A/ja
Publication of WO1999050423A2 publication Critical patent/WO1999050423A2/fr
Publication of WO1999050423A3 publication Critical patent/WO1999050423A3/fr
Anticipated expiration legal-status Critical
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8239—Externally regulated expression systems pathogen inducible
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • This invention is related to genes n the salicylic acid biosynthesis pathway, more specifically in the salicylic acid pathway through lsochoris ic acid, and their use for the induction of resistance through salicylic acid in plants. More specifically the invention is related to the use of isochorismate synthase (ICS) genes for the production of salicylic acid, specifically by a new plant isochorismate synthase gene, more specifically to the use of an isochorismate pyruvate lyase in addition to the isochorismate synthase. Further the invention is related to the use of the promoter of the new plant isochorismate synthase gene as a pathogen-mducible promoter.
  • ICS isochorismate synthase
  • plants Upon pathogen challenge, plants can react by raising a defense mechanism that acts both locally and systemically.
  • HR hypersensitive response
  • the local response consists of amongst others rapid cell necrosis/apoptosis, seen as the formation of lesions, and the accumulation of growth-inhibiting phenolic substances (phytoalexins) and pathogenesis-related (PR) proteins. Also cell wall reinforcement and cell wall thickening are observed. This combined, multifactorial defense response leads to the restriction of pathogen growth and spread.
  • This invention describes a method to induce pathogen resistance in plants, characterized m that plants are transformed with an expression cassette harboring a gene coding for an isochorismate synthase. More specifically, this method is characterized m that the gene coding for isochorismate synthase is selected from a group consisting of en tC, orfA, pchA and ICS, this last gene preferably the ICS gene from Ca tharan tus roseus . Genes which can be used for this method are depicted SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17.
  • Another embodiment of the invention is a method according to the method described above, characterized in that plants are additionally transformed with a vector carrying an expression cassette harboring a gene coding for an isochorismate pyruvate lyase, preferably on the same vector as the expression cassette comprising the gene coding for isochorismate synthase.
  • the gene coding for isochorismate pyruvate lyase is preferably selected from the group consisting of orfD and pchB .
  • a specific embodiment of the invention is a method as described above, characterized in that the gene coding for isochorismate synthase is entC and the gene coding for isochorismate pyruvate lyase is orfD.
  • a further aspect of the invention is a protein having isochorismate synthase activity whicn is isolated from Ca tharan t us roseus .
  • This protein has a Molecular Weight of 67 kD.
  • This protein preferably comprises the ammo acid sequence of SEQ ID NO: 19.
  • Also part of the invention is a nucleotide sequence encoding this protein, which is preferably a nucleoti ⁇ e sequence comprising the nucleotide sequence of SEQ ID NO: 18
  • a further aspect of tne invention is the nucleotide sequence comprising the 5' regulatory region which is naturally found to regulate the expression of the ICS gene m Ca tharan t us roseus .
  • This regulatory region can be used as a pathogen-mducible promoter, which can drive expression of a protein that has antifungal, antibacterial or antiviral properties. Examples of such proteins are chit ases, glucanases, osmot s, defens s, magamms, cecrop s, ⁇ bozymes.
  • Such a pathogen-mducible promoter can be used to express elicitor proteins or resistance genes for use a strategy aimed at the induction of a hypersensitive response (see WO 91/15585).
  • Vectors, Agroba cte ⁇ a , plant cells and plants which comprise or are transformed with the above-mentioned genes form further part of the invention.
  • Figure 1 Schematic respresentation of the vector pMOG22 GUS ICS
  • Figure 2 Northern blot of RNA isolated from transgenic plants with the indicated constructs (3 transgenic lines per construct) and control plants hybridized with a probe for PR-la.
  • Figure 3 Schematic map with restriction sites of the regulatory sequence of the Ca tharan tus roseus isochorismate synthase gene . DETAILED DESCRIPTION OF THE INVENTION
  • Biosyntnesis of salicylic acid in plants is normally thought to proceed via synthesis of transcmnamic acid and conversion to benzoic acid followed oy 2-hydroxylat ⁇ on .
  • the synthesis pathway may be altered slightly, using trans-cinnamic acid to convert it into ortho-couma ⁇ c acid which is then converted into salicylic acid.
  • the biosynthesis of salicylic acid is known to proceed from chorismate to isochorismate (catalyzed by the enzyme isochorismate synthase, ICS) .
  • chorismate pathway to produce salicylic acid can be introduced in plants by transformation of said plants with an expression cassette harboring a gene co ⁇ ing for isochorismate synthase.
  • This gene can either be derived from bacteria such as the PchA and the entC genes identified above, or the orfA gene from Pseudomonas fl uorescens, or from plants where the gene coding for isochorismate synthase has been found present, such as the ICS gene from Catharanthus roseus, as provided in this application.
  • Sucn genes can be isolated from bacteria or from plants Dy probing them with a degenerated probe derived from the sequences present in this invention.
  • nucleotide sequences coding for the enzymes may be changed freely as long as the resulting gene product still has isochorismate synthase activity. Changes which are apparent are changes to the codon usage to adapt it to the codon usage which is most similar to the plant to which the genes WJ.11 be transformed. Also the polynucleotide used for transformation may be modified that RNA instability encoding motifs and/or fortuitous splice regions may be removed so that expression of the thus modified polynucleotides yields substantially similar enzyme.
  • the genes of the invention encode enzymatically active proteins.
  • the word protein means a sequence of ammo acids connecte ⁇ trough peptide bonds. Polypeptides or peptides are also considered tc be proteins.
  • Mutems of the protein of the invention are proteins that are obtained from the proteins depicted in the sequence listing by replacing, adding and/or deleting one or more ammo acids, while still retaining their enzymatic activity. Such mutems can readily De made by protein engineering in vivo, e.g. by changing the open reading frame capable of encoding the enzyme such that the ammo aci ⁇ sequence is thereby affected. As long as the changes in the ammo acid sequences do not altogether abolish the enzymatical activity such mutems are embraced in the present invention.
  • mutations should be derivable from the proteins or the DNA sequences encoding these proteins depicted tne sequence listing while retaining biological activity, i.e. all, or a great part of the intermediates between the mutated protein and the protein depicted in the sequence listing should have enzymatical activity.
  • a great part would mean 30% or more of the intermediates, preferably 40% of more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 99% or more.
  • Tne present invention provides a chimeric DNA sequence which comprises an expression cassette according to the invention.
  • the term chimeric DNA sequence shall mean to comprise any DNA sequence which comprises DNA sequences not naturally found nature.
  • chimeric DNA shall mean to comprise DNA comprising the open reading frame coding for the enzyme m a non-natural location of the plant genome, notwithstanding the fact that said plant genome normally contains a copy of the said open reading frame in its natural chromosomal location.
  • the said open reading frame may be incorporated in the plant genome wherein it is not naturally found, or in a replicon or vector where it is not naturally found, such as a bacterial plasmid or a viral vector.
  • Chimeric DNA shall not be limited to DNA molecules which are replicable in a host, but shall also mean to comprise DNA capable of being ligated into a replicon, for instance by virtue of specific adaptor sequences, physically linked to the open reading frame according to the invention.
  • the open reading frame may or may not be linked to its natural upstream and downstream regulatory elements .
  • the open reading frame may be derived from a genomic library. In this latter it may contain one or more intron ⁇ separating the exon ⁇ making up the open reading frame that encodes a protein according to the invention.
  • the open reading frame may also be encoded by one uninterrupted exon, or by a cDNA to the mRNA encoding a protein according to the invention.
  • Open reading frames according to the invention also comprise those in which one or more introns have been artificially removed or added. Each of these variants is embraced by the present invention.
  • a chimeric DNA according to the invention will usually be provided with regulatory elements enabling it to be recognised by the biochemical machinery of the host and allowing for the open reading frame to be transcribed and/or translated in the host. It will usually comprise a transcriptional initiation region which may be suitably derived from any gene capable of being expressed in the host cell of choice, as well as a translational initiation region for ribosome recognition and attachment.
  • an expression cassette usually comprises in addition a transcriptional termination region located downstream of said open reading frame, allowing transcription to terminate and polyadenylation of the primary transcript to occur.
  • the codon usage may be adapted to accepted codon usage of the host of choice.
  • a signal sequence may be encoded, which is responsible for the targeting of the gene expression product to subcellular compartments.
  • the principles governing the expression of a chimeric DNA construct in a chosen host cell are commonly understood by those of ordinary skill in the art and the construction of expressible chimeric DNA constructs is now routine for any sort of host cell, be it prokaryotic or eukaryotic.
  • the open reading frame In order for the open reading frame to be maintained in a host cell it will usually be provided in the form of a replicon comprising said open reading frame according to the invention linked to DNA which is recognised and replicated by the chosen host cell. Accordingly, the selection of the replicon is determined largely by the host cell of choice. Such principles as govern the selection of suitable replicons for a particular chosen host are well within the realm of the ordinary skilled person in the art.
  • a special type of replicon is one capable of transferring itself, or a part thereof, to another host cell, such as a plant cell, thereby co-transferring the open reading frame according to the invention to said plant cell .
  • Replicons with such capability are herein referred to as vectors.
  • An example of such vector is a Ti- plasmid vector which, when present in a suitable host, such as Agrobac erium tumefaciens, is capable of transferring part of itself, the so-called T-region, to a plant cell.
  • Ti-plasmid vectors are now routinely being used to transfer chimeric DNA sequences into plant cells, or protoplasts, from which new plants may be generated which stably incorporate said chimeric DNA in their genomes.
  • a particularly preferred form of Ti- plasmid vectors are the so-called binary vectors as claimed in (EP 0 120 516 Bl and US 4,940,838).
  • Other suitable vectors which may be used to introduce DNA according to the invention into a plant host, may be selected from the viral vectors, e . g. non-integrative plant viral vectors , such as derivable from the double stranded plant viruses (e.g. Ca V) and single stranded viruses, gemini viruses and the like.
  • the use of such vectors may be advantageous, particularly when it is difficult to stably transform the plant host. Such may be the case with woody species, especially trees and vines.
  • host cells incorporating a chimeric DNA sequence according to the invention in their genome shall mean to comprise cells, as well as ulticellular organisms comprising such cells, or essentially consisting of such cells, which stably incorporate said chimeric DNA into their genome thereby maintaining the chimeric DNA, and preferably transmitting a copy of such chimeric DNA to progeny cells, be it through mitosis or meiosis .
  • plants are provided, which essentially consist of cells which incorporate one or more copies of said chimeric DNA into their genome, and which are capable of transmitting a copy or copies to their progeny, preferably in a Mendelian fashion.
  • chimeric DNA By virtue of the transcription and translation of the chimeric DNA according to the invention in some or all of the plant's cells, those cells that produce the enzyme will show enhanced resistance to pathogen infections.
  • the principles as indicated above govern transcription of DNA in plant cells are not always understood, the creation of chimeric DNA capable of being expressed in substantially a constitutive fashion, that is, in substantially most cell types of the plant and substantially without serious temporal and/or developmental restrictions, is now routine.
  • promoters obtainable from the cauliflower mosaic virus, notably the 35S RNA and 19S RNA transcript promoters and the so-called T-DNA promoters of Agrrojbacterium tumefaciens, in particular to be mentioned are the nopaline synthase promoter, octopine synthase promoter (as disclosed in EP 0 122 791 Bl) and the mannopine synthase promoter.
  • plant promoters may be used, which may be substantially constitutive, such as the rice actin gene promoter, or e. g. organ-specific, such as the root-specific promoter RolD, or the potato tuber specific patatin promoter.
  • inducible promoters may be used which enable induction of pathogen resistance by an external factor, which can be applied at a time point which is most suitable. Thus it prevents unwanted effects, such as for instance can occur due to the relative toxicity of the salicylic acid.
  • Inducible promoters include any promoter capable of increasing the amount of gene product produced by a given gene, in response to exposure to an inducer. In the absence of an inducer the DNA sequence will not be transcribed.
  • the factor that binds specifically to an inducible promoter to activate transcription is present in an inactive form which is then directly or indirectly converted to the active form by the inducer.
  • the inducer may be a chemical agent such as a protein, metabolite (sugar, alcohol, etc.), a growth regulator, herbicide, or a phenolic compound or a physiological stress imposed directly by heat, salt, wounding, toxic elements etc., or indirectly through the action of a pathogen or disease agent such as a virus.
  • a plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell such as by spraying, watering, heating, or similar methods.
  • Inducible promoters are known to those familiar with the art and several exist that could conceivably be used to drive expression of the genes of the invention.
  • Inducible promoters suitable for use in accordance with the present invention include, but are not limited to, the heat shock promoter, the mammalian steroid receptor system and any chemically inducible promoter.
  • inducible promoters include the inducible 70 kD heat shock promoter of Drosophila melanogaster (Freeling, M. et al . , Ann. Rev. Genet. 19, 297-323) and the alcohol dehydrogenase promoter which is induced by ethanol (Nagao, R.T. et al . , in: iflin, B.J. (ed.) Oxford Surveys of Plant Molecular and Cell Biology, Vol. 3., pp. 384-438, Oxford Univ. Press, 1986).
  • a promoter that is inducible by a simple chemical is particularly useful.
  • Examples for the last category are the promoters described in WO 90/08826, WO 93/21334, WO 93/031294 and WO 96/37609.
  • the PRP1 promoter also named gstl promoter
  • the Fisl promoter WO 96/34949
  • the Bet v 1 promoter Swoboda, I., et al . , Plant, Cell and Env. 18., 865-874, 1995
  • the Vstl promoter Fischer, R. , Dissertation, Univ.
  • high-level promoters are the light- inducible ribulose bisphosphate carboxylase small subunit (rbcSSU) promoter and the chlorophyll a/b binding protein (Cab) promoter.
  • hybrid promoters which comprise elements of different promoter regions physically linked.
  • CaMV enhanced mannopine synthase promoter US Patent 5,106,739, which comprises elements of the mannopine synthase promoter linked to the CaMV enhancer.
  • targeting of the enzymes to organelles in the plant cell can enhance the production of salicylic acid.
  • the substrate for the enzymes of the invention is abundant in special organelles.
  • a signal peptide derived from tobacco yields good results.
  • signal peptides obtained from other sources can be used.
  • a transcriptional terminator region it is generally believed that such a region enhances the reliability as well as the efficiency of transcription in plant cells. Use thereof is therefore strongly preferred in the context of the present invention.
  • transgenic tobacco and Arabidopsis plants as an example, the actual applicability being in fact not limited to these plant species.
  • Any plant species that is subject to some form of pathogen attack may be transformed with genes according to the invention, allowing the enzyme (s) to be produced in some or all of the plant's cells.
  • Methods may suitably be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., Nature 296, 72-74, 1982; Negrutiu I. et al, , Plant Mol. Biol . 8, 363-373, 1987), electroporation of protoplasts (Shillito R.D. et a2., Bio/Technol. 3 . , 1099-1102, 1985), microinj ection into plant material (Crossway A. et al . , Mol. Gen. Genet. 202, 179-185, 1986), DNA (or RNA-coated) particle bombardment of various plant material (Klein T.M. et al .
  • a preferred method according to the invention comprises Agrohacterium-mediated DNA transfer.
  • Especially preferred is the use of the so-called binary vector technology as disclosed in EP A 120 516 and U.S. Patent 4,940,838.
  • Tomato transformation is preferably done essentially as described by Van Roekel et al . (Plant Cell Rep. 12, 644-647, 1993) .
  • Potato transformation is preferably done essentially as described by Hoekema et al. (Hoekema, A. et al . , Bio/Technology 7, 273-278, 1989).
  • plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant expressible genes co-transferred with the nucleic acid sequence encoding the protein according to the invention, whereafter the transformed material is regenerated into a whole plant.
  • monocotyledonous plants are amenable to transformation and fertile transgenic plants can be regenerated from transformed cells or embryos, or other plant material.
  • preferred methods for transformation of monocots are microprojectile bombardment of embryos, explants or suspension cells, and direct DNA uptake or electroporation (Shimamoto, et al , Nature 338, 274-276, 1989) .
  • Transgenic maize plants have been obtained by introducing the
  • Streptomyces hygroscopicus bar-gene which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin) , into embryogenic cells of a maize suspension culture by microprojectile bombardment (Gordon-Ka m, , Plant Cell, 2 , 603-618, 1990).
  • Gordon-Ka m a m, Plant Cell, 2 , 603-618, 1990.
  • the introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee, Plant Mol. Biol. 13 . , 21-30, 1989).
  • Monocotyledonous plants including commercially important crops such as rice and corn are also amenable to DNA transfer by Agrobacterium strains ( vide WO 94/00977; EP 0 159 418 Bl; Gould J, et al . , Plant. Physiol. 95, 426-434, 1991).
  • putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the chimeric DNA according to the invention, copy number and/or genomic organization.
  • expression levels of the newly introduced DNA may be undertaken, using Northern and/or Western analysis, techniques well known to persons having ordinary skill in the art.
  • transformed plants showing the desired copy number and expression level of the newly introduced chimeric DNA according to the invention may be tested for resistance levels against pathogens.
  • the selected plants may be subjected to another round of transformation, for instance to introduce further genes, in order to enhance resistance levels, or broaden the resistance.
  • the transformed plants may be grown directly, but usually they may be used as parental lines in the breeding of new varieties or in the creation of hybrids and the like.
  • transgenic plants capable of constitutively expressing more than one chimeric gene
  • a number of alternatives are available including the following:
  • A. The use of DNA, e .g a T-DNA on a binary plasmid, with a number of modified genes physically coupled to a selectable marker gene.
  • the advantage of this method is that the chimeric genes are physically coupled and therefore migrate as a single Mendelian locus.
  • B. Cross-pollination of transgenic plants each already capable of expressing one or more chimeric genes, preferably coupled to a selectable marker gene, with pollen from a transgenic plant which contains one or more chimeric genes coupled to another selectable marker.
  • the seed which is obtained by this crossing, maybe selected on the basis of the presence of the two selectable markers, or on the basis of the presence of the chimeric genes themselves.
  • the plants obtained from the selected seeds can afterwards be used for further crossing.
  • the chimeric genes are not on a single locus and the genes may therefore segregate as independent loci.
  • the actual strategy may depend on several considerations as maybe easily determined such as the purpose of the parental lines (direct growing, use in a breeding programme, use to produce hybrids) but is not critical with respect to the described invention.
  • plants already containing chimeric DNA capable of encoding an enzyme of the isochorismatic pathway may form a suitable genetic background for introducing chimeric DNA according to the invention, for instance in order to enhance the production of salicylic acid, thereby enhancing the induction capability and thereby enhancing resistance levels.
  • the cloning of other genes corresponding to proteins that can suitably be used in combination with DNA, and the obtention of transgenic plants, capable of relatively over-expressing same, as well as the assessment of their effect on pathogen resistance in planta, is now within the scope of the ordinary skilled person in the art.
  • Plants, or parts thereof, which relatively over-express salicylic acid according to the invention, including plant varieties, with improved resistance against pathogens may be grown in the field, in the greenhouse, or at home or elsewhere. Plants or edible parts thereof may be used for animal feed or human consumption, or may be processed for food, feed or other purposes in any form of agriculture or industry. Agriculture shall mean to include horticulture, arboriculture, flower culture, and the like. Industries which may benefit from plant material according to the invention include but are not limited to the pharmaceutical industry, the paper and pulp manufacturing industry, sugar manufacturing industry, feed and food industry, enzyme manufacturers and the like.
  • Plants for the purpose of this invention shall mean multicellular organisms capable of photosynthesis, and subject to some form of pathogen attack. They shall at least include angiosperms as well as gymnosperms, monocotyledonous as well as dicotyledonous plants .
  • plants which relatively over-express an enzyme shall mean plants which contain cells expressing a transgene-encoded enzyme which is either not naturally present in said plant, or if it is present by virtue of an endogenous gene encoding an identical enzyme, not in the same quantity, or not in the same cells, compartments of cells, tissues or organs of the plant.
  • a further aspect of the invention is the regulatory sequence naturally occurring in the 5' untranslated region of the ICS-gene from Catharanthus roseus. It has been found that upon pathogen infection the ICS gene is highly expressed, indicating pathogen inducibility. Pathogen inducible promoters (such as the prpl-promoter described above) are of great value in biotechnological resistance engineering. Examples of proteins that may be used in combination with the
  • ICS regulatory region include, but are not limited to, ⁇ -1, 3-glucanases and chitinases which are obtainable from barley (Swegle M. et al . , Plant Mol. Biol. 12, 403-412, 1989; Balance G.M. et al . , Can. J. Plant Sci. 56, 459-466, 1976 ; Hoj P.B. et al . , FEBS Lett. 230, 67-71, 1988; Hoj P.B. et al . , Plant Mol. Biol. 13, 31- 42, 1989), bean (Boiler T. et al . , Planta 157, 22-31, 1983; Broglie K.E.
  • proteins which play a role in the gene-for-gene resistance interaction are, for example, plant proteins such as disclosed in Karrer, E.E. et al . (Plant Mol. Biol. 36, 681-690, 1998), ndrl and edsl, Cf-proteins and Pto proteins from tomato, the avr-elicitor proteins from Cladosporium fulvu , and the avrPto protein from Pseudomonas .
  • a clone harboring plasmid pMOG 1431 containing a 3kb insert which contains the ICS regulatory region according to the inventions was deposited under number 101670 with the Centraal Bureau voor Schimmelcultures at Baarn, the Netherlands on March 19, 1999.
  • Subsequently transformed plants are evaluated for the presence of the desired properties and/or the extent to which the desired properties are expressed.
  • a first evaluation may include the level of expression of the newly introduced genes, the level of salicylic acid expressed, the level of induction of pathogen-related proteins, the pathogen resistance of the transformed plants, stable heritability of the desired properties, field trials and the like.
  • the transformed plants can be crossbred with other varieties, for instance varieties of higher commercial value or varieties in which other desired characteristics have already been introduced, or used for the creation of hybrid seeds, or be subject to another round of transformation and the like.
  • the incubation mixture (total volume 250 ⁇ l) contained 0.1 M Tris-HCl pH 7,5, 2mM chorismate, 10 mM MgCl , and enzyme extract (crude extracts 125 ⁇ l, column fractions 10-100 ⁇ l) .
  • the incubation was started by addition of chorismate. After incubation for 60 min at 30°C the reaction was stopped by the addition of 62.5 ⁇ l methanol- isobutanol (1:1 v/v) .
  • the samples were centrifuged and analyzed by HPLC according to Poulsen, C. et al . Phytochem. 30 . ' 2873-2878, 1991).
  • the filtrate was concentrated and desalted using a tangential flow ultrafiltration unit (Provario, PAL- Filtron, Breda, The Netherlands) equipped with a 30 kD cut-off membrane.
  • a tangential flow ultrafiltration unit Provario, PAL- Filtron, Breda, The Netherlands
  • 30 kD cut-off membrane To the desalted extract solid ammonium sulfate was added to 30% saturation. After stirring for 20 min the precipitated protein was removed by centrifugation at 10,000g for 30 min. Additional ammonium sulfate was added to the supernatant up to 60% saturation. The precipitated protein was collected by centrifugation at 10,000g for 30 min.
  • the pellet was dissolved in 50 ml buffer A [20mM triethanolamine- HC1 pH 7.5, 10% (v/v) glycerol, 1 mM DTT and 0.2 mM PMSF] , and solid KC1 was added to a final concentration of 2M. Ammonium sulfate precipitation yielded good and reproducible fractionation without substantial loss of ICS activity. After centrifugation at 13,000g for 15 min, the supernatant was applied to a Phenyl Sepharose CL-4B column (72 ml, 2.6 x 13.5 cm) equilibrated in buffer B (A buffer + 2M KC1) .
  • ICS activity was eluted with a 700 ml linear gradient from buffer B to A, followed by 150 ml buffer A, with a flow of 1 ml/min. Fractions of 10 ml were collected. Fractions containing ICS activity were pooled and concentrated using the ultrafiltration unit. The concentrate was desalted by gel filtration over Sephadex G-25 columns (PD-10 columns, Pharmacia, Uppsala) equilibrated in buffer A and applied to a 20 ml BlueA column. After application the flow was stopped for one half-hour to allow binding. The column was washed with 40 ml buffer A, and ICS was eluted with a 160 ml gradient from buffer A to 50% buffer B.
  • ICS activity was eparated into two peaks (ICS I and ICS II).
  • the specific activities were increased 532- and 754-fold relative to the crude extract for ICS I and II, respectively.
  • ICS I and II had an activity ratio of 1 to 2, a number that was found in several independent purifications. Re-injection of either ICS resulted in the occurrence of only the injected ICS in the chromatogram.
  • Native PAGE of the MonoQ fractions showed that ICS I still contained some impurity whereas ICS II was obtained in a pure form.
  • SDS-PAGE of ICS II revealed that this protein is about 67 kD.
  • Both isoforms showed an identical pH dependency with a broad pH optimum between 7.0 and 9.0, and 50% of the maximal activity at pH 6.5 2+ and 10.
  • the presence of Mg was essential for product formation.
  • the protein band containing ICS II was isolated from a native PAGE gel and digested with trypsm, which yielded about 50 peptides. Five peptides were isolated and sequenced. One of these peptides displayed high homology to bacterial isochorismate synthase sequences.
  • a degenerate primer was developed against this peptide.
  • PCR on a cDNA library of elicited cell cultures of C. roseus using this primer and the T7 primer of pBluescript yielded a fragment of 520 bp. This fragment was cloned and sequenced.
  • a 440 bp fragment of the amplified DNA was used to screen the cDNA library of elicited C. roseus cell cultures. Screening of 450,000 plaques identified 52 independent positive plaques. Twelve of these were isolated and subjected to a second screening using the same 440 bp probe. This resulted in the identification of 7 independent positive plaques. These were in vivo excised and partially sequenced.
  • the longest clone had inserts of 2.1 kb and contained the ATG initiation codon.
  • the region around the first ATG (TCCAATGGC) closely resembles the consensus translation initiation sequence plants (Lutcke et al . EMBO J., 6, 43-48, 1987).
  • the cDNA with a complete length of 2081 bp contained an open reading frame of 1743 nucleotides encoding a protein of 581 ammo acids.
  • the calculated molecular mass was 64 kD and the isoelectric point 7.88.
  • the protein is roughly 30% identical (40% homologous) with isochorismate synthases from bacteria with most homology in the C-termmal region.
  • the ICS cDNA was cloned between the EcoRI and Xhol sites in pBluesc ⁇ pt II SK(Stratagene, CA USA) .
  • a 2 kbp BamHI-XhoI fragment containing the entire cDNA was ligated into the vector pIC-20H, digested with Bglll and Sail.
  • a further partial Hindlll digestion released the 2 kbp fragment, and this was cloned into vector pMOG843 digested with Hindlll. This places the ICS coding sequences downstream from the 35S CaMV promoter and preceding the potato PI-II terminator sequences.
  • the plasmid is named pMOG843-ICS.
  • a 35S CaMV promoter-GUS-nos terminator cassette was introduced into binary vector pMOG22. This was done by digestion of pMOGlOl with Xbal and EcoRI which releases the 2.6 kbp fragment containing the expression cassette, and ligation of this fragment into pMOG22 digested with Xbal and EcoRI. The resulting vector is pMOG22-GUS. Subsequently the ICS expression cassette was cloned into pMOG22-GUS. This was done by partially digestion of pMOG843-ICS with Xbal and ligating the 3.2 kbp fragment into pMOG22-GUS digested with Xbal.
  • the resulting plasmid is pMOG22-GUS-ICS .
  • the binary vector pMOG22-GUS-ICS was mobilized into Agrobacterium tumefaciens strain LBA4404 using tri-parental mating. Tobacco transformation was performed essentially as described (Horsch et al . , Science 227, 1229-1231, 1985) using hygromycm as a selectable marker.
  • the 35S-entC-PI cassette was then mobilized into pIC20H by Xbal digestion and cloning into the Xbal site of pIC20H.
  • a partial Xbal digest of pIC20H was used. Therefore, the cassette is in the Xbal site flanked by the EcoRV site.
  • the resultant vector is denoted pIC20H-entC
  • a chloroplast transit peptide (denoted ss) (Mazur and Chui, Nucl. Acids Res. 13, 2373-2386, 1985) was isolated from tobacco genomic DNA using primers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4) .
  • Primer 3 contains a Kpnl site that was used to introduce the transit peptide in front of the entC gene.
  • Vector pIC20H-entC was digested with Ncol, followed by blunting of the sticky sites, and then digestion with Kpnl.
  • the PCR product was digested with Kpnl and cloned into this vector.
  • the resulting vector pIC20H-entC+ss contains the transit peptide m frame with the entC coding sequences, lacking the first 36 nucleotides of the coding sequence of entC.
  • the encoded truncated entC is still fully active.
  • the orfD sequence from Pseudomonas fl uorescens was amplified using primers 5 (SEQ ID NO: 5) and 7 (SEQ ID NO: 7).
  • primers 6 SEQ ID NO: 6
  • the Rubisco chloroplast targeting signal was amplified from tobacco genomic DNA using primers 3 and 8 (SEQ ID NO: 8) .
  • the resulting PCR fragment has the orfD sequences fused m-frame to the transit peptide. This is denoted as orfD+ss .
  • Both the orfD and the orfD+ss PCR products were digested with Kpnl and BamHI and ligated into pMOG843B digested with Kpnl and BamHI.
  • the resulting expression cassettes were mobilized into binary vector pMOG800 using the EcoRI and Xbal sites. This resulted in two binary vectors denoted pMOG800-orfD and pMOG800-orfD+ss . Finally, entC sequences were added.
  • Vector pIC20H-entC+ss was digested with Xbal and Seal and the Xbal fragment containing the en C+ss expression cassette was ligated into Xbal digested pMOG800, pMOG ⁇ OO- orfD and pMOG800-orfD+ss to make the following constructs: pMOG800-entC+ss pMOG800-entC+ss + orfD pMOG800-entC+ss + orfD+ss.
  • Transgenic Samsun NN tobacco plants were grown under 16 h light regimes at 23-25°C. Leaf samples of these primary transformants were harvested and kept at -80°C until further use.
  • Isochorismate synthase activity was measured as described (Poulsen et al., Phytochem. 30, 2873-2876, 1991).
  • a fluorescence detector and integrator were linked to the HPLC to allow quantification of SA (see below) .
  • the emission wavelength detector was set at 407 nm, the excitation wavelength is 305 nm.
  • the flow rate employed was 0.9 ml/ minute.
  • TMV Tobacco Mosaic Virus
  • Transgenic tobacco plants transformed with the bacterial entC and/or orfD constructs were tested for their ability to inhibit spread of a plant pathogenic virus.
  • Three plants per construct, 8 plants per line and 3 leaves per plant were inoculated with a suspension containing 1 ⁇ g/ml TMV.
  • tobacco transgenic P12 plants were included in this assay. Inoculation was done by rubbing the plants with carborundum powder and the virus suspension. After inoculation the leaves were rinsed with water to remove the carborundum powder again. Lesion size (8 lesions per leaf) was measured at 2 , 4 and 7 days after inoculation.
  • the lesion size in the plants is expressed as the percentage of the lesion size determined in the tobacco P12 control plants .
  • Table 2 Representation of the lesion diameter measured in the transgenic tobacco plants relative to the lesion diameter measured on the P12 control plants .
  • tobacco primary transformants were selected for analysis of increased resistance to fungal infection.
  • the following lines were selected: entc+orfd SB 4, entc+orfd BB 16 and entc+orfd ss 20.
  • non-transgenic control lines wt/Nt/ssnn-1 and -2
  • Plants of 6 weeks old, 7 or 8 plants per line were taken. Plants originating from primary transformant entc+orfdssl6 were smaller in size compared to the non-transgenic control plants and the other transgenic lines.
  • the plants were inoculated with the tomato fungal pathogen Oidium lycopersicon by spraying a spore suspension of 3.5x104 sp/ml (total volume of 400 ml) .
  • the plants were tested at a temperature of 20°C, a relative humidity (RH) of 80% and a 16h light/8h dark regime.
  • Disease severity was determined by measuring the percentage of leaf area covered by powdery mildew. Disease severity was scored at 13 days, 18 days and 24 days after inoculation.
  • the Tl progeny of the transgenic lines were not selected for the presence of the genes of interest. So the population tested may have segregating T-DNA loci and therefore also segregation of resistance can be observed (as in line entc+orfd ss 4 ) .
  • RNA was isolated from 0.5 gram of leaf material.
  • the RNA was extracted by grinding the leaf material in liquid nitrogen and extraction with 0.5 ml of a buffer containing 0.35M glycin, 0.048 M NaOH, 0.34 M NaCl, 0.04 M EDTA and 4% SDS.
  • the preparation was extracted subsequently with water saturated solutions of phenol/chloroform (1:1, v:v), phenol and phenol/chloroform.
  • To the aqueous phase half a volume of 8M LiCl was added and the sample was stored overnight at 4°C. After centrifugation, the pellet was washed with 70% ethanol and dissolved in water.
  • the gel was blotted upon Hybond-N+ nylon transfer membrane, cross- linked and baked for 2 hours at 80°C (see fig. 2) .
  • a 450 bp Pstl fragment was used as a PRla probe. It was labeled by random-prime labeling using 32P-dCTP. The blot was hybridized overnight and subsequently washed with 2 x SSC, 01.% SDS at 65°C. Exposure was for 3 days at -80°C, using an mtensifier screen. Procedures are as described m Femberg and Vogelstem, Anal. Biochem 137, 266-267, 1984; Cornelissen, B. et al . , Nucl . Acids Res. 17, 6799- 6811, 1987; Payne et al . , Plant Mol. Biol. 11, 89-94, 1988; Pfitzner et al . , Mol. Gen. Genet. 211, 290-295, 1988.
  • Results are shown in Table 4. In TMV-mduced plants and transformed plants containing en tC+ss + orfD+ss accumulation of PR-la transcript is apparent.
  • RNA extraction from infected leaf tissue and cDNA synthesis RNA extraction from infected leaf tissue and cDNA synthesis.
  • Poly-A+ RNA was harvested from 100 mg of leaf tissue using the
  • FR-pUC- 257 (SEQ ID NO: 9) 5' ATA GAA ACG AGG ACA CTT CCA CGT TAA GGG ATT TTG G 3'
  • FR-pUC- 258 (SEQ ID NO: 10) 5' ATA AGC ACG GAT TAA TGG GCC GGA GCT GAA TGA AGC C 3'
  • FR-ICS-255 (SEQ ID NO: 11) 5' ATA GAA ACG AGG ACA CTT CC 3'
  • FR-ICS-256 (SEQ ID NO: 12) 5' ATA AGC ACG GAT TAA TGG GC 3' .
  • Primers FR-pUC-257 and FR-pUC-258 were used to amplify a fragment of 527 bp from the plasmid pUC18 (Yamsch-Perron, C. et al . , Gene 33, 103-119, 1985) by PCR. From this PCR product 1 ⁇ l was amplified using primers FR-ICS-255 and FR-ICS-256 by PCR to produce a large amount of PCR MIMIC. Primers FR-ICS-255 and FR-ICS-256 will amplify a band of 443 bp from the ICS cDNA so it can be distinguished easily from the 527 bp MIMIC band when seperated on a 1.5% agarose gel.
  • PCR MIMIC dilutions were made in a range of 10 ng/ ⁇ l to 0.1 ag/ ⁇ l m H20 containing 0.2 ⁇ g/ ⁇ l glycogen as a carrier.
  • the cDNA samples were analysed in a competitive PCR. Therefore 2 ⁇ l of the cDNA samples was combined in a 0.5 ml tube with 1 ⁇ l diluted MIMIC (amounts: 0.1 pg, 10 fg, 1 fg and 0.1 fg) or no MIMIC. Amplification of cDNA and MIMIC was performed using 10 ⁇ M of the primers FR-ICS-255 and FR-ICS-256, 0.5 ⁇ l of 20 M dNTP's, lx PCR buffer, MgC12 and 2.5 units Recombinant Taq DNA polymerase (Gibco BRL) and was allowed to proceed for 35 cycles, 1' 95°C, 1' 55°C, 2' 72°C.
  • Table 5 Induction levels of the ICS messenger after infection of C. roseus leaves with P. cactorum relative to the control.
  • Unmfected leaf area is the area of the inoculated leaf not infected by the fungus
  • Isolation of the isochorismate synthase promoter from Catharanthus roseus by iPCR PCR primers were developed based on the sequence of the ICS cDNA (SEQ ID NO: 18).
  • C. roseus genomic DNA was isolated using a CTAB DNA extraction procedure.
  • the genomic DNA was subjected to restriction enzyme digestion with five different enzymes, Dde I, Kpn I, Msc I, Nco I and Nla IV. After restriction enzyme digestion the DNA was extracted with phenol/chloroform/isoamylalcohol and precipitated with ethanol . The DNA pellet was dissolved n 50 ⁇ l water and 25 ⁇ l was used for furer iPCR. For this purpose the volume of the digested DNA mixture was increased to 300 ⁇ l lx ligase buffer (Gibco BRL) with 5 units of T4 DNA ligase (Gibco BRL) . This mixture was incubated at 16°C for 16 hours. After ligation the DNA was again extracted with phenol/chloroform/isoamylalcohol and precipitated with ethanol and dissolved in 50 ⁇ l water.
  • restriction enzyme digestion the DNA was extracted with phenol/chloroform/isoamylalcohol and precipitated with ethanol dissolved in 50 ⁇ l water.
  • the resulting PCR bands from the Kpn I, Nco I and Nla IV digestions were cloned into the T/A cloning vector pGEM-T (Promega) .
  • the DNA sequences of the inserts were determined.
  • New PCR primers were developed based on the DNA sequence of the cloned PCR fragments. These primers were located at the far upstream part of the promoter and at the ATG translational startcodon of the ICS open reading frame.
  • Primer FR-ICS-295 5'GCA AGC TTC ATG TAC CTT ATC TTG GCC3' (SEQ ID NO: 23) is located at the upstream end of the promoter and introduces a Hind III restriction site and primer FR- ICS-296 5 'TAG ATG CCA TGG GAT GGG AG3' (SEQ ID NO: 24) is located at the startcodon of the ICS ORF introducing a Nco I restriction site overlapping the ATG translational start.
  • primers 150 ng were used in a PCR reaction on C. roseus genomic DNA in lx Klentaq PCR buffer, 10 ⁇ M dNTP's and 2.0 ⁇ l 50x Advantage cDNA polymerase mix (Clontech, Palo Alto, CA, USA) .
  • the complete reaction mixture 100 ⁇ l was subjected to 1' at 94°C and 30 cycles of 30" 94°C, 1' 55°C, 4' 68°C.
  • a band of the correct size (3.0 Kb) was isolated from an agarose gel, purified and cloned using restriction enzymes Hind III and Nco I into a high copy cloning vector based on pUC18 (Yanisch-Perron, C, Vieira, J. and Messing, J. (1985) Gene 33, 103-119) forming plasmid pMOG 1431.
  • the DNA sequence of the complete promoter fragment was determined using automated DNA sequencing (SEQ ID NO: 25 ) .
  • the promoter was cut out using Hind III and Nco I and ligated into a Hind III, Nco I digested cloning vector containing GUSintron (Jefferson et al .
  • Example 10 Transformation of the ICS promoter-GUS binary vector to potato pMOG 1433 was transformed to potato essentially as described by Hoekema et al . (Hoekema, A. et al . , Bio/Technology 7, 273-278, 1989).
  • potatoes Solanum tuberosum cv. Kardal
  • the basic culture medium was MS30R3 medium consisting of MS salts (Murashige and Skoog (1962) Physiol. Plant. 14, 473), R3 vitamins (Ooms et al . (1987) Theor. Appl . Genet.
  • the tuber discs were washed with MS30R3 medium and transferred to solidified postculture medium (PM) .
  • PM consisted of M30R3 medium supplemented with 3.5 mg/1 zeatin riboside and 0.03 mg/1 indole acetic acid (IAA) .
  • IAA indole acetic acid
  • the tuber discs were transferred to shoot induction medium (SIM) which consisted of PM medium with 250 mg/1 carbenicillin and 100 mg/1 kanamycin.
  • SIM shoot induction medium
  • Example 11 Testing of promoter function in transgenic potato plants Transgenic potato plants harbouring the pMOG1433 ICS promoter-GUS construct were grown in tubes in vitro and assayed for expression of the GUS gene. For this purpose leaf, stem and root samples were taken and stained (results in table 7). GUS expression levels were determined visually, on a scale of 0 to 5, where 0 is no detectable expression and 5 is the highest level of GUS we have observed in leaves of a transgenic plant, of a rare tobacco 35S-GUS-transgenic (line 96306) . Samples from leaves of this plant were included in all experiments for internal reference.
  • Table 7 Expression of the GUS gene driven by the ICS promoter in leaves, stems and roots of small in vitro plantlets.
  • Table 8 Expression of the GUS gene driven by the ICS promoter in leaves of potato in vi tro plantlets infected by P. infestans
  • Promoter performance was also tested in the leaves of full grown potato plants before and after infection with P. infestans. Before inoculation leaves were detached and stained for expression of GUS. The plants were then sprayed with a spore suspension of 5x105 spores/ml and the infection was allowed to develop for 4 days (96 hours) . Again leaves were detached and stained for the expression of GUS. GUS expression levels were scored in the lesion, primary zone and in the uninfected part of the leaf (background) . The results are listed in table 9.
  • Table 9 Expression of the GUS gene driven by the ICS promoter m leaves of transgenic potato plants before and after infection with P. infestans .

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Abstract

L'invention concerne une technique qui permet d'induire la résistance aux pathogènes chez les végétaux, caractérisée par le fait que lesdits végétaux sont transformés avec une cassette d'expression hébergeant un gène codant pour une isochorismate synthase. Plus spécifiquement, cette technique est caractérisée par le fait que le gène codant pour ladite synthase est choisi dans un groupe comprenant entC, orfA, pchA et ICS, ce dernier étant de préférence le gène ICS de Catharantus roseus. Dans un autre mode de réalisation, l'invention concerne une technique conforme à la technique ci-dessus, caractérisée par le fait que les végétaux sont en outre transformés avec une cassette d'expression hébergeant un gène codant pour un isochorismate pyruvate lyase, de préférence situé sur la même cassette d'expression que le gène codant pour l'isochorismate synthase. Le gène codant pour l'isochorismate pyruvate lyase est de préférence choisi dans le groupe comprenant orfD et pchB. Un autre aspect de l'invention est une protéine présentant une activité isochorismate synthase, isolée à partir de Catharantus roseus. Un autre aspect de l'invention est la séquence nucléotidique comprenant la région régulatrice 5' qui naturellement régule l'expression du gène ICS chez Catharantus roseus.
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BR9909303-0A BR9909303A (pt) 1998-03-31 1999-03-25 Processo para induzir resistência a patógeno em plantas, proteìna que tem a atividade de isocorismato sintase, sequência de nucleotìdeo, promotor indutìvel por patógeno, uso do mesmo, vetor, cepa de agrobacterium, células de plantas, e, plantas
AU36025/99A AU746787B2 (en) 1998-03-31 1999-03-25 Salicylic acid pathway genes and their use for the induction of resistance in plants
MXPA00009573A MXPA00009573A (es) 1998-03-31 1999-03-25 Isocorismato sintasa y su uso para la induccion de resistencia en las plantas.
CA002333433A CA2333433A1 (fr) 1998-03-31 1999-03-25 Genes de la voie de l'acide salicylique et leur utilisation pour induire la resistance chez les vegetaux
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JP2000541311A JP2003513608A (ja) 1998-03-31 1999-03-25 イソコリスミ酸シンターゼおよび植物における耐性の誘導のためのその使用

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ES2199931T3 (es) * 1989-03-24 2004-03-01 Syngenta Participations Ag Plantas transgenicas resistentes a enfermedades.

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* Cited by examiner, † Cited by third party
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WO2002006447A3 (fr) * 2000-07-18 2002-05-10 Gen Hospital Corp Genes de biosynthese d'acide salicylique et utilisations associees
US7070772B2 (en) 2000-07-18 2006-07-04 The General Hospital Corporation Salicylic acid biosynthetic genes and uses thereof
US7141723B2 (en) 2001-01-29 2006-11-28 Cargill, Incorporated Transgenic plants resistant to Sclerotinia and Phoma lingam
WO2013176548A1 (fr) 2012-05-25 2013-11-28 Wageningen Universiteit Nouveau gène de résistance pour plantes
CN116286866A (zh) * 2023-02-28 2023-06-23 福建农林大学 水稻基因OsFd4在水稻抗白叶枯病中的应用
WO2025027165A1 (fr) * 2023-08-01 2025-02-06 Basf Plant Science Company Gmbh Résistance accrue par expression d'une protéine ics

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CN1295619A (zh) 2001-05-16
MA24793A1 (fr) 1999-10-01
BR9909303A (pt) 2000-11-21
MXPA00009573A (es) 2003-04-22
AR018172A1 (es) 2001-10-31
AU3602599A (en) 1999-10-18
EP1066389A2 (fr) 2001-01-10
PL343635A1 (en) 2001-08-27
PE20000358A1 (es) 2000-04-27
AU746787B2 (en) 2002-05-02
CA2333433A1 (fr) 1999-10-07
CO5050251A1 (es) 2001-06-27
JP2003513608A (ja) 2003-04-15
WO1999050423A3 (fr) 1999-12-16

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