WO2013050611A1 - Procédé de production de plantes présentant une résistance accrue à des pathogènes - Google Patents

Procédé de production de plantes présentant une résistance accrue à des pathogènes Download PDF

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WO2013050611A1
WO2013050611A1 PCT/EP2012/069893 EP2012069893W WO2013050611A1 WO 2013050611 A1 WO2013050611 A1 WO 2013050611A1 EP 2012069893 W EP2012069893 W EP 2012069893W WO 2013050611 A1 WO2013050611 A1 WO 2013050611A1
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nucleic acid
plant
acid sequence
sequence
transgenic
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PCT/EP2012/069893
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Dimitar Douchkov
Stefanie Lueck
Patrick Schweizer
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Basf Plant Science Company Gmbh
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    • 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
    • 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
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—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

Definitions

  • the present invention relates to a method of producing a transgenic plant cell, a transgenic plant or a transgenic part thereof having an increased resistance to pathogens, wherein the content and/or activity of a WRKY transcription factor is reduced, preferably by R A interference.
  • Plant diseases which are caused by various pathogens such as viruses, bacteria and fungi, may lead to significant crop losses of cultivated plants, resulting in economic consequences and in threatening human food supply. For example, infestation of cereals with Blumeria graminis, the pathogen that causes powdery mildew, may cause yield losses of up to 30%. Since the last century, chemical fungicides have been utilised for controlling fungal diseases. A different approach is to examine the natural pathogen defence of plants against different pathogens and to use the same specifically for the production of pathogen resistant plants by gene technological manipulation, e.g. by means of introducing external resistance genes or by means of manipulating the endogenous gene expression of the plants.
  • Resistance is the ability of a plant to inhibit or at least limit any infestation or population of a pest.
  • the plants have a certain degree of natural resistance which is imparted by the formation of specific defence substances, such as isoprenoids, flavonoids, enzymes and reactive oxygen species.
  • an alternative approach for producing transgenic plants having increased fungal resistance is to inhibit the expression of said plant genes which code for example for a polyphenoloxidase (WO 02/061101), NADPH oxidase
  • Another alternative for causing resistance to pathogenic fungi is to introduce gene constructs into plants which inhibit the expression and/or activity of fungal genes that are essential for the proliferation and/or development of fungi
  • the present inventors have found that the reduction of the content of a WR Y transcription factor by RNA interference leads to an enhanced resistance of barley cells to Blumeria graminis.
  • the WRKY transcription factor superfamily consists of several members in each plant species. For example, in Arabidopsis 74 members and in rice 109 members are known. The members of this family are characterized by at least one conserved DNA binding region called the WRKY domain which comprises the highly conserved amino acid sequence WRKYGQK and a zinc finger motif (CX 4 _ 7 CX 22 - 23 HXH/C). Generally, the WRKY domain binds to a sequence motif called the W box
  • the present invention provides a method of producing a transgenic plant cell, a transgenic plant or a transgenic part thereof having an increased resistance to pathogens compared to a control plant cell, plant or plant part, wherein in the transgenic plant cell, transgenic plant or transgenic part thereof the content and/or activity of a WRKY transcription factor encoded by a nucleic acid sequence selected from the group consisting of:
  • nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences
  • nucleic acid sequence comprising a sequence which is at least 70 %
  • nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences is reduced in comparison to the control plant cell, plant or plant part.
  • the present invention provides a method for increasing pathogen resistance in a plant cell, plant or part thereof, wherein the method comprises the step of reducing the content and/or activity of a WR Y transcription factor in the plant cell, plant or part thereof compared to a control plant cell, plant or plant part, wherein the WRKY transcription factor is encoded by a nucleic acid sequence selected from the group consisting of:
  • nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences
  • nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences.
  • the method comprises the steps of
  • nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences,
  • nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence which is identical to a nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence encoding a precursor micro RNA sequence comprising a micro RNA sequence which targets the nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • the method comprises the steps of:
  • nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences,
  • the method comprises the steps of:
  • nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • the method comprises the steps of:
  • nucleic acid sequence which is identical to a nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • the method comprises the steps of:
  • a promoter functional in plant cells (i) operably linked thereto a nucleic acid sequence encoding a precursor micro R A sequence comprising a micro RNA sequence which targets the nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences; (b) optionally, regenerating a transgenic plant from the transformed cell.
  • nucleic acid sequence in (ii) or (iv) is the sequence according to SEQ ID No. 1 or 2 or a fragment thereof.
  • the promoter is a tissue-specific and/or a pathogen-inducible promoter.
  • the method further comprises reducing the content and/or activity of at least one other protein which mediates pathogen susceptibility.
  • the method further comprises the step of crossing the transgenic plant produced by the above method with another plant in which the content and/or the activity of the WRKY transcription factor as defined herein is not reduced and selecting transgenic progeny in which the content and/or the activity of the WRKY transcription factor as defined herein is reduced.
  • the method is for producing true breeding plants and comprises inbreeding the transgenic progeny of the above crossing and repeating this inbreeding step until a true breeding plant is obtained.
  • the present invention relates to a method of producing or obtaining mutant plants, plant cells or plant parts having an increased resistance to pathogens compared to control plants, plant cells or plant parts, comprising the steps of: (a) mutagenizing plant material;
  • the method for producing or obtaining mutant plants, plant cells, or plant parts having an increased resistance to pathogens compared to control plants, plant cells, or plant parts, respectively further comprises step (c) of obtaining a plant, plant cell or plant part from said plant material having at least one point mutation in the endogenous nucleic acid sequence having at least 70%>, at least 80%), at least 90%>, at least 95% or even 100% sequence identity to the nucleic acid sequence according to SEQ ID No. 1 or 2 and/or the step of (d) selecting a plant, plant cell or plant part which has an increased resistance to pathogens compared to control plants, plant cells or plant parts.
  • the transgenic or mutant plant is a monocotyledonous plant and more preferably it is a wheat or barley plant.
  • the plant has an increased resistance to a fungal pathogen, more preferably to Blumeria graminis, Septoria tritici and/or Puccinia triticina.
  • nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. l or 2 or a fragment of any of these sequences;
  • nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence which is identical to a nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence encoding a precursor micro RNA sequence comprising a micro RNA sequence which targets the nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences.
  • nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence which is complementary to at least one nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • the expression construct comprises: (a) a promoter functional in plant cells;
  • nucleic acid sequence which is identical to a nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence encoding a precursor micro RNA sequence comprising a micro RNA sequence which targets the nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences.
  • the invention in another embodiment relates to a vector comprising an expression construct as defined above.
  • a preferred embodiment is the use of an expression construct or vector as described herein for the transformation of a plant, plant part, or plant cell to provide a pathogen resistant plant, plant part, or plant cell.
  • a preferred embodiment is the use of an expression construct or a vector as described herein for increasing pathogen resistance in a plant, plant part, or plant cell compared to a control plant, plant part, or plant cell.
  • the invention relates to a transgenic or mutant plant or plant cell with an increased resistance to pathogens, produced by the method of the present invention or containing an expression construct or a vector of the present invention.
  • the invention relates to the use of the transgenic or mutant plant or parts thereof as fodder material or to produce feed material.
  • the present invention also relates to transgenic or mutant seed produced from the transgenic or mutant plant and to flour produced from said transgenic or mutant seed, wherein the presence of the transgene, expression construct or the mutation which decreases the content and/or the activity of a WRKY transcription factor as defined herein can be detected in said transgenic or mutant seed or in said flour.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
  • nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences
  • nucleic acid sequence comprising a sequence which is at least 70 %
  • nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences
  • nucleic acid sequence is able to reduce the content and/or activity of the WRKY transcription factor encoded by the nucleic acid sequence according to SEQ ID No.1 or 2 when used in a suitable expression system, but not or not considerably of other proteins the expression of which should not be reduced.
  • Figure 1 Flow diagram for the high-throughput production of RNAi constructs.
  • Figure 2 Flow diagram showing how the effect of the RNAi constructs on plant resistance to Blumeria graminis is tested.
  • transgenic means that a plant cell, plant or plant part has been altered using recombinant DNA technology to contain a nucleic acid sequence which would otherwise not be present in said plant cell, plant, or plant part or which would be expressed to a considerably lower extent.
  • the transgenic plant cell, plant or plant part contains a nucleic acid sequence selected from the group consisting of
  • nucleic acid sequence comprising a sequence which is at least 70 %
  • Natural locus means the location on a specific chromosome, preferably the location between certain genes, more preferably the same sequence background as in the original plant which is transformed.
  • the nucleic acid sequence is introduced by means of a vector.
  • the nucleic acid sequence is stably integrated into the genome of the transgenic plant.
  • the transgenic plant cell, plant or plant part of the present invention contains a nucleic acid sequence which reduces the content and/or activity of a WRKY transcription factor compared to a control plant cell, plant or plant part.
  • the transgenic plant cell, plant or plant part may contain one or more other transgenic nucleic acid sequences, for example nucleic acid sequences conferring resistance to biotic or abiotic stress and/or altering the chemical composition of the transgenic plant cell, plant or plant part.
  • transgenic does not refer to plants having alterations in the genome which are the result of naturally occurring events, such as spontaneous mutations or of induced mutagenesis followed by breeding and selection.
  • mutant means that a plant cell, plant or plant part has been altered by mutagenesis so that a nucleic acid sequence selected from the group consisting of
  • nucleic acid sequence comprising a sequence which is at least 80 %
  • nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • the mutant plant contains at least one point mutation, i.e. at least one nucleotide substitution, deletion and/or addition in comparison to a control plant, plant cell or part thereof which has been used as a starting material in the mutagenesis and which has not been mutagenized.
  • the mutant plant contains at least one nucleotide substitution in the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences.
  • the transgenic or mutant plant of the present invention may be a monocotyledonous or a dicotyledonous plant.
  • Examples of monocotyledonous plants are plants belonging to the genera Avena (oat), Triticum (wheat), Secale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, Sorghum (millet), Zea (maize), and the like.
  • Dicotyledonous useful plants comprise, inter alia, cotton, legumes, like leguminous plants and in particular alfalfa, soy bean, rape, tomato, sugar beet, potato, ornamental plants, and trees.
  • Further useful plants can comprise fruit (in particular apples, pears, cherries, grapes, citrus, pineapple, and bananas), pumpkin, cucumber, wine, oil palms, tea shrubs, cacao trees, and coffee shrubs, tobacco, sisal, as well as, with medicinal plants, rauwolfia and digitalis.
  • Particularly preferred are the cereals wheat, rye, oat, barley, rice, maize and millet, sugar beet, rape, soy, tomato, potato, cotton and tobacco.
  • Further useful plants can be taken from US 6,137,030. More preferably the transgenic or mutant plants are oat, barley, rye, wheat or rice plants and most preferably the transgenic or mutant plants are barley or wheat plants.
  • transgenic plant includes the transgenic progeny of the transgenic plant and the term “mutant plant” also includes the mutant progeny of the mutant plant.
  • the transgenic progeny of the transgenic plant comprises the nucleic acid sequence and/or the expression construct which reduces the content and/or activity of the WR Y transcription factor.
  • the mutant progeny of the mutant plant comprises at least one point mutation which reduces the content and/or activity of the WRKY transcription factor.
  • transgenic or mutant progeny of the transgenic or mutant plant may be the result of a cross of the transgenic or mutant plant with another transgenic or mutant plant of the present invention or it may be the result of a cross with a wild-type plant or a transgenic plant having a transgene other than the transgene of the present invention.
  • transgenic plant also comprises true breeding transgenic plants which are obtained by repeated inbreeding steps as described below.
  • Plant parts include, but are not limited to, stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores, and the like.
  • cell refers to a single cell and also includes a population of cells.
  • the population may be a pure population comprising one cell type. Likewise, the population may comprise more than one cell type.
  • a plant cell within the meaning of the invention may be isolated (e.g., in suspension culture) or comprised in a plant tissue, plant organ or plant at any developmental stage.
  • pathogen resistance means reducing or attenuating disease symptoms of a plant as a result of attack by a pathogen, preferably by a fungus.
  • resistance also means that pests and/or a pathogen and preferably a fungus and especially preferably the fungi described below show reduced growth in a plant and reduced or absent propagation.
  • resistance also includes a so-called transient resistance, i.e. the transgenic or mutant plants or plant cells of the present invention have an increased resistance to pests and/or pathogens or fungi compared to the transient resistance, i.e. the transgenic or mutant plants or plant cells of the present invention have an increased resistance to pests and/or pathogens or fungi compared to the
  • the term "increased pathogen resistance” is understood to denote that the transgenic or mutant plants or plant cells of the present invention are infected less severely and/or less frequently by plant pathogens.
  • the reduced frequency and the reduced extent of pathogen infection, respectively, on the transgenic plants or plant cells according to the present invention is determined as compared to the corresponding control plant.
  • an increase in resistance means that an infection of the plant by the pathogen occurs less frequently or less severely by at least 5%, preferably by at least 20%, also preferably by at least 50%>, 60%> or 70%>, especially preferably by at least 80%>, 90%> or 100%, also especially preferably by the factor 5, particularly preferably by at least the factor 10, also particularly preferably by at least the factor 50, and more preferably by at least the factor 100, and most preferably by at least the factor 1000, as compared to the control plant.
  • the pathogen resistance may be described by reference to a relative susceptibility index (SI) which compares the susceptibility of a plant of the present invention to a pathogen with the susceptibility of a control plant to said pathogen, the latter being set to 100%.
  • SI relative susceptibility index
  • the relative susceptibility index of the plants of the present invention is less than 80%, preferably less than 70 or 60%, more preferably less than 50 or 40% and most preferably less than 30%.
  • control plant When used in connection with transgenic plants, the terms “control plant”, “control plant cell” and “control plant part” refer to a plant cell, an explant, seed, plant component, plant tissue, plant organ, or whole plant used to compare against a transgenic plant which has been modified by the method of the present invention for the purpose of identifying an enhanced phenotype or a desirable trait in the transgenic plant.
  • a "control plant” may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of interest that is present in the transgenic plant being evaluated, i.e. the nucleic acid sequence reducing the content and/or the activity of a WRKY transcription factor.
  • a control plant may be a plant of the same line or variety as the transgenic plant being tested, or it may be another line or variety, such as a plant known to have a specific phenotype, characteristic, or known genotype.
  • Another suitable control plant is a genetically unaltered or non-transgenic plant of the parental line used to generate the transgenic plant of the present invention, i.e. the wild-type plant.
  • control plant When used in connection with mutant plants, the terms "control plant”, “control plant cell” and “control plant part” refer to a plant cell, an explant, seed, plant component, plant tissue, plant organ, or whole plant used to compare against a mutant plant.
  • the control plant is usually the starting material used for the mutagenization and accordingly does not comprise the at least one point mutation within the nucleic acid sequence of the present invention which is introduced by mutagenization.
  • test plants with pathogens such as fungi in order to examine potential resistance phenomena are a method well-known to those skilled in the art.
  • the test plants used must be responsive to the pathogen used, i.e. they must be able to serve as a host plant for said pathogen, and the pathogen attack must be detectable by simple means.
  • Preferred test plants are wheat or barley plants, which are, for example, inoculated with the powdery mildew fungus Blumeria graminis.
  • “Inoculating” denotes contacting the plant with the fungus the plant is to be infected with, or with infectious parts thereof, under conditions in which the fungus may enter a wild-type plant.
  • the fungal infestation of the plant may then be evaluated by means of a suitable evaluation procedure.
  • the visual inspection, in which the formed fungal structures are detected in the plant and quantified, is particularly suitable.
  • a reporter gene such as the beta-glucuronidase (GUS) gene from E. coli
  • a fluorescence gene such as the green fluorescence protein (GFP) gene from Aequorea victoria, the luciferase gene from Photinus pyralis or the beta-galactosidase (lacZ) gene from E. coli, the expression of which in the plant cells may be proven by simple methods, is co -transformed in a suitable vector with the vector mediating the inhibition of the expression of the
  • the formed fungal structures may be stained by methods well-known to those skilled in the art in order to improve the
  • the number of infected plants transformed with the nucleic acid molecule to be tested is compared to the number of infected wild-type or control plants and the degree of pathogen resistance is calculated.
  • fungal resistance may be scored by determining the symptoms of fungal infection on the infected plant, for example by eye, and calculating the diseased leaf area, The diseased leaf area is the percentage of the leaf area showing symptoms of fungal infection, such as fungal pycnidia or fungal colonies.
  • the diseased leaf area of infected plants transformed with the vector reducing the content and/or activity of the WR Y transcription factor is lower than the diseased leaf area of infected control plants.
  • plant pathogens includes viral, bacterial, fungal and other pathogens.
  • plant pathogens comprises fungal pathogens.
  • the term "plant pathogens" includes biotrophic, hemibiotrophic and necrotrophic pathogens.
  • the plant pathogen is a biotrophic pathogen, more preferably a biotrophic fungal pathogen.
  • biotrophic phytopathogenic fungi such as many rusts, depend for their nutrition on the metabolism of living cells of the plants.
  • This type of fungi belong to the group of biotrophic fungi, like other rust fungi, powdery mildew fungi or oomycete pathogens like the genus Phytophthora or Peronopora.
  • the necrotrophic fungi like other rust fungi, powdery mildew fungi or oomycete pathogens like the genus Phytophthora or Peronopora.
  • phytopathogenic fungi depend for their nutrition on dead cells of the plants, e.g. species from the genus Fusarium, Rhizoctonia or Mycospaerella. Soybean rust has occupied an intermediate position, since it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic. After the penetration, the fungus changes over to an obligatory-biotrophic lifestyle.
  • the subgroup of the biotrophic fungal pathogens which follows essentially such an infection strategy is
  • Glomerella graminicola Politis Glomerella Anthracnose stalk rot tucumanensis (anamorph: Glomerella falcatum
  • Rhizoctonia solani Kuhn Rhizoctonia
  • Brown spot black spot, stalk rot
  • Cephalosporium kernel rot Acremonium strictum Cephalosporium
  • Curvularia leaf spot Curvularia clavata, C eragrostidis, C.
  • Curvularia lunata (teleomorph: Cochliobolus lunatus)
  • Curvularia pallescens (teleomorph: Cochliobolus pallescens)
  • Curvularia senegalensis C. tuberculata (teleomorph: Cochliobolus tuberculatus)
  • Diplodia ear and stalk rot Diplodia frumenti (teleomorph: Botryosphaeria festucae)
  • Dry ear rot (cob, Nigrospora oryzae
  • kernel and stalk rot (teleomorph: Khuskia oryzae)
  • Botrytis cinerea (teleomorph:
  • Botryotinia fuckeliana Cunninghamella sp.
  • Eyespot Aureobasidium zeae Kabatiella zeae
  • Gray ear rot Botryosphaeria zeae Physalospora zeae
  • Helminthosporium root rot Helminthosporium root rot
  • Exserohilum pedicellatum Helminthosporium pedicellatum (teleomorph: Setosphaeria pedicellata)
  • Hormodendrum ear rot Cladosporium cladosporioides Disease Pathogen
  • sorokinianum H. sativum
  • Epicoccum nigrum
  • Exserohilum prolatum Drechslera prolata (teleomorph: Setosphaeria prolata)
  • Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerella herpotricha, (anamorph: Scolecosporiella sp.),
  • Penicillium ear rot blue eye, blue Penicillium spp., P. chrysogenum, P. expansum, mold
  • Phaeocytostroma stalk and root rot Phaeocytostroma ambiguum, Disease Pathogen
  • Phaeosphaeria leaf spot Phaeosphaeria maydis Sphaerulina maydis
  • Red kernel disease ear mold, leaf and Epicoccum nigrum
  • Rhizoctonia ear rot (sclerotial rot) Rhizoctonia zeae (teleomorph: Waitea
  • Root rots (minor) Alternaria alternata, Cercospora sorghi,
  • Dictochaeta fertilis Fusarium acuminatum (teleomorph: Gibberella acuminata), F. equiseti (teleomorph: G. intricans), F. oxysporum, F. pallidoroseum, F. poae, F. roseum, G.
  • cyanogena (anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp., Periconia circinata, Phytophthora cactorum, P.
  • Bipolaris maydis Helminthosporium maydis
  • Trichoderma ear rot and root rot Trichoderma viride T. lignorum teleomorph:
  • fungal pathogens or fungal-like pathogens are from the group comprising Plasmodiophoramycetes, Oomycetes, Ascomycetes, Chytridiomycetes, Zygomycetes, Basidiomycetes, and Deuteromycetes (Fungi imperfecti).
  • the fungal pathogens listed in Tables 1 and 2 as well as the diseases associated therewith are to be mentioned in an exemplary, yet not limiting manner.
  • Basidiomycetes like Typhula incarnata typhula snow mold of barley, rye, and wheat
  • Ustilago maydis corn smut
  • Ustilago nuda loose smut of barley
  • Ustilago tritici loose smut of wheat and spelt
  • Ustilago avenae loose smut of oat
  • Rhizoctonia solani taproot lesions of potatoes
  • Sphacelotheca spp. head smut of sorghum
  • Melampsora lini rust of flax
  • Puccinia graminis stem rust of wheat, barley, rye, oat
  • Puccinia recondita brown rust of wheat
  • Puccinia triticina wheat leaf rust
  • Puccinia dispersa brown rust of rye
  • Puccinia hordei brown rust of barley
  • Puccinia coronata crown rust of oat
  • Puccinia striiformis yellow rust of wheat, barley, rye, and various grasses
  • Uromyces appendiculatus bean rust
  • Phakopsora pachyrhizi Asian soybean rust
  • Sclerotium rolfsii root and stem rots of many plants.
  • Deuteromycetes (Fungi imperfecti) like Septoria nodorum (glume blotch) of wheat (Septoria tritici), Pseudocercosporella herpotrichoides (stem break disease in wheat, barley, rye), Rynchosporium secalis (scald disease in rye and barley), Alternaria solani (early blight of potato and tomato), Phoma betae (black rot of beet), Cercospora beticola (Cercospora leaf spot of beet), Alternaria brassicae (dark leaf spot of rape, cabbage and other cruciferous plants), Verticillium dahliae (Verticillium wilt and stalk rot of rape),
  • Colletotrichum lindemuthianum (bean anthracnose), Phoma lingam -phoma stem canker (black leg disease of cabbage; crown and stem canker of rape),
  • Botrytis cinerea (gray mold diseases of grapevine, strawberry, tomato, hop, etc.).
  • Phytophthora infestans (late blight of tomato, root and foot rot of tomato, etc.), Microdochium nivale (formerly Fusarium nivale; snow mold of rye and wheat), Fusarium graminearum, Fusarium culmorum (head blight of wheat), Fusarium oxysporum (Fusarium wilt of tomato), Blumeria graminis (powdery mildew of barley (f. sp. hordei) and wheat (f. sp.
  • Bacterial leaf spot Xanthomonas campestris pv. holcicola Bacterial stalk rot Enterobacter dissolvens
  • the transgenic plants produced according to the present invention are resistant to the following pathogenic bacteria: Corynebacterium sepedonicum (bacterial ring rot of potato), Erwinia carotovora (black leg rot of potato), Erwinia amylovora (fire blight of pear, apple, quince), Streptomyces scabies (common scab of potato), Pseudomonas syringae pv. tabaci (wild fire disease of tobacco), Pseudomonas syringae pv. phaseolicola (halo blight disease of dwarf bean), Pseudomonas syringae pv.
  • Corynebacterium sepedonicum bacterial ring rot of potato
  • Erwinia carotovora black leg rot of potato
  • Erwinia amylovora fire blight of pear, apple, quince
  • Streptomyces scabies common scab of potato
  • tomato ("bacterial speck” of tomato), Xanthomonas campestris pv. malvacearum (angular leaf spot of cotton), and Xanthomonas campestris pv. oryzae (bacterial blight of rice and other grasses).
  • viral pathogens includes all plant viruses, like for example tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc.
  • the pathogens listed in Table 4 as well as the diseases associated therewith are to be mentioned as viral pathogens in an exemplary, yet not limiting manner.
  • AWSMV American wheat striate American wheat striate mosaic virus
  • Corn chlorotic vein banding Corn chlorotic vein banding virus (CCVBV) (Braizilian maize mosaic)
  • MCMV Maize dwarf mosaic virus
  • MDMV Maize dwarf mosaic virus
  • WSMV Wheat streak mosaic virus
  • CMV Cucumber mosaic Cucumber mosaic virus
  • Cynodon chlorotic streak virus CCSV
  • JGMV Johnsongrass mosaic Johnsongrass mosaic virus
  • MLO Maize bushy stunt Mycoplasma-like organism
  • Maize mosaic corn leaf stripe, Maize mosaic virus (MMV)
  • Maize rayado fino fine striping Maize rayado fino virus (MRFV)
  • MRMV Maize ring mottle Maize ring mottle virus
  • MRDV Maize rough dwarf Maize rough dwarf virus
  • Maize tassel abortion Maize tassel abortion virus (MTAV)
  • MVEV Maize vein enation Maize vein enation virus
  • NMV Northern cereal mosaic Northern cereal mosaic virus
  • Oat sterile dwarf Oat sterile dwarf virus (OSDV)
  • Sorghum mosaic Sorghum mosaic virus (also: sugarcane mosaic virus (SCMV) strains H, I and M)
  • SMV Sugarcane mosaic Sugarcane mosaic virus
  • insects and nematodes can also be resistant to animal pests like insects and nematodes.
  • Insects like for example beetles, caterpillars, lice, or mites are to be mentioned in an exemplary, yet not limiting manner.
  • the plants according to the present invention are resistant to insects of the species of Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera. Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc. Insects of the following species are particularly preferred:
  • EBC European corn borer
  • Diabrotica barberi Northern corn rootworm
  • the cereal leaf beetle (Oulema melanopus), the frit fly (Oscinella frit), wireworms (Agrotis lineatus), and aphids (like for example the bird cherry-oat aphid Rhopalosiphum padi, the grain aphid Sitobion avenae).
  • pathogens listed in Table 5 as well as the diseases associated therewith are to mentioned as nematode pests in an exemplary, yet not limiting manner.
  • the transgenic plants produced according to the present invention are resistant to Globodera rostochiensis and G. pallida (cyst nematodes of potato, tomato, and other solanaceae), Heterodera schachtii (beet cyst nematodes of sugar and fodder beets, rape, cabbage, etc.), Heterodera avenae (cereal cyst nematode of oat and other types of cereal), Ditylenchus dipsaci (bulb and stem nematode, beet eelworm of rye, oat, maize, clover, tobacco, beet), Anguina tritici (wheat seed gall nematode), seed galls of wheat (spelt, rye), Meloidogyne hapla (root-knot nematode of carrot, cucumber, lettuce, tomato, potato, sugar beet, lucerne).
  • the plants according to the present invention are preferably resistant to the following pathogens: In barley, the plants are resistant to the fungal, bacterial, and viral pathogens Puccinia hordei (barley stem rust), Blumeria (Erysiphe) graminis f. sp.
  • hordei barley powdery mildew
  • Rhynchosporium secalis barley scald
  • barley yellow dwarf virus BYDV
  • the pathogenic insects/nematodes Ostrinia nubilalis (European corn borer); Agrotis ipsilon (black cutworm); Schizaphis graminum (greenbug); Blissus leucopterus (chinch bug); Acrosternum hilare (green stink bug); Euschistus servus (brown stink bug); Deliaplatura (seedcorn maggot); Mayetiola destructor (Hessian fly); Petrobia latens (brown wheat mite).
  • soy bean the plants are resistant to the fungal, bacterial, or viral pathogens Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var.
  • Pseudoplusia includens (soybean looper); Anticarsia gemmatalis (velvetbean caterpillar); Plathypena scabra (green cloverworm); Ostrinia nubilalis (European corn borer); Agrotis ipsilon (black cutworm); Spodoptera exigua (beet armyworm); Heliothis virescens (cotton budworm); Helicoverpa zea (cotton bollworm);
  • Epilachna varivestis (Mexican bean beetle); Myzus persicae (green peach aphid); Empoasca fabae (potato leaf hopper); Acrosternum hilare (green stink bug);
  • Melanoplus femurrubrum (redlegged grasshopper); Melanoplus differ entialis (differential grasshopper); Hylemya platura (seedcorn maggot); Sericothrips variabilis (soybean thrips); Thrips tabaci (onion thrips); Tetranychus turkestani (strawberry spider mite); Tetranychus urticae (twospotted spider mite).
  • the plants are resistant to the fungal, bacterial, or viral pathogens Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum,
  • the plants are resistant to the fungal, bacterial, or viral pathogens
  • the plants are resistant to the fungal, bacterial, or viral pathogens
  • Pseudomonas syringae p.v. atrofaciens Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Blumeria (Erysiphe) graminis f. sp.
  • Puccinia graminis f. sp. tritici Puccinia recondita f. sp. tritici, Puccinia striiformis, Puccinia triticina, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var.
  • Diabrotica undecimpunctata howardi (southern corn rootworm); Russian wheat aphid; Schizaphis graminum (greenbug); Macrosiphum avenae (English grain aphid); Melanoplus femurrubrum (redlegged grasshopper); Melanoplus differ entialis (differential grasshopper); Melanoplus sanguinipes (migratory grasshopper);
  • Mayetiola destructor Hessian fly
  • Sitodiplosis mosellana wheat midge
  • Meromyza americana wheat stem maggot
  • Hylemya coarctata wheat bulb fly
  • Frankliniella fusca tobacco thrips
  • Cephus cinctus wheat stem sawfly
  • Aceria tulipae wheat curl mite
  • the plants are resistant to the fungal, bacterial, or viral pathogens Plasmophora halstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe dehor acearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum p.v. Carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis and to the pathogenic
  • insects/nematodes Suleima helianthana (sunflower bud moth); Homoeosoma electellum (sunflower moth); Zygogramma exclamationis (sunflower beetle);
  • Bothyrus gibbosus (carrot beetle); Neolasioptera murtfeldtiana (sunflower seed midge).
  • the plants are resistant to the fungal, bacterial, or viral pathogens Fusarium moniliforme var. subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis 0, T (Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III
  • Macrophomina phaseolina Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi p. v.
  • Peronosclerospora maydis, Peronosclerospora sacchari, Spacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium, Maize
  • Chlorotic Mottle Virus High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus (MSV, Maisstrichel- Virus), Maize Stripe Virus, Maize Rough Dwarf Virus, and the pathogenic insects / nematodes Ostrinia nubilalis (European corn borer); Agrotis ipsilon (black cutworm); Helicoverpa zea (corn earworm); Spodoptera frugiperda. (fall armyworm); Diatraea grandiosella
  • Diatraea saccharalis (surgarcane borer); Diabrotica virgifera (western corn rootworm); Diabrotica longicornis barberi (northern corn rootworm); Diabrotica undecimpunctata howardi (southern corn rootworm); Melanotus spp.
  • Anaphothrips obscurus grass thrips
  • Solenopsis milesta thief ant
  • Tetranychus urticae twospotted spider mite
  • sorghum the plants are resistant to the fungal, bacterial, or viral pathogens Exserohilum turcicum, Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v.
  • holcicola Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternata, Bipolaris sorghicola,
  • insects/nematodes Heliothis virescens (cotton budworm); Helicoverpa zea (cotton bollworm); Spodoptera exigua (beet armyworm); Pectinophora gossypiella (pink bollworm); Anthonomus grandis grandis (boll weevil); Aphis gossypii (cotton aphid); Pseudatomoscelis seriatus (cotton fleahopper); Trialeurodes abutilonea (bandedwinged whitefly); Lygus lineolaris (tarnished plant bug); Melanoplus femurrubrum (redlegged grasshopper); Melanoplus differentialis (differential grasshopper); Thrips tabaci (onion thrips); Franklinkiella fusca (tobacco thrips); Tetranychus cinnabarinus (carmine spider mite); Tetranychus urtica
  • the plants are resistant to the pathogenic insects/nematodes Diatraea saccharalis (sugarcane borer); Spodoptera frugiperda (fall armyworm); Helicoverpa zea (corn earworm); Colaspis brunnea (grape colaspis); Lissorhoptrus oryzophilus (rice water weevil); Sitophilus oryzae (rice weevil); Nephotettix nigropictus (rice leafhopper); Blissus Ieucopterus leucopterus (chinch bug); Acrosternum hilare (green stink bug).
  • the plants are resistant to the pathogenic insects/nematodes Brevicoryne brassicae (cabbage aphid); Phyllotreta cruciferae (Flea beetle); Mamestra configurata (Bertha armyworm); Plutella xylostella (Diamond-back moth); Delia ssp. (Root maggots).
  • plant pathogen comprises pathogens selected from the group consisting of Blumeria graminis f. sp. hordei, tritici, avenae, secalis, lycopersici, vitis, cucumis, cucurbitae, pisi, pruni, solani, rosae,fragariae, rhododendri, mali, and nicotianae as well as Septoria tritici and Puccinia triticina.
  • WR Y transcription factor is a protein with the amino acid sequence motif WR YGQK which regulates the expression of target genes by binding to the W box sequence motif C/TTGACT/C within the regulatory region of these genes.
  • the WRKY transcription factor is encoded by a nucleic acid sequence selected from the group consisting of:
  • nucleic acid sequence comprising the nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence comprising a sequence which is at least 70% identical to the sequence of SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences.
  • the content of a protein within a plant cell is usually determined by the expression level of the protein. Hence, in most cases the terms "content” and “expression” may be used interchangeably.
  • the content of a protein within a cell can be influenced on the level of transcription and/or the level of translation. Typically, the content is reduced on the RNA level, e.g. by RNA interference as described herein.
  • the person skilled in the art knows that the activity of a protein is not only influenced by the expression level, but also by other mechanisms such as post- translational modifications such as phosphorylations and acetylations, the interaction with other proteins such as the ML A protein of barley or the sub-cellular localisation.
  • the present invention also encompasses methods of influencing the activity of the WR Y transcription factor which do not affect the content of this protein.
  • nucleic acid sequence coding for the WRKY transcription factor may be substantially inhibited in transgenic plants for example by "silencing".
  • silencing a nucleic acid sequence which is substantially identical to the nucleic acid sequence coding for the WRKY transcription factor and/or which is
  • the nucleic acid to be transferred is normally introduced to the plant by a vector, such as a plasmid, which is able to stably replicate in the plant cell or to integrate the introduced nucleic acid into the plant genome.
  • nucleic acid sequence identical to a nucleic acid sequence is also referred to as sense nucleic acid.
  • sense sequences may also lead to suppression of the corresponding endogenous gene by means of a process called "co-suppression". If, in the scope of the present invention, sense sequences are mentioned, it is referred to those sequences which correspond to the coding strand of a nucleic acid sequence coding for the WRKY transcription factor or which comprise parts thereof. Such sequences do not have to be 100 % identical to the sequence coding for the WRKY transcription factor.
  • sequences are at least 80 %, 82 %, 84 %, 86 %, 88 %, particularly preferably at least 90 %, 91%, 92%, 93% or 94% and most preferably at least 95 %, 96 %, 97 %, 98 % or 99% identical.
  • sequences are regarded, according to the invention, as homologous to each other or comprising a homology.
  • the deviations to the nucleic acid sequence coding for the WRKY transcription factor or parts thereof may originate from deletion, substitution and/or insertion of one or more nucleotides.
  • sequences having such a low degree of identity or homology that the expression of genes other than a WRKY transcription factor, preferably the WRKY transcription factor encoded by the nucleic acid sequence according to SEQ ID No. 1 or 2, of the transgenic plant is suppressed are not specific enough for the method of the present invention, and are not suitable, since they may interfere with the metabolism of the plant.
  • sequences suppressing the expression of more than one WRKY transcription factor may be used in the method of the present invention, as long as they do not suppress the expression of genes other than those encoding a WRKY transcription factor.
  • sequences of the invention are referred to which correspond to the codogenous DNA strand of the genes coding for the WRKY transcription factor.
  • Said sequences are preferably complementary to at least 80 %, 82 %, 84 %, 86 %, 88 %, particularly preferably to at least 90 %, 92%, 94% and most preferably to at least 95 %, 96 %, 97 %, 98 %, 99% to the sequence of the WRKY transcription factor the expression of which is to be inhibited.
  • the antisense sequences are able to hybridize specifically with the mRNA of the corresponding gene coding for a WRKY transcription factor, but not with the mRNA of genes other than a WRKY transcription factor, preferably the WRKY transcription factor encoded by the nucleic acid sequence according to SEQ ID No. 1 or 2, of the transgenic plant.
  • the antisense sequence should be 100% reverse-complementary to the sense sequence to ensure optimal base-pairing.
  • the terms "complementary" and "reverse complementary” are used synonymously.
  • the at least one nucleic acid sequence is present in antisense orientation, so that upon transcription of said sequence in plant cells a RNA molecule is created, the sequence of which being complementary to the nucleic acid coding for the WRKY transcription factor.
  • the vector comprises a promoter functional in plant cells; operatively linked thereto a nucleic acid sequence which is complementary to a nucleic acid coding for the WRKY transcription factor; and, optionally, a termination sequence.
  • the nucleic acid sequence is complementary to a nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences.
  • the at least one nucleic acid sequence is present in sense orientation, so that upon transcription of said sequence in plant cells a RNA molecule is created, the sequence of which being identical to the nucleic acid sequence coding for the WRKY transcription factor.
  • antisense RNAs may be formed which may cause silencing of both the transgene, i.e. the sense sequence which was introduced, and the corresponding endogenous gene (co-suppression).
  • silencing of both the transgene, i.e. the sense sequence which was introduced, and the corresponding endogenous gene (co-suppression).
  • co-suppressing the nucleic acid sequence coding for the WRKY transcription factor in vivo the expression of the nucleic acid sequence coding for the WRKY transcription factor may be suppressed in plant cells, whereby the plant becomes pathogen resistant.
  • the vector comprises a promoter functional in plant cells; operatively linked thereto a nucleic acid sequence which is identical to a nucleic acid sequence coding for the WRKY transcription factor; and, optionally, a termination sequence.
  • the nucleic acid sequence is identical to a nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences.
  • vectors are used for introducing the nucleic acids in the plant cells which comprise in 5' - 3 '-orientation a promoter functional in plant cells, operatively linked thereto a DNA sequence coding for a ribozyme which specifically recognizes the nucleic acid sequence coding for the WRKY transcription factor, and a termination sequence.
  • a promoter functional in plant cells operatively linked thereto a DNA sequence coding for a ribozyme which specifically recognizes the nucleic acid sequence coding for the WRKY transcription factor, and a termination sequence.
  • ribozyme also refers to those RNA sequences which comprise next to the actual ribozyme leading sequences which are complementary to the nucleic acid sequence coding for the WRKY transcription factor or parts thereof, and thus direct the mRNA-specific ribozyme even more target-orientedly to the mRNA substrate of the ribozyme.
  • the recombinant nucleic acid molecule comprises a promoter which is functional in plant cells, operatively linked thereto at least one nucleic acid sequence which after transcription acts as a leading sequence, another nucleic acid sequence coding for ribonuclease P, and a termination sequence.
  • RNA molecules are formed in the cell having a leading sequence (the antisense sequence), which directs the R Ase P to the mR A of the WRKY transcription factor, thereby causing the cleavage of the mRNA by RNAse P (US Patent No. 5,168,053).
  • the leading sequence comprises 10 to 15 nucleotides which are complementary to the mRNA of the WRKY transcription factor and a 3'-NCCA nucleotide sequence, wherein N preferably is a purine.
  • the transcripts of the external leading sequence bind to the target mRNA by the formation of base pairs, thus enabling cleavage of the mRNA by RNAse P at the nucleotide 5' from the paired region. Such cleaved mRNA cannot be translated into a functional protein.
  • RNA molecules so-called small interfering RNAs, or siRNAs.
  • the double-stranded RNA molecule confers the specific degradation of the corresponding nucleic acid sequence, i.e. the nucleic acid sequence from which the double-stranded RNA sequence has been derived.
  • RNA fragments having a length of 19 - 25 nucleotides are produced from double-stranded RNA substrates.
  • Such double- stranded RNA substrates (dicer substrates) must have a length of at least 25 bp.
  • siRNAs are commonly present as double-stranded RNA.
  • the siRNAs may inhibit or prevent gene expression in many different ways:
  • R A interference Zamore et al. (2000) Cell 101 :25-33; Tang et al. (2003) Genes Dev. 17: 49-63; Smith et al. (2000) Nature 407: 319-320).
  • RNAi constructs according to the invention are based on the above- mentioned mechanisms for inhibiting gene expression of a nucleic acid sequence coding for the WR Y transcription factor. Thereby, the corresponding
  • polypeptide(s) cannot be formed.
  • the recombinant nucleic acid molecule comprises a promoter which is functional in plant cells, operatively linked thereto at least one nucleic acid sequence coding for the WRKY transcription factor, preferably the nucleic acid sequence of SEQ ID No. 1 or 2 or a fragment of any of these sequences, wherein said sequence has reverse-complementary regions, and a termination sequence.
  • nucleic acid sequence has reverse-complementary regions, so that after transcription of such a construct and self-hybridization within the nucleic acid sequence with the mentioned reverse-complementary regions, double-stranded RNA is being formed, which is a substrate for the dicer enzyme complex, for example. Accordingly, siRNA molecules are formed, which lead to the degradation of the corresponding nucleic acid.
  • reverse-complementary nucleic acid sequences are also referred to as inverted repeats.
  • the recombinant nucleic acid molecule comprises a promoter which is functional in plant cells, operatively linked thereto at least one nucleic acid sequence coding for the WR Y transcription factor, preferably the nucleic acid sequence of SEQ ID No. 1 or 2 or a fragment of any of these sequences, a "short hairpin" structure-generating nucleic acid, the nucleic acid sequence which is reverse-complementary to the at least one nucleic acid sequence, and a termination sequence.
  • the at least one nucleic acid sequence and the nucleic acid sequence reverse-complementary thereto may hybridize and form double-stranded RNA.
  • Suitable constructs and double-stranded RNA molecules are known to those skilled in the art for example as “short hairpin” RNAs or shRNAs.
  • Such constructs may be led by a U6 promoter or a CaMV35S promoter (Tuschl (2002) Nat. Biotechnol. 20: 446-448; Paul et al. (2002) Nat. Biotechnol. 20: 505- 508; Paddison et al. (2002) Genes Dev. 16(8): 948-958; Brummelkamp et al. (2002) Science 296: 550-553).
  • the recombinant nucleic acid molecule comprises a promoter which is functional in plant cells, operatively linked thereto at least one nucleic acid sequence coding for the WRKY transcription factor, preferably the nucleic acid sequence of SEQ ID No. 1 or 2 or a fragment of any of these sequences, optionally a spacer sequence, the nucleic acid sequence reverse-complementary to the at least one nucleic acid sequence, and a termination sequence.
  • the recombinant nucleic acid molecule comprises an RNAi construct, wherein the at least one nucleic acid sequence comprises the sequence according to SEQ ID NO: 1 or 2 or a fragment of any of these sequences, and a sequence reverse-complementary thereto.
  • the spacer sequence can be any sequence which is not complementary to another sequence in the construct.
  • the spacer sequence can be derived from both exons and introns.
  • the spacer sequence may also be a part of the sense or antisense sequence which is not reverse- complementary to the antisense or sense sequence, respectively.
  • the sense sequence may be extended by a certain number of nucleotides which are located 5' or 3' of the sense sequence in its natural sequence context, whereas the antisense sequence is not extended by the corresponding complementary nucleotides.
  • the spacer sequence is an intron which provides splice donor and splice acceptor sequences, such as the rgal intron from wheat. If vectors containing an intron as a spacer sequence are stably introduced in plant cells, first a pre-mRNA is formed upon transcription of said vectors which consists of a first exon comprising the at least one nucleic acid sequence of the present invention, an intron and a second exon comprising the nucleic acid sequence reverse-complementary to the at least one nucleic acid sequence. Since the intron is removed by the splicing procedure, a continuous RNA molecule is formed having regions which are complementary to each other, and thus being a substrate for specific enzyme complexes, such as the dicer enzyme complex. Those skilled in the art know that the position of the antisense (3' - 5') and sense (5' - 3') sequences may be interchanged in the vector.
  • the spacer sequence typically comprises 20 to 500 nucleotides, preferably 40 to 400 nucleotides, more preferably 60 to 300 nucleotides and most preferably 100 to 200 nucleotides.
  • the recombinant nucleic acid molecule comprises a RNAi construct, optionally comprising a spacer sequence between the at least one nucleic acid sequence and the sequence reverse- complementary thereto, wherein the at least one nucleic acid sequence comprises the nucleic acid sequence according to SEQ ID NO: 1 or 2 or a fragment of any of these sequences, and the sequence reverse-complementary thereto.
  • the RNAi construct comprises two promoters of which one regulates the expression of the sense sequence and the other one regulates the expression of the antisense sequence.
  • the two promoters may be the same or different promoters.
  • the sense and the antisense nucleic acid molecules may also be expressed under the control of a bidirectional promoter.
  • RNAi and/or PTGS the sense and antisense RNAs used for forming double-stranded RNA molecules may be of different sizes (Tuschl (2002) Nature Biotechnol. 20: 446-448).
  • microRNAs have emerged as evolutionarily conserved, RNA-based regulators of gene expression in plants and animals. MiRNAs with a length of 21 to 25 nucleotides arise from larger precursors with a stem loop structure that are transcribed from non-protein-coding genes. MiRNA targets a specific mRNA to suppress gene expression at post-transcriptional (i.e. degrades mRNA) or translational levels (i.e. inhibits protein synthesis) (Barrel (2004) Cell 116: 281-297).
  • a miRNA precursor can be engineered in such a way that endogenous miRNA encoded by pre-miRNA is replaced by a miRNA to target a gene-of-interest.
  • a native plant microR A precursor can be engineered as described in Schwab et al. (2006) Plant Cell 18(5): 1121-1133, to produce artificial miR A which specifically down-regulates target gene expression.
  • a further method for the design of functional microRNAs is the tool WMD3 as described by Ossowski et al. (2008) Plant J. 53(4): 674-690. The tool is available in the internet under the following address:
  • the present inventions further provides a method of producing a transgenic plant, plant cell or plant part having an increased resistance to pathogens compared to the control plant comprising,
  • engineered micro RNA precursors and micro-RNA for modulating the expression of a gene are well known and described e.g. in US 2004/0268441.
  • the use of engineered micro-RNA precursors and micro-RNA for modulating the expression of a gene can be combined with other methods of genetic engineering well known to the person skilled in the art.
  • synthetic double-stranded siRNAs which typically have a length of 19 - 21 nucleotides are used for inhibiting the expression of a nucleic acid coding for the WRKY transcription factor.
  • Such synthetic siRNAs may be introduced in the corresponding plant cell or plant by biolistic transformation techniques.
  • Such synthetic siRNA molecules may activate the PTGS system in plants, and trigger an R Ai effect (Hamilton and Baulcombe (1999) Science 286: 950-2).
  • the target sequence for siRNA inhibition as well as the siRNA sequence motif may be selected according to the rules and regulations known to those skilled in the art, for example according to Elbashir et al. (2001) Nature 411 : 494-8. If the target sequence for the siRNA-mediated inhibition lies within the coding regions of the gene, or within the mRNA, those skilled in the art know, for example, that the target sequence for siRNA-inhibition may typically be at least 70 nucleotides downstream from the start codon in 5' - 3' direction and at least 50 nucleotides upstream from the stop codon.
  • sequence region may then be searched for the sequence motif AA(N19), wherein N may be each nucleotide.
  • Said sequence motif typically comprises the AA dinucleotide, followed by 19 nucleotides, and preferably two additional uridine or thymidine residues.
  • the thymidine residues may be replaced by uridine residues in the siRNA sequence.
  • siRNAs which fulfil the above-mentioned criteria may be checked by appropriate search programs, e.g. BLAST, whether there are any, if possible no or only little, homologies to other nucleic acid sequences of the plant.
  • a nucleic acid sequence for reducing the content and/or the activity of a protein may be integrated into the natural locus of the sequence by targeted homologous recombination.
  • Such methods are for example described in WO 00/46386 A3, WO 01/89283A1,
  • WO 02/077246 A2 and WO 2007/ 135022 A 1.
  • a method for introducing a targeting sequence differing from the target sequence by 0.1 to 10% by homeologous recombination is described for example in WO 2006/134496 A2.
  • sequence-specific nucleases may also be used to cut the sequence of interest, thereby introducing one or more mutations into said sequence.
  • the method for producing mutant plants, plant cells or plant parts having an increased resistance to pathogens is preferably the TILLING (Targeting Induced Local Lesions IN Genomes) method.
  • plant material is mutagenized to introduce at least one mutation into the genome of the plant material.
  • This mutagenesis may be chemical mutagenesis, for example with ethyl methane sulfonate (EMS), mutagenesis by irradiation such as ionizing irradiation or mutagenesis by using sequence-specific nucleases.
  • EMS ethyl methane sulfonate
  • Single base mutations or point mutations lead to the formation of heteroduplexes which are then cleaved by single strand nucleases such as Cell at the 3' side of the mutation.
  • the precise position of the mutation within the nucleic acid sequence according to SEQ ID NO. 1 or 2 can then be determined by denaturing gel electrophoresis or the LICOR gel based system (see, e.g., McCallum et al. (2000) Plant Physiol. 123(2): 439-442; Uauy et al. (2009) BMC Plant Biol. 9: 115).
  • the expression level of the nucleic acid coding for the WR Y transcription factor may be determined in the control plants as well as in the transgenic plants, for example, by RT-PCR analysis or Northern Blot analysis with specific primers or probes. A person skilled in the art knows how to select said probes or primers in order to examine the expression of said nucleic acid.
  • the expression of the nucleic acid coding for the WRKY transcription factor is statistically significantly reduced by at least 80 %, particularly preferably by at least 90 %, also particularly preferably by at least 95 %, and most preferably by at least 98 % or 99 %.
  • the activity of the WRKY transcription factor may be determined by preparing protein extracts from cells of control and transgenic plants and analyzing the DNA binding activity of the transcription factor using known methods such as
  • EMSA electrophoretic mobility shift assay
  • the extracts are incubated with a labelled DNA probe which contains a sequence motif to which the transcription factor is known to bind. After incubation the samples are separated on a non-denaturing poly aery lamide gel and visualized. If the transcription factor has DNA binding activity, the samples show a shift in the mobility of the probe.
  • the DNA binding activity of the WRKY transcription factor may be reduced by inhibition of WRKY transcription factor expression by at least 5%, preferably at least 10%, more preferably at least 15% or 20%, even more preferably by at least 25 or 30% and most preferably by at least 35% or 40%.
  • the activity of the WRKY transcription factor may also be reduced by an antibody specific for said protein.
  • the production of monoclonal, polyclonal, or recombinant WRKY-specific antibodies follows standard protocols (Guide to Protein Purification, Meth. Enzymol. 182, pp. 663-679 (1990), M. P. Deutscher, ed.).
  • the expression of antibodies is also known from the literature (Fiedler et al. (1997) Immunotechnology 3: 205-216; Maynard and Georgiou (2000) Annu. Rev. Biomed. Eng. 2: 339-76).
  • aptamers can be used to reduce the activity of the WRKY transcription factor.
  • aptamers are overexpressed from vectors and the design and selection of aptamers is well known to the person skilled in the art (Famulok et al. (1999) Curr Top Microbiol Immunol, 243,123-36).
  • nucleic acid sequence which is selected from the group consisting of: a nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences.
  • a "fragment" of the nucleic acid sequence according to SEQ ID No. 1 or 2 is understood to refer to a smaller part of this nucleic acid sequence which consists of a contiguous nucleotide sequence found in SEQ ID No. 1 or 2 and which is able to reduce the content and/or activity of the WR Y transcription factor when used in a suitable expression system, but not or not considerably of other proteins the expression of which should not be reduced.
  • the fragment in case the fragment is described to be a fragment of a sequence with a certain degree of sequence identity to a particular sequence, the fragment shall be a fragment of the sequence which has a certain degree of sequence identity to the particular sequence.
  • the "fragment” in the second alternative refers to a fragment of the sequence which sequence is at least 70%> identical to the sequence according to SEQ ID No. 1.
  • the fragment of SEQ ID No. 1 has a length of at least 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 nucleotides, preferably of at least 60, 70 or 80, 90, 100, 110, 120, 130, 140 nucleotides, more preferably of at least 150, 160, 170, 180, 190 or 200 nucleotides, even more preferably of at least 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350 nucleotides and most preferably of at least 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500 nucleotides.
  • the fragment of SEQ ID No. 2 has a length of at least 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 nucleotides, preferably of at least 60, 70, 80, 90 or 100 nucleotides, more preferably of at least 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nucleotides, even more preferably of at least 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350 nucleotides and most preferably of at least 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480 490, 500, 510, 520, 530, 540, 550, 560 or 570 nucleotides.
  • the present invention further relates to the use of nucleic acid sequences which are at least 70%, 75% or 80 % identical, preferably at least 81, 82, 83, 84, 85 or 86% identical, more preferably at least 88, 89 or 90% identical, even more preferably at least 91, 92, 93, 94 or 95% identical and most preferably at least 96, 97, 98 or 99% identical to the complete sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences and which are able to reduce the content and/or activity of the WR Y transcription factor when used in a suitable expression system, but not or not considerably of other proteins the expression of which should not be reduced.
  • sequence identity denotes the degree of conformity with regard to the 5' - 3' sequence within a nucleic acid molecule in comparison to another nucleic acid molecule.
  • the “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over a particular region, determining the number of positions at which the identical base or amino acid is present in both sequences in order to yield the number of matched positions, dividing the number of those matched positions by the total number of positions in the segment being compared and multiplying the result by 100.
  • the sequence identity may be determined using a series of programs, which are based on various algorithms, such as BLASTN, ScanProsite, the laser gene software, etc.
  • Biotechnology Information http://www.ncbi.nlm.nih.gov/) may be used with the default parameters.
  • the program Sequencher Gene Codes Corp., Ann Arbor, MI, USA
  • the "dirtydata"-algorithm for sequence comparisons was employed.
  • sequence identity refers to the degree of the sequence identity over a length of 100 or 150 nucleotides, preferably 180 or 200 nucleotides, more preferably 220, 250 or 280 nucleotides and most preferably the whole length of the nucleic acid sequence according to SEQ ID No. 1 or 2.
  • the fragment has a length of at least 25 or 40 nucleotides, preferably of at least 50 or 80 nucleotides, more preferably of at least 100 or 120 nucleotides and most preferably of at least 150 or 200 nucleotides. If the sequence identity is to be determined in comparison to a fragment of the sequence according to SEQ ID No.2, the fragment has a length of at least 25 or 40 nucleotides, preferably of at least 50 or 80 nucleotides, more preferably of at least 100, 120 or 150 nucleotides and most preferably of at least 180, 200, 220 or 250 nucleotides.
  • the present invention further relates to the use of nucleic acid sequences which hybridize under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or 2 or a fragment of any of these sequences and which are able to reduce the content and/or activity of the WRKY transcription factor when used in a suitable expression system, but not or not considerably of other proteins the expression of which should not be reduced.
  • hybridizing under stringent conditions means that the hybridization is implemented in vitro under conditions which are stringent enough to ensure a specific hybridization. Stringent in vitro hybridization conditions are known to those skilled in the art and may be taken from the literature (e.g.
  • telomere sequence a nucleic acid sequence preferably binds to a certain nucleic acid sequence, i.e. the target sequence, if the same is part of a complex mixture of, e.g. DNA or RNA molecules, but does not, or at least very rarely, bind to other sequences.
  • stringent conditions depend on the circumstances. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are chosen such that the hybridization temperature is about 5°C below the melting point (T m ) of the specific sequence at a defined ionic strength and at a defined pH value. T m is the temperature (at a defined pH value, a defined ionic strength and a defined nucleic acid concentration), at which 50% of the molecules complementary to the target sequence hybridize to the target sequence in the state of equilibrium.
  • stringent conditions are conditions, where the salt concentration has a sodium ion concentration (or concentration of a different salt) of at least about 0.01 to 1.0 M at a pH value between 7.0 and 8.3, and the temperature is at least 30°C for small molecules (i.e.
  • stringent conditions may include the addition of substances, such as, e. g., formamide, which destabilise the hybrids.
  • substances such as, e. g., formamide, which destabilise the hybrids.
  • said stringent conditions are chosen such that sequences which are about 65%, preferably at least about 70%, and especially preferably at least about 75% or higher homologous to each other, normally remain hybridized to each other.
  • a preferred but non-limiting example of stringent hybridization conditions is hybridizations in 6 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washing steps in 0.2 x SSC, 0.1% SDS at 50 to 65°C.
  • the temperature depends on the type of the nucleic acid and is between 42°C and 58°C in an aqueous buffer having a concentration of 0.1 to 5 x SSC (pH value 7.2).
  • the temperature is about 42°C under standard conditions.
  • the temperature is about 42°C under standard conditions.
  • hybridisation conditions for DNA:DNA hybrids are, for example, 0.1 x SSC and 20°C to 45°C, preferably 30°C to 45°C.
  • the hybridisation conditions for DNA:RNA hybrids are, for example, 0.1 x SSC and 30°C to 55°C, preferably between 45°C and 55°C.
  • the above-mentioned hybridization temperatures are determined, for example, for a nucleic acid which is 100 base pairs long and has a G/C content of 50% in the absence of formamide.
  • Typical hybridization and washing buffers for example have the following composition: Pre-hybridization solution: 0.5% SDS
  • Hybridization solution pre-hybridization solution
  • Pre-hybridization at least 2 h at 50 - 55°C
  • Hybridization over night at 55 -60°C
  • the nucleic acid sequence hybridizing to a fragment of the sequence according to SEQ ID No.l under stringent conditions has a length of at least 25 or 30 nucleotides, preferably of at least 50 or 70 nucleotides, more preferably of at least 100 or 120 nucleotides, even more preferably of at least 150 or 200 nucleotides and most preferably of at least 250 or 300 nucleotides.
  • the nucleic acid sequence hybridizing to a fragment of the sequence according to SEQ ID No.2 has a length of at least 25 or 30 nucleotides, preferably of at least 50 or 70 nucleotides, more preferably of at least 100 or 120 nucleotides, even more preferably of at least 150 or 200 nucleotides and most preferably of at least 250, 300 or 320 nucleotides.
  • the expression of proteins other than a WR Y transcription factor, preferably the WR Y transcription factor encoded by the nucleic acid sequence according to SEQ ID NO. 1 or 2 is reduced by less than 10% or 8%, preferably by less than 7, 6 or 5%, more preferably by less than 4, 3 or 2% and most preferably by less than 1%.
  • a suitable expression system for reducing the expression of the WRKY transcription factor according to SEQ ID No. 1 or 2 is any expression system described herein, preferably an RNAi expression system.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence as defined herein.
  • nucleic acid molecule is separated from other nucleic acid molecules present in the natural environment of the nucleic acid sequence of the present invention.
  • An “isolated” nucleic acid preferably has no sequences naturally flanking the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (e.g. sequences located at the 5' and 3' ends of the nucleic acid).
  • the isolated nucleic acid molecule can contain, for example, less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0,5 kb, or 0,1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid originates.
  • All nucleic acid molecules mentioned herein can be e.g. RNA, DNA, or cDNA.
  • the corresponding sense or antisense nucleic acid sequences for example may be inserted into an appropriate vector by restriction digestion and subsequent ligation.
  • the corresponding sense or antisense nucleic acid sequences for example may be inserted into the vector by homologous recombination, such as by the GATEWAY ® system (Invitrogen) or the BD CreatorTM system (BD Biosciences Clontech Co.).
  • expression construct means a nucleic acid molecule which contains all elements which are necessary for the expression of a nucleic acid sequence, i.e. the nucleic acid sequence to be expressed under the control of a suitable promoter and optionally further regulatory sequences such as termination sequences.
  • An expression cassette of the present invention may be part of an expression vector which is transferred into a plant cell or may be integrated into the chromosome of a transgenic plant after transformation.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and may be used herein interchangeably with the term “recombinant nucleic acid molecule”.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and may be used herein interchangeably with the term “recombinant nucleic acid molecule”.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • Plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • a vector can be a binary vector or a T-DNA that comprises a left and a right border and that may include a gene of interest in between.
  • expression vector means a vector capable of directing expression of a particular nucleotide sequence in an appropriate host cell.
  • An expression vector comprises a regulatory nucleic acid element operably linked to a nucleic acid of interest, which is - optionally - operably linked to a termination signal and/or other regulatory element.
  • promoter refers to a DNA sequence which, when ligated to a nucleotide sequence of interest, is capable of controlling the transcription of the nucleotide sequence of interest into mRNA.
  • a promoter is typically, though not necessarily, located 5' (e.g., upstream) of a nucleotide sequence of interest (e.g., proximal to the transcriptional start site of a structural gene) whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription.
  • the promoter used in the present invention may be a constitutive promoter, an inducible promoter or a tissue-specific promoter.
  • Constitutive promoters include the 35S CaMV promoter (Franck et al. (1980) Cell 21 : 285-294), the ubiquitin promoter (Binet et al. (1991) Plant Science 79: 87-94), the Nos promoter (An et al. (1990) The Plant Cell 3: 225-233,), the MAS promoter (Velten et al. (1984) EMBO J. 3: 2723-230), the maize H3 histone promoter (Lepetit et al. (1992) Mol Gen.
  • the promoter is a regulated promoter.
  • a "regulated promoter” refers to a promoter that directs gene expression not constitutively, but in a temporally and/or spatially restricted manner, and includes both tissue-specific and inducible promoters. Different promoters may direct the expression of a
  • polynucleotide or regulatory element in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.
  • Wound-, light- or pathogen-induced promoters and other development-dependent promoters or control sequences may also be used (Xu et al. (1993) Plant Mol. Biol. 22: 573-588; Logemann et al. (1989) Plant Cell 1 : 151-158; Stockhaus et al. (1989) Plant Cell 1 : 805-813; Puente et al. (1996) EMBO J. 15: 3732-3734; Gough et al. (1995) Mol. Gen. Genet. 247: 323-337).
  • a summary of useable control sequences may be found, for example, in Zuo et al. (2000) Curr. Opin. Biotech. 11 : 146-151.
  • tissue-specific promoter refers to a regulated promoter that is not expressed in all plant cells, but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells).
  • tissue-specific promoters include, e.g., epidermis-specific promoters, such as the GSTA1 promoter (Altpeter et al. (2005) Plant Mol Biol. 57: 271-83), or promoters of photosynthetically active tissues, such as the ST-LS1 promoter
  • the promoters of phosphoenolpyruvate-carboxylase from corn (Hudspeth et al. (1989) Plant Mol. Biol. 12:579) or of fructose- 1,6- bisphosphatase from potato (WO 98/18940), which impart leaf- specific expression, are also considered to be tissue-specific promoters. Further preferred promoters are those which are in particular active in fruits. Examples of these are the promoter of a polygalacturonase gene, e. g.
  • promoters may be taken from the literature, e.g. Ward (1993, Plant Mol. Biol. 22: 361-366). The same applies to inducible and cell- or tissue-specific promoters, such as meristem-specific promoters which have also been described in the literature and which are suitable within the scope of the present invention as well.
  • Particularly suitable promoters for the method of the present invention are pathogen- inducible promoters, and especially those, which are induced by pathogenic fungi and not by useful fungi (e.g. mycorrhiza in the soil, such as the GER4 promoter (WO 2006/128882).
  • promoters which are active on the site of pathogen entry are particularly suitable in the method of the present invention.
  • promoters which are active on the site of pathogen entry such as epidermis-specific promoters.
  • Suitable epidermis-specific promoters include, but are not limited to, the GSTA1 promoter (Accession number X56012), the GLP4 promoter (Wei et al. (1998) Plant Mol. Biol. 36: 101), the GLP2a promoter (Accession number AJ237942), the Prx7 promoter (Kristensen et al. (2001) Mol. Plant Pathol. 2(6): 311), the GerA promoter (Wu et al. (2000) Plant Phys Biochem. 38: 685), the OsROCl promoter (Accession number AP004656), the RTBV promoter (Kloeti et al.
  • promoters which are inducible by fungi include promoters such as the GAFP-2 promoter (Sa et al. (2003) Plant Cell Rep. 22: 79-84), which, e.g., is induced by the fungus Trichoderma viride, or the PAL promoter which is induced by inoculation with Pyricularia oryzae (Wang et al. (2004) Plant Cell Rep. 22: 513- 518).
  • inducible promoters allows for the production of plants and plant cells which only transiently express the sequences of the present invention, and thus silence transiently.
  • Such transient expression allows for the production of plants which show only transiently increased pathogen resistance.
  • transiently increased resistance may be desired, if, for example, there is an acute risk of fungal contamination, and therefore the plants only have to be resistant to the fungus for a certain period of time.
  • transient resistance are known to those skilled in the art.
  • transient expression and thus transient silencing and transient resistance may be achieved using vectors which do not replicate stably in plant cells and which carry the respective sequences for silencing of fungal genes.
  • the actin promoter from Oryza sativa is used to express a nucleic acid sequence of the present invention.
  • the vectors which are used in the method of the present invention may further comprise regulatory elements in addition to the nucleic acid sequence to be transferred. Which specific regulatory elements must be included in said vectors depends on the procedure which is to be used for said vectors. Those skilled in the art who are familiar with the various methods for producing transgenic plants in which the expression of a protein is inhibited know which regulatory elements and also other elements said vectors must include.
  • transcription regulatory element refers to a polynucleotide that is capable of regulating the transcription of an operably linked polynucleotide. It includes, but is not limited to, promoters, enhancers, introns, 5' UTRs, and 3' UTRs.
  • operatively linked and "operably linked” mean that nucleic acid sequences are linked to each other such that the function of one nucleic acid sequence is influenced by the other nucleic acid sequence.
  • a nucleic acid sequence is operably linked to a promoter, its expression is influenced by said promoter.
  • termination sequences are sequences which ensure that the transcription or the translation is terminated properly. If the introduced nucleic acids are to be translated, said nucleic acids are typically stop codons and corresponding regulatory sequences; if the introduced nucleic acids are only to be transcribed, said nucleic acids are normally poly-A sequences.
  • the vectors of the present invention may for example also comprise enhancer elements as regulatory elements, resistance genes, replication signals and further
  • regulatory elements also comprise sequences which lead to a stabilization of the vectors in the host cells.
  • regulatory elements comprise sequences which enable a stable integration of said vector in the host genome of the plant or autonomous replication of said vector in the plant cells.
  • Such regulatory elements are known to those skilled in the art.
  • Said techniques comprise the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation means, viral infection by using viral vectors (EP 0 067 553; US 4,407,956,
  • WO 95/34668 WO 93/03161
  • the fusion of protoplasts polyethylene glycol- induced DNA uptake, liposome-mediated transformation (US 4,536,475), incubation of dry embryos in DNA-comprising solution, microinjection, the direct gene transfer of isolated DNA in protoplasts, the electroporation of DNA, the introduction of DNA by the biolistic procedure, as well as other possibilities. Thereby, stable as well as transient transformants may be produced.
  • the used plasmids do not need to fulfil special requirements per se. The same applies to direct gene transfer. Simple plasmids, such as pUC derivatives, may be used.
  • a selectable marker gene may become necessary.
  • Those skilled in the art know all commonly used selection markers, and thus there is no difficulty to select a suitable marker.
  • Common selection markers create resistance in the transformed plant cells to a biocide or antibiotic, such as kanamycin, G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonyl urea, gentamycin or phosphinotricin and the like or may confer tolerance to D-amino acids such as D- alanine.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right border, or very often both the right and the left border of the T-DNA contained in the Ti and Ri plasmid needs to be linked to the genes to be inserted.
  • the DNA to be inserted needs to be cloned into special plasmids, i.e. either into an intermediate vector or into a binary vector.
  • the intermediate vectors may be integrated into the Ti or Ri plasmid of the agrobacteria by means of homologous recombination due to sequences which are homologous to sequences in the T-DNA, which contains the vir region required for the transfer of the T-DNA. Intermediate vectors are not able to replicate in agrobacteria.
  • the intermediate vector may be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors are able to replicate in both E.
  • Said vectors contain a selection marker gene and a linker or polylinker located between the right and left T-DNA border region.
  • the vector may be transformed directly into the agrobacteria (Holsters et al. (1978) Molecular and General Genetics 163: 181-187).
  • the agrobacterium, serving as host cell is to contain a plasmid which includes a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. In addition, T-DNA may be present.
  • the agrobacterium transformed in such a manner is used for the transformation of plant cells.
  • plant explants may be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Agrobacterium tumefaciens or Agrobacterium rhizogenes From the infected plant material (e.g. leaf cuttings, stem sections, roots, but also protoplasts or suspension- cultivated plant cells) whole plants may be regenerated in a suitable medium which may contain antibiotics, biocides or D-amino acids for the selection of transformed cells, if a selection marker was used in the transformation.
  • the regeneration of the plants is performed according to standard regeneration procedures using well-known culture media.
  • the plants or plant cells obtained this way may then be examined for the presence of the introduced DNA.
  • Other possibilities for introducing foreign DNA using the biolistic method or by protoplast transformation are well-known to those skilled in the art (see L.
  • Monocotyledonous plants or the cells thereof may also be transformed using vectors which are based on agrobacteria (see e.g. Chan et al. (1993) Plant Mol. Biol. 22: 491-506).
  • Alternative systems for the transformation of monocotyledonous plants or the cells thereof are transformation by biolistic approach (Wan and Lemaux (1994) Plant Physiol. 104: 37-48; Vasil et al. (1993) Bio/Technology 11 : 1553-1558; Ritala et al. (1994) Plant Mol. Biol. 24: 317-325; Spencer et al. (1990) Theor. Appl. Genet. 79: 625-631), the protoplast transformation, the electroporation of partially permeabilized cells, and the insertion of DNA by means of glass fibres.
  • the vectors described herein can be directly transformed into the plastid genome. Plastid expression, in which genes are inserted by homologous recombination into the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number over nuclear-expressed genes to permit high expression levels.
  • the nucleotides are inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplasmic for plastid genomes containing the nucleotide sequences are obtained, and are preferentially capable of high expression of the nucleotides.
  • Plastid transformation technology is for example extensively described in U.S. Pat. Nos. 5,451,513; 5,545,817; 5,545,818 and 5,877,462, in WO 95/16783 and
  • the transformed cells grow within the plant in the usual manner (see also
  • the resulting plants may be cultivated in the usual manner, and may be crossed with plants which have the same transformed genes or other genes.
  • the hybrid individuals resulting therefrom have the respective phenotypical properties.
  • the method of the present invention may further comprise the step of crossing the transgenic plant produced by the method of the present invention with another plant in which the content and/or the activity of the WRKY transcription factor is not decreased and selecting transgenic progeny in which the content and/or the activity of the WRKY transcription factor is decreased.
  • the other plant in which the content and/or the activity of the WRKY transcription factor is not decreased is preferably from the same species as the transgenic plant and may be a wild-type plant, i.e. a plant which does not contain any transgenic nucleic acid sequence, or it may be a transgenic plant which contains a transgenic nucleic acid sequence other than the nucleic acid sequences disclosed herein, e.g.
  • transgenic nucleic acid sequence coding for another protein involved in pathogen resistance or a protein conferring resistance to abiotic stress is preferably an elite variety which is characterized by at least one favourable agronomic property which is stably present in said elite variety. Methods for determining whether the content and/or activity of the WRKY transcription factor is decreased are discussed above.
  • An "elite variety" within the meaning of the present invention is a variety which is adapted to specific environmental conditions and/or which displays at least one superior characteristic such as an increased yield compared to non-elite varieties.
  • the transgenic progeny of the above crossing step can be further crossed with each other to produce true breeding lines.
  • the transgenic progeny of the above cross is inbred and the transgenic progeny of this crossing step in which the content and/or the activity of the WRKY transcription factor is decreased is selected and again inbred.
  • This inbreeding step is repeated until a true breeding line is established, for example at least five times, six times or seven times.
  • a "true breeding" plant or "inbred plant” is a plant which upon self-pollination produces only offspring which is identical to the parent with respect to at least one trait, in the present case the transgene which decreases the content and/or the activity of the WRKY transcription factor.
  • the true breeding lines can then be used in hybrid breeding yielding Fl hybrids which can be marketed. This method is particularly suitable for example for maize and rice plants. Alternatively, the true breeding lines can be further inbred in a linebreeding process. This method is particularly suitable for example for wheat and barley plants.
  • transgenic lines which are homozygous for the introduced nucleic acid molecules may also be identified and examined with respect to pathogen resistance compared to the pathogen resistance of hemizygous lines.
  • plant cells which contain the recombinant nucleic acid molecules of the present invention may also be further cultivated as plant cells (including protoplasts, calli, suspension cultures and the like).
  • the method of the present invention may additionally comprise the reduction of the content and/or the activity of at least one, for example two or three, other plant proteins which mediate pathogen susceptibility.
  • Suitable genes include the Mlo gene (WO 00/01722), the Bax inhibitor- 1 gene (Eichmann et al. (2010) Mol. Plant Microbe Interact. 23(9): 1217-1227) and the Pmr genes (Vogel and Somerville (2000) Proc. Natl. Acad. Sci. USA 97(4): 1897-1902).
  • the transgenic or mutant plants of the present invention or parts thereof can be used as fodder plants or for producing feed.
  • Fodder is intended to mean any agricultural foodstuff which is specifically used to feed domesticated animals such as cattle, goats, sheep and horses. It includes includes hay, straw, silage and also sprouted grains and legumes. The person skilled in the art knows that it may be necessary to treat the transgenic or mutant plants of the present invention to make them suitable for use as fodder.
  • feed is intended to mean a dry feed which can be blended from various raw materials and additives such as soybean shred or barley shred in a feed mill.
  • the transgenic or mutant seed of the transgenic or mutant plants of the present invention can be used to prepare flour, in particular if the transgenic or mutant plants are monocotyledonous plants such as barley or wheat.
  • another embodiment of the present invention is a method for the production of a product comprising the steps of:
  • the product produced by said methods of the invention is flour comprising the nucleic acid sequence which reduces the content and/or activity of the WRKY transcription factor or the mutation which reduces the content and/or activity of the WRKY transcription factor.
  • the flour prepared from the transgenic seed of the present invention can be distinguished from the flour prepared from other plants by the presence of the transgenic nucleic acid sequence, the expression construct or the vector of the present invention.
  • the transgenic nucleic acid sequence is expressed under the control of a promoter which is not endogenous to the transgenic plant, the presence of the promoter can be detected in the flour prepared from the transgenic seed.
  • an antisense sequence is part of the transgene mediating the reduction of the content and/or the activity of the WRKY transcription factor, the presence of this antisense sequence can be detected in the flour prepared from the transgenic seed.
  • the flour prepared from the mutant seed can be distinguished from the flour prepared from other plants by the presence of the at least one point mutation within the nucleic acid sequence defined herein.
  • Harvestable parts of the transgenic plants of the present invention are also a subject of the invention.
  • the harvestable parts comprise a nucleic acid sequence which reduces the content and/or activity of the WRKY transcription factor, i.e. this nucleic acid sequence is detectable in the harvestable parts by conventional means.
  • the harvestable plants may be seeds, roots, leaves, stems, and/or flowers comprising the nucleic acid sequence which reduces the content and/or activity of the WRKY transcription factor.
  • Preferred harvestable parts are seeds comprising the nucleic acid sequence which reduces the content and/or activity of the WRKY transcription factor.
  • Entry vector (pIPKTA38) preparation The pIPKTA38 plasmid (Douchkov et al. (2005) Mol. Plant Microbe Interact. 18(8): 755-761) with a kanamycvin resistance gene was used as Gateway Entry vector.
  • Plasmid DNA was prepared with the Jetstar midi DNA kit (Genomed).
  • the plasmid was digested with the restriction enzyme Apal, yielding bands of 1257 bp and 1054 bp. Then the DNA concentration was measured and adjusted to 150 ng/ ⁇ .
  • the pIPKTA30 plasmid (Douchkov et al. (2005) Mol. Plant Microbe Interact. 18(8): 755-761) was used as the RNAi vector. It contains an ampicillin resistance gene, a ccdB negative selection marker gene which requires the propagation of the plasmid in DB3.1 cells and a chloramphenicol resistance gene. Plasmid DNA was prepared with the Jetstar midi DNA kit (Genomed). The plasmid preparations were digested as a control with EcoRI (correct bands - 687, 1007, 2641, and 2857 bp) or Sal I (601, 1589, and 5002 bp). The DNA concentration was measured and adjusted to 150 ng/ ⁇ .
  • Specific primers (SEQ ID Nos. 3 and 4) were designed to amplify -500 bp fragments from the EST clone. Tm of the primers is ⁇ 65°C.
  • a "PCR master mix” (see Table 6) was prepared of which 11,0 were dispensed to each well of a 96-well PCR plate. 4,25 ⁇ , of each EST-specific primer and 0,5 ⁇ , EST DNA as template were added to each well.
  • Target-specific sense primer ( ⁇ ) 4,25 ⁇ , per well
  • Target-specific antisense primer ( ⁇ ) 4,25 ⁇ , per well
  • a ligation master mix was prepared (see Table 7), 6 ⁇ each of this ligation mix were added to each well and 4 ⁇ , of the purified PCR product were added. The samples were incubated for 1 h at 25°C and the reaction was then stopped by heating up to 65°C for 10 min. 5 ⁇ , Swal master mix (see Table 8) was added to each well, followed by an incubation at 25°C for 1 h. Next, the ligation samples were transformed into competent bacteria and suitable clones were isolated after miniprep and control digestion.
  • H 2 100 ⁇ 200 ⁇ 400 ⁇ pIPKTA38 (150 ng ⁇ L) 100 ⁇ 200 ⁇ 400 ⁇
  • Figure 2 shows a schematic drawing of the test procedure for the RNAi constructs.
  • Vacuum was applied for biolistic transformation, wherein the bombardment was made at a pressure of 27.5 mm Hg.
  • the leaves were collected, the leaf tips were cut off and the resulting leaves were transferred to Greiner tubes containing 10 mL of X-glucose solution (100 mM sodium phosphate , pH 7,0;_10 mM sodium EDTA ⁇ 1,4 mM K- hexacyanoferrate(II); 1 ,4 mM K-hexacyanoferrate(III); 0, 1 % Triton X- 100; 20% methanol and 1 mg / ml X-Gluc).
  • the tubes were placed in a suction bottle and vacuum was applied thereto 2 - 3 times. The infiltration is complete when the leaves become transparent and start to sink.
  • the X-glucose solution was refilled to 14 mL and the tubes were sealed. The tubes were incubated over night at 37 °C in the incubator.
  • the leaves were placed in destaining solution (7.5 % TCA, 50 % methanol) for 5 min. Then the leaves were washed with distilled water. Then, the leaves were carefully removed from the tube and were placed onto an object slide with their adaxial side facing upwards.
  • destaining solution 7.5 % TCA, 50 % methanol
  • the leaves were transferred to 1% phytoagar with 2% benzimidazole.
  • a nylon net (mesh width of 200 ⁇ ) was stretched over the leaves, and they were inoculated with a conidia density of about 200 conidia/mm 2 .
  • the conidia originated from barley plants (cultivar ,Golden Promise'), which had been inoculated 6 - 7 days before.
  • the leaves were stored in closed Petri dishes with holes for ventilation at 20 °C at a north-facing window.
  • GUS staining was performed. Said staining was stopped after 24 h by incubation in 7.5 % trichloroacetic acid, 50 % v/v methanol, and the leaves were bleached.
  • each experiment contained 3 parallel bombardments to 7 leaf sections each of the negative control (empty vector pIPKTA30N). Further, each experiment contained 2 parallel bombardments of the positive control pIPKTA36, which causes resistance by inhibiting the Mlo gene of barley. Data per experiment are based on the comparison of the effect of the test constructs with the average value of the 3 negative controls of the respective experiment.
  • Table 10 shows the relative susceptibility index (Rel. SI) of barley cells transiently transformed with an R Ai construct inhibiting the expression of the WR Y transcription factor according to SEQ IDNo. 1. The susceptibility index relative to the empty vector control was determined in five independent transformation experiments. As the cells transformed with the RNAi construct have a susceptibility index of less than 100% compared to the control cells transformed with the empty vector (pIPKTA30N), the RNAi construct suppresses putative susceptibility genes in barley. Table 10

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Abstract

La présente invention concerne un procédé de production d'une cellule végétale transgénique, d'une plante transgénique ou d'une partie transgénique de celle-ci présentant une résistance accrue à des pathogènes, comprenant l'étape de réduction de la teneur et/ou de l'activité d'un facteur de transcription WRKY, de préférence par interférence par ARN.
PCT/EP2012/069893 2011-10-07 2012-10-08 Procédé de production de plantes présentant une résistance accrue à des pathogènes WO2013050611A1 (fr)

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CN111635904A (zh) * 2020-06-29 2020-09-08 山东农业大学 一种增强黄瓜靶斑病抗性的基因CsWRKY10及其应用
CN116064586A (zh) * 2022-11-01 2023-05-05 广东省农业科学院果树研究所 一种番木瓜CpWRKY50基因及其提高番木瓜炭疽病抗性的用途

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CN111635904A (zh) * 2020-06-29 2020-09-08 山东农业大学 一种增强黄瓜靶斑病抗性的基因CsWRKY10及其应用
CN116064586A (zh) * 2022-11-01 2023-05-05 广东省农业科学院果树研究所 一种番木瓜CpWRKY50基因及其提高番木瓜炭疽病抗性的用途
CN116064586B (zh) * 2022-11-01 2024-04-02 广东省农业科学院果树研究所 一种番木瓜CpWRKY50基因及其提高番木瓜炭疽病抗性的用途

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