WO2014076659A1 - Procédé de production de plantes dotées d'une résistance augmentée aux agents pathogènes - Google Patents

Procédé de production de plantes dotées d'une résistance augmentée aux agents pathogènes Download PDF

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Publication number
WO2014076659A1
WO2014076659A1 PCT/IB2013/060144 IB2013060144W WO2014076659A1 WO 2014076659 A1 WO2014076659 A1 WO 2014076659A1 IB 2013060144 W IB2013060144 W IB 2013060144W WO 2014076659 A1 WO2014076659 A1 WO 2014076659A1
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plant
nucleic acid
acid sequence
transgenic
seq
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PCT/IB2013/060144
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English (en)
Inventor
Holger Schultheiss
Tobias MENTZEL
Patrick Schweizer
Dimitar Douchkov
Axel Himmelbach
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Basf Plant Science Company Gmbh
Basf (China) Company Limited
Basf Schweiz Ag
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Publication of WO2014076659A1 publication Critical patent/WO2014076659A1/fr

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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)
    • C12N15/8241—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 receptor-like protein kinase is increased.
  • 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%.
  • 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. Therefore, one approach for producing pathogen resistant plants is the (over)expression of a transgene in said plants, resulting in the formation of specific defence substances.
  • chitinase WO 92/17591
  • pathogenesis-related genes WO 92/20800
  • genes for various oxidizing enzymes such as glucose oxidase (WO 95/21924) and oxalate oxidase (WO 99/04013), have already been overexpressed in plants, thus creating plants having increased fungal resistance.
  • transgenic plants having increased fungal resistance is to inhibit the expression of said plant genes which code for example for a polyphenoloxidase (WO 02/061 101 ), NADPH oxidase (WO 2004/009820) and the Mlo gene (WO 00/01722) in transgenic plants.
  • 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 (US 2007/0061918).
  • nonhost resistance is usually defined as the durable resistance of all known genotypes of a plant species to all known races or isolates of a pathogen species. However, it may also operate at the subspecies level, for example with respect to formae speciales of Blumeria graminis.
  • barley Hadeum vulgare
  • wheat Triticum aestivum
  • wheat shows resistance to B. graminis f.sp. hordei, but is susceptible to B. graminis f.sp. tritici.
  • the present inventors have found that the transgenic expression of a receptor-like kinase leads to an enhanced resistance of wheat cells to Blumeria graminis f.sp. tritici.
  • RLKs Receptor-like protein kinases
  • 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, the transgenic plant or the transgenic part thereof the content and/or activity of a receptor-like protein kinase which is encoded by a nucleic acid sequence selected from the group consisting of:
  • nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 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 any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1 -13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence encoding the same receptor-like kinase as any of the nucleic acid sequences of (a) to (d) above, but differing from the nucleic acid sequences of (a) to (d) above due to the degeneracy of the genetic code
  • the present invention also provides a method for increasing pathogen resistance in a transgenic plant cell, a transgenic plant or a transgenic part thereof compared to a control plant cell, plant or plant part, wherein in the transgenic plant cell, the transgenic plant or the transgenic part thereof the content and/or activity of a receptor-like protein kinase which is encoded by a nucleic acid sequence selected from the group consisting of:
  • nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1-5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences; b) a nucleic acid sequence encoding a protein comprising the amino acid sequence according to any of SEQ ID Nos. 6, 10, 14, 18, 22 and 25;
  • nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1 -13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence encoding the same receptor-like kinase as any of the nucleic acid sequences of (a) to (d) above, but differing from the nucleic acid sequences of (a) to (d) above due to the degeneracy of the genetic code
  • the method comprises the steps of
  • the method further comprises step (b) or, if step (b) is present in the above method, step (c) of selecting transgenic plant cells or transgenic plants.
  • 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 protein which mediates pathogen susceptibility or increasing the content and/or activity of at least one further protein which mediates pathogen resistance.
  • 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 receptor-like protein kinase as defined herein is not increased and selecting transgenic progeny in which the content and/or the activity of the receptor-like protein kinase as defined herein is increased.
  • 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.
  • Another embodiment of 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:
  • 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 any of SEQ ID Nos. 1-5, 7-9, 1 1- 13, 15-17, 19-21 , 23 and 24 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, more preferably it is a barley, a maize or a wheat plant, most preferably it is a wheat or a maize plant.
  • the transgenic or mutant plant has an increased resistance to a fungal pathogen, more preferably to Blumeria graminis, Septoria tritici, Puccinia triticina, Fusarium verticilloides, Fusarium proliferatum, Fusarium subglutinans, Fusarium graminaearum, Diplodia maydis, Macrophomina phaseolina and/or Colletotrichum graminicola.
  • a fungal pathogen more preferably to Blumeria graminis, Septoria tritici, Puccinia triticina, Fusarium verticilloides, Fusarium proliferatum, Fusarium subglutinans, Fusarium graminaearum, Diplodia maydis, Macrophomina phaseolina and/or Colletotrichum graminicola.
  • the transgenic or mutant plant is a wheat plant and the pathogen is Blumeria graminis f.sp. tritici or the transgenic or mutant plant is a maize plant and the pathogen is Fusarium verticilloides, Fusarium proliferatum, Fusarium subglutinans, Fusarium
  • the resistance conferred by the present invention is non-host resistance.
  • the present invention relates to an expression construct comprising at least one nucleic acid sequence selected from the group consisting of:
  • nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence encoding a protein comprising the amino acid sequence according to any of SEQ ID Nos. 6, 10, 14, 18, 22 and 25;
  • nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1 -13,
  • nucleic acid sequence encoding the same receptor-like kinase as any of the nucleic acid sequences of (a) to (d) above, but differing from the nucleic acid sequences of (a) to (d) above due to the degeneracy of the genetic code
  • the expression construct further comprises regulatory sequences which can act as termination and/or polyadenylation signal in the plant cell and which are operably linked to the DNA sequence as defined herein.
  • the promoter is a tissue-specific and/or a pathogen-inducible promoter.
  • the invention relates to a vector comprising the 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, plant cell or plant part with an increased resistance to pathogens compared to a control plant, plant cell or plant part, which plant is produced by the method of the present invention or contains 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, the expression construct, the vector or the mutation which increases the content and/or the activity of the receptor-like protein kinase as defined herein can be detected in said transgenic or mutant seed or in said flour.
  • the term "obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. a plant is defined to be obtainable by a specific method, this is also to be understood to disclose a plant which is obtained by this method.
  • 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 the sequence according to any of SEQ ID Nos. 1-5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 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 any of SEQ ID Nos 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1 -13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence encoding the same receptor-like kinase as any of the nucleic acid sequences of (a) to (d) above, but differing from the nucleic acid sequences of (a) to (d) above due to the degeneracy of the genetic code, which is not present at the natural locus of this sequence in the genome of the control plant and/or which has been linked to sequences to which the nucleic acid sequence is not linked in the genome of the control plant and/or which is present in another 5' to 3' orientation compared to the orientation of this sequence in the natural locus of the control plant.
  • 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 increases the content and/or the activity of the receptor-like protein kinase 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 an endogenous nucleic acid sequence selected from the group consisting of
  • nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence encoding the same receptor-like kinase as any of the nucleic acid sequences of (a) to (d) above, but differing from the nucleic acid sequences of (a) to (d) above due to the degeneracy of the genetic code
  • the mutant plant contains at least one point mutation, i.e. at least one nucleotide substitution, deletion and/or addition, in comparison to the corresponding nucleic acid sequence in 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 any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences.
  • the transgenic 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.
  • Useful dicotyledonous 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.
  • transgenic or mutant plants are oat, barley, rye, maize, wheat or rice plants, even more preferably the transgenic or mutant plants are barley, maize or wheat plants and most preferably the transgenic or mutant plants are wheat or maize plants.
  • transgenic plant also 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 which increases the content and/or activity of the receptor-like protein kinase of the present invention.
  • the mutant progeny of the mutant plant comprises at least one point mutation which increases the content and/or activity of the receptor-like protein kinase of the present invention.
  • the 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.
  • the term "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, seeds 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. While said symptoms can be manifold, they preferably comprise such symptoms directly or indirectly leading to impairment of plant quality, yield quantity, or suitability for use as feed or food, or impeding sowing, cultivation, harvest, or processing of the crop.
  • resistance also means that pests and/or a pathogen and preferably a fungus and especially preferably the fungi described below display reduced growth in a plant and reduced or absent propagation.
  • the term “resistance” also includes a so-called transient resistance, i.e. the transgenic plants or plant cells of the present invention have an increased resistance to pests and/or pathogens or fungi compared to the corresponding control plants only for a limited period of time.
  • the resistance conferred is a nonhost resistance which is the durable resistance of all known genotypes of a plant species to all known races or isolates of a pathogen species.
  • the resistance is transferred from a plant species which is resistant to a specific pathogen to a plant species which is susceptible to said pathogen.
  • the term "increased pathogen resistance” is understood to denote that the transgenic 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 90%, preferably less than 85 or 80%, more preferably less than 75 or 70% and most preferably less than 68%.
  • 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 increasing the content and/or the activity of receptor-like protein kinase.
  • a control plant may be a plant of the same line or variety as the transgenic plant being tested, or it may be of 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 and which has been used as starting material for the mutagenization and which does not contain the at least one point mutation of the mutant plant in the nucleic acid sequence of the present invention.
  • test plants The infection of test plants with pathogens such as fungi in order to examine potential resistance phenomena is 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 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, preferably with the corresponding forma specialis of the plant to be inoculated, i.e. the pathogen which is adapted to the specific host used.
  • wheat is preferably inoculated with Blumeria graminis f.sp.
  • tritici and barley is preferably inoculated with Blumeria graminis f.sp. hordei.
  • “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 £. coli, a fluorescence gene, the green fluorescence protein (GFP) gene from Aequorea victoria, the luciferase gene from Photinus pyralis or the beta-galactosidase (lacZ) gene from E.
  • 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 expression of the receptor-like protein kinase.
  • the formed fungal structures may be stained by methods well-known to those skilled in the art in order to improve the determination thereof, e.g. by staining with coomassie or trypan blue. Then, 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 mediating the expression of the receptor-like protein kinase is lower than the diseased leaf area of infected control plants.
  • the diseased leaf area of infected plants transformed with the vector mediating the expression of the receptor-like protein kinase is 90%, 85%, 80%, 75% or 70%, more preferably it is 65%, 60%, 55% or 50%, even more preferably it is 45%, 40%, 35% or 30% and most preferably it is 25%, 20%, 15% or 10% of the diseased leaf area of the infected control plants.
  • plant pathogens includes viral, bacterial, fungal and other pathogens.
  • the term "plant pathogens" comprises fungal pathogens.
  • plant pathogens includes biotrophic
  • the plant pathogen is a biotrophic pathogen, more preferably a biotrophic fungal pathogen.
  • the biotrophic phytopathogenic fungi such as many rusts, depend for their nutrition on the metabolism of living cells of the plants.
  • This type of fungi belongs to the group of biotrophic fungi, like other rust fungi, powdery mildew fungi or oomycete pathogens like the genus Phytophthora or Peronospora.
  • the necrotrophic phytopathogenic fungi e.g. species from the genus Fusarium, Rhizoctonia or Mycospaerella, depend for their nutrition on dead cells of the plants. Soybean rust has occupied an intermediate position, since it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic.
  • Rhizoctonia solani Kuhn Rhizoctonia
  • Brown spot black spot, stalk rot
  • Cephalosporium kernel rot Acremonium strictum Cephalosporium
  • Curvularia leaf spot Curvularia clavata, C. eragrostidis, C.
  • Diplodia ear and stalk rot Diplodia frumenti (teleomorph: Botryosphaeria festucae)
  • Diplodia ear and stalk rot, seed rot Diplodia maydis Disease Pathogen
  • Green ear downy mildew (graminicola Sclerospora graminicola
  • 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
  • sorokinianum H. sativum
  • Epicoccum nigrum
  • Leptosphaeria maydis, Leptothyrium zeae,
  • Penicillium ear rot blue eye, blue Penicillium spp., P. chrysogenum
  • Phaeocytostroma stalk and root rot Phaeocytostroma ambiguum,
  • Phaeosphaeria leaf spot Phaeosphaeria maydis Sphaerulina maydis
  • Red kernel disease ear mold, leaf 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.
  • Helminthosporium carbonum, Diplodia maydis, Exserohilum pedicillatum, Exserohilum turcicum Helminthosporium turcicum, Fusarium avenaceum, F. culmorum, F. moniliforme, Gibberella zeae (anamorph: F. graminearum), Macrophomina phaseolina, Penicillium spp., Phomopsis sp., Pythium spp., Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicaria sp.
  • 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 Plasmodiophoromycetes, 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. Particularly preferred are:
  • Spongospora subterranea pausing potato tubers
  • Polymyxa graminis root disease of cereals and grasses
  • Oomycetes like Bremia lactucae (downy mildew of lettuce), Peronospora (downy mildew) of snapdragon (P. antirrhini), onion (P. destructor), spinach (P. effusa), soy bean ⁇ P. manchurica), tobacco ("blue mold” , P. tabacina) alfalfa and clover (P. trifolium),
  • Pseudoperonospora humuli downy mildew of hop
  • Plasmopara downy mildew of grapes
  • sun flower P. halstedii
  • Sclerophthora macrospora downy mildew of cereals and grasses
  • Pythium seed rot, seedling damping-off, and root rot and all types of plants, for example black root disease of beet caused by
  • Pseudopeziza medicaginis (leaf spot diseases of lucerne, white and red clover).
  • 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
  • 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.
  • the transgenic plants produced according to the present invention 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.
  • 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.
  • Cynodon chlorotic streak virus CCSV
  • JGMV Johnsongrass mosaic Johnsongrass mosaic virus
  • MLO Maize bushy stunt Mycoplasma-like organism
  • MDMV Maize dwarf mosaic Maize dwarf mosaic virus
  • Maize mosaic corn leaf stripe, Maize mosaic virus (MMV)
  • Maize rayado fino fine striping Maize rayado fino virus (MRFV) disease
  • 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) (also: sugarcane).
  • SCMV mosaic virus
  • SMV Sugarcane mosaic Sugarcane mosaic virus
  • Wheat spot mosaic Wheat spot mosaic virus (WSMV)
  • 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: Coleoptera and Lepidoptera, like, for example, the European corn borer (ECB), Diabrotica barberi (Northern corn rootworm), Diabrotica undecimpunctata (Southern corn rootworm), Diabrotica virgifera (Western corn rootworm), Agrotis ipsilon (black cutworm), Crymodes devastator (glassy cutworm), Feltia prisens (dingy cutworm), Agrotis gladiaria (claybacked cutworm), Melanotus spp., Aeolus mellillus (wireworm), Aeolus mancus (wheat wireworm), Horistonotus uhlerii (sand wireworm), Sphenophorus maidis (maize billbug), Sphenophorus zeae (timothy billbug), Sphenophorus parvulus (bluegrass billbug), Sphenophorus callosus (southern corn billbug), Phyllog
  • 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 resistant to Globodera
  • 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.
  • 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, Peronospora parasitica, Fusarium roseum and Alternaria alternata.
  • the plants are resistant to the fungal, bacterial, or viral pathogens Clavibacter michiganensis subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae.
  • 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.
  • 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 cichoracearum, 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.
  • Anuraphis maidiradicis corn root aphid
  • Blissus leucopterus leucopterus corninch bug
  • Rhopalosiphum maidis corn leaf aphid
  • Siphaflava yellow sugarcane aphid
  • Blissus leucopterus leucopterus chinch bug
  • Contarinia sorghicola sorghum midge
  • Tetranychus cinnabarinus carmine spider mite
  • Tetranychus urticae two-spotted spider mite
  • 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 leucopterus 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).
  • the term "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, Puccinia triticina, Fusarium verticilloides, Fusarium proliferatum, Fusarium subglutinans, Fusarium graminaearum, Diplodia maydis, Macrophomina phaseolina and Colletotrichum graminicola .
  • a "receptor-like protein kinase” is a protein having an extracellular domain, a transmembrane domain and an intracellular kinase domain which protein catalyzes the transfer of phosphate to a substrate protein.
  • the receptor-like kinase of the present invention has an amino acid sequence selected from the group consisting of:
  • the receptor-like kinase of the present invention is encoded by a nucleic acid sequence selected from the group consisting of:
  • nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence encoding a protein comprising the amino acid sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24;
  • nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos. 1-5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence encoding the same receptor-like kinase as any of the nucleic acid sequences of (a) to (d) above, but differing from the nucleic acid sequences of (a) to (d) above due to the degeneracy of the genetic code.
  • the protein with the amino acid sequence according to SEQ ID No. 6 is encoded by a nucleic acid sequence according to any of SEQ ID NOs. 1-5, preferably by the nucleic acid sequence according to SEQ ID No. 1 or 3.
  • the protein with the amino acid sequence according to SEQ ID No. 10 is encoded by a nucleic acid sequence according to any of SEQ ID NOs. 7-9, preferably by the nucleic acid sequence according to SEQ ID No. 7 or 8.
  • the protein with the amino acid sequence according to SEQ ID No. 14 is encoded by a nucleic acid sequence according to any of SEQ ID NOs. 1 1-13, preferably by the nucleic acid sequence according to SEQ ID No. 1 1 or 12.
  • the protein with the amino acid sequence according to SEQ ID No. 22 is encoded by a nucleic acid sequence according to any of SEQ ID NOs. 19-21 , preferably by the nucleic acid sequence according to SEQ ID No. 19 or 20.
  • the protein with the amino acid sequence according to SEQ ID No. 25 is encoded by a nucleic acid sequence according to any of SEQ ID NOs. 23 and 24, preferably by the nucleic acid sequence according to SEQ ID No. 23.
  • 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
  • the content of a protein within a cell can be influenced on the level of transcription and/or the level of translation.
  • 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 or the interaction with other proteins.
  • the present invention also encompasses methods of increasing the activity of the receptor-like protein kinase which do not affect the content of this protein, such as the expression of a protein which modifies the receptor-like protein kinase by, e.g., phosphorylation, and thereby increases its activity.
  • the expression level of the nucleic acid coding for the receptor-like protein kinase 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 protein can also be quantified by determining the strength of the signal in the Northern Blot analysis or by performing a quantitative PCR.
  • the expression of the nucleic acid coding for the receptor-like protein kinase is statistically significantly increased by at least the factor 2, 3 or 4, preferably by at least the factor 5, 7 or 10, more preferably by at least the factor 12, 15 or 18, even more preferably by at least the factor 20, 22 or 25 and most preferably by at least the factor 30, 35, 40, 45 or 50.
  • the expression level of the receptor-like protein kinase protein may also be determined by Western Blot analysis using suitable antibodies.
  • the amount of the receptor-like protein kinase protein is statistically significantly increased by at least the factor 2, 3 or 4, preferably by at least the factor 5, 7 or 10, more preferably by at least the factor 12, 15 or 18, even more preferably by at least the factor 20, 22 or 25 and most preferably by at least the factor 30, 35, 40, 45 or 50.
  • the activity of the receptor-like protein kinase may be determined by isolating the receptor-like protein kinase protein from a cell containing it, e.g. by immunoprecipitation, and incubating the protein with a target protein which is phosphorylated by the receptor-like protein kinase and radiolabeled ATP. Then, a sample of the reaction is separated on an SDS-PAGE gel, dried and examined by autoradiography. If the kinase is active, the target protein was phosphorylated and the radiogram will show a corresponding signal which can be quantified and compared to the signal in the control plant.
  • the increased activity of the receptor-like protein kinase will lead to an increase in target protein phosphorylation by at least the factor 1 .5 or 2, preferably by at least the factor 3 or 4, more preferably by at least the factor 5 or 6, even more preferably by at least the factor 7 or 8 and most preferably by at least the factor 9 or 10.
  • the method involves introducing into a plant or plant cell a vector which comprises:
  • endogenous receptor-like protein kinase inherent to the plant/s. This can, for example, be achieved by altering the promoter DNA sequence of a nucleic acid sequence coding for the receptor-like protein kinase. Such a modification, which leads to an increased expression rate of at least one endogenous receptor-like protein kinase, can be effected by deleting or inserting DNA sequences in the promoter region.
  • an increased expression of at least one endogenous receptor-like protein kinase can be achieved by means of a regulator protein, which is not present in the control plant and which interacts with the promoter of the gene encoding the endogenous receptor-like protein kinase.
  • a regulator protein which is not present in the control plant and which interacts with the promoter of the gene encoding the endogenous receptor-like protein kinase.
  • a regulator can be a chimeric protein, which consists of a DNA binding domain and a transcription activator domain, as is described, for example, in WO 96/06166.
  • a further possibility for increasing the activity and/or the content of the endogenous receptor-like protein kinase is to upregulate transcription factors, which are involved in the transcription of the endogenous genes coding for the receptor-like protein kinase, for example by overexpression.
  • the measures for overexpressing transcription factors are known to the person skilled in the art and within the scope of the present invention are also disclosed for the receptor-like protein kinase.
  • An alteration of the activity of the endogenous receptor-like protein kinase can also be achieved by influencing the post-translational modifications of the receptor-like protein kinase protein. This can, for example, be done by regulating the activity of enzymes like kinases or
  • phosphatases which are involved in the post-translational modification of the receptor-like protein kinase, by means of corresponding measures like overexpression or gene silencing.
  • the expression of the endogenous receptor-like protein kinase can also be regulated via the expression of aptamers specifically binding to the promoter sequences of the receptor-like protein kinase. If the aptamers bind to stimulating promoter regions, the amount and thus, in this case, the activity of the endogenous receptor-like protein kinase is increased.
  • the skilled person also knows other methods for increasing the content and/or activity of a protein, such as the receptor-like protein kinase encoded by the nucleic acid sequence according to any of SEQ ID Nos.
  • a nucleic acid sequence for increasing the content and/or the activity of a protein may be integrated into the natural locus of the sequence by targeted homologous recombination.
  • 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 A1 .
  • 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.
  • nucleic acid sequences within the genomic DNA for introducing a nucleic acid sequence for increasing the content and/or the activity of a protein different enzymes such as meganucleases (WO 2009/1 14321 A2), zink finger nucleases (WO 2009/042164 A1 ), transcription activator-like effector nucleases (WO 201 1/072246 A2) and chimeric nucleases which comprise a DNA binding domain targeting the nuclease to a specific sequence within the genome
  • meganucleases WO 2009/1 14321 A2
  • zink finger nucleases WO 2009/042164 A1
  • transcription activator-like effector nucleases WO 201 1/072246 A2
  • chimeric nucleases which comprise a DNA binding domain targeting the nuclease to a specific sequence within the genome
  • 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.
  • TILLING Targeting induced Local Lesions IN_ Genomes
  • 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
  • mutagenesis by irradiation such as ionizing irradiation or mutagenesis by using sequence- specific nucleases.
  • Single base mutations or point mutations lead to the formation of heteroduplexes which are then cleaved by single strand nucleases such as Ce/I at the 3' side of the mutation.
  • Single base mutations or point mutations lead to the formation of heteroduplexes which are then cleaved by single strand nucleases such as Ce/I at the 3' side of the mutation.
  • Ce/I single strand nucleases
  • 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 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. 9:1 15). If necessary, it can then be determined whether the mutant plant having the at least one point mutation within the nucleic acid sequence according to any of SEQ ID Nos. 1-5, 7-9, 1 1- 13, 15-17, 19-21 , 23 and 24 has an increased resistance to pathogens and optionally, suitable plants can be selected.
  • nucleic acid sequence is used which is selected from the group consisting of:
  • nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 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 any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos. 1-5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences;
  • nucleic acid sequence encoding the same receptor-like kinase as any of the nucleic acid sequences of (a) to (d) above, but differing from the nucleic acid sequences of (a) to (d) above due to the degeneracy of the genetic code.
  • a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 1 , 3, 7, 8, 1 1 , 12, 15, 16, 19, 20 and 23 is used.
  • 1 -5, 7-9, 1 1 -13, 15- 17, 19-21 , 23 and 24 is understood to refer to a smaller part of this nucleic acid sequence which consists of a contiguous nucleotide sequence found in SEQ ID Nos. 1-5, 7-9, 1 1-13, 15-17, 19- 21 and 23-25 and which encodes a protein having the activity of a receptor-like protein kinase.
  • 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 any of SEQ ID Nos. 1 and 2 has a length of at least 1000 or 1300 nucleotides, preferably of at least 1500, 1800 or 2000 nucleotides, more preferably of at least 2300, 2600 and 2900 nucleotides and most preferably of at least 3100, 3200 or 3300 nucleotides.
  • the fragment of any of SEQ I D Nos. 4, 5, 17 and 24 has a length of at least 3000 nucleotides, preferably of at least 3500 or 3800 nucleotides, more preferably of at least 4000, 4300 or 4600 nucleotides and most preferably of at least 4700, 4800 or 5000 nucleotides.
  • the fragment of any of SEQ ID Nos. 9 and 13 has a length of at least 12000, 12500, 13000 or 13500
  • nucleotides preferably of at least 14000, 14500, 15000, 15500 or 16000 nucleotides, more preferably of at least 16200, 16400, 16600, 16800 or 17000 nucleotides and most preferably of at least 17200, 17400, 17600, 17800, 18000, 18200, 18400 or 18600 nucleotides.
  • the fragment of any of SEQ ID Nos. 7, 15 and 19 has a length of at least 1000, 1 100, 1200, 1300, 1400 or 1500 nucleotides, preferably of at least 1600, 1700 or 1800 nucleotides, more preferably of at least 1850, 1900 or 1950 nucleotides and most preferably of at least 2000, 2050, 2080 or 2100 nucleotides.
  • the fragment of any of SEQ ID Nos. 8, 16 and 20 has a length of at least 800, 850, 900 or 950 nucleotides, preferably of at least 1000, 1050, 1 100, 1 150, 1200, 1250 or 1300 nucleotides, more preferably of at least 1350, 1400, 1450, 1500 or 1550 nucleotides and most preferably of at least 1600, 1650, 1700 or 1750 nucleotides.
  • 3 and 1 1 has a length of at least 1200, 1300, 1350, 1400, 1450 or 1500 nucleotides, preferably of at least 1550, 1600, 1650, 1700, 1750 or 1800 nucleotides, more preferably of at least 1850, 1900, 1950, 2000, 2050, 2100, 2150 or 2200 nucleotides and most preferably of at least 2250, 2300, 2350, 2400, 2450 or 2500 nucleotides.
  • 12 has a length of at least 700, 750, 800, 850, 900 or 950 nucleotides, preferably of at least 1000, 1050, 1 100, 1 150, 1200 or 1250 nucleotides, more preferably of at least 1300, 1320, 1340, 1360, 1380, 1400, 1420 or 1440 nucleotides and most preferably of at least 1460, 1480 or 1500 nucleotides.
  • 21 has a length of at least 5000, 5500, 6000, 6500, 7000 or 7500 nucleotides, preferably of at least 8000, 8200, 8400, 8600, 8800 or 9000 nucleotides, more preferably of at least 9200, 9400, 9600 or 9800 nucleotides and most preferably of at least 10000, 10100, 10200 or 10300 nucleotides.
  • 23 has a length of at least 500, 550, 600, 650 or 700 nucleotides, preferably of at least 720, 740, 760, 780, 800, 820, 840, 860 or 880 nucleotides, more preferably of at least 900, 910, 920, 930, 940 or 950 nucleotides and most preferably of at least 960, 970, 980, 990, 1000, 1010 or 1020 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 87, 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, 99 or 100% identical to the complete sequence according to any of SEQ ID Nos. 1 -5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences and which encode a protein having the activity of a receptor-like protein kinase.
  • 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.
  • the BLAST program package of the National Center for Biotechnology Information
  • sequence identity refers to the degree of the sequence identity over a length of 1000 or 1300 nucleotides, preferably of 1500, 1800 or 2000 nucleotides, more preferably of 2300, 2600, 2900, 3100, 3200 or 3300 nucleotides and most preferably the whole length of any of SEQ ID Nos. 1 and 2.
  • sequence identity refers to the degree of the sequence identity over a length of 3000 nucleotides, preferably of 3500 or 3800 nucleotides, more preferably of 4000, 4300, 4600, 4700, 4800 or 5000 nucleotides and most preferably over the whole length of any of SEQ ID Nos. 4, 5, 17 and 24.
  • sequence identity refers to the degree of the sequence identity over a length of 12000, 12500, 13000 or 13500 nucleotides, preferably of 14000, 14500, 15000, 15500 or 16000 nucleotides, more preferably of 16200, 16400, 16600, 16800, 17000, 17200, 17400, 17600, 17800, 18000, 18200, 18400 or 18600 nucleotides and most preferably the whole length of any of SEQ ID Nos. 9 and 13.
  • sequence identity refers to the degree of the sequence identity over a length of 1000, 1 100, 1200, 1300, 1400 or 1500 nucleotides, preferably of 1600, 1700 or 1800 nucleotides, more preferably of 1850, 1900, 1950, 2000, 2050, 2080 or 2100 nucleotides and most preferably the whole length of any of SEQ ID Nos. 7, 15 and 19.
  • sequence identity refers to the degree of the sequence identity over a length of 800, 850, 900 or 950 nucleotides, preferably of 1000, 1050, 1 100, 1 150, 1200, 1250 or 1300 nucleotides, more preferably of 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700 or 1750 nucleotides and most preferably the whole length of any of SEQ ID Nos. 8, 16 and 20.
  • sequence identity refers to the degree of the sequence identity over a length of 1200, 1300, 1350, 1400, 1450 or 1500 nucleotides, preferably of 1550, 1600, 1650, 1700, 1750 or 1800 nucleotides, more preferably of 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450 or 2500 nucleotides and most preferably the whole length of any of SEQ ID Nos. 3 and 1 1.
  • sequence identity refers to the degree of the sequence identity over a length of 700, 750, 800, 850, 900 or 950 nucleotides, preferably of 1000, 1050, 1 100, 1 150, 1200 or 1250 nucleotides, more preferably of 1300, 1320, 1340, 1360, 1380, 1400, 1420, 1440, 1460, 1480 or 1500 nucleotides and most preferably the whole length of SEQ ID No. 12.
  • sequence identity refers to the degree of the sequence identity over a length of 5000, 5500, 6000, 6500, 7000 or 7500 nucleotides, preferably of 8000, 8200, 8400, 8600, 8800 or 9000 nucleotides, more preferably of 9200, 9400, 9600, 9800, 10000, 10100, 10200 or 10300 nucleotides and most preferably the whole length of SEQ ID No. 21.
  • sequence identity refers to the degree of the sequence identity over a length of 500, 550, 600, 650 or 700 nucleotides, preferably of 750, 800, 850 or 900 nucleotides, more preferably of 920, 940, 960, 980, 1000, 1010, 1020 or 1030 nucleotides and most preferably the whole length of SEQ ID No. 23.
  • the present invention further relates to the use of nucleic acid sequences which hybridize under stringent conditions with a complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos. 1-5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24 or a fragment of any of these sequences and which encode an amino acid sequence having the activity of a receptor-like protein kinase.
  • hybridizing under stringent conditions denotes in the context of the present invention 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. Sambrook and Russell (2001 ) Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY).
  • specific hybridization refers to the circumstance that a molecule, under stringent conditions, 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 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
  • the nucleic acid sequence hybridizing to a fragment of the complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos. 1 and 2 under stringent conditions has a length of at least 1000 or 1300 nucleotides, preferably of at least 1500, 1800 or 2000 nucleotides, more preferably of at least 2300, 2600, 2900 nucleotides and most preferably of at least 3100, 3200 or 3300 nucleotides.
  • 4, 5, 17 and 24 under stringent conditions has a length of at least 3000 nucleotides, preferably of at least 3500 or 3800 nucleotides, more preferably of at least 4000, 4300, 4600 nucleotides and most preferably of at least 4700, 4800 or 5000 nucleotides.
  • the nucleic acid sequence hybridizing to a fragment of the complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos.
  • 9 and 13 under stringent conditions has a length of at least 12000, 12500, 13000 or 13500 nucleotides, preferably of at least 14000, 14500, 15000, 15500 or 16000 nucleotides, more preferably of at least 16200, 16400, 16600, 16800, 17000, 17200, 17400 nucleotides and most preferably of at least 17600, 17800, 18000, 18200, 18400 or 18600 nucleotides.
  • the nucleic acid sequence hybridizing to a fragment of the complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos.
  • 7, 15 and 19 under stringent conditions has a length of at least 1000, 1 100, 1200, 1300, 1400 or 1500 nucleotides, preferably of at least 1600, 1700 or 1800 nucleotides, more preferably of at least 1850, 1900, 1950, 2000 nucleotides and most preferably of at least 2050, 2080 or 2100 nucleotides.
  • the nucleic acid sequence hybridizing to a fragment of the complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos.
  • 16 and 20 under stringent conditions has a length of at least 800, 850, 900 or 950 nucleotides, preferably of at least 1000, 1050, 1 100, 1 150, 1200, 1250 or 1300 nucleotides, more preferably of at least 1350, 1400, 1450, 1500 nucleotides and most preferably of at least 1550, 1600, 1650, 1700 or 1750 nucleotides.
  • the nucleic acid sequence hybridizing to a fragment of the complementary sequence of a nucleic acid sequence according to any of SEQ ID Nos.
  • 3 and 1 1 under stringent conditions has a length of at least 1200, 1300, 1350, 1400, 1450 or 1500 nucleotides, preferably of at least 1550, 1600, 1650, 1700, 1750 or 1800 nucleotides, more preferably of at least 1850, 1900, 1950, 2000, 2050, 2100 nucleotides and most preferably of at least 2150, 2200, 2250, 2300, 2350, 2400, 2450 or 2500 nucleotides.
  • the nucleic acid sequence hybridizing to a fragment of the complementary sequence of a nucleic acid sequence according to SEQ ID No.
  • 12 under stringent conditions has a length of at least 700, 750, 800, 850, 900 or 950 nucleotides, preferably of at least 1000, 1050, 1 100, 1 150, 1200 or 1250 nucleotides, more preferably of at least 1300, 1320, 1340, 1360, 1380, 1400 nucleotides and most preferably of at least 1420, 1440, 1460, 1480 or 1500 nucleotides.
  • the nucleic acid sequence hybridizing to a fragment of the complementary sequence of a nucleic acid sequence according to SEQ ID No.
  • 21 under stringent conditions has a length of at least 5000, 5500, 6000, 6500, 7000 or 7500 nucleotides, preferably of at least 8000, 8200, 8400, 8600, 8800 or 9000 nucleotides, more preferably of at least 9200, 9400, 9600, 9800 or 10000 nucleotides and most preferably of at least 10100, 10200 or 10300 nucleotides.
  • the nucleic acid sequence hybridizing to a fragment of the complementary sequence of a nucleic acid sequence according to SEQ ID No.
  • 23 under stringent conditions has a length of at least 500, 550, 600, 650, 700 or 750 nucleotides, preferably of at least 800, 820, 840, 860, 880 or 900 nucleotides, more preferably of at least 920, 940, 960, 980 oM OOO nucleotides and most preferably of at least 1010, 1020 or 1030 nucleotides.
  • the term "encodes a protein having the activity of a receptor-like protein kinase” means that the encoded protein has essentially the same activity as the receptor-like protein kinase encoded by a nucleic acid sequence of any of SEQ ID Nos. 1 -5, 7- 9, 1 1-13, 15-17, 19-21 , 23 and 24.
  • "Essentially the same activity” means that the protein has at least 5 or 10%, preferably at least 20, 30 or 40%, more preferably 50, 60 or 70% and most preferably at least 80, 85, 88, 90, 95, or 98% of the activity of the receptor-like protein kinase encoded by a sequence of any of SEQ ID Nos. 1-5, 7-9, 1 1-13, 15-17, 19-21 , 23 and 24.
  • the activity of the receptor-like protein kinase can be determined as described above.
  • a suitable nucleic acid sequence may for example be inserted into an appropriate expression construct or vector by restriction digestion and subsequent ligation using techniques well-known to the person skilled in the art and described in the textbooks referred to herein.
  • the terms "expression construct” or "expression cassette” mean 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”.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • 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 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. 1 1 : 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 (Stockhaus et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7943-7947; Stockhaus et al. (1989) EMBO J. 8: 2445-2451 ).
  • 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. from tomato, which mediates expression during the ripening process of tomato fruits (Nicholass et al. (1995) Plant Mol. Biol. 28: 423-435), the promoter of an ACC oxidase, e.g. from apples, which mediates ripening and fruit specificity in transgenic tomatoes (Atkinson et al. (1998) Plant Mol. Biol. 38: 449-460), or the 2A1 1 promoter from tomato (van Haaren et al. (1991 ) Plant Mol. Biol. 17: 615-630). Further, the chemically inducible Tet repressor system (Gatz et al. (1991 ) Mol. Gen. Genet. 227: 229-237) may be used.
  • 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.
  • 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).
  • Further 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).
  • promoters which are active on the site of pathogen entry are also particularly suitable in the method of the present invention.
  • 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): 31 1 ), the GerA promoter (Wu et al. (2000) Plant Phys Biochem.
  • the OsROCI promoter (Accession number AP004656), the RTBV promoter (Kloeti et al. (1999) PMB 40: 249); the chitinase ChtC2 promoter (Ancillo et al. (2003) Planta 217(4): 566), the AtProT3 promoter (Grallath et al. (2005) Plant Physiol. 137(1 ): 1 17) and the SHN promoters from Arabidopsis (Aaron et al. (2004) Plant Cell 16(9): 2463). Furthermore, those skilled in the art are able to isolate further suitable promoters by means of routine procedures.
  • inducible promoters allows for the production of plants and plant cells which only transiently express the sequences of the present invention.
  • 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 is desirable, are known to those skilled in the art.
  • transient expression 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 providing constitutive transgene expression 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.
  • the regulatory elements which are contained in the vectors ensure the transcription and, if desired, the translation in the plant cell.
  • 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. For example, if 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 DNA regions which allow for a propagation of the vectors in bacteria, such as E.coli.
  • Regulatory elements also comprise sequences which lead to a stabilization of the vectors in the host cells.
  • such 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.
  • plasmids do not need to fulfil special requirements per se.
  • Simple plasmids such as pUC derivatives, may be used. If, however, whole plants are to be regenerated from cells which were transformed in such manner, the presence of a selectable marker gene may become necessary.
  • selectable marker gene may become necessary.
  • 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.
  • 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.
  • 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
  • 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. 5,451 ,513; U.S. 5,545,817; U.S. 5,545,818 and U.S. 5,877,462, in WO 95/16783 and WO 97/32977, and in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 7301 -7305.
  • the transformed cells grow within the plant in the usual manner (see also McCormick et al. (1986) Plant Cell Reports 5: 81-84).
  • 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 receptor-like protein kinase is not increased and selecting transgenic progeny in which the content and/or the activity of the receptor-like protein kinase is increased.
  • the other plant in which the content and/or the activity of the receptor-like protein kinase is not increased 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 receptor-like protein kinase is increased 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 in which the content and/or the activity of the receptor-like protein kinase is increased is inbred and the transgenic progeny of this crossing step 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 increases the content and/or the activity of the receptor-like protein kinase.
  • the true breeding lines can then be used in hybrid breeding yielding F1 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 expression constructs, vectors or 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, 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 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 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.
  • 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 increases the content and/or activity of a receptor-like protein kinase.
  • 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. For example, if 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.
  • the flour prepared from the mutant seed of the present invention 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 increases the content and/or activity of a receptor-like protein kinase, 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 increases the content of a receptor-like protein kinase.
  • Preferred harvestable parts are seeds comprising the nucleic acid sequence which increases the content of a receptor-like protein kinase.
  • Transformation of wheat leaves with BAC clones 1.1 Transformation of wheat leaves with BAC clones 1.1 . Transformation
  • BAC clones from a barley BAC library (Yu et al. (2000) TAG 101 : 1093-1099) which carry genomic DNA encoding the receptor-like protein kinases of the present invention were transformed into wheat leaves using biolistic transformation with a gene gun (Bio-Rad-model PDS-1000/He, hepta adapter) and the method according to Douchkov et al. (2005) Mol. Plant- Microbe Interact 18: 755-761.
  • the following plasmid mixtures were used for transformation (Table 6):
  • the bombarded leaves were transferred to large, square Petri dishes containing 1 % w/v phytoagar with 20 ppm of benzimidazole.
  • the inoculation with wheat mildew conidia was performed in an inoculation tower by shaking conidia from strongly infected wheat leaves (about 200 conidia/mm 2 ) into the tower. After five minutes the dishes were removed, closed and incubated at 20°C and indirect daylight for 40 hours.
  • GUS staining for staining the transformed cells 40 h after inoculation, the leaves were contacted with the GUS detection solution (100 mM sodium phosphate , pH 7,0; 10 mM EDTA; 5 mM K 3 [Fe(CN) 6 ], 5 mM K 4 [Fe(CN) 6 ]; 0,1 % Triton X-100; 20% methanol and 1 mg/ml 3-bromo-4-chloro-3-indolyl ⁇ -D-glucuronic acid) under vacuum and incubated in this solution over night at 37°C. After removing the detection solution the leaves were destained with a solution containing 7.5% TCA and 50% (v/v) methanol for 15 minutes at 20°C.
  • the GUS detection solution 100 mM sodium phosphate , pH 7,0; 10 mM EDTA; 5 mM K 3 [Fe(CN) 6 ], 5 mM K 4 [Fe(CN) 6 ]; 0,1 % Trit
  • RLK_compl_3Hcluster_2 sequence was amplified using the BAC clone BAC
  • PHENOME_WP3_103 as PCR template.
  • specific primers were designed each containing a gene specific part as well as a nucleotide overhang for the addition of an restriction endonuclease recognition site (forward primer according to SEQ ID NO. 26 including A/oil recognition site and the start codon; reverse primer according to SEQ ID NO. 27 including Xma ⁇ recognition site and the stop codon).
  • the PCR reaction was run using 100ng of BAC DNA-template, 0.2 mM of each dNTP, 50 pmol forward primer, 50 pmol reverse primer, 1 U Phusion DNA polymerase (NEB) and 1 x Phusion HF reaction buffer, following a cycle protocol as follows: 1 cycle of 60 seconds at 98°C, followed by 35 cycles of in each case 10 seconds at 98°C, 30 seconds at 55°C and 60 at 72°C, followed by 1 cycle of 10 minutes at 72°C, then 4°C.
  • the resulting fragment was purified on an agarose gel, and subjected to a restriction digest using the restriction endonucleases Xma ⁇ and A/oil.
  • the plPKTA9 plasmid (Dong et al. (2006) Plant Cell 18(1 1 ): 3321-3331 ) was used.
  • This vector is based on the pUC18-Vector and contains a CaMV 35S promotor and a 35S terminator which are separated by a multiple cloning site.
  • the vector was cut within the multiple cloning site using the restriction endonuclease A/oil and Xma ⁇ , followed by a purification on a agarose gel. Vector and PCR fragment were combined and subjected to ligation. After isolation of the resulting vector, correctness of sequence was confirmed by standard sequencing techniques. 2.2. Cloning of an expression vector encoding RLK7
  • the RLK_7 DNA sequence was amplified using the BAC clone BAC PHENOME_WP3_104 as PCR template.
  • specific primers were designed each containing a gene specific part as well as a nucleotide overhang for the addition of an restriction endonuclease recognition site (forward primer according to SEQ ID No. 28, including a A/oil recognition site and the start codon; reverse primer according to SEQ ID No. 29 including a Xma ⁇ recognition site and the stop codon).
  • the PCR reaction was run using 100ng of BAC DNA-template, 0.2 mM of each dNTP, 50 pmol forward primer, 50 pmol reverse primer, 1 U Phusion DNA polymerase (NEB) and 1 x Phusion HF reaction buffer, following a cycle protocol as follows: 1 cycle of 60 seconds at 98°C, followed by 35 cycles of in each case 10 seconds at 98°C, 30 seconds at 55°C and 60 at 72°C, followed by 1 cycle of 10 minutes at 72°C, then 4°C.
  • the resulting fragment was purified on an agarose gel, and subjected to a restriction digest using the restriction endonucleases Xma ⁇ and A/oil.
  • target vector the plPKTA9 plasmid (Dong et al. (2006) Plant Cell 18(1 1 ): 3321-3331 ) was used.
  • This vector is based on the pUC18- Vector and contains a CaMV 35S promotor and a 35S terminator which are separated by a multiple cloning site.
  • the vector was cut within the multiple cloning site using the restriction endonucleases A/oil and Xma ⁇ , followed by a purification on a agarose gel. Vector and PCR fragment were combined and subjected to ligation. After isolation of the resulting vector, correctness of sequence was confirmed by standard sequencing techniques.
  • the genomic DNAs encoding the receptor-like kinases RLK_compl_3Hcluster_2 and RLK7 were generated by DNA synthesis (Geneart, Regensburg, Germany) in a way that an attB5- recombination site (Gateway system, (Invitrogen, Life Technologies, Carlsbad, California, USA)) is located upstream of the start-ATG and an attB4 recombination site is located downstream of the stop-codon.
  • the synthesized DNAs were transferred to a pENTRY-B vector by using the BP reaction (Gateway system (Invitrogen, Life Technologies, Carlsbad, California, USA)) according to the protocol provided by the supplier.
  • pENTRY-A vector containing a maize ubiquitine promoter p- ZmUbi
  • the pENTRY-B vector containing the DNA coding for the receptor-like protein kinase and a pENTRY-C vector containing a Agrobacterium octopine synthase promoter (t-ocs) were used.
  • t-ocs Agrobacterium octopine synthase promoter
  • the optimal harvesting time is 12-20 days post-anthesis.
  • transformation lEs should be 0.8 - 1.5 mm in length and translucent in appearance.
  • Donor plants used for harvesting should be at peak vigour to ensure optimal transformation and regeneration frequencies.
  • Immature seeds are surface sterilized by rinsing them 30-60 sec. in 70% (v/v) aqueous ethanol followed by 15 minutes 10% (v/v) Domestos bleach solution (Lever) gentle shaking. Then the immature seeds are rinsed 3-4 times with sterile distilled water and transferred to a sterile Petri dish, avoiding extreme dehydration. Immature seeds are ready for use. 3.3. Aqrobacterium culture
  • Agrobacterium cultures containing the vector harbouring a selectable marker (SM) cassette and the gene(s) of interests (GOI) described above are grown for 24-72 hours in a 28°C incubator on LB agar plates with appropriate selection.
  • SM selectable marker
  • GOI gene(s) of interests
  • liquid Agrobacterium culture To obtain a liquid Agrobacterium culture one colony is picked from a 1-3 days old plate and re- suspended in liquid medium (5 g mannitol, 1 g L-glutamic acid, 250 mg KH 2 P0 4 , 100 mg NaCI, 100 mg MgS0 4 x 7H 2 0, 5 g tryptone, 2.5 g yeast extract, pH 7.0, add after autoclave 1 g biotin incl. appropriate antibiotics). Liquid culture is grown at 28°C for ⁇ 16h to reach an OD 6 oo of ⁇ 1.
  • the Agrobacterium culture is centrifuged at 4.500 g for 10 minutes and resuspended in 4 ml inoculation medium (1/10 MS complete (30g maltose, 100mg MES; adjusted to pH 5.8 and add after autoclave 0.01 % Pluronic, 200 ⁇ acetosyringone to an OD 6 oo of ⁇ 1 .
  • the Agrobacterium inoculation medium is ready to use. 3.4. Isolation of immature embryos (lEs)
  • the lEs are isolated from the immature seed followed by removing and discarding the embryo axis.
  • the lEs are directly transferred in the Agrobacterium inoculation culture.
  • the tube is vortexed at full speed for 10 seconds and lEs are allowed to settle in the solution for 30 - 60 minutes.
  • the Agrobacterium solution is removed and the lEs are placed on sterile Whatman filter paper #1 (4-5 pieces) to blot excess Agrobacterium solution.
  • the top filter paper containing the lEs are transferred onto a plate containing approx. 20 ml of solidified co-culture media (1/10
  • MScomplete (30g maltose, 0.69g proline, 100mg MES, 10g Agar, adjust to pH 5.8, add after autoclave, 4mg 2,4-D, 200 ⁇ acetosyringone, 100mg ascorbic acid)).
  • the plates are sealed with parafilm and incubated for 2-3 days at 24°C in the dark.
  • Calli are transferred to shoot regeneration medium (MS full complete (30g maltose, 20mg thiamine, 100mg myo-inositol, 750mg glutamine, 5 ⁇ CuS0 4 , 1.95g MES; 8g agar (Plant TC), adjust to pH 5.8 and add after autoclave, 0.5mg TDZ, 200mg timentin, 1 1 mM D-alanine) and are cultivated under light conditions at 21-25°C for 3-4 weeks.
  • the explants are transferred to rooting media (1 ⁇ 2 MS complete, sucrose 30g, agar 7g and adjust to pH 5.8, add after autoclave, NAA 0.5mg, timentin 200mg, D-alanine 1 1 mM) in 100x20 plates and are cultivated for 4-5 weeks at 21 -25°C under light conditions.
  • Putative transgenic shoots that develop roots are planted out into a nursery soil mix consisting of peat and sand (1 :1 ) and maintained at 22-24°C with elevated humidity (>70%) After two weeks, plants are removed from the humidity chamber and are further cultivated under greenhouse conditions.
  • Transgenic plants are grown in the greenhouse at 19°C and 60-80% humidity. After 1 1 days plants are inoculated with Septoria tritici spores (1 ,3x10 6 Spores/ml in 0.1 % Tween20 solution). Plants are incubated for 4 days at 19°C and 80-90% humidity under long day conditions (16h light). Plants are then grown for approx. 3 weeks at 19°C and 60-80% humidity under long day conditions.
  • the diseased leaf area is scored by eye by trained personal. The percentage of the leaf area showing fungal pycnidia or strong yellowing/browning is considered as diseased leaf area. Per experiment the diseased leaf area of 16 transgenic plants (and 16 WT plants as control) is scored. For analysis the average of the diseased leaf area of the non-transgenic mother plant is set to 100% to calculate the relative diseased leaf area of the transgenic lines. The expression of the receptor-like protein kinase will lead to enhanced resistance of wheat against Septoria tritici.
  • the expression of the receptor-like protein kinase will lead to enhanced resistance of wheat against rust fungi.
  • Transgenic plants are grown in the phytochamber at 22°C and 75% humidity (16/8 h light/dark rhythm) for 2 weeks.
  • the 2 weeks old plants are inoculated with spores of the powdery mildew fungus (Blumeria graminis f.sp. tritci).
  • spores of the powdery mildew fungus Bolumeria graminis f.sp. tritci.
  • spores of the powdery mildew fungus Bolumeria graminis f.sp. tritci
  • Generally inoculations with powdery mildew are performed with dry spores using an inoculation tower to a density of approx. 10 spores/mm 2 .
  • Plants are incubated for 7 days at 20°C, 75% humidity and a 16/8 hours light/dark rhythm.
  • Diseased leaf area is scored by eye by trained personal. The percentage of the leaf area showing white fungal colonies is considered as diseased leaf area. Per experiment the diseased leaf area of 16 transgenic plants (and 16 WT plants as control) is scored. For analysis the average of the diseased leaf area of the non-transgenic mother plant is set to 100% to calculate the relative diseased leaf area of the transgenic lines.
  • the nucleic acid sequence encoding the receptor like kinase from barley as described in Example 2 was synthesized in a way that enables further cloning.
  • the expression cassettes were then assembled in a vector by cloning the synthesized or cloned DNA encoding the receptor like kinase from barley downstream of a maize ubiquitin promoter and upstream of a t- nos terminator (3'UTR of Nopaline Synthase from Agrobacterium tumefaciens).
  • Plant transformation binary vectors such as pBi-nAR were used (Hofgen & Willmitzer 1990, Plant Sci. 66:221 -230). Construction of the binary vectors was performed by ligation of the expression cassette, as described above, into the binary vector. Further examples for plant binary vectors are the pSUN300 or pSUN2-GW vectors and the pPZP vectors (Hajdukiewicz et al., Plant Molecular Biology 25: 989-994, 1994). These binary vectors contain an antibiotic resistance gene under the control of the NOS promoter.
  • Expression cassettes were cloned into the multiple cloning site of the pEntry vector using standard cloning procedures.
  • pEntry vectors were combined with a pSUN destination vector to form a binary vector by the use of the GATEWAY technology (Invitrogen, webpage at invitrogen.com) following the manufacturer's instructions.
  • the recombinant vector containing the expression cassette was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected on LB agar containing 50 ⁇ g/ml kanamycin grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Analysis of subsequent clones and restriction mapping was performed according to standard molecular biology techniques (Sambrook et al., 1989,
  • Agrobacterium cells harboring a plasmid containing the gene of interest and the mutated maize AHAS gene were grown in YP medium supplemented with appropriate antibiotics for 1 -2 days.
  • One loop of Agrobacterium cells was collected and suspended in 1.8 ml M-LS-002 medium (LS- inf). The cultures were incubated while shaking at 1 ,200 rpm for 5 min-3 hrs.
  • Corn cobs were harvested at 8-1 1 days after pollination. The cobs were sterilized in 20% Clorox solution for 5 min, followed by spraying with 70% Ethanol and then thoroughly rinsed with sterile water.
  • Immature embryos 0.8-2.0 mm in size were dissected into the tube containing Agrobacterium cells in LS-inf solution.
  • the constructs were transformed into immature embryos by a protocol modified from Japan Tobacco Agrobacterium mediated plant transformation method (US Patent Nos. 5,591 ,616; 5,731 ,179; 6,653,529; and U.S. Patent Application Publication No. 2009/0249514).
  • Two types of plasmid vectors were used for transformation. One type had only one T-DNA border on each of left and right side of the border, and selectable marker gene and gene of interest were located between the left and right T-DNA borders. The other type was so called "two T-DNA constructs" as described in Japan Tobacco U.S. Patent No. 5,731 ,179. In the two DNA constructs, the selectable marker gene was located between one set of T-DNA borders and the gene of interest was included in between the second set of T-DNA borders.
  • the plasmid vector was
  • Agrobacterium infection of the embryos was carried out by inverting the tube several times. The mixture was poured onto a filter paper disk on the surface of a plate containing co-cultivation medium (M-LS-01 1 ). The liquid agro-solution was removed and the embryos were checked under a microscope and placed scutellum side up. Embryos were cultured in the dark at 22°C for 2-4 days, and transferred to M-MS-101 medium without selection and incubated for four to seven days. Embryos were then transferred to M-LS-202 medium containing 0.75 ⁇
  • imazethapyr and grown for three weeks at 27°C to select for transformed callus cells.
  • Plant regeneration was initiated by transferring resistant calli to M-LS-504 medium
  • Transgenic maize plant production is also described, for example, in U.S. Patent No. 5,591 ,616 and 6,653,529; U.S. Patent Application Publication No. 2009/0249514; and WO/2006136596, each of which are hereby incorporated by reference in their entirety.
  • Transformation of maize may be made using Agrobacterium transformation, as described in U.S. Patent Nos. 5,591 ,616; 5,731 ,179; U.S. Patent Application Publication No. 2002/0104132, and the like. Transformation of maize (Zea mays L.) can also be performed with a modification of the method described by Ishida et al. (Nature Biotech., 1996, 14:745-750). The inbred line A188 (University of
  • Ears were harvested from corn plants at approximately 1 1 days after pollination (DAP) when the length of immature embryos was about 1 to 1.2 mm. Immature embryos were co-cultivated with Agrobacterium tumefaciens that carry "super binary" vectors and transgenic plants were recovered through organogenesis. The super binary vector system is described in WO
  • Vectors were constructed as described.
  • Various selection marker genes were used including the maize gene encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Patent No. 6,025,541 ).
  • AHAS mutated acetohydroxy acid synthase
  • various promoters were used to regulate the trait gene to provide constitutive, developmental, inducible, tissue or environmental regulation of gene transcription.
  • Excised embryos can be used and can be grown on callus induction medium, then maize regeneration medium, containing imidazolinone as a selection agent.
  • the Petri dishes were incubated in the light at 25°C for 2-3 weeks, or until shoots develop.
  • the green shoots were transferred from each embryo to maize rooting medium and incubated at 25°C for 2-3 weeks, until roots developed.
  • the rooted shoots were transplanted to soil in the greenhouse.
  • T1 seeds were produced from plants that exhibit tolerance to the imidazolinone herbicides and which were PCR positive for the transgenes. 9. Fusarium and Colletotrichum resistance screening
  • Transgenic maize plants were grown in greenhouse or phyto-chamber under standard growing conditions in a controlled environment (20-25°C, 60-90% humidity).
  • transgenic plants Shortly after plants entered the reproductive phase the transgenic plants were inoculated near the base of the stalk using a fungal suspension of spores (105 spores in PBS solution) of Fusarium ssp. or Colletotrichum graminicola. Plants were incubated for 2-4 weeks at 20-25°C and 60-90% humidity.
  • stalks were split and the progression of the disease was scored by observation of the characteristic brown to black color of the fungus as it grows up the stalk.
  • Disease ratings were conducted by assigning a visual score.

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Abstract

L'invention porte sur un procédé de production d'une cellule végétale transgénique, d'une plante transgénique ou d'une partie de plante transgénique dotée d'une résistance augmentée aux agents pathogènes, la teneur en une protéine kinase de type récepteur et/ou l'activité d'une protéine kinase de type récepteur étant augmentée.
PCT/IB2013/060144 2012-11-15 2013-11-14 Procédé de production de plantes dotées d'une résistance augmentée aux agents pathogènes WO2014076659A1 (fr)

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Publication number Priority date Publication date Assignee Title
US10231397B2 (en) 2013-01-29 2019-03-19 Basf Plant Science Company Gmbh Fungal resistant plants expressing EIN2
US10435705B2 (en) 2013-01-29 2019-10-08 Basf Plant Science Company Gmbh Fungal resistant plants expressing HCP6
US10462994B2 (en) 2013-01-29 2019-11-05 Basf Plant Science Company Gmbh Fungal resistant plants expressing HCP7
US10465204B2 (en) 2013-03-08 2019-11-05 Basf Plant Science Company Gmbh Fungal resistant plants expressing MybTF
CN114480488A (zh) * 2022-02-17 2022-05-13 河南农业大学 一种拟轮枝镰孢菌相关的ZmWAX基因的应用

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WO2002046439A2 (fr) * 2000-12-04 2002-06-13 Universiteit Utrecht Nouveaux promoteurs specifiques des racines activant l'expression d'une nouvelle kinase de type recepteur du domaine lrr
WO2007138070A2 (fr) * 2006-05-30 2007-12-06 Cropdesign N.V. Plantes possédant des caractéristiques de rendement amélioré et procédé de fabrication
WO2010080589A2 (fr) * 2008-12-18 2010-07-15 The Regents Of The University Of California Méthodes de criblage de nouvelles fonctions de kinases de type récepteur
WO2011104153A1 (fr) * 2010-02-23 2011-09-01 Basf Plant Science Company Gmbh Plantes transgéniques résistantes aux nématodes

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WO2002046439A2 (fr) * 2000-12-04 2002-06-13 Universiteit Utrecht Nouveaux promoteurs specifiques des racines activant l'expression d'une nouvelle kinase de type recepteur du domaine lrr
WO2007138070A2 (fr) * 2006-05-30 2007-12-06 Cropdesign N.V. Plantes possédant des caractéristiques de rendement amélioré et procédé de fabrication
WO2010080589A2 (fr) * 2008-12-18 2010-07-15 The Regents Of The University Of California Méthodes de criblage de nouvelles fonctions de kinases de type récepteur
WO2011104153A1 (fr) * 2010-02-23 2011-09-01 Basf Plant Science Company Gmbh Plantes transgéniques résistantes aux nématodes

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10231397B2 (en) 2013-01-29 2019-03-19 Basf Plant Science Company Gmbh Fungal resistant plants expressing EIN2
US10435705B2 (en) 2013-01-29 2019-10-08 Basf Plant Science Company Gmbh Fungal resistant plants expressing HCP6
US10462994B2 (en) 2013-01-29 2019-11-05 Basf Plant Science Company Gmbh Fungal resistant plants expressing HCP7
US10925223B2 (en) 2013-01-29 2021-02-23 Basf Plant Science Company Gmbh Fungal resistant plants expressing EIN2
US10465204B2 (en) 2013-03-08 2019-11-05 Basf Plant Science Company Gmbh Fungal resistant plants expressing MybTF
CN114480488A (zh) * 2022-02-17 2022-05-13 河南农业大学 一种拟轮枝镰孢菌相关的ZmWAX基因的应用
CN114480488B (zh) * 2022-02-17 2023-08-01 河南农业大学 一种拟轮枝镰孢菌相关的ZmWAX基因的应用

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