WO2013050318A1 - Method of producing plants having increased resistance to pathogens - Google Patents

Method of producing plants having increased resistance to pathogens Download PDF

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Publication number
WO2013050318A1
WO2013050318A1 PCT/EP2012/069328 EP2012069328W WO2013050318A1 WO 2013050318 A1 WO2013050318 A1 WO 2013050318A1 EP 2012069328 W EP2012069328 W EP 2012069328W WO 2013050318 A1 WO2013050318 A1 WO 2013050318A1
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Prior art keywords
plant
transgenic
nucleic acid
acid sequence
plants
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PCT/EP2012/069328
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French (fr)
Inventor
Dimitar Douchkov
Stefanie Lueck
Patrick Schweizer
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Basf Plant Science Company Gmbh
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Publication of WO2013050318A1 publication Critical patent/WO2013050318A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • 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 the activity of an mRNA export protein 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,
  • 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.
  • a transgene for example, chitinase (WO 92/17591) and pathogenesis-related genes (WO 92/20800) as well as 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 02/061101 ), NADPH oxidase (WO 2004/009820) and the Mlo gene (WO 00/0 722) 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).
  • the present inventors have found that the reduction of the content of an mRNA export protein by RNA interference leads to an enhanced
  • this mRNA export protein is considered to be involved in mediating pathogen resistance.
  • MRNA export proteins are involved in regulating the nucleocytoplasmic distribution of mRNAs and may be a component of the nuclear pore complex or may be nucleocytoplasmic shuttling proteins which together with the mRNA form an export-competent mRNA ribonucleoprotein.
  • nucleocytoplasmic shuttling also play a role in regulating plant immunity (Cheng et ai. (2009) Plant Cell 21 : 2503-2516; Palma et ai. (2005) Current Biology 15: 1129-1135; Germain et al. (2010) PLoS Genetics 6(12): e1001250). However, so far this has not been shown for monocotyledonous plants.
  • 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 the activity of an mRNA export protein 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 -6 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 -6 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences;
  • the present invention provides a method for increasing pathogen resistance in a plant cell, plant or part thereof, wherein the method comprises the step of increasing the content and/or activity of an mRNA export protein in the plant cell, plant or part thereof compared to a control plant cell, plant or plant part, wherein the mRNA export protein is encoded by a nucleic acid sequence which is selected from the group consisting of:
  • nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; b) a nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; and
  • nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences.
  • the method comprises the steps of
  • 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 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 mRNA export protein as defined herein is not increased and selecting transgenic progeny in which the content and/or the activity of the mRNA export protein 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 -6 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 or a wheat plant.
  • the transgenic or mutant plant has an increased resistance to a fungal pathogen, more preferably to Blumeria graminis, Septoria tritici and/or Puccinia triticina.
  • 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. 1-6 or a fragment of any of these sequences; (b) a nucleic acid sequence comprising a sequence which is at least
  • 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 in another embodiment 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, produced by the method of the present invention or containing an expression construct or a vector of the present invention. in another embodiment 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 or the mutation which increases the content and/or the activity of an mRNA export protein as defined herein can be detected in said transgenic or mutant seed or in said flour.
  • FIG. 1 Flow diagram for the high-throughput production of RNAi
  • I PCR amplification of cDNA fragments of interest
  • I la Ligation of the PCR fragments in the intermediate vector plPKTA38 in the presence of the restriction endonuclease, Swa I, which inhibits the re-ligation of the vector
  • Figure 2 Flow diagram showing how the effect of the RNAi constructs on plant resistance to Blumeria graminis is tested.
  • 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 contains a 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. 1-6 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences;
  • 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 activity of an mRNA export protein compared to a control plant cell, plant or plant part.
  • the transgenic plant cell, plant or plant part may contain one or more other transgenic nucleic acid sequences, for example nucleic acid sequences conferring resistance to biotic or abiotic stress and/or altering the chemical composition of the transgenic plant cell, plant or plant part.
  • transgenic does not refer to plants having alterations in the genome which are the result of naturally occurring events, such as spontaneous mutations, or of induced mutagenesis followed by breeding and selection.
  • mutant means that a plant cell, plant or plant part has been altered by mutagenesis so that a nucleic acid sequence selected from the group consisting of
  • nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences;
  • the mutant plant contains at least one point mutation, i.e. at least one nucleotide substitution, deletion and/or addition, in comparison to a control plant, plant cell or part thereof which has been used as a starting material in the mutagenesis and which has not been mutagenized.
  • the mutant plant contains at least one nucleotide substitution in the nucleic acid sequence according to any of SEQ ID Nos. 1-6 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.
  • Dicotyledonous useful plants comprise, inter alia, cotton, legumes, like leguminous plants and in particular alfalfa, soy bean, rape, tomato, sugar beet, potato, ornamental plants, and trees.
  • Further useful plants can comprise fruit (in particular apples, pears, cherries, grapes, citrus, pineapple, and bananas), pumpkin, cucumber, wine, oil palms, tea shrubs, cacao trees, and coffee shrubs, tobacco, sisal, as well as, with medicinal plants, rauwolfia and digitalis.
  • 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.
  • transgenic or mutant progeny of the transgenic or mutant plant may be the result of a cross of the transgenic or mutant plant with another transgenic or mutant plant of the present invention or it may be the result of a cross with a wild- type plant or a transgenic plant having a transgene other than the transgene of the present invention.
  • transgenic plant also comprises true breeding transgenic plants which are obtained by repeated inbreeding steps as described below.
  • Plant parts include, but are not limited to, stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores, seeds and the like.
  • the term "cell” or "plant cell” as used herein 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
  • 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.
  • 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 term "increased pathogen resistance” is understood to denote that the transgenic plants or plant cells of the present invention are infected iess severely and/or less frequently by plant
  • 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 ieast the factor 50, and more preferably by at Ieast the factor 100, and most preferably by at Ieast the factor 1000, as compared to the control plant.
  • the pathogen resistance may be described by reference to a relative susceptibility index (SI) which compares the susceptibility of a plant of the present invention to a pathogen with the susceptibility of a control plant to said pathogen, the latter being set to 100%.
  • SI relative susceptibility index
  • the relative susceptibility index of the plants of the present invention is less than 80%, preferably less than 70 or 60%, more preferably less than 50 or 40% and most preferably less than 30%.
  • control plant When used in connection with transgenic plants, the terms "control plant”, “control plant cell” and “control plant part” refer to a plant cell, an explant, seed, plant component, plant tissue, plant organ, or whole plant used to compare against a transgenic plant which has been modified by the method of the present invention for the purpose of identifying an enhanced
  • 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 an mRNA export protein.
  • a control plant may be a plant of the same line or variety as the transgenic plant being tested, or it may be another line or variety, such as a plant known to have a specific phenotype, characteristic, or known genotype.
  • Another suitable control plant is a genetically unaltered or non-transgenic plant of the parental line used to generate the transgenic plant of the present invention, i.e. the wild-type plant.
  • control plant When used in connection with mutant plants, the terms "control plant”, “control plant cell” and “control plant part” refer to a plant cell, an explant, seed, plant component, plant tissue, plant organ, or whole plant used to compare against a mutant plant 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.
  • test plants with pathogens such as fungi in order to examine potential resistance phenomena are a method well-known to those skilled in the art.
  • the test plants used must be responsive to the pathogen used, i.e. they must be able to serve as 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.
  • “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 wiid-type plant.
  • the fungal infestation of the plant may then be evaluated by means of a suitable evaluation procedure.
  • the visual inspection, in which the formed fungal structures are detected in the plant and quantified, is particularly suitable.
  • a reporter gene such as the beta-glucuronidase (GUS) gene from E. coli, a fluorescence gene, the green fluorescence protein (GFP) gene from Aequorea victoria, the luciferase gene from Photinus pyralis or the beta- galactosidase (lacZ) gene from E. coli, the expression of which in the plant cells may be proven by simple methods, is co-transformed in a suitable vector with the vector mediating the expression of the mRNA export protein.
  • GUS beta-glucuronidase
  • GFP green fluorescence protein
  • lacZ beta- galactosidase
  • 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 increasing the content and/or the activity of the mRNA export protein is lower than the diseased leaf area of infected control plants.
  • plant pathogens includes viral, bacterial, fungal and other pathogens.
  • plant pathogens comprises fungal pathogens.
  • the term "plant pathogens" includes biotrophic, hemibiotrophic and necrotrophic pathogens.
  • the plant pathogen is a biotrophic pathogen, more preferably a biotrophic fungal pathogen.
  • 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 belong to the group of biotrophic fungi, like other rust fungi, powdery mildew fungi or oomycete pathogens like the genus Phytophthora or Peronopora.
  • the necrotrophic phytopathogenic fungi depend for their nutrition on dead cells of the plants, e.g. species from the genus Fusarium, Rhizoctonia or Mycospaerella. Soybean rust has occupied an intermediate position, since it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic.
  • biotrophic fungal pathogens which follows essentially such an infection strategy is hemibiotrophic.
  • Table 1 Diseases caused by biotrophic phytopathogenic fungi
  • Glomerella graminicola Politis Glomerella Anthracnose stalk rot tucumanensis (anamorph: Glomerella falcatum Went)
  • Rhizoctonia solani Kuhn Rhizoctonia microsclerotia J. Matz (telomorph:
  • Brown spot black spot, stalk rot
  • Cephalosporium kernel rot Acremonium strictum Cephalosporium acremonium
  • Curvularia leaf spot Curvularia clavata, C. eragrostidis, C.
  • Cochliobolus pallescens Curvularia senegalensis, C. tubercuiata (teleomorph: Cochliobolus tuberculatus)
  • Dry ear rot (cob, Nigrospora oryzae
  • kernel and stalk rot (teleomorph: Khuskia oryzae)
  • Eyespot Aureobasidium zeae Kabatiella zeae
  • Gray ear rot Botryosphaeria zeae Physalospora zeae
  • Hormodendrum ear rot Cladosporium cladosporioides
  • herbarum (teleomorph: Mycosphaerella tassiana)
  • Exserohilum prolatum Drechslera prolata (teleomorph: Setosphaeria prolata) Graphium penicillioides,
  • Leptosphaeria maydis, Leptothynum zeae, Ophiosphaerella herpotricha, (anamorph: Scolecosporiella sp.),
  • Exserohilum turcicum Helminthosporium turcicum
  • Bipolaris zeicola Helminthosporium
  • Penicillium ear rot blue eye, blue Penicillium spp., P. chrysogenum, mold
  • P. expansum P. oxalicum Disease Pathogen
  • Phaeocytostroma stalk and root Phaeocytostroma ambiguum
  • Botryosphaeria festucae Physalospora (Botryosphaeria ear rot) zeicola (anamorph: Diplodia frumenti)
  • Red kernel disease ear mold, leaf Epicoccum nigrum
  • Rhizoctonia ear rot (sclerotial rot) Rhizoctonia zeae (teleomorph: Waitea circinata)
  • Root rots (minor) Alternaria alternata, Cercospora sorghi,
  • Dictochaeta fertilis Fusarium acuminatum (teleomorph: Gibberella acuminata), F. equiseti (teleomorph: G. intricans), F. oxysporum, F. pallidoroseum, F. poae, F. roseum, G. cyanogena, (anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp., Periconia circinata, Phytophthora cactorum, P. drechsleri, P. nicotianae var. Disease Pathogen
  • Exserohilum turcicum Helminthosporium turcicum, Fusarium avenaceum, F.
  • Macrophomina phaseolina Penicillium spp., Phomopsis sp., Pythium spp., Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicaria sp. Disease Pathogen
  • Trichoderma ear rot and root rot Trichoderma viride T. lignorum
  • fungal pathogens or fungal-like pathogens are from the group comprising Plasmodiophoromycetes,
  • Plasmodiophoromycetes like Plasmodiophora brassicae (clubroot of crucifers), Spongospora subterranea (powdery scab of potato tubers), Polymyxa graminis (root disease of cereals and grasses),
  • 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 tracheiphila (grapevine red fire disease), Claviceps purpurea (ergot on, for example, rye and grasses), Gaeumannomyces graminis (black leg disease of wheat, rye and, inter alia, grasses), Magnaporthe grisea (rice blast disease), Pyrenophora graminea (leaf stripe disease of barley), Pyrenophora teres (net blotch disease of barley), Pyrenophora tritici-repentis (tan spot disease) Septoria tritici (leaf spot of wheat), Venturia inaequalis (apple scab disease),
  • Scierotinia sclerotium (white moid, stem canker of rape),
  • 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
  • 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 are resistant to the following pathogenic bacteria: Corynebacterium sepedonicum (bacterial ring rot of potato), Erwinia
  • carotovora black leg rot of potato
  • Erwinia amylovora fire blight of pear, apple, quince
  • Streptomyces scabies common scab of potato
  • Pseudomonas syringae pv. phaseolicola halo blight disease of dwarf bean
  • Pseudomonas syringae pv. tomato ("bacterial speck" of tomato)
  • Xanthomonas campestris pv. malvacearum angular leaf spot of cotton
  • 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.
  • AWSMV American wheat striate American wheat striate mosaic virus
  • Corn chlorotic vein banding Corn chlorotic vein banding virus (CCVBV) (Braizilian maize mosaic)
  • MCMV Maize dwarf mosaic virus
  • MDMV Maize dwarf mosaic virus
  • WSMV Wheat streak mosaic virus
  • CMV Cucumber mosaic Cucumber mosaic virus
  • Cynodon chlorotic streak virus CCSV
  • JGMV Johnsongrass mosaic Johnsongrass mosaic virus
  • MLO Maize bushy stunt Mycoplasma-like organism
  • Maize mosaic corn leaf stripe, Maize mosaic virus (MMV)
  • Maize pellucid ringspot Maize pellucid ringspot virus MPRV
  • Maize raya gruesa Maize raya gruesa virus
  • MRGV Maize raya gruesa virus
  • MRFV Maize rayado fino
  • 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
  • Sorghum mosaic Sorghum mosaic virus (also: sugarcane mosaic virus (SCMV) strains H, 1 and M)
  • SMV Sugarcane mosaic Sugarcane mosaic virus
  • Wheat spot mosaic Wheat spot mosaic virus (WSMV)
  • the plants and plant cells according to the present invention can also be resistant to animal pests like insects and nematodes. Insects, like for
  • 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),
  • EBCB European corn borer
  • Diabrotica barberi Northern corn rootworm
  • Diabrotica undecimpunctata Southern corn rootworm
  • Diabrotica virgifera Western corn rootworm
  • Agrotis ipsilon black cutworm
  • Sphenophorus maidis (maize billbug), Sphenophorus zeae (timothy billbug), Sphenophorus parvulus (bluegrass billbug), Sphenophorus callosus
  • the cereal leaf beetle (Oulema melanopus), the frit fly (Oscinella frit), wireworms (Agrotis lineatus), and aphids (like for example the bird cherry-oat aphid Rhopalosiphum padi, the grain aphid Sitobion avenae).
  • pathogens listed in Table 5 as well as the diseases associated therewith are to be mentioned as nematode pests in an exemplary, yet not limiting manner.
  • the transgenic plants produced according to the present invention are resistant to Globodera rostochiensis and G. pallida
  • the plants according to the present invention are preferably resistant to the following pathogens:
  • the plants are resistant to the fungal, bacterial, and viral pathogens Puccinia hordei (barley stem rust), Blumeria (Erysiphe) graminis f. sp. hordei (barley powdery mildew), Rhynchosporium secalis (barley scald), barley yellow dwarf virus (BYDV), and 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 sen/us (brown stink bug); Deliaplatura
  • soy bean the plants are resistant to the fungal, bacterial, or viral pathogens Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium
  • Diaporthe phaseolorum var. sojae Phomopsis sojae
  • Diaporthe phaseolorum var. caulivora Sclerotium rolfsii
  • Cercospora kikuchii Sclerotium rolfsii
  • Cercospora sojina Peronospora manshurica, Colletotrichum dematium (Colletotrichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p. .
  • glycinea Xanthomonas campestris p. v. phaseoli, Microsphaera diffussa, Fusarium semitectum, Phialophora gregata, soy bean mosaic virus,
  • Glomerella glycines glycines, tobacco ring spot virus, tobacco streak virus,
  • Phakopsora pachyrhizi Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, tomato spotted wilt virus, Heterodera glycines, Fusarium solani and the pathogenic insects / nematodes
  • Pseudoplusia includens (soybean looper); Anticarsia gemmatalis (velvetbean caterpillar); Plathypena scabra (green cloverworm); Ostrinia nubilalis (European corn borer); Agrotis ipsilon (black cutworm); Spodoptera exigua (beet armyworm); Heliothis virescens (cotton budworm); Helicoverpa zea (cotton bollworm); Epilachna varivestis (Mexican bean beetle); Myzus persicae (green peach aphid); Empoasca fabae (potato leaf hopper); A
  • the plants are resistant to the fungai, 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
  • the plants are resistant to the fungal, bacterial, or viral pathogens Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternana alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici,
  • Carotovora Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis and to the pathogenic insects/nematodes Suieima heiianthana (sunflower bud moth); Homoeosoma electellum (sunflower moth);
  • Zygogramma exclamationis (sunflower beetle); Bothyrus gibbosus (carrot beetle); Neolasioptera murtfeldtiana (sunflower seed midge).
  • the plants are resistant to the fungal, bacterial, or viral pathogens Fusarium moniliforme var. subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium
  • aphanidermatum, Aspergillus flavus, Bipolaris maydis 0, T (Cochliobolus heterostrophus), Helminthosporium carbon urn I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp.
  • Cephalosporium acremonium Maize Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus (MSV, Maisstrichel-Virus), Maize Stripe Virus, Maize Rough Dwarf Virus, and the pathogenic insects / nematodes Ostrinia nubilalis (European corn borer); Agrotis ipsilon (black cutworm); Helicoverpa zea (corn earworm); Spodoptera frugiperda.
  • Cyclocephala borealis (northern masked chafer; white grub); Cyclocephala immaculata (southern masked chafer; white grub); Popillia japonica
  • Solenopsis milesta (thief ant); Tetranychus urticae (twospotted spider mite).
  • sorghum the plants are resistant to the fungal, bacterial, or viral pathogens Exserohilum turcicum, Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi, Qloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea,
  • Macrophomina phaseolina Perconia circinata, Fusarium moniliforme, Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvulaha lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca omenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum,
  • 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 iineolahs (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 urticae two-spotted spider mite. ln rice, 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 leaf hopper); Blissus leucopterus leucopterus (chinch bug); Acrostemum hilare (green stink bug).
  • the plants are resistant to the pathogenic insects/nematodes
  • Brevicoryne brassicae cabbage aphid
  • Phyllotreta cruciferae Felea 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, mail, and nicotianae as well as Septoria tritici and Puccinia triticina.
  • an "mRNA export protein” is a protein which is involved in the transport of mRNA from the nucleus of a cell to the cytoplasm.
  • the mRNA export protein is encoded by a nucleic acid sequence selected from the group consisting of:
  • a nucleic acid sequence comprising a sequence which is at least 70% identical to the sequence of any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; and (c) a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence any of SEQ ID Nos. 1 -6 or a fragment of any of these sequences.
  • the content of a protein within a plant cell is usually determined by the expression level of the protein. Hence, in most cases the terms "content” and “expression” may be used interchangeably.
  • the content of a protein within a cell can be influenced on the level of transcription and/or the level of translation.
  • 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 mRNA export protein which do not affect the content of these proteins.
  • the expression level of the nucleic acid coding for the mRNA export protein 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 PGR.
  • the expression of the nucieic acid coding for the mRNA export 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 expression level of the mRNA export protein may also be determined by Western Blot analysis using suitable antibodies.
  • the amount of the mRNA export 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 mRNA export protein may for example be determined by incubating seedlings from control and transgenic plants with 5' end-labeled oligo d(T) oligonucleotide and determining the fluorescence in the sample, as described for example in Germain et al. (2010) PLoS Genetics 6(12):
  • the increased activity of the mRNA export protein will lead to an accumulation of the fluorescence in the cytoplasm.
  • Another possibility is to produce nuciear and cytoplasmic extracts from control and transgenic plants and to compare the expression of suitable proteins in the nucleus and cytoplasm of the control and the transgenic plants, e.g. by Western Blot using antibodies directed against these proteins.
  • the increased activity of the mRNA export protein will lead to an increased accumulation of one or more of these proteins in the cytoplasm.
  • the activity of the mRNA export protein may be increased by the method of the present invention 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 person skilled in the art is familiar with methods for increasing the content of a given protein.
  • the method involves introducing into a plant or plant cell a vector which comprises:
  • increasing the content and/or the activity of an mRNA export protein is also understood to denote the manipulation of the expression of the endogenous mRNA export protein 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 mRNA export protein. Such a modification, which leads to an increased expression rate of at least one endogenous mRNA export protein, can be effected by deleting or inserting DNA sequences in the promoter region.
  • an increased expression of at ieast one endogenous mRNA export protein 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 mRNA export protein.
  • a regulator protein 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 mRNA export protein is to upregulate transcription factors, which are involved in the transcription of the endogenous genes coding for the mRNA export protein, 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 mRNA export protein.
  • An alteration of the activity of the endogenous mRNA export protein can also be achieved by influencing the post-translational modifications of the mRNA export 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 mRNA export protein, by means of corresponding measures like overexpression or gene silencing.
  • the expression of the endogenous mRNA export protein can also be regulated via the expression of aptamers specifically binding to the promoter sequences of the mRNA export protein. If the aptamers bind to stimulating promoter regions, the amount and thus, in this case, the activity of the endogenous mRNA export protein is increased.
  • 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.
  • 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.
  • a nucieic acid sequence for increasing the content and/or the activity of a protein different enzymes such as meganucleases (WO 2009/114321 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 (WO 2009/130695 A2) may be used.
  • sequence-specific nucleases may also be used to cut the sequence of interest, thereby introducing one or more mutations into said sequence.
  • the method for producing mutant plants, plant cells or plant parts having an increased resistance to pathogens is preferably the TILLING (Targeting induced Local Lesions IN Genomes) method.
  • plant material is mutagenized to introduce at least one mutation into the genome of the plant material.
  • This mutagenesis may be chemical mutagenesis, for example with ethyl methane sulfonate (EMS), mutagenesis by irradiation such as ionizing irradiation or mutagenesis by using sequence-specific nucleases.
  • EMS ethyl methane sulfonate
  • nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; a nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences;
  • nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences.
  • a “fragment” of the nucleic acid sequence according to any of SEQ ID Nos. 1-6 is understood to refer to a smaller part of this nucleic acid sequence which consists of a contiguous nucleotide sequence found in SEQ ID No. 1-6 and which encodes a protein having the activity of an mRNA export protein.
  • 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. 2-6 has a length of at least 200, 250 or 300 nucleotides, preferably of at least 350, 400, 450, 500, 550 or 600 nucleotides, more preferably of at least 650, 700, 750, 800, 850 or 900 nucleotides, even more preferably of at least 950, 1000, 1050, 00, 1150 or 1200 nucleotides and most preferably of at least 1250, 1300 or 1350 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 or 99% identical to the complete sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these
  • 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.
  • 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.
  • 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 (http://www.ncbi.nlm.nih.gov/) may be used, employing the default parameters.
  • the program may be used, employing the default parameters.
  • sequence identity refers to the degree of the sequence identity over a length of 300, 350, 400, 450 or 500 nucleotides and most preferably the whole length of the nucleic acid sequence according to SEQ ID No. 1.
  • sequence identity refers to the degree of the sequence identity over a length of 300, 350, 400, 450 or 500 nucleotides, preferably over a length of 550, 600, 650, 700, 750 or 800 nucleotides, more preferably over a length of 850, 900, 950, 1000, 1050 or 1 100 nucleotides, even more preferably over a length of 150, 1200, 1250, 1300, 1350 or 1400 nucleotides and most preferably the whole length of the nucleic acid sequence according to any of SEQ ID Nos. 2-6.
  • sequence identity may also refer to the degree of the sequence identity over a length of 1500, 1600, 1700, 1800, 1900, 2000 or 2100 nucleotides. If the sequence identity is to be determined with respect to a fragment of the sequence according to SEQ ID No. 1 , the fragment has a length of at least 300, 350, 400, 450 or 500 nucleotides. If the sequence identity is to be determined with respect to a fragment of the sequence according to any of SEQ ID Nos.
  • the fragment has a length of at ieast 300, 350, 400, 450 or 500 nucleotides, preferably of at Ieast 550, 600, 650, 700, 750 or 800 nucleotides, more preferably of at Ieast 850, 900, 950, 1000, 1050 or 1 100 nucleotides and most preferably of at Ieast 1150, 1200, 1250, 1300, 1350 or 1400 nucleotides.
  • the fragment may also have a length of 1500, 1600, 1700, 1800, 1900, 2000 or 2100 nucleotides.
  • the present invention further relates to the use of nucleic acid sequences which hybridize under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these
  • 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 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).
  • 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. 10 to 50 nucleotides, for example).
  • stringent conditions may include the addition of substances, such as, e. g., formamide, which destabilise the hybrids.
  • normally nucleotide sequences which are at least 60%
  • stringent hybridization 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.
  • 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.
  • Those skilled in the art know how to determine the required hybridization conditions using text books such as those mentioned above or the following textbooks: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), Hames and Higgins (publ.) 1985, Nucleic
  • Typical hybridization and washing buffers for example have the following composition:
  • 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 sequence according to any of SEQ ID Nos.1 -6 under stringent conditions has a length of at least 300, 350, 400, 450 or 500 nucleotides, preferably a length of at least 550, 600, 650, 700, 750 or 800 nucleotides, more preferably a length of at least 850, 900, 950, 1000, 1050 or 1 100 nucleotides and most preferably a length of at least 1 150, 1200, 1250, 1300, 1350 or 1400 nucleotides.
  • the nucleic acid sequence may also have a length of 1500, 1600, 1700, 1800, 1900, 2000 or 2100 nucleotides.
  • the term "encodes a protein having the activity of an imRNA export protein” means that the encoded protein has essentially the same activity as the mRNA export protein encoded by a sequence of any of SEQ ID Nos. 1 -6.
  • "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 mRNA export protein encoded by a sequence of any of SEQ ID Nos. 1 -6.
  • the activity of the mRNA export protein 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.
  • 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
  • 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).
  • epidermis-specific promoters such as the GSTA1 promoter (Altpeter et al. (2005) Plant Mol Biol. 57: 271- 83)
  • 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)
  • tissue-specific promoters 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.
  • 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).
  • epidermis-specific promoters which are active on the site of pathogen entry, such as epidermis- specific promoters.
  • Suitable epidermis-specific promoters include, but are not limited to, the GSTA1 promoter (Accession number X56012), the GLP4 promoter (Wei et al. (1998) Plant Mol. Biol. 36: 101), the GLP2a promoter (Accession number AJ237942), the Prx7 promoter (Kristensen et al. (2001) Mol. Plant Pathol. 2(6): 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): 117) 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, and thus silence transiently.
  • Such transient expression allows for the production of plants which show only transiently increased pathogen resistance.
  • transiently increased resistance may be desired, if, for example, there is an acute risk of fungal contamination, and therefore the plants only have to be resistant to the fungus for a certain period of time.
  • transient resistance is desirable, are known to those skilled in the art.
  • transient expression and thus transient silencing and transient resistance may be achieved using vectors which do not replicate stably in plant cells and which carry the respective sequences for silencing of fungal genes.
  • the actin promoter from Oryza sativa 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. Typically, 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.
  • nucleic acid sequences or DNA sections in expression constructs or vectors the terms "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 operabiy 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 ceils.
  • 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
  • the used plasmids do not need to fulfil special requirements per se. The same applies to direct gene transfer. Simple plasmids, such as pUC derivatives, may be used. 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. Those skilled in the art know all commonly used selection markers, and thus there is no difficulty to select a suitable marker.
  • Common selection markers create resistance in the transformed plant cells to a biocide or antibiotic, such as kanamycin, G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonyl urea, gentamycin or phosphinotricin and the like or may confer tolerance to D-amino acids such as D-alanine.
  • kanamycin G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonyl urea, gentamycin or phosphinotricin and the like
  • 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 repiicate 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. ( 978) 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 ceils thereof may also be transformed using vectors which are based on agrobacteria (see e.g. Chan et al. (1993) Plant
  • the vectors described herein can be directly transformed into the plastid genome. Plastid expression, in which genes are inserted by homologous recombination into the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number over nuclear-expressed genes to permit high expression levels.
  • the nucleotides are inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplasmic for plastid genomes containing the nucleotide sequences are obtained, and are preferentially capable of high expression of the
  • Plastid transformation technology is for example extensively described in U.S. Pat. NOs. 5,451 ,513; 5,545,817; 5,545,818 and 5,877,462, in WO 95/16783 and WO 97/32977, and in McBride er a/. (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 mRNA export protein is not increased and selecting transgenic progeny in which the content and/or the activity of the r mRNA export protein is increased.
  • the other plant in which the content and/or the activity of the mRNA export protein 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 piant 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 mRNA export protein 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 mRNA export protein is increased is inbred and the transgenic progeny of this crossing step is selected and again inbred.
  • This inbreeding step is repeated untii 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 mRNA export protein.
  • 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.
  • the true breeding lines can be further inbred in a linebreeding process. This method is particularly suitable for example for wheat and bariey plants.
  • transgenic lines which are homozygous for the introduced nucleic acid molecules may also be identified and examined with respect to pathogen resistance compared to the pathogen resistance of hemizygous lines.
  • plant cells which contain the recombinant nucleic acid molecules of the present invention may also be further cultivated as plant cells
  • 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.
  • transgenic plants of the present invention includes includes hay, straw, silage and also sprouted grains and legumes.
  • feed is intended to mean a dry feed which can be blended from various raw materials and additives such as soybean shred or barley shred in a feed mill.
  • the transgenic or mutant seed of the transgenic or mutant plants of the present invention can be used to prepare flour, in particular if the transgenic or mutant plants are monocotyledonous plants such as barley or wheat.
  • another embodiment of the present invention is a method for the production of a product comprising the steps of:
  • the product produced by said methods of the invention is flour comprising the nucleic acid sequence coding for the mRNA export protein and/or comprising the mRNA export protein.
  • 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 coding for the mRNA export protein, i.e. this nucleic acid sequence is detectable in the harvestable parts by conventional means, and/or the mRNA export protein.
  • the harvestable plants may be seeds, roots, leaves, stems, and/or flowers comprising the nucleic acid sequence coding for the mRNA export protein and/or comprising the mRNA export protein.
  • Preferred harvestable parts are seeds comprising the nucleic acid sequence coding for the mRNA export protein and/or comprising the mRNA export protein.
  • Entry vector (plPKTA38) preparation The plPKTA38 plasmid (Douchkov et al. (2005) Mol. Plant Microbe Interact. 18(8): 755-761 ) with a kanamycvin resistance gene was used as Gateway Entry vector. Bacteria which contained the plasmid were grown in LB + kanamycin (50 Mg/mL). Plasmid DNA was prepared with the Jetstar midi DNA kit (Genomed).
  • the plasmid was digested with the restriction enzyme Apa I, yielding bands of 1257 bp and 1054 bp. Then the DNA concentration was measured and adjusted to 150 ng/ ⁇ .
  • the plPKTA30 plasmid (Douchkov et al. (2005) Mol. Plant Microbe Interact. 18(8): 755-761) was used as the RNAi vector. It contains an ampicillin resistance gene, a ccdB negative selection marker gene which requires the propagation of the plasmid in DB3.1 cells and a chloramphenicol resistance gene.
  • Plasmid DNA was prepared with the Jetstar midi DNA kit (Genomed). The plasmid preparations were digested as a control with EcoRI (correct bands - 687, 1007, 2641 , and 2857 bp) or Sal I (601 , 1589, and 5002 bp). The DNA concentration was measured and adjusted to 150 ng/ ⁇ . 1 .3. PCR amplification of the cDNA clones
  • a "PCR master mix” (see Table 6) was prepared of which 1 1 ,0 pL were dispensed to each well of a 96-well PCR plate. 4,25 pl_ of each EST-specific primer and 0,5 pL EST DNA as template were added to each well.
  • a ligation master mix was prepared (see Table 7), 6 ⁇ _ each of this ligation mix were added to each well and 4 ⁇ _ of the purified PCR product were added.
  • T4 DNA ligase (5 U/pL) 50 pL 100 pL 200 pL (Fermentas)
  • Figure 2 shows a schematic drawing of the test procedure for the RNAi constructs.
  • Barley was cultivated in IPK cereal soil for 7 days without fertilization in a Sanyo phyto-cultivator, at constantly 20 °C, 60 - 70 % relative humidity and a 16 h light-cycle.
  • Primary leaves (about 7 cm) were cut off and were arranged in parallel on a phytoagar Petri dish (adaxial side up). Thereby, magnetic stirrers were put onto the leaves such that they repel each other.
  • Vacuum was applied for biolistic transformation, wherein the bombardment was made at a pressure of 27.5 mm Hg.
  • the leaves were collected, the leaf tips were cut off and the resulting leaves were transferred to Greiner tubes containing 10 mL of X- glucose solution (100 mM sodium phosphate , pH 7,0;_10 m sodium EDTA; 1 ,4 mM K-hexacyanoferrate(ll); 1 ,4 mM K-hexacyanoferrate(lll); 0,1 % Triton X-100; 20% methanol and 1 mg / ml X-Gluc).
  • the tubes were placed in a suction bottle and vacuum was applied thereto 2 - 3 times. The infiltration is complete When the leaves become transparent and start to sink.
  • the X- glucose solution was refilled to 14 mL and the tubes were sealed. The tubes were incubated over night at 37 °C in the incubator.
  • the leaves were placed in destaining solution (7.5 % TCA, 50 % methanol) for 5 min. Then the leaves were washed with distilled water. Then, the leaves were carefully removed from the tube and were placed onto an object slide with their adaxial side facing upwards.
  • destaining solution 7.5 % TCA, 50 % methanol
  • RNAi constructs The effect of the RNAi constructs on plant resistance to the fungal pathogen Blumeria graminis was tested in transient experiments.
  • pUbiGUS reporter gene vector
  • the leaves were stored in closed Petri dishes at 20 °C until inoculation. Three days after bombardment, the leaves were transferred to 1 % phytoagar with 2 % benzimidazole. A nylon net (mesh width of 200 pm) was stretched over the leaves, and they were inoculated with a conidia density of about 200 conidia/mm 2 .
  • the conidia originated from barley plants (cultivar ,Golden Promise'), which had been inoculated 6 - 7 days before.
  • the leaves were stored in closed Petri dishes with holes for ventilation at 20 °C at a north-facing window. About 45 h after inoculation, GUS staining was performed. Said staining was stopped after 24 h by incubation in 7.5 % trichloroacetic acid, 50 % v/v methanol, and the leaves were bleached.
  • Each experiment contained 3 parallel bombardments to 7 leaf sections each of the negative control (empty vector plPKTA30N). Further, each experiment contained 2 parallel bombardments of a TIGS positive control plPKTA36, which causes resistance by inhibiting the MIo gene of barley. Data per experiment are based on the comparison of the effect of the test constructs with the average value of the 3 negative controls of the respective experiment.
  • Table 10 shows the relative susceptibility index (Rel SI) of barley cells transiently transformed with the RNAi constructs comprising SEQ ID No.1 and the sequence reverse complementary thereto. These RNAi constructs inhibit the expression of the mRNA export protein. The susceptibility index was determined in five independent transformation experiments. As the cells transformed with the RNAi construct have a susceptibility index of more than 100% compared to the control cells transformed with empty vector (plPKTA30N), the RNAi construct suppresses putative resistance genes in barley.
  • Rel SI relative susceptibility index
  • the binary vector for transformations is constructed such that the cDNA/ functional fragment is located in sense direction between the parsley ubiquitine promoter (PcUbi) and a Agrobacterium tOCS terminator.
  • the synthesized cDNA/functional fragments are subcloned into a Gateway pENTRY vector (Invitrogen, Life Technologies, Carlsbad, California, USA).
  • the binary plant transformation vector is made by a triple LR reaction (Gateway system, (Invitrogen, Life Technologies,
  • a binary pDEST vector is used which is composed of: (1 ) a Kanamycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a pBR322 origin of replication for stable maintenance in E.
  • 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.
  • Agrobacterium 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
  • 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 -7H 2 O, 5 g tryptone, 2.5 g yeast extract, pH 7.0, add after autoclave 1 pg Biotin incl. appropriate antibiotics).
  • liquid medium 5 g mannitol, 1 g L-glutamic acid, 250 mg KH 2 P0 4> 100 mg NaCI, 100 mg MgS0 4 -7H 2 O, 5 g tryptone, 2.5 g yeast extract, pH
  • Liquid culture is grown at 28°C for ⁇ 16h to reach an OD 6 oo ⁇ 1 ⁇
  • the Agrobacterium culture is centrifuged at 4.500 g for 10 minutes and resuspended in 4 ml inoculation medium ((1/ 0 MS complete) 30g maltose, 100mg MES; adjusted to pH 5.8 and add after autoclave 0.01 % Pluronic, 200 ⁇ acetosyringone) to an OD 60 o of ⁇ 1 .
  • the Agrobacterium inoculation medium is ready to use.
  • lEs immature embryos
  • 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 mi of solidified co-culture media (1/10 MS complete, 30g maitose, 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.
  • the explants are placed with the embryo axis facing down on recovery media (MS full complete, 30g maltose, 0.69g proline, 20mg thiamine, g casein hydrolysate, 100mg myo-inositol, 5 ⁇ CuS0 4 ; 2.4g NH 4 N0 3 , .95g MES, 8g agar (Plant TC), adjust to pH 5.8 and add after autoclave 2mg 2,4-D, 200mg timentin, l OOmg ascorbic acid) for 4 weeks at 24°C in the dark. The calli are transferred to fresh recovery medium after two weeks.
  • the explants are transferred to rooting media (1 ⁇ 2 MS complete, 30g sucrose, 7g agar and adjust to pH 5.8, add after autoclave, 0.5mg NAA, 200mg timentin, 11 mM D-alanine) 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.
  • Plants are grown in the greenhouse at 19°C and 60-80% humidity. After 11 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 overexpression of the mRNA export protein will lead to enhanced resistance of wheat against Septoria tritici.
  • Diseased leaf area is scored by eye by trained personal. The percentage of the leaf area showing fungal colonies 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 the 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 overexpression of the mRNA export protein will lead to enhanced resistance of wheat against rust fungi.
  • Powdery mildew screening assay 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). 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 overexpression of the mRNA export protein will lead to enhanced resistance of wheat to powdery mildew fungus.

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Abstract

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, comprising the step of increasing the content and/or activity of an mRNA export protein.

Description

Method of producing plants having increased resistance to pathogens
FIELD OF THE INVENTION
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 the activity of an mRNA export protein is increased.
BACKGROUND OF THE INVENTION
Plant diseases, which are caused by various pathogens such as viruses, bacteria and fungi, may lead to significant crop losses of cultivated plants, resulting in economic consequences and in threatening human food supply. For example, infestation of cereals with Blumeria graminis, the pathogen that causes powdery mildew, may cause yield losses of up to 30%.
Since the last century, chemical fungicides have been utilised for controlling fungal diseases. A different approach is to examine the natural pathogen defence of plants aqainst different nathnoens and to use the same
specifically for the production of pathogen resistant plants by gene
technological manipulation, e.g. by means of introducing external resistance genes or by means of manipulating the endogenous gene expression of the plants. Resistance is the ability of a plant to inhibit or at least limit any infestation or population of a pest. The plants have a certain degree of natural resistance which is imparted by the formation of specific defence substances, such as isoprenoids, flavonoids, enzymes and reactive oxygen species,
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. For example, chitinase (WO 92/17591) and pathogenesis-related genes (WO 92/20800) as well as 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.
Conversely, it could be shown that some of the plant genes help a fungus to enter the plant. Thus, an alternative approach for producing transgenic plants having increased fungal resistance is to inhibit the expression of said plant genes which code for example for a polyphenoloxidase (WO 02/061101 ), NADPH oxidase (WO 2004/009820) and the Mlo gene (WO 00/0 722) 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).
Nevertheless, there is still a need to identify further genes which code for polypeptides involved in pathogen resistance and to develop methods for producing transgenic plants with increased pathogen resistance by using these genes. OBJECT AND SUMMARY OF THE INVENTION
It is thus an object of the present invention to identify genes which are involved in the pathogen resistance of plants.
It is a further object of the present invention to provide a method for producing transgenic plants with increased pathogen resistance, preferably resistance to fungal pathogens such as Blumeria graminis, Septoria tritici and/or Puccinia triticina.
These and further objects of the invention, as will become apparent from the description, are attained by the subject-matter of the independent claims.
Some of the preferred embodiments of the present invention form the subject-matter of the dependent claims.
The present inventors have found that the reduction of the content of an mRNA export protein by RNA interference leads to an enhanced
susceptibility of barley cells to Blumeria graminis. Hence, this mRNA export protein is considered to be involved in mediating pathogen resistance.
MRNA export proteins are involved in regulating the nucleocytoplasmic distribution of mRNAs and may be a component of the nuclear pore complex or may be nucleocytoplasmic shuttling proteins which together with the mRNA form an export-competent mRNA ribonucleoprotein. in Arabidopsis it has been shown that proteins involved in nucleocytoplasmic shuttling also play a role in regulating plant immunity (Cheng et ai. (2009) Plant Cell 21 : 2503-2516; Palma et ai. (2005) Current Biology 15: 1129-1135; Germain et al. (2010) PLoS Genetics 6(12): e1001250). However, so far this has not been shown for monocotyledonous plants.
Accordingly, 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 the activity of an mRNA export protein which is encoded by a nucleic acid sequence selected from the group consisting of:
a) a nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1 -6 or a fragment of any of these sequences;
b) a nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1 -6 or a fragment of any of these sequences; and
c) a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences;
is increased in comparison to the control plant cell, plant or plant part.
Thus, the present invention provides a method for increasing pathogen resistance in a plant cell, plant or part thereof, wherein the method comprises the step of increasing the content and/or activity of an mRNA export protein in the plant cell, plant or part thereof compared to a control plant cell, plant or plant part, wherein the mRNA export protein is encoded by a nucleic acid sequence which is selected from the group consisting of:
a) a nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; b) a nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; and
c) a nucleic acid sequence comprising a sequence which hybridizes
under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences.
Preferably, the method comprises the steps of
(a) introducing into a plant cell a vector which comprises:
(i) a promoter functional in plant cells,
(ii) operatively linked thereto at least one nucleic acid sequence as defined above;
(iii) optionally, a termination sequence; to produce a transformed plant ceii; and
(b) optionally, regenerating a transgenic plant from the transformed cell.
Preferably, the promoter is a tissue-specific and/or a pathogen-inducible promoter. In another preferred embodiment, 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 protein which mediates pathogen resistance. In another embodiment 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 mRNA export protein as defined herein is not increased and selecting transgenic progeny in which the content and/or the activity of the mRNA export protein as defined herein is increased. ln a preferred embodiment 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:
(a) mutagenizing plant material;
(b) identifying plant material having at least one point mutation in an 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 -6.
In a preferred embodiment, 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 -6 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.
In a further preferred embodiment the transgenic or mutant plant is a monocotyledonous plant, more preferably it is a barley or a wheat plant. Preferably, the transgenic or mutant plant has an increased resistance to a fungal pathogen, more preferably to Blumeria graminis, Septoria tritici and/or Puccinia triticina. In another embodiment the present invention relates to an expression construct comprising at least one nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; (b) a nucleic acid sequence comprising a sequence which is at least
70 % identical to the sequence according to any of SEQ ID Nos. 1- 6 or a fragment of any of these sequences; and
(c) a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences,
operatively linked to a promoter functional in plant cells.
In a preferred embodiment 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.
In another preferred embodiment the promoter is a tissue-specific and/or a pathogen-inducible promoter.
In another embodiment 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. Thus, 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.
In still a further embodiment 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, produced by the method of the present invention or containing an expression construct or a vector of the present invention. in another embodiment 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 or the mutation which increases the content and/or the activity of an mRNA export protein as defined herein can be detected in said transgenic or mutant seed or in said flour.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 : Flow diagram for the high-throughput production of RNAi
constructs.
I, PCR amplification of cDNA fragments of interest; I la, Ligation of the PCR fragments in the intermediate vector plPKTA38 in the presence of the restriction endonuclease, Swa I, which inhibits the re-ligation of the vector;
lib, Re-cutting all re-ligated vector molecules;
III, Recombination of the cloned cDNA fragments in the RNAi vector plPKTA30 by means of LR clonase.
Figure 2: Flow diagram showing how the effect of the RNAi constructs on plant resistance to Blumeria graminis is tested.
DETAILED DESCRIPTION OF THE INVENTION
The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.
The present invention will be described with respect to particular
embodiments, but the invention is not limited thereto, but only by the claims. Where the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.
For the purposes of the present invention, 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.
Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.
The term "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. Within the present invention, the transgenic plant contains a nucleic acid sequence selected from the group consisting of
(a) a nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences;
(b) a nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; and
(c) a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences;
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 contains one or more mutations with respect to the sequence present at the natural locus of this sequence in the genome of the control plant and/or which encodes an imRNA export protein having an increased activity compared to the mRNA export protein encoded by the sequence present at the natural locus of this sequence in the genome 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. Preferably, the nucleic acid sequence is introduced by means of a vector. Also preferably, the nucleic acid sequence is stably integrated into the genome of the transgenic plant. In particular, the transgenic plant cell, plant or plant part of the present invention contains a nucleic acid sequence which increases the content and/or activity of an mRNA export protein compared to a control plant cell, plant or plant part. In addition to the nucleic acid sequence which increases the content and/or activity of an mRNA export protein, 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. The term
"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. The term "mutant" means that a plant cell, plant or plant part has been altered by mutagenesis so that a nucleic acid sequence selected from the group consisting of
(a) a nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences;
(b) a nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; (c) a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences;
contains at least one point mutation, i.e. at least one nucleotide substitution, deletion and/or addition, in comparison to a control plant, plant cell or part thereof which has been used as a starting material in the mutagenesis and which has not been mutagenized. Preferably, the mutant plant contains at least one nucleotide substitution in the nucleic acid sequence according to any of SEQ ID Nos. 1-6 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.
Dicotyledonous useful plants comprise, inter alia, cotton, legumes, like leguminous plants and in particular alfalfa, soy bean, rape, tomato, sugar beet, potato, ornamental plants, and trees. Further useful plants can comprise fruit (in particular apples, pears, cherries, grapes, citrus, pineapple, and bananas), pumpkin, cucumber, wine, oil palms, tea shrubs, cacao trees, and coffee shrubs, tobacco, sisal, as well as, with medicinal plants, rauwolfia and digitalis.
Particularly preferred are the cereals wheat, rye, oat, barley, rice, maize and millet, sugar beet, rape, soy, tomato, potato, cotton and tobacco. Further useful plants can be taken from US 6,137,030. More preferably the transgenic or mutant plants are oat, barley, rye, wheat or rice plants and most preferably the transgenic or mutant plants are barley or wheat plants. Within the meaning of the present invention the term "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 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. In particular, 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. The term "cell" or "plant cell" as used herein 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.
According to the present invention, "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. Furthermore, "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.
According to the present invention, the term "increased pathogen resistance" is understood to denote that the transgenic plants or plant cells of the present invention are infected iess severely and/or less frequently by plant
pathogens.
In one embodiment 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. According to the present invention, 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 ieast the factor 50, and more preferably by at Ieast the factor 100, and most preferably by at Ieast the factor 1000, as compared to the control plant. Alternatively, 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%. The relative susceptibility index of the plants of the present invention is less than 80%, preferably less than 70 or 60%, more preferably less than 50 or 40% and most preferably less than 30%.
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 an mRNA export protein. A control plant may be a plant of the same line or variety as the transgenic plant being tested, or it may be another line or variety, such as a plant known to have a specific phenotype, characteristic, or known genotype. Another suitable control plant is a genetically unaltered or non-transgenic plant of the parental line used to generate the transgenic plant of the present invention, i.e. the wild-type plant.
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.
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. "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 wiid-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. In order to identify successfully transformed cells in transient experiments, a reporter gene, such as the beta-glucuronidase (GUS) gene from E. 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. coli, the expression of which in the plant cells may be proven by simple methods, is co-transformed in a suitable vector with the vector mediating the expression of the mRNA export protein. Optionally, 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.
Alternatively, 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 increasing the content and/or the activity of the mRNA export protein is lower than the diseased leaf area of infected control plants.
According to the present invention, the term "plant pathogens" includes viral, bacterial, fungal and other pathogens. Preferably, the term "plant pathogens" comprises fungal pathogens.
According to the present invention, the term "plant pathogens" includes biotrophic, hemibiotrophic and necrotrophic pathogens. Preferably, 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 belong to the group of biotrophic fungi, like other rust fungi, powdery mildew fungi or oomycete pathogens like the genus Phytophthora or Peronopora. The necrotrophic phytopathogenic fungi depend for their nutrition on dead cells of the plants, e.g. species from the genus Fusarium, Rhizoctonia or Mycospaerella. Soybean rust has occupied an intermediate position, since it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic. After the penetration, the fungus changes over to an obligatory- biotrophic lifestyle. The subgroup of the biotrophic fungal pathogens which follows essentially such an infection strategy is hemibiotrophic. Table 1 : Diseases caused by biotrophic phytopathogenic fungi
Figure imgf000020_0001
Table 2: Diseases caused by necrotrophic and/or hemibiotrophic fungi and Oomycetes
Disease Pathogen
Plume blotch Septoria (Stagonospora) nodorum
Leaf blotch Septoria tritici
Ear fusarioses Fusarium spp.
Eyespot Pseudocercosporella herpotrichoides
Smut Ustilago spp.
Late blight Phytophthora infestans Disease Pathogen
Bunt Tilletia caries
Take-all Gaeumannomyces graminis
Anthrocnose leaf blight Colletotrichum graminicola (teleomorph:
Glomerella graminicola Politis); Glomerella Anthracnose stalk rot tucumanensis (anamorph: Glomerella falcatum Went)
Aspergillus ear and Aspergillus flavus
kernel rot
Banded leaf and sheath spot Rhizoctonia solani Kuhn = Rhizoctonia microsclerotia J. Matz (telomorph:
Thanatephorus cucumeris)
Black bundle disease Acremonium strictum W. Gams - alosporium acremonium Auct. non Corda
Black kernel rot Lasiodiplodia theobromae =
Botryodiplodia theobromae
Borde bianco Marasmiellus sp.
Brown spot (black spot, stalk rot) Physoderma maydis
Cephalosporium kernel rot Acremonium strictum = Cephalosporium acremonium
Charcoal rot Macrophomina phaseolina
Corticium ear rot Thanatephorus cucumeris =
Corticium sasakii
Curvularia leaf spot Curvularia clavata, C. eragrostidis, = C.
maculans (teleomorph: Cochliobolus eragrostidis), Curvularia inaequalis, C. intermedia (teleomorph: Cochliobolus intermedius), Curvularia lunata Disease Pathogen
(teleomorph: Cochliobolus lunatus), Curvularia pallescens (teleomorph:
Cochliobolus pallescens), Curvularia senegalensis, C. tubercuiata (teleomorph: Cochliobolus tuberculatus)
Didymella leaf spot Didymella exitalis
Diplodia ear and stalk rot Diplodia frumenti (teleomorph:
Botryosphaeria festucae)
Diplodia ear and stalk rot, seed rot Diplodia maydis =
and seedling blight Stenocarpella maydis
Diplodia leaf spot or streak Stenocarpella macrospora =
Diplodialeaf macrospora
Brown stripe downy Sclerophthora rayssiae var. zeae mildew
Crazy top downy mildew Sclerophthora macrospora =
Sclerospora macrospora
Green ear downy mildew Sclerospora graminicola
(graminicola downy mildew)
Dry ear rot (cob, Nigrospora oryzae
kernel and stalk rot) (teleomorph: Khuskia oryzae)
Ear rots (minor) Alternaria alternata = A. tenuis,
Aspergillus glaucus, A. niger,
Aspergillus spp., Botrytis cinerea
(teleomorph: Botryotinia fuckeliana), Cunninghamella sp.,
Curvularia pallescens,
Doratomyces stemonitis - Disease Pathogen
Cephalotrichum stemonitis,
Fusarium culmorum,
Gonatobotrys simplex,
Pithomyces maydicus,
Rhizopus microsporus Tiegh.,
R. stolonifer = R. nigricans,
Scopulariopsis brumptii
Ergot (horse's tooth) Claviceps gigantea
(anamorph: Sphacelia sp.)
Eyespot Aureobasidium zeae = Kabatiella zeae
Fusarium ear and stalk rot Fusarium subglutinans =
F. monili forme var.subglutinans
Fusarium kernel, root and stalk Fusarium moniliforme
rot, seed rot and seedling blight (teleomorph: Gibberella fujikuroi)
Fusarium stalk rot, Fusarium avenaceum
seedling root rot (teleomorph: Gibberella avenacea)
Gibberella ear and stalk rot Gibberella zeae
(anamorph: Fusarium graminearum)
Gray ear rot Botryosphaeria zeae = Physalospora zeae
(anamorph: Macrophoma zeae)
Gray leaf spot Cercospora sorghi = C. sorghi var. maydis, (Cercospora leaf spot) C. zeae-maydis
Helminthosporium root rot Exserohilum pedicellatum =
Helminthosporium pedicellatum
(teleomorph: Setosphaeria pedicellate)
Hormodendrum ear rot Cladosporium cladosporioides =
(Cladosporium rot) Hormodendrum cladosporioides, C. Disease Pathogen
herbarum (teleomorph: Mycosphaerella tassiana)
Leaf spots, minor Alternaria alternata,
Ascochyta maydis, A. tritici,
A. zeicola, Bipolaris victoriae =
Helminthosporium victoriae
(teleomorph: Cochliobolus victoriae), C. sativus (anamorph: Bipolaris sorokiniana = H. sorokinianum = H. sativum), Epicoccum nigrum,
Exserohilum prolatum = Drechslera prolata (teleomorph: Setosphaeria prolata) Graphium penicillioides,
Leptosphaeria maydis, Leptothynum zeae, Ophiosphaerella herpotricha, (anamorph: Scolecosporiella sp.),
Paraphaeosphaeria michotii, Phoma sp., Septoria zeae, S. zeicola,
S. zeina
Northern corn leaf blight (white Setosphaeria turcica (anamorph:
blast, crown stalk rot, stripe) Exserohilum turcicum = Helminthosporium turcicum)
Northern corn leaf spot Cochliobolus carbonum (anamorph:
Helminthosporium ear rot (race 1) Bipolaris zeicola = Helminthosporium
carbonum)
Penicillium ear rot (blue eye, blue Penicillium spp., P. chrysogenum, mold) P. expansum, P. oxalicum Disease Pathogen
Phaeocytostroma stalk and root Phaeocytostroma ambiguum, =
rot Phaeocytosporella zeae
Phaeosphaeria leaf spot Phaeosphaeria maydis - Sphaerulina maydis
Physalospora ear rot Botryosphaeria festucae = Physalospora (Botryosphaeria ear rot) zeicola (anamorph: Diplodia frumenti)
Purple leaf sheath Hemiparasitic bacteria and fungi
Pyrenochaeta stalk and root rot Phoma terrestris =
Pyrenochaeta terrestris
Pythium root rot Pythium spp., P. arrhenomanes,
P. graminicola
Pythium stalk rot Pythium aphanidermatum =
P. butleri L
Red kernel disease (ear mold, leaf Epicoccum nigrum
and seed rot)
Rhizoctonia ear rot (sclerotial rot) Rhizoctonia zeae (teleomorph: Waitea circinata)
Rhizoctonia root and stalk rot Rhizoctonia solani, Rhizoctonia zeae
Root rots (minor) Alternaria alternata, Cercospora sorghi,
Dictochaeta fertilis, Fusarium acuminatum (teleomorph: Gibberella acuminata), F. equiseti (teleomorph: G. intricans), F. oxysporum, F. pallidoroseum, F. poae, F. roseum, G. cyanogena, (anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp., Periconia circinata, Phytophthora cactorum, P. drechsleri, P. nicotianae var. Disease Pathogen
parasitica, Rhizopus arrhizus
Rostratum leaf spot Setosphaeria rostrata, (anamorph:
(Helminthosporium leaf disease, xserohilum rostratum = Helminthosporium ear and stalk rot) rostratum)
Java downy mildew Peronosclerospora maydis =
Sclerospora maydis
Philippine downy mildew Peronosclerospora philippinensis =
Sclerospora philippinensis
Sorghum downy mildew Peronosclerospora sorghi =
Sclerospora sorghi
Spontaneum downy mildew Peronosclerospora spontanea =
Sclerospora spontanea
Sugarcane downy mildew Peronosclerospora sacchari =
Sclerospora sacchari
Sclerotium ear rot (southern blight) Sclerotium rolfsii Sacc. (teleomorph: Athelia rolfsii)
Seed rot-seedling blight Bipolaris sorokiniana, B. zeicola =
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. Disease Pathogen
Selenophoma leaf spot Selenophoma sp.
Sheath rot Gaeumannomyces graminis
Shuck rot Myrothecium gramineum
Silage mold Monascus purpureus, M ruber
Smut, common Ustilago zeae = U. maydis
Smut, false Ustilaginoidea virens
Smut, head Sphacelotheca reiliana = Sporisorium
holcisorghi
Southern corn leaf blight and stalk Cochliobolus heterostrophus (anamorph: rot Bipolaris maydis = Helminthosporium
maydis)
Southern leaf spot Stenocarpella macrospora = Diplodia
macrospora
Stalk rots (minor) Cercospora sorghi, Fusarium episphaeria,
F. merismoides, F. oxysporum
Schlechtend, F. poae, F. roseum, F. solani (teleomorph: Nectria haematococca), F. tricinctum, Mariannaea elegans, Mucor sp., Rhopographus zeae, Spicaria sp.
Storage rots Aspergillus spp., Penicillium spp. und
weitere Pilze
Tar spot Phyllachora maydis
Trichoderma ear rot and root rot Trichoderma viride = T. lignorum
teleomorph: Hypocrea sp.
White ear rot, root and stalk rot Stenocarpella maydis = Diplodia zeae
Yellow leaf blight Ascochyta ischaemi, Phyllosticta maydis
(teleomorph: Mycosphaerella zeae-maydis) Disease Pathogen
Zonate leaf spot Gloeocercospora sorghi
Preferably, fungal pathogens or fungal-like pathogens (like for example Chromista) 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:
- Plasmodiophoromycetes like Plasmodiophora brassicae (clubroot of crucifers), Spongospora subterranea (powdery scab of 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 (P. viticola) and 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
P. debaryanum), Phytophthora Infestans (potato light blight, tomato late blight, etc.), Albugo spec, (white rust on cruciferous plants) Ascomycetes like Microdochium nivale (snow mold of rye and wheat), Fusarium graminearum, Fusarium culmorum (head blight, in particular of wheat), Fusarium oxysporum (fusarium wilt of tomato), Blumeria graminis (powdery mildew of barley (f. sp. hordei) and wheat (f. sp. tritici)), Erysiphe pisi (pea mildew), Nectria galligena (Nectria canker of fruit trees), Unicnula necator (grapevine powdery mildew),
Pseudopeziza tracheiphila (grapevine red fire disease), Claviceps purpurea (ergot on, for example, rye and grasses), Gaeumannomyces graminis (black leg disease of wheat, rye and, inter alia, grasses), Magnaporthe grisea (rice blast disease), Pyrenophora graminea (leaf stripe disease of barley), Pyrenophora teres (net blotch disease of barley), Pyrenophora tritici-repentis (tan spot disease) Septoria tritici (leaf spot of wheat), Venturia inaequalis (apple scab disease),
Scierotinia sclerotium (white moid, stem canker of rape),
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), Sphacelotheca spp. (head smut of sorghum), Melampsora lini (rust of flax), Puccinia graminis (stem rust of wheat, barley, rye, oat), Puccinia recondita (brown rust of wheat), Puccinia triticina (wheat leaf rust), Puccinia dispersa (brown rust of rye), Puccinia hordei (brown rust of barley), Puccinia coronata (crown rust of oat), Puccinia striiformis (yellow rust of wheat, bariey, rye, and various grasses), Uromyces appendiculatus (bean rust), Phakopsora pachyrhizi (Asian soybean rust), Sclerotium rolfsii (root and stem rots of many plants).
- Deuteromycetes (Fungi imperfecti) like Septoria nodorum (glume
blotch) of wheat (Septoria tritici), Pseudocercosporella herpotrichoides
(stem break disease in wheat, barley, rye), Rynchosporium secalis (scald disease in rye and barley), Alternaria solani (early blight of potato and tomato), Phoma betae (black rot of beet), Cercospora beticola (Cercospora leaf spot of beet), Alternaria brassicae (dark leaf spot of rape, cabbage and other cruciferous plants), Verticillium dahliae (Verticillium wilt and stalk rot of rape), Colietotrichum 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.).
Likewise preferred are: 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. tritici)), Puccinia triticina (wheat leaf rust), Magnaporthe grisea (rice blast disease), Sclerotinia sclerotium (white mold, stem canker of rape), Septoria nodorum and Septoria tritici (glume blotch of wheat), Alternaria brassicae (dark leaf spot of rape, cabbage and other cruciferous plants), Phakopsora pachyrhizi (Asian soybean rust), Phoma lingam (phoma stem canker, black leg disease of cabbage; crown and stem canker of rape). The pathogens listed in Table 3 as well as the diseases associated therewith are to be mentioned as bacterial pathogens in an exemplary, yet not limiting manner.
Table 3: Bacterial diseases
Disease Pathogen
Bacterial leaf blight and stalk rot Pseudomonas avenae subsp. avenae
Bacterial leaf spot Xanthomonas campestris pv. holcicola
Bacterial stalk rot Enterobacter dissolvens =
Erwinia dissolvens
Bacterial stalk and top rot Erwinia carotovora subsp. carotovora,
Erwinia chrysanthemi pv. zeae
Bacterial stripe Pseudomonas andropogonis
Chocolate spot Pseudomonas syringae pv. coronafaciens
Goss's bacterial wilt and blight Clavibacter michiganensis subsp.
(leaf freckles and wilt) nebraskensis = Corynebacterium
michiganense pv.andnebraskense
Holcus spot Pseudomonas syringae pv. syringae
Purple leaf sheath Hemiparasitic bacteria
Seed rot-seedling blight Bacillus subtilis
Stewart's disease Pantoea stewartii =
(bacterial wilt) Erwinia stewartii
Corn stunt Spiroplasma kunkelii
(achapparramiento, maize stunt, Mesa Central or Rio Grande
maize stunt)
Particularly preferably, the transgenic plants produced according to the present invention are resistant to the following pathogenic bacteria: Corynebacterium sepedonicum (bacterial ring rot of potato), Erwinia
carotovora (black leg rot of potato), Erwinia amylovora (fire blight of pear, apple, quince), Streptomyces scabies (common scab of potato),
Pseudomonas syringae pv. tabaci (wild fire disease of tobacco),
Pseudomonas syringae pv. phaseolicola (halo blight disease of dwarf bean), Pseudomonas syringae pv. 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). The term "viral pathogens" includes all plant viruses, like for example tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc.
The pathogens listed in Table 4 as well as the diseases associated therewith are to be mentioned as viral pathogens in an exemplary, yet not limiting manner.
Table 4: Viral diseases
Disease Pathogen
American wheat striate American wheat striate mosaic virus (AWSMV) (wheat striate mosaic)
Barley stripe mosaic Barley stripe mosaic virus (BSMV)
Barley yellow dwarf Barley yellow dwarf virus (BYDV)
Brome mosaic Brome mosaic virus (BMV)
Cereal chlorotic mottle Cereal chlorotic mottle virus (CCMV)
Corn chlorotic vein banding Corn chlorotic vein banding virus (CCVBV) (Braizilian maize mosaic)
Corn lethal necrosis Virus complex from Maize chlorotic mottle virus
(MCMV) and Maize dwarf mosaic virus (MDMV) A or B or Wheat streak mosaic virus(WSMV)
Cucumber mosaic Cucumber mosaic virus (CMV)
Cynodon chlorotic streak Cynodon chlorotic streak virus (CCSV)
Johnsongrass mosaic Johnsongrass mosaic virus (JGMV)
Maize bushy stunt Mycoplasma-like organism (MLO) associated
Maize chlorotic dwarf Maize chlorotic dwarf virus (MCDV)
Maize chlorotic mottle Maize chlorotic mottle virus (MCMV)
Maize dwarf mosaic Maize dwarf mosaic virus (MDMV)
strains A, D, E and F
Maize leaf fleck Maize leaf fleck virus (MLFV)
Maize line Maize line virus (MLV)
Maize mosaic (corn leaf stripe, Maize mosaic virus (MMV)
enanismo rayado)
Maize mottle and chlorotic Maize mottle and chlorotic stunt virus
stunt
Maize pellucid ringspot Maize pellucid ringspot virus (MPRV) Maize raya gruesa Maize raya gruesa virus (MRGV) maize rayado fino (fine striping Maize rayado fino virus (MRFV) disease)
Maize red leaf and red stripe Mollicute
Maize red stripe Maize red stripe virus (MRSV)
Maize ring mottle Maize ring mottle virus (MRMV)
Maize rio IV Maize rio cuarto virus (MRCV)
Maize rough dwarf Maize rough dwarf virus (MRDV) (nanismo ruvido) (Cereal tillering disease virus)
Maize sterile stunt Maize sterile stunt virus
(strains of barley yellow striate virus)
Maize streak Maize streak virus (MSV)
Maize stripe (maize chlorotic Maize stripe virus
stripe, maize hoja blanca)
Maize stunting Maize stunting virus
Maize tassel abortion Maize tassel abortion virus (MTAV)
Maize vein enation Maize vein enation virus (MVEV)
Maize wallaby ear Maize wallaby ear virus (MWEV)
Maize white leaf Maize white leaf virus
Maize white line mosaic Maize white line mosaic virus (MWLMV)
Mi!!et red leaf Mi!!et red leaf virus (MRLV)
Northern cereal mosaic Northern cereal mosaic virus (NCMV)
Oat pseudorosette Oat pseudorosette virus
(zakuklivanie) Oat sterile dwarf Oat sterile dwarf virus (OSDV)
Rice black-streaked dwarf Rice black-streaked dwarf virus (RBSDV)
Rice stripe Rice stripe virus (RSV)
Sorghum mosaic Sorghum mosaic virus (SrMV) (also: sugarcane mosaic virus (SCMV) strains H, 1 and M)
Sugarcane Fiji disease Sugarcane Fiji disease virus (FDV)
Sugarcane mosaic Sugarcane mosaic virus (SCMV) strains A, B,
D, E, SC, BC, Sabi and MB (formerly MDMV-B)
Wheat spot mosaic Wheat spot mosaic virus (WSMV)
The plants and plant cells according to the present invention can also be resistant to animal pests like insects and nematodes. Insects, like for
example beetles, caterpiiiars, iice, or mites are to be mentioned in an
exemplary, yet not limiting manner.
Preferably, 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 ducens (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), Phyllogphaga spp. (white grubs), Anuraphis maidiradicis (corn root aphid), Delia platura (seedcorn maggot), Colaspis brunnea (grape colaspis), Stenolophus lecontei (seedcorn beetle), and Clivinia impressifrons (lender seedcorn beetle).
Furthermore, there are to be mentioned: the cereal leaf beetle (Oulema melanopus), the frit fly (Oscinella frit), wireworms (Agrotis lineatus), and aphids (like for example the bird cherry-oat aphid Rhopalosiphum padi, the grain aphid Sitobion avenae).
The pathogens listed in Table 5 as well as the diseases associated therewith are to be mentioned as nematode pests in an exemplary, yet not limiting manner.
Table 5: Parasitic nematodes
Damage Pathogenic nematode
Awl Dolichodorus spp., D. heterocephalus
Bulb and stem nematode, Ditylenchus dipsaci
beet eelworm
("Bulb and stem"; Europe)
Burrowing Radopholus similis
Cereal cyst nematode Heterodera avenae, H. zeae, ("Cyst") Punctodera chalcoensis
Dagger Xiphinema spp., X. americanum,
X. mediterraneum
False root-knot Nacobbus dorsalis
Lance, Columbia Hoplolaimus columbus
Lance Hoplolaimus spp., H. galeatus
Lesion Pratylenchus spp., P. brachyurus,
P. crenatus, P. hexincisus, P. neglectus,
P. penetrans, P. scribneri, P. thornei, P. zeae
Needle Longidorus spp., L. breviannulatus
Ring Criconemella spp., C. ornata
Root-knot nematode Me!oidogyne spp., M. chitwoodi,
M. incognita, M. javanica
Spiral Helicotylenchus spp.
Sting Belonolaimus spp., B. longicaudatus
Stubby-root Paratrichodorus spp., P. christiei, P. minor,
Quinisulcius acutus, Trichodorus spp.
Stunt Tylenchorhynchus dubius
Particularly preferably, the transgenic plants produced according to the present invention are resistant to Globodera rostochiensis and G. pallida
(cyst nematodes of potato, tomato, and other so!anaceae), 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).
In individual species of particular agricultural importance, the plants according to the present invention are preferably resistant to the following pathogens:
In barley, the plants are resistant to the fungal, bacterial, and viral pathogens Puccinia hordei (barley stem rust), Blumeria (Erysiphe) graminis f. sp. hordei (barley powdery mildew), Rhynchosporium secalis (barley scald), barley yellow dwarf virus (BYDV), and 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 sen/us (brown stink bug); Deliaplatura
(seedcorn maggot); Mayetiola destructor (Hessian fly); Petrobia latens (brown wheat mite).
In 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. caulivora, Sclerotium rolfsii, Cercospora kikuchii,
Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotrichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p. .
glycinea, Xanthomonas campestris p. v. phaseoli, Microsphaera diffussa, Fusarium semitectum, Phialophora gregata, soy bean mosaic virus,
Glomerella glycines, tobacco ring spot virus, tobacco streak virus,
Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, tomato spotted wilt virus, Heterodera glycines, Fusarium solani and the pathogenic insects / nematodes Pseudoplusia includens (soybean looper); Anticarsia gemmatalis (velvetbean caterpillar); Plathypena scabra (green cloverworm); Ostrinia nubilalis (European corn borer); Agrotis ipsilon (black cutworm); Spodoptera exigua (beet armyworm); Heliothis virescens (cotton budworm); Helicoverpa zea (cotton bollworm); Epilachna varivestis (Mexican bean beetle); Myzus persicae (green peach aphid); Empoasca fabae (potato leaf hopper); Acrosternum hilare (green stink bug); Melanoplus femurrubrum (redlegged grasshopper); Melanoplus differentialis (differential grasshopper); Hylemya platura (seedcom maggot); Sericothrips variabilis (soybean thrips); Thrips tabaci (onion thrips); Tetranychus turkestani
(strawberry spider mite); Tetranychus urticae (twospotted spider mite).
In canoia, the plants are resistant to the fungai, 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.
In alfalfa, 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. alfaifae, Aphanomyces euteiches,
Stemphylium herbarum, Stemphylium alfaifae.
In wheat, the plants are resistant to the fungal, bacterial, or viral pathogens Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternana alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici,
Cephalosporium gramineum, Collotetrichum graminicola, Blumeria (Erysiphe) graminis f. sp. tritici, Puccinia graminis f. sp. tritici, Puccinia recondita f. sp. tritici, Puccinia striiformis, Puccinia triticina, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle Streak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Pythium gramicoia, High Piains Virus, European wheat striate virus and to the pathogenic insects / nematodes Pseudaletia unipunctata (army worm);
Spodoptera frugiperda (fall armyworm); Elasmopalpus lignosellus (lesser cornstalk borer); Agrotis orthogonia (western cutworm); Elasmopalpus Zignosellus (lesser cornstalk borer); Oulema melanopus (cereal leaf beetle); Hypera punctata (clover leaf weevil); Diabrotica undecimpunctata howardi (southern corn rootworm); Russian wheat aphid; Schizaphis graminum (greenbug); Macrosiphum avenae (English grain aphid); Melanoplus femurrubrum (redlegged grasshopper); Melanoplus differentialis (differential grasshopper); Melanoplus sanguinipes (migratory grasshopper); Mayetiola destructor (Hessian fly); Sitodiplosis mosellana (wheat midge); Meromyza americana (wheat stem maggot); Hyiemya coarciata (wheat buib fiy);
Frankliniella fusca (tobacco thrips); Cephus cinctus (wheat stem sawfly); Aceria tulipae (wheat curl mite). ln sun flower, 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 Suieima heiianthana (sunflower bud moth); Homoeosoma electellum (sunflower moth);
Zygogramma exclamationis (sunflower beetle); Bothyrus gibbosus (carrot beetle); Neolasioptera murtfeldtiana (sunflower seed midge).
In maize, the plants are resistant to the fungal, bacterial, or viral pathogens Fusarium moniliforme var. subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium
aphanidermatum, Aspergillus flavus, Bipolaris maydis 0, T (Cochliobolus heterostrophus), Helminthosporium carbon urn I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi p.v. Zea, Erwinia corotovora, Cornstunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinesis, Peronosclerospora maydis, Peronosclerospora sacchari, Spacelotheca reiliana, Physopella zeae, Cephalosporium maydis,
Cephalosporium acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus (MSV, Maisstrichel-Virus), Maize Stripe Virus, Maize Rough Dwarf Virus, and the pathogenic insects / nematodes Ostrinia nubilalis (European corn borer); Agrotis ipsilon (black cutworm); Helicoverpa zea (corn earworm); Spodoptera frugiperda. (fall armyworm); Diatraea grandiosella (southwestern corn borer); Elasmopalpus lignosellus (lesser cornstalk borer); Diatraea saccharalis (surgarcane borer); Diabrotica virgifera (western corn rootworm); Diabrotica longicornis barberi (northern corn rootworm); Diabrotica undecimpunctata howardi (southern corn rootworm); Melanotus spp. (wireworms);
Cyclocephala borealis (northern masked chafer; white grub); Cyclocephala immaculata (southern masked chafer; white grub); Popillia japonica
(Japanese beetle); Chaetocnema pulicaria (corn flea beetle); Sphenophorus maidis (maize billbug); Rhopalosiphum maidis (corn leaf aphid); Anuraphis maidiradicis (corn root aphid); Blissus leucopterus leucopterus (chinch bug); Melanoplus femurrubrum (redlegged grasshopper); Melanoplus sanguinipes (migratory grasshopper); Hylemva platura (seedcom maggot); Agromyza parvicornis (corn blot leafminer); Anaphothrips obscurus (grass thrips);
Solenopsis milesta (thief ant); Tetranychus urticae (twospotted spider mite).
In sorghum, the plants are resistant to the fungal, bacterial, or viral pathogens Exserohilum turcicum, Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi, Qloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea,
Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvulaha lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca omenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum,
Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium graminicola and to the pathogenic insects / nematodes Chilo parteilus (sorghum borer); Spodoptera frugiperda (fall armyworm); Helicoverpa zea (corn earworm); Elasmopalpus lignosellus (lesser cornstalk borer); Feltia subterranea (granulate cutworm); Phyllophaga crinita (white grub); Eleodes, Conoderus und Aeolus spp.
(wireworm); Oulema melanopus (cereal leaf beetle); Chaetocnema pulicaria (corn flea beetle); Sphenophorus maidis (maize bilibug); Rhopaiosiphum 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). In cotton, the plants are resistant to the pathogenic 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 iineolahs (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 urticae (two-spotted spider mite). ln rice, 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 leaf hopper); Blissus leucopterus leucopterus (chinch bug); Acrostemum hilare (green stink bug).
In rape, 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).
Particularly preferably, 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, mail, and nicotianae as well as Septoria tritici and Puccinia triticina. Within the meaning of the present invention an "mRNA export protein" is a protein which is involved in the transport of mRNA from the nucleus of a cell to the cytoplasm. Preferably, the mRNA export protein is encoded by a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence comprising the nucleic acid sequence
according to any of SEQ iD Nos. i-6 or a fragment of any of these sequences;
(b) a nucleic acid sequence comprising a sequence which is at least 70% identical to the sequence of any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; and (c) a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence any of SEQ ID Nos. 1 -6 or a fragment of any of these sequences. The content of a protein within a plant cell is usually determined by the expression level of the protein. Hence, in most cases the terms "content" and "expression" may be used interchangeably. The content of a protein within a cell can be influenced on the level of transcription and/or the level of translation.
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 mRNA export protein which do not affect the content of these proteins.
The expression level of the nucleic acid coding for the mRNA export protein 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 PGR. Preferably, the expression of the nucieic acid coding for the mRNA export 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 expression level of the mRNA export protein may also be determined by Western Blot analysis using suitable antibodies. Preferably, the amount of the mRNA export 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 mRNA export protein may for example be determined by incubating seedlings from control and transgenic plants with 5' end-labeled oligo d(T) oligonucleotide and determining the fluorescence in the sample, as described for example in Germain et al. (2010) PLoS Genetics 6(12):
e1001250. The increased activity of the mRNA export protein will lead to an accumulation of the fluorescence in the cytoplasm. Another possibility is to produce nuciear and cytoplasmic extracts from control and transgenic plants and to compare the expression of suitable proteins in the nucleus and cytoplasm of the control and the transgenic plants, e.g. by Western Blot using antibodies directed against these proteins. The increased activity of the mRNA export protein will lead to an increased accumulation of one or more of these proteins in the cytoplasm. The activity of the mRNA export protein may be increased by the method of the present invention 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 person skilled in the art is familiar with methods for increasing the content of a given protein. Typically, the method involves introducing into a plant or plant cell a vector which comprises:
(i) a promoter functional in plant cells, (ii) operatively linked thereto at least one nucleic acid sequence encoding the mRNA export protein as defined herein, and
(iii) optionally, a termination sequence. According to the present invention, increasing the content and/or the activity of an mRNA export protein is also understood to denote the manipulation of the expression of the endogenous mRNA export protein 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 mRNA export protein. Such a modification, which leads to an increased expression rate of at least one endogenous mRNA export protein, can be effected by deleting or inserting DNA sequences in the promoter region.
Furthermore, an increased expression of at ieast one endogenous mRNA export protein 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 mRNA export protein. Such 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 mRNA export protein is to upregulate transcription factors, which are involved in the transcription of the endogenous genes coding for the mRNA export protein, 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 mRNA export protein. An alteration of the activity of the endogenous mRNA export protein can also be achieved by influencing the post-translational modifications of the mRNA export 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 mRNA export protein, by means of corresponding measures like overexpression or gene silencing.
The expression of the endogenous mRNA export protein can also be regulated via the expression of aptamers specifically binding to the promoter sequences of the mRNA export protein. If the aptamers bind to stimulating promoter regions, the amount and thus, in this case, the activity of the endogenous mRNA export protein is increased.
The skilled person also knows other methods for increasing the content and/or activity of a protein, such as the mRNA export protein encoded by the nucleic acid sequence according to any of SEQ ID Nos. 1-6. For example, 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. 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. To cleave DNA sequences within the genomic DNA for introducing a nucieic acid sequence for increasing the content and/or the activity of a protein different enzymes such as meganucleases (WO 2009/114321 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 (WO 2009/130695 A2) may be used. Such sequence-specific nucleases may also be used to cut the sequence of interest, thereby introducing one or more mutations into said sequence.
Within the scope of the present invention, 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. In a first step of this method, plant material is mutagenized to introduce at least one mutation into the genome of the plant material. This mutagenesis may be chemical mutagenesis, for example with ethyl methane sulfonate (EMS), mutagenesis by irradiation such as ionizing irradiation or mutagenesis by using sequence-specific nucleases. Single base mutations or point mutations lead to the formation of heterodupiexes which are then cleaved by single strand nucleases such as Ce/I at the 3' side of the mutation. The precise position of the at least one mutation within the nucleic acid sequence according to any of SEQ ID NOs. 1-6 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. 1-6 has an increased resistance to pathogens and optionally, suitable plants can be selected. In the method and the recombinant nucleic acid molecule of the present invention a nucleic acid sequence is used which is selected from the group consisting of:
a) a nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; a nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; and
a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences.
A "fragment" of the nucleic acid sequence according to any of SEQ ID Nos. 1-6 is understood to refer to a smaller part of this nucleic acid sequence which consists of a contiguous nucleotide sequence found in SEQ ID No. 1-6 and which encodes a protein having the activity of an mRNA export protein. In case the fragment is described to be a fragment of a sequence with a certain degree of sequence identity to a particular sequence, the fragment shall be a fragment of the sequence which has a certain degree of sequence identity to the particular sequence. Thus, for instance, in expressions like "a nucleic acid sequence comprising a sequence which is at least 70% identical to the sequence according to SEQ ID No. 1 or a fragment of this 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. 2-6 has a length of at least 200, 250 or 300 nucleotides, preferably of at least 350, 400, 450, 500, 550 or 600 nucleotides, more preferably of at least 650, 700, 750, 800, 850 or 900 nucleotides, even more preferably of at least 950, 1000, 1050, 00, 1150 or 1200 nucleotides and most preferably of at least 1250, 1300 or 1350 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 or 99% identical to the complete sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these
sequences and which encode a protein having the activity of an mRNA export protein. Within the meaning of the present invention, "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. Preferably, 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. As an alternative, the BLAST program package of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) may be used, employing the default parameters. Here, in addition, the program
Sequencher (Gene Codes Corp., Ann Arbor, Ml, USA) using the "dirtydata"- algorithm for sequence comparisons was employed. The sequence identity refers to the degree of the sequence identity over a length of 300, 350, 400, 450 or 500 nucleotides and most preferably the whole length of the nucleic acid sequence according to SEQ ID No. 1. Alternatively, the sequence identity refers to the degree of the sequence identity over a length of 300, 350, 400, 450 or 500 nucleotides, preferably over a length of 550, 600, 650, 700, 750 or 800 nucleotides, more preferably over a length of 850, 900, 950, 1000, 1050 or 1 100 nucleotides, even more preferably over a length of 150, 1200, 1250, 1300, 1350 or 1400 nucleotides and most preferably the whole length of the nucleic acid sequence according to any of SEQ ID Nos. 2-6. For SEQ ID No. 5 the sequence identity may also refer to the degree of the sequence identity over a length of 1500, 1600, 1700, 1800, 1900, 2000 or 2100 nucleotides. If the sequence identity is to be determined with respect to a fragment of the sequence according to SEQ ID No. 1 , the fragment has a length of at least 300, 350, 400, 450 or 500 nucleotides. If the sequence identity is to be determined with respect to a fragment of the sequence according to any of SEQ ID Nos. 2-6, the fragment has a length of at ieast 300, 350, 400, 450 or 500 nucleotides, preferably of at Ieast 550, 600, 650, 700, 750 or 800 nucleotides, more preferably of at Ieast 850, 900, 950, 1000, 1050 or 1 100 nucleotides and most preferably of at Ieast 1150, 1200, 1250, 1300, 1350 or 1400 nucleotides. For SEQ ID No. 5 the fragment may also have a length of 1500, 1600, 1700, 1800, 1900, 2000 or 2100 nucleotides.
The present invention further relates to the use of nucleic acid sequences which hybridize under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these
sequences and which encode an amino acid sequence having the activity of an mRNA export protein.
The term "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). The term "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 (Tm) of the specific sequence at a defined ionic strength and at a defined pH value. Tm 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. Typically, 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. 10 to 50 nucleotides, for example). In addition, stringent conditions may include the addition of substances, such as, e. g., formamide, which destabilise the hybrids. At hybridization under stringent conditions, as used herein, normally nucleotide sequences which are at least 60%
homologous to each other hybridize to each other. Preferably, 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).
If an organic solvent, e.g. 50% formamide, is present in the above-mentioned buffer, the temperature is about 42°C under standard conditions. Preferably, 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. Preferably, 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. Those skilled in the art know how to determine the required hybridization conditions using text books such as those mentioned above or the following textbooks: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), Hames and Higgins (publ.) 1985, Nucleic
Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (publ.) 1991 , Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.
Typical hybridization and washing buffers for example have the following composition:
Pre-hybridization solution: 0.5 % SDS
5x SSC
50 mM NaPO4, pH 6.8
0.1 % sodium pyrophosphate 5x Denhardt's solution
100 pg/mL salmon sperm DNA
Hybridization solution: pre-hybridization solution
1x106 cpm/mL probe (5 - 10 min
20x SSC: 3 M NaCI
0.3 M sodium citrate
ad pH 7 with HCI
50x Denhardt's reagent: 5 g Ficoll
5 g polyvinylpyrrolidone
5 g bovine serum albumin
ad 500 mL aqua destillata
A typical procedure for hybridization is as follows:
Optional: wash blot 30 min in 1x SSC/ 0.1 % SDS at 65 °C Pre-hybridization: at least 2 h at 50 - 55 °C
Hybridization: over night at 55 - 60 °C
Washing: 05 min 2x SSC/ 0.1 % SDS hybridization temp.
30 min 2x SSC/ 0.1 % SDS hybridization temp.
30 min 1x SSC/ 0.1 % SDS hybridization temp.
45 min 0.2x SSC/ 0.1 % SDS 65 °C
5 min O.lx SSC room temperature Those skilled in the art know that the given solutions and the presented protocol may be modified or have to be modified, depending on the application. The nucleic acid sequence hybridizing to a fragment of the sequence according to any of SEQ ID Nos.1 -6 under stringent conditions has a length of at least 300, 350, 400, 450 or 500 nucleotides, preferably a length of at least 550, 600, 650, 700, 750 or 800 nucleotides, more preferably a length of at least 850, 900, 950, 1000, 1050 or 1 100 nucleotides and most preferably a length of at least 1 150, 1200, 1250, 1300, 1350 or 1400 nucleotides. For SEQ ID No. 5 the nucleic acid sequence may also have a length of 1500, 1600, 1700, 1800, 1900, 2000 or 2100 nucleotides. in the context of the above, the term "encodes a protein having the activity of an imRNA export protein" means that the encoded protein has essentially the same activity as the mRNA export protein encoded by a sequence of any of SEQ ID Nos. 1 -6. "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 mRNA export protein encoded by a sequence of any of SEQ ID Nos. 1 -6. The activity of the mRNA export protein can be determined as described above.
In order to produce the expression constructs or vectors of the present invention, 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. Within the scope of the present invention, 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.
As used herein, the term "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". One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. in the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. A vector can be a binary vector or a T-DNA that comprises a left and a right border and may include a gene of interest in between. The term "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.
The term "promoter" as used herein 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. Genet 231 : 276-285), the ALS promoter (WO 96/30530), the 19S CaMV promoter (US 5,352,605), the super-promoter (US 5,955,646), the figwort mosaic virus promoter (US 6,051 ,753), the Rubisco small subunit promoter (US 4,962,028) and the actin promoter (McElroy et al. (1990) Plant Cell 2: 163-171 ).
In another embodiment, 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.
A "tissue-specific promoter" or "tissue-preferred 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). Suitable 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 chemicaiiy inducible Tet repressor system (Gatz et al. (1991 ) Mol. Gen. Genet. 227: 229-237) may be used. Other suitable promoters may be taken from the literature, e.g. Ward ((1993) Plant Mol. Biol. 22: 361-366). The same applies to inducible and cell- or tissue-specific promoters, such as meristem-specific promoters which have also been described in the literature and which are suitable within the scope of the present invention as well.
Particularly suitable promoters for the method of the present invention are pathogen-inducible promoters, and especially those, which are induced by pathogenic fungi and not by useful fungi (e.g. mycorrhiza in the soil, such as the GER4 promoter (WO 2006/128882). 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).
Also particularly suitable in the method of the present invention are
promoters which are active on the site of pathogen entry, such as epidermis- specific promoters. Suitable epidermis-specific promoters include, but are not limited to, the GSTA1 promoter (Accession number X56012), the GLP4 promoter (Wei et al. (1998) Plant Mol. Biol. 36: 101), the GLP2a promoter (Accession number AJ237942), the Prx7 promoter (Kristensen et al. (2001) Mol. Plant Pathol. 2(6): 31 1), the GerA promoter (Wu et al. (2000) Plant Phys Biochem. 38: 685), 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): 117) 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.
The skilled person knows that the use of inducible promoters allows for the production of plants and plant cells which only transiently express the sequences of the present invention, and thus silence transiently. Such transient expression allows for the production of plants which show only transiently increased pathogen resistance. Such 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. Further situations, in which transient resistance is desirable, are known to those skilled in the art. The skilled person also knows that transient expression and thus transient silencing and transient resistance may be achieved using vectors which do not replicate stably in plant cells and which carry the respective sequences for silencing of fungal genes.
In a preferred embodiment of the method of the invention, 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. Typically, the regulatory elements which are contained in the vectors ensure the transcription and, if desired, the translation in the plant cell. The term "transcription regulatory element" as used herein 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. With respect to nucleic acid sequences or DNA sections in expression constructs or vectors the terms "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 operabiy linked to a promoter, its expression is influenced by said promoter.
So-called 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 ceils. In particular, 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.
A number of well-known techniques are available for introducing DNA into a plant host cell, and those skilled in the art may easily determine the suitable technique for each case. 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 weli as other possibilities. Thereby, stable as well as transient transformants may be produced.
For injection and electroporation of DNA in plant cells, the used plasmids do not need to fulfil special requirements per se. The same applies to direct gene transfer. Simple plasmids, such as pUC derivatives, may be used. 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. Those skilled in the art know all commonly used selection markers, and thus there is no difficulty to select a suitable marker. Common selection markers create resistance in the transformed plant cells to a biocide or antibiotic, such as kanamycin, G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonyl urea, gentamycin or phosphinotricin and the like or may confer tolerance to D-amino acids such as D-alanine. However, it is also possible to select transformed cells by PCR, i.e. without the use of selection markers. Depending on the introduction method of the desired genes into the plant cell, further DNA sequences may become necessary. If, for example, 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.
If agrobacteria are used for the transformation, 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 repiicate in agrobacteria. By means of a heiper plasmid, the intermediate vector may be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors are able to replicate in both E. coli and in agrobacteria. 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. ( 978) 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.
For the transfer of the DNA into the plant cell, plant explants may be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (e.g. leaf cuttings, stem sections, roots, but also protoplasts or suspension-cultivated plant cells) whole plants may be regenerated in a suitable medium which may contain antibiotics, biocides or D-amino acids for the selection of transformed cells, if a selection marker was used in the transformation. The regeneration of the plants is performed according to standard regeneration procedures using well-known culture media. The plants or plant cells obtained this way may then be examined for the presence of the introduced DNA.
Other possibilities for introducing foreign DNA using the biolistic method or by protoplast transformation are well-known to those skilled in the art (see L. Willmitzer (1993) Transgenic Plants in: Biotechnology, A Multi-Volume
Comprehensive Treatise (publisher: H.J. Rehm et al.), volume 2, 627 - 659, VCH Weinheim, Germany).
Monocotyledonous plants or the ceils thereof may also be transformed using vectors which are based on agrobacteria (see e.g. Chan et al. (1993) Plant
Mol. Biol. 22: 491 -506). Alternative systems for the transformation of monocotyledonous plants or the cells thereof are transformation by biolistic approach (Wan and Lemaux (1994) Plant Physiol. 104: 37-48; Vasil et al.
(1993) Bio/Technology 1 1 : 1553-1558; Ritala et al. (1994) Plant Mol. Biol. 24: 317-325; Spencer et al. (1990) Theor. Appl. Genet. 79: 625-631), the protoplast transformation, the electroporation of partially permeabilized cells, and the insertion of DNA by means of glass fibres.
The vectors described herein can be directly transformed into the plastid genome. Plastid expression, in which genes are inserted by homologous recombination into the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number over nuclear-expressed genes to permit high expression levels. In one embodiment, the nucleotides are inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplasmic for plastid genomes containing the nucleotide sequences are obtained, and are preferentially capable of high expression of the
nucleotides.
Plastid transformation technology is for example extensively described in U.S. Pat. NOs. 5,451 ,513; 5,545,817; 5,545,818 and 5,877,462, in WO 95/16783 and WO 97/32977, and in McBride er a/. (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.
Hence, 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 mRNA export protein is not increased and selecting transgenic progeny in which the content and/or the activity of the r mRNA export protein is increased. The other plant in which the content and/or the activity of the mRNA export protein 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 piant which contains a transgenic nucleic acid sequence other than the nucleic acid sequences disclosed herein, e.g. a transgenic nucleic acid sequence coding for another protein involved in pathogen resistance or a protein conferring resistance to abiotic stress. The other plant 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 mRNA export protein 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. For this purpose the transgenic progeny of the above cross in which the content and/or the activity of the mRNA export protein is increased is inbred and the transgenic progeny of this crossing step is selected and again inbred. This inbreeding step is repeated untii 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 mRNA export protein. 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 bariey plants.
According to common procedures, 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.
Of course, plant cells which contain the recombinant nucleic acid molecules of the present invention may also be further cultivated as plant cells
(including protoplasts, calli, suspension cultures and the like).
The method of the present invention may additionally comprise the reduction of the content and/or the activity of at least one, for example two or three, 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. The term feed is intended to mean a dry feed which can be blended from various raw materials and additives such as soybean shred or barley shred in a feed mill. The transgenic or mutant seed of the transgenic or mutant plants of the present invention can be used to prepare flour, in particular if the transgenic or mutant plants are monocotyledonous plants such as barley or wheat. Hence, another embodiment of the present invention is a method for the production of a product comprising the steps of:
(a) growing the plants of the present invention or plants obtainable by the methods of the present invention; and
(b) producing said product from or by the plants of the invention and/or parts, e.g. seeds, of these plants.
In a further embodiment the method comprises the steps of:
(a) growing the plants of the present invention or plants obtainable by the methods of the present invention;
(b) removing the harvestable parts from the plants and
(c) producing said product from or by the harvestable parts of the plants of the invention. In one embodiment the product produced by said methods of the invention is flour comprising the nucleic acid sequence coding for the mRNA export protein and/or comprising the mRNA export protein.
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. Preferably, the harvestable parts comprise a nucleic acid sequence coding for the mRNA export protein, i.e. this nucleic acid sequence is detectable in the harvestable parts by conventional means, and/or the mRNA export protein. The harvestable plants may be seeds, roots, leaves, stems, and/or flowers comprising the nucleic acid sequence coding for the mRNA export protein and/or comprising the mRNA export protein. Preferred harvestable parts are seeds comprising the nucleic acid sequence coding for the mRNA export protein and/or comprising the mRNA export protein.
The identification of an mRNA export protein as a protein involved in pathogen resistance and the use thereof for producing transgenic plants with increased pathogen resistance wili be described in the following. The following examples shall not limit the scope of the present invention. The content of all literature references, patent applications, patent specifications and patent publications, which are cited in this patent application, is incorporated herein by reference. EXAMPLES
1) A practical protocol for construction of a RNAi library
A schematic overview of the steps for RNAi library construction is shown in Figure 1.
1 .1. Entry vector (plPKTA38) preparation The plPKTA38 plasmid (Douchkov et al. (2005) Mol. Plant Microbe Interact. 18(8): 755-761 ) with a kanamycvin resistance gene was used as Gateway Entry vector. Bacteria which contained the plasmid were grown in LB + kanamycin (50 Mg/mL). Plasmid DNA was prepared with the Jetstar midi DNA kit (Genomed).
As a control the plasmid was digested with the restriction enzyme Apa I, yielding bands of 1257 bp and 1054 bp. Then the DNA concentration was measured and adjusted to 150 ng/μΙ.
1.2. Destination vector (plPKTA30) preparation The plPKTA30 plasmid (Douchkov et al. (2005) Mol. Plant Microbe Interact. 18(8): 755-761) was used as the RNAi vector. It contains an ampicillin resistance gene, a ccdB negative selection marker gene which requires the propagation of the plasmid in DB3.1 cells and a chloramphenicol resistance gene.
Plasmid DNA was prepared with the Jetstar midi DNA kit (Genomed). The plasmid preparations were digested as a control with EcoRI (correct bands - 687, 1007, 2641 , and 2857 bp) or Sal I (601 , 1589, and 5002 bp). The DNA concentration was measured and adjusted to 150 ng/μΙ. 1 .3. PCR amplification of the cDNA clones
"Master mixes" for 20-pL PCR reactions with two specific EST-primers Specific primers (SEQ ID Nos. 9 and 10) were designed to amplify ~500 bp fragments from the EST clone. Tm of the primers is ~65°C.
A "PCR master mix" (see Table 6) was prepared of which 1 1 ,0 pL were dispensed to each well of a 96-well PCR plate. 4,25 pl_ of each EST-specific primer and 0,5 pL EST DNA as template were added to each well.
Table 6: "PCR master mix" content ( 1 μΙ_ "Master mix" + 4.25 pL of each specific primer + 0,5 pL template per reaction):
Figure imgf000072_0001
PCR cycle conditions
r 30 cycles
Figure imgf000073_0001
Purification of the PCR product
30 pL H2O were added to each PCR reaction to obtain 50-pL volumes followed by purification using the Qiagen MinElute UF 96-well kit. The PCR product was eluted with 20 μΙ_ H20 (according to Qiagen's MinElute protocol). As a control, 2 μΐ_ each of the purified PCR product were separated by agarose gel electrophoresis. 1.4. Cloning of the PCR products
A ligation master mix was prepared (see Table 7), 6 μΙ_ each of this ligation mix were added to each well and 4 μΙ_ of the purified PCR product were added.
The samples were incubated for 1 h at 25 °C and the reaction was then stopped by heating up to 65 °C for 10 min. 5 μΙ_ Swa I master mix (see Table 8) was added to each well, followed by an incubation at 25 °C for 1 h. Next, the ligation samples were transformed into competent bacteria and suitable clones were isolated after miniprep and control digestion. Table 7: Ligation Master mix for 10-pL reactions (6 pL master mix + 4 pL PCR product per reaction)
Components For 96 For 2 plates For 4 plates samples
H20 100 pL 200 pL 400 pL plPKTA38 (150 ng/pL) 100 pL 200 pL 400 pL
Ligation buffer (10x) 100 pL 200 pL 400 pL
50% PEG 4000 100 pL 200 pL 400 pL
NaCI (0,5 M) 100 pL 200 pL 400 pL
Swa\ (10 U/pL) 50 pL 100 pL 200 pL
(New England Biolabs)
T4 DNA ligase (5 U/pL) 50 pL 100 pL 200 pL (Fermentas)
PCR product (purified) 4 pL per reaction
Table 8: Swa\ Master mix (add 5 pL per reaction)
Components For 96 For 2 plates For 4 plates samples
Swa\ buffer (1 Ox) 50 pL 100 pL 200 pL
NaCI (0,5 M) 100 pL 200 pL 400 pL
H20 300 pL 600 pL 1200 pL
Swa\ (10 U/pL) 50 pL 100 pL 200 pL 1.5. LR Reaction
Master mixes for 6 μΙ_ LR reactions
Table 9: LR-master mix (5 pL master mix + 1 pL plPKTA38::EST DNA per 5 reaction)
Figure imgf000075_0001
ncubation at room temperature over night (or at least 6 h).
2. TransGen test and TIGS protocol
0
Figure 2 shows a schematic drawing of the test procedure for the RNAi constructs.
2.1. Preparation of the plant material
J c
Barley was cultivated in IPK cereal soil for 7 days without fertilization in a Sanyo phyto-cultivator, at constantly 20 °C, 60 - 70 % relative humidity and a 16 h light-cycle. Primary leaves (about 7 cm) were cut off and were arranged in parallel on a phytoagar Petri dish (adaxial side up). Thereby, magnetic stirrers were put onto the leaves such that they repel each other.
2.2. Coating of gold particles with DNA or RNA
7 μΙ_ (= 7 pg) DNA (plasmid) of the gene to be tested and 7 μΙ of the vector pUbiGUS containing the GUS reporter gene were used per bombardment. A Bio-Rad Hepta-Adaptor (7 macro carrier slides) was used. Per bombardment, 87.5 μΙ_ 1 M Ca(N03)2 pH 10 were added drop wise to 87.5 μΙ_ coating suspension (gold particles, 25 mg/mL in 50 % of glycerol; storage at 4 °C) while vortexing. The particle suspension was left for 10 min at room temperature and was occasionally tipped. The suspension was centrifuged (15 sec, 14000 rpm) and the supernatant was removed with a pipette and discarded. The pellet was washed with 1 ml ethanol, and the ethanol was removed with a pipette. Then, the pellet was resuspended in 30 μΙ_ ethanol (absolute).
2.3. Coating of macro carrier
Tensile disks and macro carriers were placed in ethanol (absolute) for 30 sec, subsequently dried at room temperature, and placed in the macro carrier holder using a pipette. The tube containing the coating suspension
(DNA/particle mixture) was placed in an ultrasonic bath for 10 sec, and then the coating suspension was mixed with a pipette. 3 μί_ of the coating suspension were applied to each macro carrier and the suspension was left to dry for 2 to 5 min. 2.4. Biolistic transformation
Leaves and macro carrier holder with the treated macro carriers as well as grids (Hepta Stop Screen) were placed in the chamber for biolistic
transformation. Vacuum was applied for biolistic transformation, wherein the bombardment was made at a pressure of 27.5 mm Hg.
2.5. Incubation and inoculation of the leaves with mildew Bombarded leaves were first incubated for 4 h in slightly opened Petri dishes. Then, the leaves were transferred in large, square Petri dishes containing 1 % w/v phytoagar with 20 ppm of benzimidazole. Thereby, leaves of all preparations were mixed. For inoculation, open Petri dishes were put in dishes with nylon nets (100 m mesh width) stretched thereover. The leaves were inoculated with mildrew (about 200 conidia/mm2). For inoculation, conidia as fresh as possible were used, i.e. either from older plants, which were shaken 24 h - 48 h prior to inoculation, or from fresh plants, which had been inoculated seven days before. The dishes were then placed in the incubation chamber.
2.6. GUS staining (for staining the transformed cells)
40 h after inoculation, the leaves were collected, the leaf tips were cut off and the resulting leaves were transferred to Greiner tubes containing 10 mL of X- glucose solution (100 mM sodium phosphate , pH 7,0;_10 m sodium EDTA; 1 ,4 mM K-hexacyanoferrate(ll); 1 ,4 mM K-hexacyanoferrate(lll); 0,1 % Triton X-100; 20% methanol and 1 mg / ml X-Gluc). The tubes were placed in a suction bottle and vacuum was applied thereto 2 - 3 times. The infiltration is complete When the leaves become transparent and start to sink. The X- glucose solution was refilled to 14 mL and the tubes were sealed. The tubes were incubated over night at 37 °C in the incubator.
2.7. TCA destaininq
The leaves were placed in destaining solution (7.5 % TCA, 50 % methanol) for 5 min. Then the leaves were washed with distilled water. Then, the leaves were carefully removed from the tube and were placed onto an object slide with their adaxial side facing upwards.
Distilled water was added to each object slide and the cover glass was carefully applied. The GUS-staining and the fungal structures were then analyzed in the microscope. 3. Experiment for primary data acquisition
The effect of the RNAi constructs on plant resistance to the fungal pathogen Blumeria graminis was tested in transient experiments. The barley plants used {Hordeum vulgare, cultivar, 'Golden promise) were cultivated in soil without fertilisation in a phyto-cultivator (20 °C, 70 % rel. humidity). On the day of bombardment, plants were 7 days old. The primary leaves were cut off, placed on 0.5 % phytoagar with 200 ppm benzimidazole and bombarded with 2.2 mg of gold particles, which were coated with a mixture of 7 μg reporter gene vector (pUbiGUS) and 7 pg of a control vector PIPKTA30 or a RNAi construct. The leaves were stored in closed Petri dishes at 20 °C until inoculation. Three days after bombardment, the leaves were transferred to 1 % phytoagar with 2 % benzimidazole. A nylon net (mesh width of 200 pm) was stretched over the leaves, and they were inoculated with a conidia density of about 200 conidia/mm2. The conidia (from the pathogen Blumeria graminis hordei) originated from barley plants (cultivar ,Golden Promise'), which had been inoculated 6 - 7 days before. Until GUS staining, the leaves were stored in closed Petri dishes with holes for ventilation at 20 °C at a north-facing window. About 45 h after inoculation, GUS staining was performed. Said staining was stopped after 24 h by incubation in 7.5 % trichloroacetic acid, 50 % v/v methanol, and the leaves were bleached.
Each experiment contained 3 parallel bombardments to 7 leaf sections each of the negative control (empty vector plPKTA30N). Further, each experiment contained 2 parallel bombardments of a TIGS positive control plPKTA36, which causes resistance by inhibiting the MIo gene of barley. Data per experiment are based on the comparison of the effect of the test constructs with the average value of the 3 negative controls of the respective experiment.
Table 10 shows the relative susceptibility index (Rel SI) of barley cells transiently transformed with the RNAi constructs comprising SEQ ID No.1 and the sequence reverse complementary thereto. These RNAi constructs inhibit the expression of the mRNA export protein. The susceptibility index was determined in five independent transformation experiments. As the cells transformed with the RNAi construct have a susceptibility index of more than 100% compared to the control cells transformed with empty vector (plPKTA30N), the RNAi construct suppresses putative resistance genes in barley.
Table 10
Sequenz ID BlastX Screen Rel SI (%) p t-Test
Contig13183_s_at mRNA export protein PHENOME 5H 139,8 0,0354
4. Cloning of overexpression vector constructs for stable
stransformatlon
Based on the nucleic acid sequences coding for an mRNA export protein from barley the corresponding sequences in wheat were identified using BLAST search (Altschul et al, (1990) Journal of Molecular Biology 215: 403- 410) (SEQ ID Nos. 5 and 6).
The cDNAs and functional fragments of all genes mentioned in this
application were generated by DNA synthesis (Geneart, Regensburg,
Germany). The binary vector for transformations is constructed such that the cDNA/ functional fragment is located in sense direction between the parsley ubiquitine promoter (PcUbi) and a Agrobacterium tOCS terminator.
To obtain the binary vector the synthesized cDNA/functional fragments are subcloned into a Gateway pENTRY vector (Invitrogen, Life Technologies, Carlsbad, California, USA). The binary plant transformation vector is made by a triple LR reaction (Gateway system, (Invitrogen, Life Technologies,
Carlsbad, California, USA), which is performed according to manufacturer's protocol by using a pENTRY-A vector containing a parsley ubiquitine promoter, the above described pENTRY-B vector containing the cDNA/functional fragment and a pENTRY-C vector containing a tOCS terminator. As target a binary pDEST vector is used which is composed of: (1 ) a Kanamycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a pBR322 origin of replication for stable maintenance in E. coli and (4) between the right and left border an D-amino acid oxidase (GenBank U60066) under control of a pcUbi-promoter as D- aminoacid tolerance marker. The recombination reaction is transformed into E. coli (DH5alpha), mini-prepped and screened by specific restriction digestions. A positive clone from each vector construct is sequenced and used for wheat transformation.
5. Wheat transformation
5.1. Plant Material and Surface Sterilisation
A comprehensive discussion about wheat transformation methods and a protocol for the Agrobacterium-med'iated transformation of wheat can be found in Jones et al. (2005) Plant Methods 1 : 5. Immature embryos (lEs) from Triticum aestivum (variety 'Bobwhite') are used as explant for
Figure imgf000081_0001
transformation. Donor plants are grown at 18-20°C day and 14-16°C night temperatures under a 16 h photoperiod (500 - 1000 molm-2s-1 photosynthetically active radiation (PAR)) with relative air humidity of 50-70% for approximately 8 to 11 weeks.
The optimal harvesting time is 12-20 days post-anthesis. For 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.
5.2. Agrobacterium 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. 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 KH2P04> 100 mg NaCI, 100 mg MgS04-7H2O, 5 g tryptone, 2.5 g yeast extract, pH 7.0, add after autoclave 1 pg Biotin incl. appropriate antibiotics). Liquid culture is grown at 28°C for ~16h to reach an OD6oo ~1■ The Agrobacterium culture is centrifuged at 4.500 g for 10 minutes and resuspended in 4 ml inoculation medium ((1/ 0 MS complete) 30g maltose, 100mg MES; adjusted to pH 5.8 and add after autoclave 0.01 % Pluronic, 200μΜ acetosyringone) to an OD60o of ~1 . The Agrobacterium inoculation medium is ready to use.
5.3. 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. 5.4. Co-culture
Following isolation of immature embryos (lEs), 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 mi of solidified co-culture media (1/10 MS complete, 30g maitose, 0.69g proline, 100mg MES, 10g agar, adjust to pH 5.8, add after autoclave, 4mg 2,4-D, 200μΜ acetosyringone, 100mg ascorbic acid). The plates are sealed with parafilm and incubated for 2-3 days at 24°C in the dark.
5.5. Callus Induction
Following co-culture, the explants are placed with the embryo axis facing down on recovery media (MS full complete, 30g maltose, 0.69g proline, 20mg thiamine, g casein hydrolysate, 100mg myo-inositol, 5μΜ CuS04; 2.4g NH4N03, .95g MES, 8g agar (Plant TC), adjust to pH 5.8 and add after autoclave 2mg 2,4-D, 200mg timentin, l OOmg ascorbic acid) for 4 weeks at 24°C in the dark. The calli are transferred to fresh recovery medium after two weeks.
5.6. Shoot Regeneration, Rooting and Selection Calli are transferred to shoot regeneration medium (MS full complete, 30g maltose, 20mg thiamine, 100mg myo-inositol, 750mg glutamine, 5μΜ CuS04, 1.95g MES; 8g agar (Plant TC), adjust to pH 5.8 and add after autoclave, 0.5mg TDZ, 200mg timentin, 11mM D-alanine) and are cultivated under light conditions at 21-25°C for 3-4 weeks.
After shoot induction the explants are transferred to rooting media (½ MS complete, 30g sucrose, 7g agar and adjust to pH 5.8, add after autoclave, 0.5mg NAA, 200mg timentin, 11 mM D-alanine) 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.
6. Wheat Septoria screening assay Transgenic plants are grown in the greenhouse at 19°C and 60-80% humidity. After 11 days plants are inoculated with Septoria tritici spores (1 ,3x106 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 overexpression of the mRNA export protein will lead to enhanced resistance of wheat against Septoria tritici.
7. Wheat rust screening assay 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 wheat brown rust {Puccinia triticina) spores. Generally plants are inoculated with a 0.2% (w/v) spore suspension in HFE (Hydrofluoroether). Plants are incubated for 24 h in darkness under 100% humidity and 24°C. After the dark phase, plants are grown at 23°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 fungal colonies 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 the 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 overexpression of the mRNA export protein will lead to enhanced resistance of wheat against rust fungi.
8. Powdery mildew screening assay 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). Generally inoculations with powdery mildew are performed with dry spores using an inoculation tower to a density of approx. 10 spores/mm2. 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 overexpression of the mRNA export protein will lead to enhanced resistance of wheat to powdery mildew fungus.

Claims

Claims
1. 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 an mRNA export protein which is encoded by a nucleic acid sequence selected from the group consisting of:
a) a nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences;
b) a nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences; and
c) a nucieic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1-6 or a fragment of any of these sequences,
is increased in comparison to the control plant cell, plant or plant part.
2. Method of claim 1 , comprising the steps of
(a) introducing into a plant cell a vector which comprises:
(i) a promoter functional in plant cells,
(ii) operatively linked thereto at least one nucleic acid sequence as defined in claim 1 ;
(iii) optionally, a termination sequence; and
(b) optionally, regenerating a transgenic plant from the transformed ceii.
3. Method of claim 2, wherein the promoter is a tissue-specific and/or a pathogen-inducible promoter.
4. Method of any of the preceding claims, further comprising reducing the content and/or activity of at least one plant protein mediating pathogen susceptibility or increasing the content and/or activity of at least one other plant protein mediating pathogen resistance.
5. Method of any of the preceding claims, further comprising the step of crossing the transgenic plant produced by the method of claim 1 with another plant in which the content and/or the activity of the mRNA export protein as defined in claim 1 is not increased and selecting transgenic progeny in which the content and/or the activity of the mRNA export protein as defined in claim 1 is increased.
6. Method of producing mutant plants, plant cells or plant parts having an increased resistance to pathogens compared to control plants, plant cells or plant parts, comprising the steps of:
(a) mutagenizing plant material;
(b) identifying plant material having at least one point mutation in a nucleic acid sequence comprising the nucleic acid sequence according to any of SEQ ID Nos. 1-6.
7. Method of any of the preceding claims, wherein the transgenic or mutant plant is a monocotyledonous plant.
8. Method of claim 7, wherein the monocotyledonous plant is a barley or a wheat plant.
9. Method of any of the preceding claims, wherein the transgenic or mutant plant has an increased resistance to a fungal pathogen.
10. Method of claim 9, wherein the plant has an increased resistance to Blumeria graminis, Septoria tritici and/or Puccinia triticina.
1 1 . Expression construct comprising at least one nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence comprising the sequence according to any of SEQ ID Nos. 1 -6 or a fragment of any of these sequences;
(b) a nucleic acid sequence comprising a sequence which is at least 70 % identical to the sequence according to any of SEQ ID Nos. 1 - 6 or a fragment of any of these sequences; and
(c) a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to any of SEQ ID Nos. 1 -6 or a fragment of any of these sequences;
operatively linked to a promoter functional in plant cells.
12. Expression construct of claim 1 1 , further comprising 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 in claim 1 1 .
13. Expression construct of claim 1 1 or 12, wherein the promoter is a tissue-specific and/or a pathogen-inducible promoter.
14. Vector comprising the expression construct of any of claims 1 to 13.
15. 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, produced according to the method of any of claims 1 to 10 or containing an expression construct of any of claims 11 to 13 or containing a vector of claim 14.
16. Use of the transgenic or mutant plant of claim 15 or parts thereof as fodder or to produce feed material.
17. Transgenic or mutant seed produced from the transgenic or mutant plant of claim 15.
18. Flour produced from the transgenic or mutant seed of claim 7, wherein the presence of the transgene or the mutation which increases the content and/or the activity of the mRNA export protein as defined in claim 1 can be detected in said flour.
19. Method for producing true breeding plants comprising inbreeding the transgenic progeny of the crossing step of claim 5 and repeating this inbreeding step until a true breeding plant is obtained.
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