WO2013050593A1 - 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
WO2013050593A1
WO2013050593A1 PCT/EP2012/069842 EP2012069842W WO2013050593A1 WO 2013050593 A1 WO2013050593 A1 WO 2013050593A1 EP 2012069842 W EP2012069842 W EP 2012069842W WO 2013050593 A1 WO2013050593 A1 WO 2013050593A1
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Prior art keywords
plant
nucleic acid
sequence
acid sequence
transgenic
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PCT/EP2012/069842
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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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Priority to EP11184411.4 priority
Priority to US61/544,313 priority
Priority to EP11184411 priority
Application filed by Basf Plant Science Company Gmbh filed Critical Basf Plant Science Company Gmbh
Publication of WO2013050593A1 publication Critical patent/WO2013050593A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279Phenotypically 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279Phenotypically 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
    • C12N15/8282Phenotypically 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

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, wherein the content of an RNA of a powdery mildew susceptibility factor is reduced, preferably by RNA interference.

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 of an R A of a powdery mildew susceptibility factor is reduced, preferably by RNA interference.

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 against different pathogens and to use the same specifically for the production of pathogen resistant plants by gene technological manipulation, e.g. by means of introducing external resistance genes or by means of manipulating the endogenous gene expression of the plants.

Resistance is the ability of a plant to inhibit or at least limit any infestation or population of a pest. The plants have a certain degree of natural resistance which is imparted by the formation of specific defence substances, such as isoprenoids, flavonoids, enzymes and reactive oxygen species.

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 00/01722) in transgenic plants.

Another alternative for causing resistance to pathogenic fungi is to introduce gene constructs into plants which inhibit the expression and/or activity of fungal genes that are essential for the proliferation and/or development of fungi

(US 2007/0061918).

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 R A of a powdery mildew susceptibility factor from barley by RNA interference leads to an enhanced resistance of barley cells to Blumeria graminis.

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 of an RNA of a powdery mildew susceptibility factor transcribed from a nucleic acid sequence which is selected from the group consisting of:

a) a nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or a fragment of this sequence;

b) 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; and

c) a nucleic acid sequence comprising a sequence which hybridizes under

stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence

is reduced 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 reducing the content of an RNA of a powdery mildew susceptibility factor in the plant cell, plant or part thereof compared to a control plant cell, plant or plant part, wherein the RNA of the powdery mildew susceptibility factor is transcribed from a nucleic acid sequence which is selected from the group consisting of:

a) a nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or a fragment of this sequence;

b) 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; and

c) a nucleic acid sequence comprising a sequence which hybridizes under

stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence.

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, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence,

(iii) optionally a spacer sequence,

(iv) the nucleic acid sequence being reverse-complementary to the nucleic acid sequence of (ii);

(v) a termination sequence;

or which comprises:

(i) a promoter functional in plant cells; (ii) operatively linked thereto a nucleic acid sequence which is

complementary to a nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence; and

(iii) optionally, a termination sequence;

or which comprises:

(i) a promoter functional in plant cells;

(ii) operatively linked thereto a nucleic acid sequence which is identical to a nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence;

(iii) optionally, a termination sequence;

or which comprises:

(i) a promoter functional in plant cells;

(ii) operably linked thereto a nucleic acid sequence encoding a precursor micro RNA sequence comprising a micro RNA sequence which targets the nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence;

(b) optionally, regenerating a transgenic plant from the transformed cell.

In one embodiment, 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, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence,

(iii) optionally a spacer sequence,

(iv) the nucleic acid sequence being reverse-complementary to the nucleic acid sequence mentioned in (ii);

(v) a termination sequence;

(b) optionally, regenerating a transgenic plant from the transformed cell. In an alternative embodiment, 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 a nucleic acid sequence which is

complementary to a nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence; and

(iii) optionally, a termination sequence;

(b) optionally, regenerating a transgenic plant from the transformed cell. In another alternative embodiment, 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 a nucleic acid sequence which is identical to a nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence;

(iii) optionally, a termination sequence;

(b) optionally, regenerating a transgenic plant from the transformed cell.

In a further alternative embodiment, the method comprises the steps of:

(a) introducing into a plant cell a vector which comprises:

(i) a promoter functional in plant cells;

(ii) operably linked thereto a nucleic acid sequence encoding a precursor micro RNA sequence comprising a micro RNA sequence which targets the nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence;

(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.

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 of an RNA of a powdery mildew susceptibility factor as defined herein is not reduced and selecting transgenic progeny in which the content of the RNA of a powdery mildew susceptibility factor as defined herein is reduced. In 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.

In another embodiment 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 SEQ ID No. l .

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 SEQ ID No. 1 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.

Also 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.

Preferably, the transgenic or mutant plant is a monocotyledonous plant. More preferably it is a wheat or barley plant.

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 SEQ ID No. 1 or a fragment of this sequence;

(b) 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;

(c) a nucleic acid sequence comprising a sequence which hybridizes under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence; and

(d) a nucleic acid sequence which is reverse-complementary to a nucleic acid sequence of any of (a) to (c),

operatively linked to a promoter functional in plant cells. In a preferred embodiment the expression construct comprises:

(a) a promoter functional in plant cells,

(b) operatively linked thereto at least one nucleic acid sequence as defined above, preferably at least one nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence;

(c) optionally a spacer sequence, (d) the nucleic acid sequence being reverse-complementary to the nucleic acid sequence of (b); and

(e) a termination sequence;

or:

(a) a promoter functional in plant cells;

(b) operatively linked thereto at least one nucleic acid sequence which is

complementary to a nucleic acid sequence as defined above, preferably at least one nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence; and

(c) optionally, a termination sequence;

or:

(a) a promoter functional in plant cells;

(b) operatively linked thereto at least one nucleic acid sequence which is identical to a nucleic acid sequence as defined above, preferably at least one nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence;

(c) optionally, a termination sequence;

or:

(a) a promoter functional in plant cells;

(b) operably linked thereto a nucleic acid sequence encoding a precursor micro RNA sequence comprising a micro RNA sequence which targets the nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence.

In a preferred embodiment the expression construct comprises:

(a) a promoter functional in plant cells,

(b) operatively linked thereto at least one nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence;

(c) optionally a spacer sequence,

(d) the nucleic acid sequence being reverse-complementary to the nucleic acid sequence mentioned in (b); and (e) a termination sequence.

In an alternative embodiment the expression construct comprises:

(a) a promoter functional in plant cells;

(b) operatively linked thereto a nucleic acid sequence which is complementary to at least one nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence; and

(c) optionally, a termination sequence. In another alternative embodiment the expression construct comprises:

(a) a promoter functional in plant cells;

(b) operatively linked thereto at least one nucleic acid sequence which is identical to a nucleic acid as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence;

(c) optionally, a termination sequence.

In still another alternative embodiment the expression construct comprises:

(a) a promoter functional in plant cells;

(b) operably linked thereto a nucleic acid sequence encoding a precursor micro RNA sequence comprising a micro RNA sequence which targets the nucleic acid sequence as defined above, preferably the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence.

In another embodiment the present invention relates to a vector comprising an expression construct as defined above.

A preferred embodiment is the use of an expression construct or vector as described herein for the transformation of a plant, plant part, or plant cell to provide a pathogen resistant plant, plant part, or plant cell. 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 another embodiment the invention relates to a transgenic or mutant plant or plant cell with an increased resistance to pathogens, produced by the method of the present invention or containing an expression construct or a vector of the present invention.

In another embodiment the invention relates to the use of the transgenic or mutant plant or parts thereof as feed or as fodder material.

The present invention also relates to transgenic or mutant seed produced from the transgenic or mutant plant and to flour produced from said transgenic or mutant seed, wherein the presence of the transgene, expression construct or the mutation which reduces the content of an RNA of a powdery mildew susceptibility factor 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;

Ila, Ligation of the PCR fragments in the intermediate vector pIPKTA38 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 pIPKTA30 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 scope of the present invention, the transgenic plant cell, plant or plant part contains a nucleic acid sequence selected from the group consisting of

(a) a nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or a fragment of this sequence;

(b) 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;

(c) a nucleic acid sequence comprising a sequence hybridizing under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence; and

(d) a nucleic acid sequence which is reverse-complementary to a nucleic acid sequence of any of (a) to (c),

which is not present at the natural locus of this sequence in the genome of the control plant and/or which has been linked to sequences to which the nucleic acid sequence is not linked in the genome of the control plant and/or which is present in another 5' to 3' orientation compared to the orientation of this sequence in the natural locus of the control plant. Natural locus means the location on a specific chromosome, preferably the location between certain genes, more preferably the same sequence background as in the original plant which is transformed.

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 reduces the content of an RNA of a powdery mildew susceptibility factor compared to a control plant cell, plant or plant part. In addition to the nucleic acid sequence which reduces the content of an RNA of a powdery mildew susceptibility factor, 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 SEQ ID No. 1 or a fragment of this sequence;

(b) 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;

(c) a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence; 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 SEQ ID No. 1 or a fragment of this sequence.

The transgenic or mutant plant of the present invention may be a monocotyledonous or a dicotyledonous plant.

Examples of monocotyledonous plants are plants belonging to the genera Avena (oat), Triticum (wheat), Secale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, Sorghum (millet), Zea (maize), and the like.

Dicotyledonous useful plants comprise, inter alia, cotton, legumes, like leguminous plants and in particular alfalfa, soy bean, rape, tomato, sugar beet, potato, ornamental plants, and trees. Further useful plants can comprise fruit (in particular apples, pears, cherries, grapes, citrus, pineapple, and bananas), pumpkin, cucumber, wine, oil palms, tea shrubs, cacao trees, and coffee shrubs, tobacco, sisal, as well as, with medicinal plants, rauwolfia and digitalis. Particularly preferred are the cereals wheat, rye, oat, barley, rice, maize and millet, sugar beet, rape, soy, tomato, potato, cotton and tobacco. Further useful plants can be taken from US 6,137,030.

More preferably the transgenic or mutant plants are oat, barley, rye, wheat or rice plants and most preferably the transgenic or mutant plants are barley or wheat plants.

Within the meaning of the present invention the term "transgenic plant" includes the transgenic progeny of the transgenic plant and the term "mutant plant" also includes the mutant progeny of the mutant plant. The transgenic progeny of the transgenic plant comprises a nucleic acid sequence which reduces the content of an RNA of a powdery mildew susceptibility factor and the mutant progeny comprises a mutation which reduces the content of an RNA of a powdery mildew susceptibility factor. 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, respectively, 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, 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 no propagation. The term "resistance" also includes a so-called transient resistance, i.e. the transgenic or mutant plants or plant cells of the present invention have an increased resistance to pests and/or pathogens or fungi compared to the

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 or mutant plants or plant cells of the present invention are infected less 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 least the factor 50, and more preferably by at least the factor 100, and most preferably by at least the factor 1000, as compared to the control plant. 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 35% or 30%.

When used in connection with transgenic plants, the terms "control plant", "control plant cell" or "control plant part" refers to a plant cell, an explant, seed, plant component, plant tissue, plant organ, or whole plant used to compare against a transgenic plant which has been modified by the method of the present invention for the purpose of identifying an enhanced phenotype or a desirable trait in the transgenic plant. A "control plant" may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of interest that is present in the transgenic plant being evaluated, i.e. the nucleic acid sequence reducing the content of an RNA of a powdery mildew susceptibility factor. A control plant may be a plant of the same line or variety as the transgenic plant being tested, or it may be another line or variety, such as a plant known to have a specific phenotype, characteristic, or known genotype. Another suitable control plant is a genetically unaltered or non-transgenic plant of the parental line used to generate the transgenic plant of the present invention, i.e. the wild-type plant.

When used in connection with mutant plants, the terms "control plant", "control plant cell" or "control plant part" refers to a plant cell, an explant, seed, plant component, plant tissue, plant organ, or whole plant 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 a host plant for said pathogen, and the pathogen attack must be detectable by simple means. Preferred test plants are wheat or barley plants, which are, for example, inoculated with the corresponding forma specialis of the powdery mildew fungus Blumeria graminis. "Inoculating" denotes contacting the plant with the fungus the plant is to be infected with, or with infectious parts thereof, under conditions in which the fungus may enter a wild-type plant.

The fungal infestation of the plant may then be evaluated by means of a suitable evaluation procedure. The visual inspection, in which the formed fungal structures are detected in the plant and quantified, is particularly suitable. 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, such as the green fluorescence protein (GFP) gene from Aequorea victoria, the luciferase gene from Photinus pyralis or the beta-galactosidase (lacZ) gene from E. coli, the expression of which in the plant cells may be proven by simple methods, is co -transformed in a suitable vector with the vector mediating the reduction of the content of an RNA of a powdery mildew susceptibility factor. 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 reducing the content of an R A of a powdery mildew susceptibility factor 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 imgf000021_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

Bunt Tilletia caries

Take-all Gaeumannomyces graminis

Anthrocnose leaf blight Colletotrichum graminicola (teleomorph:

Glomerella graminicola Politis); Glomerella Anthracnose stalk rot tucumanensis (anamorph: Glomerella falcatum Disease Pathogen

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 striatum 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 (teleomorph: Cochliobolus lunatus), Curvularia pallescens (teleomorph: Cochliobolus pallescens), Curvularia senegalensis, C. tuberculata (teleomorph: Cochliobolus tuberculatus)

Didymella leaf spot Didymella exitalis

Diplodia ear and stalk rot Diplodia frumenti (teleomorph: Botryosphaeria Disease Pathogen

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 =

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.) Disease Pathogen

Eyespot Aureobasidium zeae = Kabatiella zeae

Fusarium ear and stalk rot Fusarium subglutinans =

F. moniliforme var. subglutinans

Fusarium kernel, root and stalk rot, Fusarium moniliforme

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, C. (Cercospora leaf spot) zeae-maydis

Helminthosporium root rot Exserohilum pedicellatum = Helminthosporium pedicellatum (teleomorph: Setosphaeria pedicellata)

Hormodendrum ear rot Cladosporium cladosporioides =

(Cladosporium rot) Hormodendrum cladosporioides, C 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 Disease Pathogen

(teleomorph: Setosphaeria prolata)

Graphium penicillioides,

Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerella herpotricha, (anamorph: Scolecosporiella sp.),

Paraphaeosphaeria michotii, Phoma sp., Septoria zeae, S. zeicola,

S. zeina

Northern corn leaf blight (white blast, Setosphaeria turcica (anamorph: Exserohilum crown stalk rot, stripe) turcicum = Helminthosporium turcicum)

Northern corn leaf spot Cochliobolus carbonum (anamorph: Bipolaris Helminthosporium ear rot (race 1) zeicola = Helminthosporium carbonum)

Penicillium ear rot (blue eye, blue Penicillium spp., P. chrysogenum, P. expansum, mold) P. oxalicum

Phaeocytostroma stalk and root rot Phaeocytostroma ambiguum, =

Phaeocytosporella zeae

Phaeosphaeria leaf spot Phaeosphaeria maydis = Sphaerulina maydis

Physalospora ear rot (Botryosphaeria Botryosphaeria festucae = Physalospora 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 and Epicoccum nigrum

seed rot) Disease Pathogen

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. parasitica, Rhizopus arrhizus

Rostratum leaf spot Setosphaeria rostrata, (anamorph:

(Helminthosporium leaf disease, ear xserohilum rostratum = Helminthosporium 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) Disease Pathogen

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.

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 rot Cochliobolus heterostrophus (anamorph:

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. Disease Pathogen

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)

Zonate leaf spot Gloeocercospora sorghi

Preferably, fungal pathogens or fungal-like pathogens (like for example Chromista) are from the group comprising Plasmodiophoramycetes, Oomycetes, Ascomycetes, Chytridiomycetes, Zygomycetes, Basidiomycetes, and Deuteromycetes (Fungi imperfecti). The fungal pathogens listed in Tables 1 and 2 as well as the diseases associated therewith are to be mentioned in an exemplary, yet not limiting manner.

Particularly preferred are:

Plasmodiophoramycetes 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), Sclerotinia sclerotium (white mold, 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, barley, rye, and various grasses), Uromyces appendiculatus (bean rust), Phakopsora pachyrhizi (Asian soybean rust), Sclerotium rolfsii (root and stem rots of many plants).

Deuteromycetes (Fungi imperfecti) like Septoria nodorum (glume blotch) of wheat (Septoria tritici), Pseudocercosporella herpotrichoides (stem break disease in wheat, barley, rye), Rynchosporium secalis (scald disease in rye and barley), Alternaria solani (early blight of potato and tomato), Phoma betae (black rot of beet), Cercospora beticola (Cercospora leaf spot of beet), Alternaria brassicae (dark leaf spot of rape, cabbage and other cruciferous plants), Verticillium dahliae (Verticillium wilt and stalk rot of rape),

Colletotrichum lindemuthianum (bean anthracnose), Phoma lingam - phoma stem canker (black leg disease of cabbage; crown and stem canker of rape), Botrytis cinerea (gray mold diseases of grapevine, strawberry, tomato, hop, etc.).

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

Figure imgf000031_0001

Particularly preferably, the transgenic plants produced according to the present invention are resistant to the following pathogenic bacteria: Corymb acterium 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. phaseohcola (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 stunt Maize mottle and chlorotic stunt virus

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)

Millet red leaf Millet red leaf virus (MRLV)

Northern cereal mosaic Northern cereal mosaic virus (NCMV)

Oat pseudorosette (zakuklivanie) Oat pseudorosette virus

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, I 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,

caterpillars, lice, 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 Meloidogyne 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 solanaceae), Heterodera schachtii (beet cyst nematodes of sugar and fodder beets, rape, cabbage, etc.), Heterodera avenae (cereal cyst nematode of oat and other types of cereal), Ditylenchus dipsaci (bulb and stem nematode, beet eelworm of rye, oat, maize, clover, tobacco, beet), Anguina tritici (wheat seed gall nematode), seed galls of wheat (spelt, rye), Meloidogyne hapla (root-knot nematode of carrot, cucumber, lettuce, tomato, potato, sugar beet, lucerne).

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 servus (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.v. 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 differ entialis (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 canola, the plants are resistant to the fungal, bacterial, or viral pathogens Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum,

Peronospora parasitica, Fusarium roseum and Alternaria alternata.

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. alfalfae, Aphanomyces euteiches,

Stemphylium herbarum, Stemphylium alfalfae. 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, Alternaria 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 gramicola, High Plains 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); Hyper a 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 differ entialis (differential grasshopper); Melanoplus sanguinipes (migratory grasshopper);

Mayetiola destructor (Hessian fly); Sitodiplosis mosellana (wheat midge); Meromyza americana (wheat stem maggot); Hylemya coarctata (wheat bulb fly); Frankliniella fusca (tobacco thrips); Cephus cinctus (wheat stem sawfly); Aceria tulipae (wheat curl mite). In 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 dehor acearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum p.v. Carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis and to the pathogenic

insects/nematodes Suleima helianthana (sunflower bud moth); Homoeosoma electellum (sunflower moth); Zygogramma exclamationis (sunflower beetle);

Bothyrus gibbosus (carrot beetle); Neolasioptera murtfeldtiana (sunflower seed midge).

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 carbonum 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, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p. v. syringae, Xanthomonas campestris p. v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternata, Bipolaris sorghicola,

Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, 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 partellus (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 billbug); Rhopalosiphum maidis (corn leaf aphid); Siphaflava (yellow sugarcane aphid); Blissus leucopterus leucopterus (chinch bug); Contarinia sorghicola

(sorghum midge); Tetranychus cinnabarinus (carmine spider mite); Tetranychus urticae (two-spotted spider mite).

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 lineolaris (tarnished plant bug); Melanoplus femurrubrum (redlegged grasshopper); Melanoplus differentialis (differential grasshopper); Thrips tabaci (onion thrips); Franklinkiella fusca (tobacco thrips); Tetranychus cinnabarinus (carmine spider mite); Tetranychus urticae (two-spotted spider mite).

In 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 leafhopper); Blissus leucopterus leucopterus (chinch bug); Acrosternum 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, mali, and nicotianae as well as Septoria tritici and Puccinia triticina.

Within the meaning of the present invention the term "RNA of a powdery mildew susceptibility factor" refers to an RNA which is transcribed from a

nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence comprising the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence;

(b) a nucleic acid sequence comprising a sequence which is at least 70% identical to the sequence of SEQ ID No. 1 or a fragment of this sequence; and

(c) a nucleic acid sequence comprising a sequence which hybridizes under

stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence

or a complement of any of these sequences. The reduction of the content of this RNA leads to an increased pathogen resistance of plants.

Most preferably, the RNA is transcribed from a nucleic acid sequence comprising the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence.

The nucleic acid sequence comprising any of the nucleic acid sequences of alternatives a) to c) above is preferably no longer than 5.000 or 4.000 nucleotides, more preferably no longer than 3.000, 2.500 or 2.000 nucleotides, even more preferably no longer than 1.500 or 1.200 nucleotides and most preferably no longer than 1.000 or 700 nucleotides. In any case, the nucleic acid sequence comprising the sequence according to SEQ ID No. 1 does not comprise the whole chromosome on which the nucleic acid sequence according to SEQ ID No.l is located in the genome of the control plant.

The person skilled in the art knows methods for reducing the content of an RNA.

The content of the RNA of the powdery mildew susceptibility factor may be substantially inhibited in transgenic plants for example by "silencing". For silencing, a nucleic acid sequence which is substantially identical to the nucleic acid sequence from which the RNA of the powdery mildew susceptibility factor is transcribed and/or which is substantially complementary thereto is transferred to the plant. To ensure that the plants are transgenic for the introduced nucleic acids, the nucleic acid to be transferred is normally introduced to the plant by a vector, such as a plasmid, which is able to stably replicate in the plant cell or to integrate the introduced nucleic acid into the plant genome.

In this context, those skilled in the art refer to a nucleic acid sequence

complementary to a nucleic acid sequence as antisense nucleic acid, wherein the same is typically antisense RNA. Use of antisense RNA leads to suppression of the corresponding endogenous gene. A nucleic acid sequence identical to a nucleic acid sequence is also referred to as sense nucleic acid. The use of sense RNA may also lead to suppression of the corresponding endogenous gene by means of a process called "co-suppression".

If, in the scope of the present invention, sense sequences are mentioned, it is referred to those sequences which correspond to the coding strand of the nucleic acid sequence from which the RNA of the powdery mildew susceptibility factor is transcribed or which comprise parts thereof. Such sequences do not have to be 100 % identical to the nucleic acid sequence from which the RNA of the powdery mildew susceptibility factor is transcribed. It should be sufficient, if said sequences are at least 80 %, 82 %, 84 %, 86 %, 88 %, particularly preferably at least 90 % and most preferably at least 95 %, 96 %, 97 %, 98 % or 99% identical. In case of such degrees of identity, the sequences are regarded, according to the invention, as homologous to each other or comprising a homology. The deviations to the nucleic acid sequence from which the R A of the powdery mildew susceptibility factor is transcribed or parts thereof may originate from deletion, substitution and/or insertion of one or more nucleotides. The skilled person surely knows that with decreasing identity, it becomes more likely that multiple nucleic acids are suppressed in a silencing manner. Sequences having such a low degree of identity or homology that the expression of genes other than the RNA of the powdery mildew susceptibility factor, preferably the expression of nucleic acid sequences other than the nucleic acid sequence comprising the sequence according to SEQ ID No. l , of the transgenic plant is suppressed, are not specific enough for the method of the present invention, and are not suitable, since they may interfere with the metabolism of the plant.

If antisense sequences are mentioned, those sequences of the invention are referred to which correspond to the codogenous DNA strand of the nucleic acid sequence from which the RNA of the powdery mildew susceptibility factor is transcribed. Said sequences are preferably complementary to at least 80 %>, 82 %>, 84 %>, 86 %>, 88 %>, particular preferably to at least 90 %>, 92%>, 94%> and most preferably to at least 95 %>, 96 %>, 97 %>, 98 %>, 99% to the sequence the expression of which is to be inhibited. As mentioned above, it is sufficient, if the antisense sequences are able to hybridize specifically with the RNA of the powdery mildew susceptibility factor, but not with the mRNA of other nucleic acid sequences of the plant. However, if a double- stranded RNA is to be produced, the antisense sequence should be 100% reverse- complementary to the sense sequence to ensure optimal base-pairing. Within the meaning of the present invention the terms "complementary" and "reverse

complementary" are used synonymously. In one embodiment of the present invention, the at least one nucleic acid sequence is present in antisense orientation, so that upon transcription of said sequence in plant cells a RNA molecule is created, the sequence of which being complementary to the RNA of the powdery mildew susceptibility factor. By hybridizing the antisense sequence with the RNA of the powdery mildew susceptibility factor in vivo, the expression of the RNA may be suppressed in plant cells, whereby the plant becomes pathogen resistant. Hence, in this case the vector comprises a promoter functional in plant cells; operatively linked thereto a nucleic acid sequence which is

complementary to the RNA of the powdery mildew susceptibility factor or a fragment of this sequence; and, optionally, a termination sequence. Preferably, the nucleic acid sequence is complementary to a nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or a fragment of this sequence. In another preferred embodiment of the present invention, the at least one nucleic acid sequence is present in sense orientation, so that upon transcription of said sequence in plant cells a RNA molecule is created, the sequence of which being identical to the RNA of the powdery mildew susceptibility factor. Those skilled in the art know that upon expression of such sense sequences in plants, antisense RNAs (asRNAs) may be formed which may cause silencing of both the transgene, i.e. the sense sequence which was introduced and the corresponding endogenous gene (co- suppression). By co-suppressing the RNA of the powdery mildew susceptibility factor in vivo, the expression of the RNA transcribed of the powdery mildew susceptibility factor may be suppressed in plant cells, whereby the plant becomes pathogen resistant. Hence, in this case the vector comprises a promoter functional in plant cells; operatively linked thereto a nucleic acid sequence which is identical to the RNA of the powdery mildew susceptibility factor or a fragment thereof; and, optionally, a termination sequence. Preferably, the nucleic acid sequence is identical to the nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or a fragment of this sequence. In another embodiment of the method of the present invention, vectors are used for introducing the nucleic acids in the plant cells which comprise in 5' - 3 '-orientation a promoter functional in plant cells, operatively linked thereto a DNA sequence coding for a ribozyme which specifically recognizes the RNA of the powdery mildew susceptibility factor, and a termination sequence. Those skilled in the art know how ribozymes which exhibit an endonuclease activity directed against a certain nucleic acid sequence, for example an RNA, may be produced. In the scope of the invention, the term "ribozyme" also refers to those RNA sequences which comprise next to the actual ribozyme leading sequences which are complementary to the RNA of the powdery mildew susceptibility factor or parts thereof, and thus direct the RNA- specific ribozyme even more target-orientedly to the RNA substrate of the ribozyme.

In another preferred embodiment of the above-mentioned method, the recombinant nucleic acid molecule comprises a promoter which is functional in plant cells, operatively linked thereto at least one nucleic acid sequence which after transcription acts as a leading sequence, another nucleic acid sequence coding for ribonuclease P, and a termination sequence. Upon transcription of such vectors, RNA molecules are formed in the cell having a leading sequence (the antisense sequence), which leading sequence directs the RNAse P to the RNA of the powdery mildew susceptibility factor, thereby causing the cleavage of the RNA by RNAse P (US 5,168,053).

Preferably, the leading sequence comprises 10 to 15 nucleotides which are complementary to the RNA of the powdery mildew susceptibility factor and a 3'- NCCA nucleotide sequence, wherein N preferably is a purine. The transcripts of the external leading sequence bind to the target RNA by the formation of base pairs, thus enabling cleavage of the RNA by RNAse P at the nucleotide 5' from the paired region. Such cleaved mRNA cannot be translated into a functional protein. The skilled person knows diverse techniques for suppressing or inhibiting the expression of an endogenous gene by small double-stranded RNA molecules, so- called small interfering RNAs, or siRNAs. Thereby, the double-stranded RNA molecule confers the specific degradation of the corresponding nucleic acid sequence, i.e. the nucleic acid sequence from which the double-stranded RNA sequence has been derived. By enzymatic cleavage, e.g. by the dicer enzyme complex, RNA fragments having a length of 19 - 25 nucleotides, the so-called siRNAs, are produced from double-stranded RNA substrates. Such double- stranded RNA substrates (dicer substrates) must have a length of at least 25 bp.

However, those skilled in the art know that also substantially longer double-stranded RNA molecules are suitable substrates. Due to the very high sequence identity of both siRNAs, the siRNAs are commonly present as double-stranded RNA. The siRNAs may inhibit or prevent gene expression in many different ways:

a) transcription (transcriptional gene silencing or TGS)

b) degradation of the mRNA (post-transcriptional gene silencing or PTGS) c) translation.

Said method for suppressing the expression of an endogenous nucleic acid sequence by sequence-specific double-stranded RNA is known to those skilled in the art as

RNA interference or RNAi (Zamore et al. (2000) Cell 101 :25-33; Tang et al. (2003) Genes Dev. 17: 49-63; Smith et al. (2000) Nature 407: 319-320).

The use of RNAi constructs according to the invention is based on the above- mentioned mechanisms for inhibiting expression of an RNA of a powdery mildew susceptibility factor.

Those skilled in the art know that various strategies may be chosen to make double- stranded RNA available as dicer substrate in the cell, and thus to trigger a specific RNAi effect. In a preferred embodiment of the above-mentioned method, the recombinant nucleic acid molecule comprises a promoter which is functional in plant cells, operatively linked thereto at least one nucleic acid sequence from which the R A of the powdery mildew susceptibility factor is transcribed, wherein said sequence has reverse-complementary regions, and a termination sequence. The skilled person knows constructs in which a nucleic acid sequence has reverse-complementary regions, so that after transcription of such a construct and self-hybridization within the nucleic acid sequence with the mentioned reverse-complementary regions, double-stranded RNA is being formed, which is a substrate for the dicer enzyme complex, for example. Accordingly, siRNA molecules are formed, which lead to the degradation of the corresponding nucleic acid. The above-mentioned reverse- complementary nucleic acid sequences are also referred to as inverted repeats. In another preferred embodiment, the recombinant nucleic acid molecule comprises a promoter which is functional in plant cells, operatively linked thereto at least one nucleic acid sequence from which the RNA of the powdery mildew susceptibility factor is transcribed, a "short hairpin" structure-generating nucleic acid, the nucleic acid sequence which is reverse-complementary to the at least one nucleic acid sequence, and a termination sequence.

By refolding the "short hairpin" structure, the at least one nucleic acid sequence and the nucleic acid sequence reverse-complementary thereto may hybridize and form double-stranded RNA. Suitable constructs and double-stranded RNA molecules are known to those skilled in the art for example as "short hairpin" RNAs or shRNAs. Such constructs may be led by a U6 promoter or a CaMV35S promoter (Tuschl (2002) Nat. Biotechnol. 20: 446-448; Paul et al. (2002) Nat. Biotechnol. 20: 505- 508; Paddison et al. (2002) Genes Dev. 16(8): 948-958; Brummelkamp et al. (2002) Science 296: 550-553). In a preferred embodiment of the method of the present invention using the RNAi methodology, the recombinant nucleic acid molecule comprises a promoter which is functional in plant cells, operatively linked thereto at least one nucleic acid sequence from which the RNA of the powdery mildew susceptibility factor is transcribed, optionally a spacer sequence, the nucleic acid sequence reverse-complementary to the at least one nucleic acid sequence, and a termination sequence.

In an especially preferred embodiment, the recombinant nucleic acid molecule comprises an RNAi construct, wherein the at least one nucleic acid sequence comprises a sequence selected from the group consisting of:

a) a nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or a fragment of this sequence;

b) 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; and

c) a nucleic acid sequence comprising a sequence which hybridizes under

stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence,

and a sequence reverse-complementary thereto. Even more preferably, the recombinant nucleic acid molecule comprises a sequence according to SEQ ID No. 1 or a fragment thereof.

If a spacer sequence is used in the RNAi construct, the spacer sequence can be any sequence which is not complementary to another sequence in the construct. The spacer sequence can be derived from both exons and introns. The spacer sequence may also be a part of the sense or antisense sequence which is not reverse- complementary to the antisense or sense sequence, respectively. For example, the sense sequence may be extended by a certain number of nucleotides which are located 5' or 3' of the sense sequence in its natural sequence context, whereas the antisense sequence is not extended by the corresponding complementary nucleotides. In another embodiment, the spacer sequence is an intron which provides splice donor and splice acceptor sequences, such as the rgal intron from wheat. If vectors containing an intron as a spacer sequence are stably introduced in plant cells, first a pre-mRNA is formed upon transcription of said vectors which consists of a first exon comprising the at least one nucleic acid sequence of the present invention, an intron and a second exon comprising the nucleic acid sequence reverse-complementary to the at least one nucleic acid sequence. Since the intron is removed by the splicing procedure, a continuous RNA molecule is formed having regions which are complementary to each other, and thus being a substrate for specific enzyme complexes, such as the dicer enzyme complex. Those skilled in the art know that the position of the antisense (3' - 5') and sense (5' - 3') sequences may be interchanged in the vector. The spacer sequence typically comprises 20 to 500 nucleotides, preferably 40 to 400 nucleotides, more preferably 60 to 300 nucleotides and most preferably 100 to 200 nucleotides.

In another embodiment of the present invention, the RNAi construct comprises two promoters of which one regulates the expression of the sense sequence and the other one regulates the expression of the antisense sequence. The two promoters may be the same or different promoters. The sense and the antisense nucleic acid molecules may also be expressed under the control of a bidirectional promoter. Those skilled in the art know that for RNAi and/or PTGS the sense and antisense RNAs used for forming double-stranded RNA molecules may be of different sizes (Tuschl (2002) Nature Biotechnol. 20: 446-448).

In another embodiment the content of the RNA of the powdery mildew susceptibility factor is reduced by microRNA. MicroRNAs (miRNAs) have emerged as evolutionarily conserved, R A-based regulators of gene expression in plants and animals. MiRNAs with a length of 21 to 25 nucleotides arise from larger precursors with a stem loop structure that are transcribed from non-protein-coding genes.

MiRNA targets a specific mRNA to suppress gene expression at post-transcriptional (i.e. degrades mRNA) or translational levels (i.e. inhibits protein synthesis) (Bartel (2004) Cell 116: 281-297).

A miRNA precursor (pre-miRNA) can be engineered in such a way that endogenous miRNA encoded by pre-miRNA is replaced by a miRNA to target a gene-of-interest. A native plant microRNA precursor can be engineered as described in Schwab et al. (2006) Plant Cell 18(5): 1121-1133, to produce artificial miRNA which specifically down-regulates target gene expression. A further method for the design of functional microRNAs is the tool WMD3 as described by Ossowski et al. (2008) Plant J. 53(4): 674-690. This tool is available in the internet under the following URL:

http://wmd3.weigelworld.org/cgi-bin/webapp.cgi.

Hence, the present inventions further provides a method of producing a transgenic plant, plant cell or plant part having an increased resistance to pathogens compared to the control plant comprising the steps of,

(a) transforming a plant cell with a miRNA precursor construct, and

(b) generating from the plant cell the transgenic plant, wherein said construct contains a promoter that drives expression in a plant cell operably linked to a nucleotide sequence encoding a precursor micro RNA sequence comprising a micro RNA sequence which targets the RNA of the powdery mildew susceptibility factor.

The use of engineered micro RNA precursors and micro-RNA for modulating the expression of a gene is well known and described e.g. in US 2004/0268441. The use of engineered micro-RNA precursors and micro-RNA for modulating the expression of a gene can be combined with other methods of genetic engineering well known to the person skilled in the art. In another embodiment of the present invention synthetic double-stranded siR As which typically have a length of 19 - 21 nucleotides are used for inhibiting the expression of a nucleic acid sequence from which the R A of the powdery mildew susceptibility factor is transcribed. Such synthetic siRNAs may be introduced in the corresponding plant cell or plant by biolistic transformation techniques. Such synthetic siRNA molecules may activate the PTGS system in plants, and trigger an RNAi effect (Hamilton and Baulcombe (1999) Science 286: 950-2). The target sequence for siRNA inhibition as well as the siRNA sequence motif may be selected according to the rules and regulations known to those skilled in the art, for example according to Elbashir et al. (2001) Nature 411 : 494-8. If the target sequence for the siRNA-mediated inhibition lies within the coding regions of the gene, or within the mRNA, those skilled in the art know, for example, that the target sequence for siRNA-mediated inhibition may typically be at least 70 nucleotides downstream from the start codon in 5' - 3' direction and at least 50 nucleotides upstream from the stop codon.

The sequence region may then be searched for the sequence motif AA (N19), wherein N may be any nucleotide. Said sequence motif typically comprises the AA dinucleotide, followed by 19 nucleotides, and preferably two additional uridine or thymidine residues. In general, the thymidine residues may be replaced by uridine residues in the siRNA sequence. Further, those skilled in the art know the rules and regulations, established by Reynolds et al. ((2004) Nature Biotechnol. 22: 326-30):

1. a guanine/cytosine content of 30 - 50 %

2. at least three adenine or uracil groups at positions 15 to 19 of the sense strand 3. no intermolecular "hairpin" structures 4. one adenine residue at position 19 of the sense strand

5. one adenine residue at position 3 of the sense strand

6. one uracil residue at position 10 of the sense strand

7. no guanine or cytosine residue at position 19 of the sense strand

8. no guanine residue at position 13 of the sense strand. eight criteria may be rated as follows:

1 point each for criteria 1, 3, 4, 5 and 6

1 point each for each adenine or uridine residue at position 15 to 19, at least 3 corresponding bases (criterion 2)

(iii) minus 1 point each for not fulfilling the criteria 7 - 8.

According to Reynolds et al., only those siRNAs should be considered which have at least 6 points. Such siRNAs which fulfil the above-mentioned criteria may be checked by appropriate search programs, e.g. BLAST, whether there are any, if possible no or only little, homologies to other nucleic acid sequences of the plant.

The skilled person also knows other methods for reducing the content of an RNA, such as the RNA of the powdery mildew susceptibility factor transcribed from the nucleic acid sequence according to SEQ ID No. 1. For example, a nucleic acid sequence for reducing the content of a RNA 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 Al . 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 nucleic acid sequence for reducing the content and/or the activity of a RNA and/or a protein different enzymes such as meganucleases (WO 2009/114321 A2), zink finger nucleases (WO 2009/042164 Al), transcription activator-like effector nucleases (WO 2011/072246 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 heteroduplexes which are then cleaved by single strand nucleases such as Cell at the 3' side of the mutation. The precise position of the mutation within the nucleic acid sequence according to SEQ ID NO. 1 can then be determined by denaturing gel electrophoresis or the LICOR gel based system (see, e.g., McCallum et al. (2000) Plant Physiol. 123(2): 439-442; Uauy et al. (2009) BMC Plant Biol. 9: 115).

The expression level of the RNA of the powdery mildew susceptibility factor may be determined in the control plants as well as in the transgenic plants, for example, by RT-PCR analysis or Northern Blot analysis with specific primers or probes. A person skilled in the art knows how to select said probes or primers in order to examine the expression of said nucleic acid. Preferably, the expression of the RNA of the powdery mildew susceptibility factor is statistically significantly reduced by at least 80 %, particularly preferably by at least 90 %, also particularly preferably by at least 95 %, and most preferably by at least 98 % or 99 %. Preferably, in the method and the expression construct 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 SEQ ID No. 1 or a fragment of this sequence;

b) 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; and

c) a nucleic acid sequence comprising a sequence which hybridizes under

stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence.

A "fragment" of the nucleic acid sequence according to SEQ ID No. 1 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 and which is able to reduce the content of the RNA of the powdery mildew susceptibility factor transcribed from the nucleic acid sequence according to SEQ ID No. l when used in a suitable expression system, but not or not considerably of other RNAs the expression of which should not be reduced. 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 has a length of at least 19, 20, 21, 22, 23, 24, 25, 30 or 40 nucleotides, preferably 50, 60 or 70 nucleotides, more preferably of at least 80, 90, 100, 110, 120, 130, 140 or 150 nucleotides, even more preferably of at least 160, 170, 180, 190, 200 or 210 nucleotides and most preferably of at least 220, 230, 240, 250, 260, 270, 280 or 290 nucleotides. The present invention further relates to the use of nucleic acid sequences which are at least 70, 72, 74, 76, 78%, preferably at least 80, 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 SEQ ID No. 1 or a fragment of this sequence and which are able to reduce the content of the RNA of the powdery mildew susceptibility factor transcribed from a nucleic acid sequence comprising the sequence according to SEQ ID No. l when used in a suitable expression system, but not or not considerably of other RNAs the expression of which should not be reduced.

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 applying the default parameters. Here, in addition, the program Sequencher (Gene Codes Corp., Ann Arbor, MI, 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 150 or 180 nucleotides, preferably 200 or 220 nucleotides, more preferably 250 or 280 nucleotides and most preferably the whole length of the nucleic acid sequence according to SEQ ID No .1.

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 120 or 150 nucleotides, preferably 180 or 200 nucleotides, more preferably 220 or 250 nucleotides and most preferably of 280 nucleotides.

The present invention further relates to the use of nucleic acid sequences which hybridize under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 and which are able to reduce the content of the RNA of the powdery mildew susceptibility factor transcribed from a nucleic acid sequence comprising the sequence according to SEQ ID No. l when used in a suitable expression system, but not or not considerably of other RNAs the expression of which should not be reduced. In the context of the present invention the term "hybridizing under stringent conditions" means that the hybridization is implemented in vitro under conditions which are stringent enough to ensure a specific hybridization. Stringent in vitro hybridization conditions are known to those skilled in the art and may be taken from the literature (e.g. Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY). The term "specific hybridization" means that under stringent conditions a molecule 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 μg/mL salmon sperm DNA

Hybridization solution: pre-hybridization solution

lxlO6 cpm/mL probe (5 - 10 min 95 °C)

20x SSC: 3 M NaCl

0.3 M sodium citrate

ad pH 7 with HC1

5 Ox 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 lx 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 lx SSC/ 0.1% SDS hybridization temp

45 min 0.2x SSC/ 0.1( ½ 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.

A nucleic acid sequence hybridizing to a fragment of the sequence according to SEQ ID No. 1 under stringent conditions has a length of at least 25 or 30 nucleotides, preferably of at least 50 or 70 nucleotides, more preferably of at least 100 or 120 nucleotides, even more preferably of at least 150 or 200 nucleotides and most preferably of at least 250 or 300 nucleotides.

The expression of RNAs other than the RNA of the powdery mildew susceptibility factor transcribed from the nucleic acid sequence comprising the sequence according to SEQ ID No.1 is reduced by less than 10%> or 8%, preferably by less than 7, 6 or 5%, more preferably by less than 4, 3 or 2% and most preferably by less than 1%.

In the context of the above, a suitable expression system for reducing the expression of the RNA of the powdery mildew susceptibility factor transcribed from the nucleic acid sequence comprising the sequence according to SEQ ID No. 1 is any expression system described herein, preferably an RNAi expression system.

In order to produce RNAi constructs, the corresponding sense or antisense nucleic acid sequences for example may be inserted into an appropriate vector by restriction digestion and subsequent ligation.

Alternatively, the corresponding sense or antisense nucleic acid sequences for example may be inserted into the vector by homologous recombination, such as by the GATEWAY® system (Invitrogen) or the BD Creator™ system (BD Biosciences Clontech Co.).

Within the scope of the present invention, the term "expression construct" means a nucleic acid molecule which contains all elements which are necessary for the expression of a nucleic acid sequence, i.e. the nucleic acid sequence to be expressed under the control of a suitable promoter and optionally further regulatory sequences such as termination sequences. An expression cassette of the present invention may be part of an expression vector which is transferred into a plant cell or may be integrated into the chromosome of a transgenic plant after transformation.

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 that 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. 11 : 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-LS 1 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 2A11 promoter from tomato (van Haaren et al. (1991) Plant Mol. Biol. 17: 615-630). Further, the chemically inducible Tet repressor system (Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237) may be used. 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).

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): 311), the GerA promoter (Wu et al. (2000) Plant Phys Biochem. 38: 685), the OsROCl promoter (Accession number AP004656), the RTBV promoter (Kloeti et al. (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).

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). 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 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 operably 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 cells. 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 well 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 a 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 replicate in agrobacteria. By means of a helper 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. (1978) Molecular and General Genetics 163: 181-187). The agrobacterium, serving as host cell, is to contain a plasmid which includes a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. In addition, T-DNA may be present. The agrobacterium transformed in such 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 cells thereof may also be transformed using vectors which are based on agrobacteria (see e.g. Chan et al. (1993) Plant Mol. Biol. 22: 491-506). Alternative systems for the transformation of monocotyledonous plants or the cells thereof are transformation by biolistic approach (Wan and Lemaux (1994) Plant Physiol. 104: 37-48; Vasil et al. (1993) Bio/Technology 11 : 1553-1558; Ritala et al. (1994) Plant Mol. Biol. 24: 317-325; Spencer et al. (1990) Theor. Appl. Genet. 79: 625-631), the protoplast transformation, the electroporation of partially permeabilized cells, and the insertion of DNA by means of glass fibres. The vectors described herein can be directly transformed into the plastid genome. Plastid expression, in which genes are inserted by homologous recombination into the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number over nuclear-expressed genes to permit high expression levels. 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 et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 7301 - 7305.

The transformed cells grow within the plant in the usual manner (see also

McCormick et al. (1986) Plant Cell Reports 5 : 81-84). The resulting plants may be cultivated in the usual manner, and may be crossed with plants which have the same transformed genes or other genes. The hybrid individuals resulting therefrom have the respective phenotypical properties. 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 of the RNA of the powdery mildew susceptibility factor is not decreased and selecting transgenic progeny in which the content of the RNA of the powdery mildew susceptibility factor is decreased. The other plant in which the content of the RNA of the powdery mildew susceptibility factor is not decreased is preferably from the same species as the transgenic plant and may be a wild-type plant, i.e. a plant which does not contain any transgenic nucleic acid sequence, or it may be a transgenic plant which contains a transgenic nucleic acid sequence other than the nucleic acid sequences disclosed herein, e.g. 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 of the RNA of the powdery mildew susceptibility factor is decreased are discussed above. An "elite variety" within the meaning of the present invention is a variety which is adapted to specific environmental conditions and/or which displays at least one superior characteristic such as an increased yield compared to non-elite varieties. The transgenic progeny of the above crossing step can be further crossed with each other to produce true breeding lines. For this purpose the transgenic progeny of the above cross in which the content of a RNA of a powdery mildew susceptibility factor is decreased is inbred and the transgenic progeny of this crossing step is selected and again inbred. This inbreeding step is repeated until a true breeding line is established, for example at least five times, six times or seven times. A "true breeding plant" or "inbred plant" is a plant which upon self-pollination produces only offspring which is identical to the parent with respect to at least one trait, in the present case the transgene which decreases the content of the RNA of the powdery mildew

susceptibility factor. The true breeding lines can then be used in hybrid breeding yielding Fl hybrids which can be marketed. This method is particularly suitable for example for maize and rice plants. Alternatively, the true breeding lines can be further inbred in a linebreeding process. This method is particularly suitable for example for wheat and barley plants.

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 which reduces the content of an RNA of the powdery mildew susceptibility factor.

The flour prepared from the transgenic seed of the present invention can be distinguished from the flour prepared from other plants by the presence of the transgenic nucleic acid sequence, the expression construct or the vector of the present invention. 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. Alternatively or additionally, if an antisense sequence is part of the transgene mediating the reduction of the content of the R A of the powdery mildew susceptibility factor, the presence of this antisense sequence can be detected in the flour prepared from the transgenic seed. The flour prepared from the mutant seed 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 which reduces the content of an RNA of the powdery mildew susceptibility factor, i.e. this nucleic acid sequence is detectable in the harvestable parts by conventional means. The harvestable plants may be seeds, roots, leaves, stems, and/or flowers comprising the nucleic acid sequence which reduces the content of an RNA of the powdery mildew susceptibility factor. Preferred harvestable parts are seeds comprising the nucleic acid sequence which reduces the content of an RNA of the powdery mildew susceptibility factor.

The identification of a nucleic acid sequence comprising the sequence according to SEQ ID No.l as being involved in pathogen resistance and the use thereof for producing transgenic plants with increased pathogen resistance will 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 (pIPKTA38) preparation

The pIPKTA38 plasmid (Douchkov et al. (2005) Mol. Plant Microbe Interact. 18(8): 755-761) with a kanamycvin resistance gene was used as Gateway Entry vector.

Bacteria which contained the plasmid were grown in LB medium containing 50 μg/mL kanamycin. Plasmid DNA was prepared with the Jetstar midi DNA kit (Genomed).

As a control the plasmid was digested with the restriction enzyme Apal, yielding bands of 1257 bp and 1054 bp. Then the DNA concentration was measured and adjusted to 150 ng/μΕ.

1.2. Destination vector (pIP TA30) preparation

The pIPKTA30 plasmid (Douchkov et al. (2005) Mol. Plant Microbe Interact. 18(8): 755-761) was used as the RNAi vector. It contains an ampicillin resistance gene, a ccdB negative selection marker gene which requires the propagation of the plasmid in DB3.1 cells and a chloramphenicol resistance gene.

Plasmid DNA was prepared with the Jetstar midi DNA kit (Genomed). The plasmid preparations were digested as a control with EcoRI (correct bands - 687, 1007, 2641, and 2857 bp) or Sal I (601 , 1589, and 5002 bp). The DNA concentration was measured and adjusted to 150 ng^L.

1.3. PCR amplification of the cDNA clones

"Master mixes" for 20-μί PCR reactions with two specific EST-primers

Specific primers (SEQ ID Nos. 2 and 3) 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 were dispensed to each well of a 96-well PCR plate. 4,25 of each EST-specific primer and 0,5 μΐ, EST DNA as template were added to each well.

Table 6: PCR master mix content (1 1 μΕ Master mix + 4,25 μΐ^ of each specific primer + 0,5 μΐ^ template per reaction):

Component For 96 For 2 For 4 samples plates plates

Thermal Ace buffer (lOx) 200 μΕ 400 μΕ 800 μΕ dNTPs (50x, 10 mM each) 50 μΕ 100 μΕ 200 μΕ

Thermal Ace DNA Polymerase (2υ/μΕ) 25 μΕ 50 μΕ 100 μΕ

Water 825 μΕ 1650 μΕ 3300 μΕ

Dispense 1 1 ,0 μΐ^ PCR master mix per well

Target-specific sense primer (Ι μΜ) 4,25 μΐ, per well

Target-specific antisense primer (Ι μΜ) 4,25 μΐ, per well

Template (EST clone DNA or genomic 0,5 μΐ, per well

DNA) PCR cycle conditions

30 cycles

Figure imgf000077_0001

Purification of the PCR product

30 μΙ_, H20 were added to each PCR reaction to obtain 50-μί 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 μΙ_, 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 μί 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 minutes. 5 μΙ_, Swal master mix (see Table 8) was added to each well, followed by an incubation at 25 °C for 1 h. Next, the ligation samples were transformed into competent bacteria and suitable clones were isolated after miniprep and control digestion using EcoRI. Table 7: Ligation Master mix for ΙΟ-μί reactions (6 μί master mix + 4 μΐ^ PCR product per reaction)

Components For 96 samples For 2 plates For 4 plates

H20 100 μΐ. 200 μΐ. 400 μΐ. pIPKTA38 (150 ng^L) 100 μΕ 200 μΕ 400 μΕ

Ligation buffer (lOx) 100 μΕ 200 μΕ 400 μΕ

50% PEG 4000 100 μΕ 200 μΕ 400 μΕ

NaCl (0,5 M) 100 μΕ 200 μΕ 400 μΕ

5νναΙ (10 υ/μί) 50 μΕ 100 μΕ 200 μΕ (New England Bio labs)

T4 DNA ligase (5 υ/μΐ,) 50 μΕ 100 μΕ 200 μΕ (Fermentas)

PCR product (purified) 4 μΕ per reaction

Table 8: Swal Master mix (add 5 μΐ, per reaction)

Components For 96 samples For 2 plates For 4 plates

Swal buffer (1 Ox) 50 μΕ 100 μΕ 200 μΕ

NaCl (0,5 M) 100 μΕ 200 μΕ 400 μΕ

H20 300 μΕ 600 μΕ 1200 μΕ

Swal (10υ/μΐ,) 50 μΕ 100 μΕ 200 μΕ

1.5. LR Reaction

Master mixes for 6 μΐ^ LR reactions

Table 9: LR-master mix (5 μΐ, master mix + 1 μΐ, pIPKTA38: :EST DNA per reaction)

Component For 96 samples For 2 plates For 4 plates

LR Buffer (5x) 120 μΐ, 240 μΐ, 480 μΐ, pIPKTA30 (150 ng^L) 100 μΐ, 200 μΐ, 400 μΐ,

H20 200 μΐ, 400 μΐ, 800 μΐ,

LR Clonase Mix 80 μΐ, 160 μΐ, 320 μΐ,

(Invitrogen)

Dispense 5 μΙ_, Master mix per well pIPKTA38::EST DNA 1 μΐ^ per reaction

Incubation at room temperature over night (or at least 6 h).

2. Trans(¾¾ test and TIGS protocol Figure 2 shows a schematic drawing of the test procedure for the RNAi constructs. 2.1. Preparation of the plant material

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 μg) DNA (plasmid) of the gene to be tested and 7 μΐ 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 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 μιη mesh width) stretched thereover. The leaves were inoculated with mildew (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 mM sodium EDTA; 1,4 mM K- hexacyanoferrate(II); 1 ,4 mM K-hexacyanoferrate(III); 0, 1 % Triton X- 100; 20% methanol and 1 mg / ml X-Gluc). The tubes were placed in a suction bottle and vacuum was applied thereto 2 - 3 times. The infiltration is complete when the leaves become transparent and start to sink. The X-glucose solution was refilled to 14 mL and the tubes were sealed. The tubes were incubated over night at 37 °C in the incubator.

2.7. TCA destaining

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.

200 μΐ of 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 R Ai constructs on plant resistance to the fungal pathogen

Blumeria graminis was tested in transient experiments.

The barley plants used (Hordeum vulgare, cultivar, 'Golden promise1) 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 μg of a control vector PIPKTA30 or of an 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 μιη) 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 pIPKTA30N). Further, each experiment contained 2 parallel bombardments of the positive control pIPKTA36, which causes resistance by inhibiting the Mlo gene of barley. Data per experiment are based on the comparison of the effect of the test constructs with the average value of the 3 negative controls of the respective experiment.

Table 10 shows the relative susceptibility index (Rel SI) of barley cells transiently transformed with an RNAi construct inhibiting the expression of the RNA of the powdery mildew susceptibility factor transcribed from the nucleic acid sequence comprising the sequence according to SEQ ID No.l . The susceptibility index relative to the empty vector control was determined in five independent transformation experiments. As the cells transformed with the RNAi construct have a susceptibility index of less than 100% compared to the control cells transformed with the empty vector (pIPKTA30N), they the RNAi construct suppresses putative susceptibility genes in barley.

Table 10

Sequenz ID BlastX Rel SI (%) p t-Test

Unknown 1 Unknown genomic barley sequence 26,8 0,0057

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 of an R A of a powdery mildew susceptibility factor transcribed from a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence comprising the sequence according to SEQ ID No. 1 or a fragment of this sequence;
(b) 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; and
(c) a nucleic acid sequence comprising a sequence which hybridizes under stringent conditions with a nucleic acid sequence according to SEQ ID
No. 1 or a fragment of this sequence
is reduced in comparison to the control plant cell, plant or plant part.
2. Method of claim 1, wherein the method is selected from:
(A) a method 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 a nucleic acid sequence as defined in claim 1 ;
(iii) optionally a spacer sequence,
(iv) the nucleic acid sequence being reverse-complementary to the nucleic acid sequence of (ii);
(v) a termination sequence;
(b) optionally, regenerating a transgenic plant from the transformed cell; (B) a method 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 a nucleic acid sequence which is
complementary to a nucleic acid as defined in claim 1 ; and
(iii) optionally, a termination sequence;
(b) optionally, regenerating a transgenic plant from the transformed cell;
(C) a method 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 a nucleic acid sequence which is identical to a nucleic acid as defined in claim 1 ;
(iii) optionally, a termination sequence;
(b) optionally, regenerating a transgenic plant from the transformed cell; and
(D) a method comprising the steps of:
(a) introducing into a plant cell a vector which comprises:
(i) a promoter functional in plant cells;
(ii) operably linked thereto a nucleic acid sequence encoding a precursor micro RNA sequence comprising a micro RNA sequence which targets the nucleic acid sequence as defined in claim 1 ;
(b) optionally, regenerating a transgenic plant from the transformed cell.
3. Method according to claim 2, wherein the nucleic acid sequence in (ii) or (iv) is the nucleic acid sequence according to SEQ ID No. 1 or a fragment thereof.
4. Method of any of the preceding claims, wherein the promoter is a tissue-specific and/or a pathogen-inducible promoter.
5. Method of any of the preceding claims, further comprising reducing the content and/or activity of at least one plant protein mediating pathogen susceptibility.
6. 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 of a RNA of the powdery mildew susceptibility factor as defined in claim 1 is not reduced and selecting transgenic progeny in which the content of the RNA of the powdery mildew susceptibility factor as defined in claim 1 is reduced.
7. 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% sequence identity to the nucleic acid sequence according to SEQ ID No. l .
8. Method of any of the preceding claims, wherein the transgenic or mutant plant has an increased resistance to a fungal pathogen.
9. Method of claim 8, wherein the transgenic or mutant plant has an increased resistance to Blumeria graminis, Septoria tritici and/or Puccinia triticina.
10. Method of any of the preceding claims, wherein the transgenic or mutant plant is a monocotyledonous plant.
11. Method of claim 10, wherein the transgenic or mutant plant is a wheat or a barley plant.
12. 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 SEQ ID No. 1 or a fragment of this sequence;
(b) 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;
(c) a nucleic acid sequence comprising a sequence which hybridizes under stringent conditions with a nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence; and
(d) a nucleic acid sequence which is reverse-complementary to a nucleic acid sequence of any of (a) to (c),
operatively linked to a promoter functional in plant cells.
13. Expression construct of claim 12, selected from the group consisting of:
(a) an expression construct comprising:
(i) a promoter functional in plant cells,
(ii) operatively linked thereto at least one nucleic acid sequence as defined in claim 12;
(iii) optionally a spacer sequence,
(iv) the nucleic acid sequence being reverse-complementary to the nucleic acid sequence of (ii); and
(v) optionally, a termination sequence;
(b) an expression construct comprising:
(i) a promoter functional in plant cells;
(ii) operatively linked thereto at least one nucleic acid sequence which is
complementary to a nucleic acid sequence as defined in claim 12; and
(iii) optionally, a termination sequence; (c) an expression construct comprising:
(i) a promoter functional in plant cells;
(ii) operatively linked thereto at least one nucleic acid sequence which is identical to a nucleic acid sequence as defined in claim 12;
(iii) optionally, a termination sequence; and
(d) an expression construct comprising:
(i) a promoter functional in plant cells;
(ii) operably linked thereto a nucleic acid sequence encoding a precursor micro RNA sequence comprising a micro RNA sequence which targets the nucleic acid sequence in claim 12.
14. Expression construct of claim 12 or 13, wherein the nucleic acid sequence in (ii) or (iv) is the nucleic acid sequence according to SEQ ID No. 1 or a fragment of this sequence.
15. Vector comprising the expression construct of any of claims 12 to 14.
16. Transgenic or mutant plant or plant cell with an increased resistance to pathogens, produced according to the method of any of claims 1 to 11 or containing an expression construct of any of claims 12 to 14 or a vector of claim 15.
17. Use of the transgenic or mutant plant of claim 16 or parts thereof as fodder or to produce feed material.
18. Transgenic or mutant seed produced from the transgenic plant of claim 16.
19. Flour produced from the transgenic or mutant seed of claim 18, wherein the presence of the transgene or the mutation which reduces the content of the RNA of the powdery mildew susceptibility factor as defined in claim 1 can be detected in said flour.
20. Method for producing true breeding plants comprising inbreeding the transgenic progeny of the crossing step of claim 6 and repeating this inbreeding step until a true breeding plant is obtained.
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