DE10063986A1 - Plants with improved resistance - Google Patents

Abstract

The invention relates to transgenic plants with improved resistance to pathogens, whereby the improved resistance is a result of an increased expression of one or more nucleic acid sequences according to SEQ ID NO:1 to 11, or homologues, fragments or derivatives thereof, or by a change in the biological activity of the genetic product coded by one or more nucleic acid sequences according to SEQ ID NO:1 to 11, or the homologues, derivatives or fragments thereof. The invention further relates to transgenic plants with at least one regulatory nucleic acid sequence according to SEQ ID NO:12 to 23, integrated into the genome in a stable manner and a nucleic acid sequence coding for a genetic product functionally linked to the above nucleic acid sequence. The invention furthermore relates to a method for the production of the said transgenic plants and the nucleic acid sequence according to SEQ ID NO:1 to 23.

Description

The present invention relates to transgenic plants with improved resistance against pathogens, the improved resistance through increased expression one or more nucleic acid sequences according to SEQ ID NO: 1 to 11, or their homologs or derivatives or fragments, or by changing the biological activity of one or more nucleic acid sequences according to SEQ ID NO: 1 to 11, or their homologs or derivatives or fragments encoded gene products. The present The invention further relates to transgenic plants with at least one stably in the genome integrated regulatory nucleic acid sequence according to SEQ ID NO: 12 to 23, and one with this nucleic acid sequence functionally linked for coding a gene product Nucleic acid sequence. Furthermore, the present invention relates to methods for producing the transgenic plants according to the invention and the nucleic acid sequences according to SEQ ID NO: 1 until 23.

The attack of certain phytopathogens induces a wide range in infected host plants Defense response spectrum. Common defense strategies include, for example rapid death of plant cells at the infection site (hypersensitive response), the structural cell wall reinforcement by incorporating polymeric components around the site of intrusion, the production of hydrolytic antipathogenic enzymes as well as the Synthesis of phytoalexins with antimicrobial activity. At the beginning of an infection As a rule, specific molecular pathogen components, so-called elicitors, are developed by Plant receptors bound, whereby the plant is informed about the infection and Initiates countermeasures that involve numerous enzymatic processes.

Under the generic term WRKY genes a group of genes is summarized, which for encode structurally related putative transcription factors in plants. The members of the The WRKY family regulate a wide variety of plant physiological processes such as B. Defense against pathogens, wound healing, trichome development and senescence. So far, however, is only of Very few WRKY proteins know in which specific metabolic processes they are involved  intervention. The current state of WRKY research can be found in T. Eulgem, P. J. Rushton, S. Robatzek and I. E. Somssich (2000): The WRKY superfamily of plant transcription factors, Trends in Plant Sciences 5, 199-206. Currently the WRKY- Protein superfamily in the "thale cress" Arabidopsis thaliana about 70 different ones Transcription factors that have in common an approximately 60 amino acid DNA-binding WRKY domain (with the amino acid sequence WRKYG (QK)). DNA binding is conveyed by a zinc finger motif of this domain.

Due to the number of WRKY domains and the structure of the zinc fingers, WRKY proteins are classified into three groups. Group I proteins contain two, group 11 proteins only one WRKY domain. Group III WRKYs also have only one WRKY domain, but differ from the members of group 11 by a different zinc finger motif (type C ₂ -HC). It is not possible to infer the physiological function of the WRKYs based on the group membership.

To date, there are more than 500 WRKY ESTs from various plant species in databases in front. Many are tissue-specific and have been isolated from roots, leaves, flowers or seeds or are only used under certain conditions, e.g. B. salt or dry stress expressed. At the moment there is only concrete evidence of this for some of these WRKY genes what metabolic pathway they are involved in. Few have been shown to work at Defense against plant pathogens are involved: WRKY1 and WRKY3 from Petroselinum crispum (Eulgem, T. et al .: Early nuclear events in plant defense signaling: rapid gene activation by WRKY transcription factors. EMBO Journal 18, 4689-4699, 1999) as well as WRKY3 and WRKY4 from Nicotiana tabacum (Chen, C. and Chen, Z: Isolation and characterization of two pathogenic and salicylic acid induced genes encoding WRKY DNA binding proteins from tobacco. Plant Molecular Biology 42, 387-396, 2000). A pathogen defense function of the in this patent application called WRKY proteins 30, 33, 41, 46, 54, 55, 62, 63, 64, 67 and 70 was not yet known.

The role of WRKY proteins in defense against pathogens is presumably that transcriptional activation of special genes. So z. B. in cell suspension cultures Induction of the PR1-1 and PR1-2 genes of Petroselinum crispum by secreted by the fungus Elicitors caused, among other things, by a defined fungal oligopeptide (Pep25). The Binding of Pep25 to a plant plasma membrane receptor leads to an increase  different ion concentrations, numerous for phosphorylation / dephosphorylation Proteins and ultimately for the activation of genes involved in the defense against pathogens, to which also PR1-1 and PR1-2 belong. So-called W- Boxes (W1, W2 and W3) with the sequence (T) (T) TGAC (C / T) that act as binding sites for sequence-specific DNA-binding WRKY proteins act. The WRKY proteins thus represent transcription factors for genes whose promoters are some copies of the W boxes contain. Genes whose promoters have W-boxes can e.g. B. for pathogen damaging Encode proteins such as antimicrobial chitinases or glucanases. The promoters of the most WRKY genes contain W boxes. These genes are therefore in turn from other WRKY genes regulated.

Almost all modern crops are affected by pathogenic organisms and some badly damaged. These pathogens include various fungi, bacteria, nematodes and viruses. The plants have various natural defense mechanisms, but they often do only give limited protection against the pathogens. For this reason, the intensive use of arable land in modern agriculture and horticulture frequent use of chemical pesticides, if they are available .. Regardless of all crop protection agents and methods developed to date, as well as the Successful classic resistance breeding in crops is through today Infestation with pathogens resulting crop loss is still significant and causes one worldwide economic damage of several billion marks annually. A development new plants with improved resistance or a wider range of resistance could help reduce the problems mentioned.

The present invention is therefore based on the object of novel pathogen-resistant To provide plants and processes for their production.

The object is achieved by the subject-matter defined in the patent claims.

The invention is illustrated by the following figures.

Figure 1 shows the genomic nucleic acid sequence of the WRKY30 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Fig. 2 shows the genomic nucleic acid sequence of the WRKY33 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 3 shows the genomic nucleic acid sequence of the Arabidopsis thaliana WRKY41 gene. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 4 shows the genomic nucleic acid sequence of the WRKY46 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 5 shows the genomic nucleic acid sequence of the Arabidopsis thaliana WRKY54 gene. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 6 shows the genomic nucleic acid sequence of the Arabidopsis thaliana WRKY55 gene. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 7 shows the genomic nucleic acid sequence of the Arabidopsis thaliana WRKY62 gene. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 8 shows the genomic nucleic acid sequence of the Arabidopsis thaliana WRKY63 gene. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 9 shows the genomic nucleic acid sequence of the WRKY64 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 10 shows the genomic nucleic acid sequence of the Arabidopsis thaliana WRKY67 gene. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 11 shows the genomic nucleic acid sequence of the Arabidopsis thaliana WRKY70 gene. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 12 shows the promoter region of the Arabidopsis thaliana WRKY33 gene. The 1270 bp promoter fragment (SEQ ID NO: 12) according to FIG. 21 is highlighted in italics and the 361 bp promoter fragment (SEQ ID NO: 13) according to FIG. 21 is underlined. The forward primers for the 1270 bp, 361 bp, 331 bp and 193 bp fragment according to FIG. 21 and the reverse primer of the four fragments are highlighted in bold. The start ATG is in the reverse primer. Lower case letters denote the coding region of the WRKY33 gene in the reverse primer.

Fig. 13 shows a RT-PCR analysis to demonstrate the induction of WRKY genes 30, 41, 46, 62, 63, 64 and 67 after infection of Arabidopsis thaliana with the Peronospora parasitica Blattpathogen cala2 pv. Samples were taken at the time of infection (0) and 2, 4, 6, 12 and 24 hours after the infection (hpi = hours post induction). To prove that the induction was caused by the pathogen infections, control infections with pure water (H ₂ O) were carried out instead of a pathogen application.

Fig. 14 shows a RT-PCR analysis to demonstrate the induction of WRKY genes 54, 55 and 70 after infection of Arabidopsis thaliana with the Peronospora parasitica Blattpathogen cala2 pv. Gene-specific primers were used for the analysis. Samples were taken at the time of infection (0) and 2, 4, 6, 12 and 24 hours after the infection (hpi = hours post induction). To prove that the induction was caused by the pathogen infections, control infections with pure water (H ₂ O) were carried out instead of a pathogen application.

Fig. 15 shows a Northern blot for detecting the induction of WRKY33 after infection of Arabidopsis thaliana with the Blattpathogen Peronospora parasitica parasiticum cala2 pv. Samples were taken at the time of infection (0) and 2, 4, 6 and 24 hours after the infection (hpi = hours post induction). To prove that the induction was caused by the pathogen infections, control infections with pure water (H ₂ O) were carried out instead of a pathogen application.

Fig. 16 shows the local induction of GUS staining WRKY33 means of Peronospora parasitica infected (right) and non-infected Arabidopsis thaliana plant (left). Part A shows GUS-colored primary leaves, part B corresponding cotyledons. Staining was carried out for 2 hours 2 days after infection.

Fig. 17 shows the local induction of GUS staining of WRKY33 by Alternaria alternata-infected Arabidopsis thaliana plants. Part A shows an overall view of the mycelium infection on Arabidopsis leaves, part B shows a 2.5-fold enlargement of an infection site. Admission was made 9 days after infection.

Fig. 18 shows the local induction of GUS staining of WRKY33 means Sclerotina sclerotiorum-infected Arabidopsis thaliana plants. Part A shows an overall view of the mycelium infection on Arabidopsis leaves two or three days after the infection, part B shows the 5-fold enlargement of an infection site. Admission was made 9 days after infection.

Fig. 19 shows the induction of WRKY33 by GUS staining of Arabidopsis thaliana plants after contact with the Wurzelpathogen Pythium sylvaticum. Part A shows an overall view of uninfected plants (control), Part B plants that were infected with Pythium sylvaticum. The partial images C and D represent detailed images of the root area of infected plants. All images were taken two weeks after the infection.

Fig. 20 shows a table listing the occurring transcription factor binding sites in the promoters of Arabidopsis thaliana WRKY genes 33, 41, 46, 54, 55, 62, 63, 64, 67 and 70. For the investigation, 1.5 kilobase-long promoter sections before the respective transcription start point were taken into account and these were searched with computer support for known transcription factor binding sites.

Fig. 21 shows a schematic representation of Example 3 carried out in accordance promoter analysis. Constructs consisting of WRKY33 promoter portions of different lengths and the beta-glucoronidase reporter gene (uidA) were used for expression studies. A strong induction was found with a 1270 bp promoter portion, while 361 bp was sufficient for a basal induction. No induction was achieved with 331 and 193 long promoter parts.

The terms "homologs" or "homologous sequences" used here denote non-Arabidopsis thaliana-derived nucleic acid or amino acid sequences with significant similarity to the comparison sequence or parts thereof and identical physiological function in other plants. Homologous sequences are therefore nucleic acid sequences with identical physiological function in other plants which hybridize with the comparison sequences or parts of these sequences under stringent conditions (for stringent conditions see Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989), ISBN 0- 87969-309-6). An example of stringent hybridization conditions is: Hybridization in 4 × SSC at 65 ° C (alternatively in 50% formamide and 4 × SSC at 42 ° C), followed by several washing steps in 0.1 × SSC at 65 ° C for a total of about one Hour. Furthermore, nucleic acid or amino acid sequences or parts thereof with identical physiological function in other plants should be considered as homologous sequences, which with the aid of the BLAST similarity algorithm (Basic Local Alignment Search Tool, Altschul et al., Journal of Molecular Biology 215, 403-410 ( 1990) have a significant similarity to comparison sequences: Sequences are described as significantly similar, as used here, which are used, for example, using standard parameters in the BLAST service of the NCBI (http: / /www.ncbi.nlm.nih.govBLAST /) have a level of significance (E-Value or Probability) of P <10 ^-5 when compared with the comparison sequences.

The term "homologs" or "homologous sequences" used here denotes the further nucleic or amino acid sequences or parts thereof, which with the Comparative sequences of at least 70%, preferably 80%, particularly preferably 90% and particularly preferably are 95% identical. The deviations from the Comparison sequences can be by deletion, substitution, insertion, inversion or Recombination occurred.

The term "derivatives" used here denotes nucleic acids, which are also the Carry genetic information for the protein encoded by the comparison sequence, although their Base sequence differs from that of the comparison sequence. In this sense, the The expression "derivatives" includes both introns containing genomic equivalents of RNA, EST or cDNA sequences as well as equivalents of the comparison sequence due to the degeneration  of the genetic code have arisen.

The term "fragments" used here denotes parts of nucleic acid or amino acid sequences, provided that they have at least one functionally important region (domain, sequence or structural motif) of the comparison sequence. Corresponding functionally important areas are in particular elements of DNA binding, e.g. B. (1) a WRKY protein domain which contains the consensus sequence WRKYG, (2) zinc finger motifs with the consensus sequences CX _4-5 -CX _22-23 -HX ₁ -H or CX ₇ -CX ₂₃ -HX ₁ -C, ( 3) Structural elements that contribute to binding to so-called W boxes with the base sequence (T) (T) TGAC (C / T). Other functionally important areas are binding sites for transcription factors, catalytic centers, amino acid motifs interacting with proteins, e.g. B. receptor or ligand binding sites, and phosphorylation acetylation or myristylation sites.

The term "expression" used here means the transcriptional transcription of a genetic information in RNA and, in the case of protein-coding genes, the subsequent one Translation into polypeptides.

The term "enhanced expression" or "enhanced gene expression" as used herein means an increase in the amount of transcript formed to more than 100% compared to that corresponding amount of transcripts in wild type plants.

The term "endogenous" used here denotes biological components such as. B. Gene sequences that come naturally from the very organism in which they are located are integrated or are to be integrated.

The term "exogenous" used here denotes biological components such as. B. Gene sequences that naturally originate from a body other than the organism in which they are integrated or should be integrated.

The term "functionally linked" as used herein means that a regulatory Sequence like a promoter expression of a gene or other functional Controls nucleic acid or that a nucleic acid sequence starting from the promoter is expressed.  

The term "functional nucleic acid" or "functional." Nucleic acid sequence "means a nucleic acid sequence that is not natural for any occurring gene product encoded, e.g. B. a ribozyme, an antisense DNA or RNA or a sequence composed of different exons.

The term "vector" used here means naturally occurring or artificial created constructs for the uptake, multiplication, expression or transfer of Nucleic acids, e.g. B. plasmids, phagemids, cosmids, artificial chromosomes, bacteriophages, Viruses, retroviruses.

The term "transgenic plant" used here refers to plants that are produced using recombinant Genetic engineering and / or microbiological processes and not using conventional ones Breeding methods were produced.

The term "expression system" as used herein means any combination of Vectors, restriction enzymes, transformation methods, cell extracts, living cells e.g. B. prokaryotic or eukaryotic cells or organisms with the purpose of expression of Genes.

It was based on infection experiments with various phytopathogens Surprisingly found that Arabidopsis thaliana a number in the thale cress of WRKY genes with hitherto completely unexplained physiological function by the Infection of the plant can be induced with a pathogen. The induction starts short Time after pathogen contact and is essentially local to the site of the infection limited. In particular, a corresponding pathogen-induced activation of the genes coding for WRKY30, WRKY33, WRKY41, WRKY54, WRKY55, WRKY62, WRKY63, WRKY64, WRKY67 and WRKY70 detected. It was found that the Induction is not limited to a pathogen, but is triggered by various pathogens can be.

The present invention thus relates to transgenic plants with one or more nucleic acid sequence (s) stably integrated into the genome according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, or their homologue or derivative or fragment, or a nucleic acid sequence with identical physiological function which hybridizes with one of these nucleic acid sequences, preferably hybridizes under stringent conditions, and leads to the development of an improved resistance to pathogens in the plants concerned, and methods for the production of the transgenic plants. The above-mentioned SEQ ID NO: 1 to 11 can be assigned to the WRKY genes described in this application according to the following list:
SEQ ID NO: 1 corresponds to the sequence of the gene coding for WRKY30.
SEQ ID NO: 2 corresponds to the sequence of the gene coding for WRKY33.
SEQ ID NO: 3 corresponds to the sequence of the gene coding for WRKY41.
SEQ ID NO: 4 corresponds to the sequence of the gene coding for WRKY46.
SEQ ID NO: 5 corresponds to the sequence of the gene coding for WRKY54.
SEQ ID NO: 6 corresponds to the sequence of the gene coding for WRKY55.
SEQ ID NO: 7 corresponds to the sequence of the gene coding for WRKY62.
SEQ ID NO: 8 corresponds to the sequence of the gene coding for WRKY63.
SEQ ID NO: 9 corresponds to the sequence of the gene coding for WRKY64.
SEQ ID NO: 10 corresponds to the sequence of the gene coding for WRKY67.
SEQ ID NO: 11 corresponds to the sequence of the gene coding for WRKY70.

One or more of the following methods can be used to improve resistance to pathogens, for example:

• a) an increased expression of one or more endogenously present WRKY genes,
• b) introduction and expression of additional WRKY gene copies into the plant genome,
• c) a change in the biological activity of WRKY gene products.

In the case groups a) and b) is compared to wild type plants temporarily or constitutively increased expression intracellularly a larger amount of WRKY- Proteins formed. The WRKY transcription factors interact in the further course of the Reaction chain with promoter elements (so-called W-boxes) that are in front locate and increase genes relevant for defense against pathogens (so-called "defense related genes") by binding their transcription. Due to a higher cellular concentration of WRKY Proteins are also formed and the subsequent products of this metabolic pathway Immune defense of the plant significantly improved. Transgenic plants that are caused by a  increased gene expression or by additional WRKY gene copies one over the Wild-type increased amount of WRKY transcripts or their fragments or homologues or produce derivatives show a significantly improved pathogen defense.

An increased expression can accordingly z. B. by combining the nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or whose homolog or derivative or fragment can be achieved with a strong promoter. Examples of suitable promoters which lead to an increased constitutive expression of a CaMV are the WRKY gene or its homolog or fragment or derivative 35S promoter from the cauliflower mosaic virus and the ubiquitin promoter from maize. Either the endogenous promoter that expresses one of the Nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their homolog or derivative or fragment controls by the strong Promoter can be replaced, or there can be at least one additional copy of one of the Nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their homolog or derivative or fragment integrated into the genome with the additional copy (s) functionally linked to the strong promoter Is (are).

Enhanced gene expression of the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their fragment or homolog or derivative can also by changing or adding functional components of the naturally the expression of the WRKY gene regulating promoter can be achieved. Essential components of a typical eukaryotic promoter are state of the art and can be found in relevant textbooks. Examples of corresponding functional Components are TATA-Box, CAAT-Box, GC-Box, enhancers or binding sites for others Transcription factors. Suitable methods for changing the nucleic acid sequence of the naturally the expression of the WRKY gene regulating promoter are the expert known. Known mutagenesis techniques are, for example, the generation of Deletion mutants or the introduction of point mutations through "Site Directed  Mutagenesis ". Preferred methods for mutagenesis or for changing expression are furthermore chimeric RNA / DNA oligonucleotides, homologous recombination or RNA interference (RNAi).

Enhanced expression can also be achieved by adding more than one additional copy the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their homolog or derivative or fragment, in particular two, three, four, five or more copies to be integrated into the genome. These copies can be made individually or be integrated together. Will be more than one of the listed Integrated nucleic acid sequences, so several identical copies or a mixture of the nucleic acid sequences mentioned are used. Furthermore, the copies are marked with a Functionally linked promoter, which is a strong promoter, an endogenous or an exogenous one Expression of the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their homolog or derivative-controlling promoter, or a can be tissue-specific or inducible promoter.

Constructs comprising a corresponding promoter and one functional with it linked nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their homologue or derivative or fragment can by Common methods for the transformation of plant cells and subsequently for the regeneration of whole plants can be used. The introduction of nucleic acid sequences in Vegetable organisms and cells are state of the art and used, for example of T-DNA vectors, easy to do. Due to the increased expression of Nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their homolog or derivative or fragment is added accordingly Multiple of proteins encoded by these nucleic acid sequences.

According to case group c) there is a change in the biological activity of the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their homolog or derivative or fragment-encoded gene product due to changes in gene segments which are necessary for functional protein domains such as e.g. , B. encode receptor binding sites, phosphorylation domains or DNA binding sites. Modifications in the WRKY DNA binding domain in particular have a considerable influence on the properties of the protein. In this way, proteins can be formed with one or more amino acids which have been modified compared to the wild type and which, for. B. have a changed substrate specificity or a changed K _m value or are no longer subject to the regulatory mechanisms normally present in the cell via allosteric regulation or covalent modification. Correspondingly modified WRKY proteins could accordingly. B. perform the functions of the WRKY wild type protein in an improved manner. In this sense, in particular modifications which result in the replacement of one or more of the following amino acids by others, the loss of one or more of the following amino acids or the integration of one or more amino acids in the following protein regions can lead to a correspondingly improved function of the protein :

Suitable methods for changing the sequence of the WRKY gene are known to the person skilled in the art known. Known mutagenesis techniques are, for example, the generation of Deletion mutants or the introduction of point mutations through "Site Directed Mutagenesis ". Preferred methods for mutagenesis or for changing expression are chimeric RNA / DNA oligonucleotides, homologous recombination or RNA Interference (RNAi). Show transgenic plants with such modified WRKY genes also improved resistance to pathogens.

Those used in the method according to the invention and in transgenic plants  Nucleic acid sequences can be of natural origin or have been produced artificially his.

Nucleic acid sequences with an identical physiological function to the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 can easily from other organisms with the help of special, belonging to the state of the art PCR, hybridization or screening methods are isolated. For example, the nucleic acid sequences listed above as a probe for homology screening in DNA Libraries, more similar with the help of hybridization to single-stranded nucleic acids Base sequence can be used. Furthermore, by knowing the base sequence of the nucleic acid sequences listed above primers are constructed with the help of which Amplification of functionally identical PCR fragments from other organisms is possible.

The with the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their homolog or derivative or fragment functional linked regulatory DNA sequence, e.g. B. a promoter, can both with Nucleic acid sequence endogenously present regulatory DNA sequence according to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 as well as its homolog or fragment as well as any other regulatory DNA sequence. The regulatory DNA sequence can be either an endogenous promoter of the to be transformed organism or an exogenous promoter which z. B. on the vector used. In principle, any regulatory sequence is the promoter suitable for the expression of foreign genes in organisms, e.g. B. plants can control, e.g. B. the CaMV 355 promoter from the cauliflower mosaic virus; see. Franck et al., Cell 21, 285- 294 (1980). The expression of the nucleic acid sequences can also by a chemical inducible promoter can be achieved. Examples of chemically inducible promoters are the PRPI promoter (Ward et al., Plant Molecular Biology 22, 361-366 (1993)), a by Salicylic acid inducible promoter (WO 95/19443), an inducible by benzenesulfonamide Promoter (EP-A 388186), a tetracycline-inducible promoter (Gatz et al., Plant Journal 2, 397-404 (1992)), an abscisic acid inducible promoter (EP-A 335528) or a promoter inducible by ethanol or cyclohexanone (WO 93/21334). It can  promoters are also used which, for. B. in certain plant tissues or Plant parts are active. Examples of corresponding promoters are the phaseolin promoter (US 5504200), the isoflavone reductase promoter (US 5750399), a seed-specific Promoter, e.g. B. from tobacco (US 5824863) or the ST-LSI promoter from potato (Stockhaus et al., (1989) EMBO J 8, 2445-2452).

The method according to the invention for time-controlled pathogen defense is in this sense suitable by the WRKY-specific promoter against an inducible promoter is exchanged. Through a time-controlled induction of the pathogen defense z. B. on the seasonal occurrence of pathogens.

Endogenous or exogenous nucleic acid sequences can be used for the methods according to the invention according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or whose fragments or derivatives or homologs are used. Endogenous means that the Nucleic acid sequence comes from the same organism in which it is associated with the the inventive method is integrated, for. B. a WRKY33 gene from Arabidopsis thaliana is integrated into Arabidopsis thaliana using the method according to the invention. Exogenous means that the nucleic acid sequence comes from another organism, e.g. B. a WRKY33 gene from Arabidopsis thaliana with the method according to the invention in z. B. Wheat integrated. After the stable integration of the transformed nucleic acid sequence there are two or more WRKY33 genes in the genome, so that an increased amount of corresponding Gene products is expected. In a preferred embodiment, the Nucleic acid sequences compared to the naturally occurring nucleic acid sequences Deletions, substitutions, additions, insertions and / or inversions.

For the purposes of the invention, the transgenic plants can not only be produced Polynucleotides with the cDNA sequences of the WRKY genes according to SEQ ID NO: 1 to 11 or their fragments or derivatives or homologs are used, but also Polynucleotides that also contain the introns. The introns can do that naturally have occurring or other sequences.

The present invention further relates to a transformed cell, in particular a transformed plant cell or a transformed plant tissue in which the  Nucleic acid sequence according to the invention according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their fragments or derivatives or homologs, stable are integrated. Furthermore, the present invention relates to one with the invention Nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their fragments or homologs or derivatives Plant cell or a transformed plant tissue that or to a fertile plant is regenerable. In particular, the present invention relates to a plant according to the method according to the invention is available. The present invention further relates to seeds, which is obtained from plants obtained by the process according to the invention. The invention further relates to the fruits, seeds, leaves and shoots produced by the plants and storage devices, e.g. B. fruits, berries, grapes, cereals and potatoes.

In a preferred embodiment of the method according to the invention, a Nucleic acid sequence used that consists of nucleic acid fragments of various WRKY genes is composed. This composite nucleic acid sequence can also also Contain modifications such as deletions, additions or substitutions. The procedures for Preparation of such composite nucleic acid sequences is state of the art and of Expert easy to do.

The method according to the invention can be applied to all plant organisms. In particular, the method according to the invention for the production of pathogen-resistant mono- and dicotyledonous plants can be used. Plants are particularly preferred Cultivated plants z. B. coffee bush, tea bush, soybean, cotton, flax, hemp, sunflower, Potato, tobacco, tomato, bell pepper, zucchini, eggplant, cucumber, summer rape, winter rape, Alfalfa, lettuce, chicory, asparagus, salsify, pea, bean, lentils, carrot, onion, Garlic, leek, olive, radish, radish, beetroot, turnip, kohlrabi, sugar beet, Beet, sugar cane, spinach, chard, the various tree, nut and wine species, the various types of cabbage such as Brussels sprouts, cauliflower, broccoli, white cabbage, red cabbage, savoy, Chinese cabbage, also cereals e.g. B. barley, hops, wheat, rye, oats, corn, rice, Forage grasses, also types of fruit z. B. mango, apple, pear, peach, plum, raspberry, Blueberry, blackberry, gooseberry, strawberry, cherry, currant, guava, banana, Melon, pumpkin and citrus z. As lemon, orange, grapefruit or tangerine as well  Ornamental plants such as B. cyclamen, aster, bougainvillea, chrysanthemum, petunia, marguerite, Narcissus, snowdrops, delphinium, sunflower, geranium, goose cress, gerbera, Gladiolus, iris, hyacinth, lily, magnolia, orchid, rhododendron, rose, bleeding heart, Tulip, poinsettia, and finally all timber, coniferous and deciduous tree species such as B. Maple, birch, beech, yew, oak, ash, spruce, pine, linden, mahogany, poplar, fir, teak. The plants transformed with the nucleic acid sequence can be unmodified Wild type plants or plants obtained by breeding or already modified otherwise Plants z. B. be transgenic plants. The method according to the invention can also in Plant tissues and in plant cells, e.g. B. in a cell culture or in expression systems, can be used to increase pathogen resistance.

Since the pathogen defense based on WRKY proteins causes not only specific but also specific defense reactions, the method according to the invention can be used to improve the general resistance to a large number of plant pathogens. In particular, the method according to the invention can be used to protect against the following plant diseases, but is not limited to these:
Cabbage hernia, powdery scab of potatoes, powdery mildew, downy mildew, late blight on herbs and tubers, powdery mildew, snow mold in cereals, ergot, root rot on rapeseed, net blotch disease, Septoria leaf drought, Septoria leaf blemishes and browning, brown rust, yellow stone, brown stone, Zwandanderg rust Flying barley, corn bump fire, typhula rot on barley, root killers on potatoes, parasitic stalk fracture, Rhynchosporium leaf spots, Cercospora leaf spots, scab, gray mold, Alternaria leaf spot disease / rot, stem rot, core rot, leaf and stem cancer, potato blight Botrytis fruit rot, Colletotrichum fruit rot, strawberry powdery mildew, Gnomonia fruit rot, Phytophthora Lederbeerenfäule, Pseudomonas bacterial leaf spot, phytophthora crown rot, black scurf, Red root rot, Rotfleckenkrankheit, slime mold, verticillium wilt, soft rot of strawberry, white spot disease, cauliflower disease e blotchy leaf blotch disease and all viroses.

Examples of corresponding pests which cause these and similar diseases and can be controlled by the method according to the invention are

• A) Phytopathogenic nematode species, especially root nematodes, e.g. B. bile-forming root nematodes from the genus Meloidogyne, freely migrating root nematodes from the genera Pratylenchus and Paratylenchus, cyst-forming root nematodes from the genera Heterodera and Globodera; Stick and stem nematodes, e.g. B. from the genus Ditylenchus, leaf nematodes, z. B. from the genus Aphelenchoides and flower nematodes, for. B. from the genus Anguina, also virus-transmitting nematodes, for. B. from the genus Xiphinema and nematodes from the genus Trichodorus.
  Relevant representatives of these genera, to which the method according to the invention can be applied, are e.g. B. Aphelenchoides ritzemabosi, fragariae, Aphelenchoides, Anguina gram ines, Anguina tritici, Globodera rostochiensis, Globodera pallida, Heterodera avenae, Heterodera cruciferae, Heterodera glycines, Heterodera schachtii, Heterodera rostochiensis, Meloidogyne javanica, Meloidogyne incognita, Pratylenchus zeae, Pratylenchus hexincisus, Pratylenchus penetrans, Pratylenchus fallax, Pratylenchus pseudocoffae, Pratylenchus coffae, Pratylenchus gutierrezi, Pratylenchus loosi, Pratylenchus neglectus, Pratylenchus vulnus, Pratylenchus thornei, Pratylenchus scribneri, Trichodorus primitivus, Xiphinema.
• B) Phytopathogenic mushrooms, e.g. As from the genera Albugo, Alternaria, Aphanomyces, Ascochyta, Aureobasidium, Botrytis, Cephalosporium, Cercospora, Cladosporium, Claviceps, Colletotrichum, Cylindrosporium, Diachea, Diplocarpon, Erysiphe, Fusarium, Gnomonia, Helminthosporium, Leptosphaeria, Macrophomina, Microthecium, Microdochium, Mucor , Mycosphaerella, Oidium, Ophiobolus, Peronospora, Plasmodiophora, Plasmopora, Pseudocercosporella, Puccinia, Pyrenophora, Pythium, Phytophtora, Ramularia, Rhizoctonia, Rhynchosporium, Sclerophtora, Sclerhaophops, Sporia, Sporia , Venturia, Verticillium.
  Relevant representatives of these genera, to which the method according to the invention can be applied, are e.g. B. Albugo candida, Alternaria alternata, Alternaria brassicae, Alternaria brassicicola, Alternaria solani, Aphanomyces cochlioides, Ascochyta hordei, Botrytis cinerea, Cephalosporium gramineum, Cercospora beticola, Claviceps purpurea, Colletotrichum trifolioriaaroparodirocarpus, cruciformia, cephalopods, diarrhea, diarrhea, diarrhea, diarrhea, diarrhea, diarrhea, diarrhea Erysiphe graminis, Fusarium ambrosium, Fusarium culmorum, Fusarium graminearum, Fusarium moniliforme, Fusarium oxysporum, Fusarium solani, Gnomonia fruticola, Helminthosporium avenae, Helminthosporium gramineum, Helminthosporium sativumporium Mucor piriformis, Mycosphaerella brassicola, Mycospaerella fragariae, Myrothecium roridum, Oidium cyclaminis, Oidium hortensiae, Oidium lycopersici, Oidium chugging, Ophiobolus graminis, Peronospora parasitica, Plasmodccorccinora pomorodinora pore, puodophora pellora hora ia recondita, Puccinia coronata, Puccinia graminis, Puccinia striifomis, Phytophthora cactorum, Phytophthora fragariae, Phytophthora infestans, Pseudocercosporella caps harboring Pseudocercosporella herpotrichoides, Pyrenophora teres, Ramularia armoraciae, Ramularia beticola, Rhynchosporium secalis, Rhizoctonia cerealis, Rhizoctonia solani, Sclerophtora macrospora, Sclerotinia sclerotiorum , Septoria avenae, Septoria tritici, Septoria nodorum, Sphaeropsis sapinea, Sphaerotheca humuli, Sphaerotheca pannosa, Spongospora subteranea, Thielaviopsis basicola, Tilletia caries, Tilletia contraversa, Typhula gyrans, Typhula incarnula, Uagoiagostilago Uagoiagooragostilago, Uagoiago nuda, Ustilago tritici, Venturia inaequalis, Verticillium dahliae.
• C) Phytopathogenic bacteria, e.g. B. from the genera Agrobacterium, Erwinia, Pseudomonas, Ralstonia, Xanthomonas.
  Relevant representatives of these genera, to which the method according to the invention can be applied, are e.g. B. Agrobacterium tumefaciens, Agrobacterium rhizobacter, Erwinia chrysanthemi, Pseudomonas syringae, Ralstonia solanacearum, Xanthomonas campestris.
• D) Phytopathogenic viruses, including single-stranded RNA viruses, viruses with double-stranded RNA (wound tumor viruses), plant viruses with circular single-stranded DNA (Gemini viruses), plant viruses with double-stranded DNA and viroids.
  As typical representatives of phytopathogenic viruses z. B. Abutilon mosaic virus, arabic mosaic virus, barley yellow dwarf virus (BYDV), barley yellow mosaic virus (BaYMY, BaYMY 2), bean golden mosaic virus, cassava latend virus, beet western yellows virus, cauliflower mosaic virus, grapevine fanleaf virus, maize streak virus, potato spindle tuber virus, raspberry ringspot virus, rice yellow mottle virus, strawberry crinkle virus, soil-borne wheat mosaic virus (SBWMV), strawberry yellow edge virus, strawberry mottle virus, tabac mosaic virus, tomato golden mosaic virus, turnip mosaic virus, wheat spindle streak mosaic virus (WSSMV), wheat yellow mosaic virus (WYMV).
• E) Other animal pests in crops, z. B. Psylliodes chrysocephala, Ceutorhynchus pleurostigma, Ceutorhynchus napi, Ceutorhynchus quadridens, Ceutorhynchus picitaris, Ceutorhynchus assimilis, Melighetes aeneus, Delia brassicae, Dasineura brassicae and all types of insect pests in cereals.

The present invention further relates to the nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 or their homologue or derivative or fragment.

The present invention further relates to vectors comprising a nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their Homologous or derivative or fragment, and a regulatory DNA sequence.

Suitable vectors for recording and transferring the nucleic acid sequences can be the Propagation and / or expression of the nucleic acids taken up in single cells such as. B. Escherichia coli or Agrobacterium tumefaciens or in plant cells, plant tissues or Ensure plants. Corresponding vectors can of course occur or else be made artificially. The vectors can contain selection markers, terminator sequences, Polylinkers, promoter elements, enhancers, polyadenylation sites and other genetic Include items. Vectors suitable for cloning are e.g. B. Plasmids of pBluescript Series, plasmids of the pUC series, plasmids of the pGEM series or on the bacteriophage λ based vectors. A plasmid vector used in Agrobacterium is e.g. B. pBin19; see. Bevan et al., (1984), Nucleic Acids Research 12, 8711-8721. For transformation and expression in plants are based on or from the Ti plasmid of Agrobacterium species Constructs that construct plant viruses are usable vectors and are known to the person skilled in the art known. Furthermore, certain transformation methods such. B. Microinjection, protoplast transformation and injection (microprojectile bombardment) instead of Ti plasmids also the above-mentioned cloning vectors or linearized DNA used. A summary description of vectors used so far can be found in Guerineau and Mullineaux, Plant Transformation and Expression Vectors, in: Plant Molecular Biology Labfax, edited by Croy, Oxford, BIOS Scientific Publishers, 121-148 (1993).  

Some of those used in commercial transformation and expression systems Transformation methods for the transfer of foreign genes (transformation) into the genome of plants are presented below. Choosing the method of contributing The nucleic acid sequence according to the invention and that which is functionally linked to it for a However, gene product-encoding nucleic acid sequences in plant cells is not based on this Limited list. So far, transformation processes used in plants are e.g. B. the Gene transfer using Agrobacterium tumefaciens (e.g. bathing seeds, inflorescences or leaf fragments in an agrobacterial solution), by means of plant viruses Electroporation, by microprojectile bombardment or injection (Microinjection) and the incubation of dry embryos in DNA-containing liquids and the transformation of protoplasts with the help of polyethylene glycol. more accurate Descriptions of the mentioned methods can be found e.g. B. in Jens et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by Kung and Wu, Academic Press 128-143 (1993).

Another aspect of the present invention relates to regulatory Nucleic acid sequences (hereinafter also referred to as promoters), which are naturally found in Arabidopsis thaliana control the expression of the WRKY genes. This invention Nucleic acid sequences are under SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

The above-mentioned SEQ ID NO: 12 to 23 can be assigned to the sequences for WRKY promoters described in this application according to the following list:
SEQ ID NO: 12 corresponds to a 1270 bp fragment of the WRKY33 promoter.
SEQ ID NO: 13 corresponds to a 361 bp fragment of the WRKY33 promoter.
SEQ ID NO: 14 corresponds to the overall sequence of the WRKY33 promoter.
SEQ ID NO: 15 corresponds to the overall sequence of the WRKY41 promoter.
SEQ ID NO: 16 corresponds to the overall sequence of the WRKY46 promoter.
SEQ ID NO: 17 corresponds to the overall sequence of the WRKY54 promoter.
SEQ ID NO: 18 corresponds to the overall sequence of the WRKY55 promoter.
SEQ ID NO: 19 corresponds to the overall sequence of the WRKY62 promoter.
SEQ ID NO: 20 corresponds to the overall sequence of the WRKY63 promoter.
SEQ ID NO: 21 corresponds to the overall sequence of the WRKY64 promoter.
SEQ ID NO: 22 corresponds to the overall sequence of the WRKY67 promoter.
SEQ ID NO: 23 corresponds to the overall sequence of the WRKY70 promoter.

The invention further relates to fragments or homologs of the nucleic acid sequence according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23, which have the biological function of a promoter. The invention further relates to Nucleic acid sequences, which with one of the nucleic acid sequences according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 hybridize and have the biological form of a promoter. Are preferred Nucleic acid sequences under stringent conditions with either of these Hybridize nucleic acid sequences.

The regulatory nucleic acid sequences according to the invention according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their Fragments or homologs are suitable for the specific control of the expression of genes or other functional nucleic acids in organisms or cells, preferably for specific control of genes or genes involved in pathogen defense, coding for toxic components. After the stable integration of the transformed nucleic acid sequence then there are two or more such promoters in the genome, so that the downstream ones Nucleic acid sequences have an expression regulation corresponding to these promoters.

Due to the immediate and local induction of the WRKY Transcription factors can be obtained by combining the regulatory Nucleic acid sequences according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their fragments or homologues with a toxic component or other candidate for defense against pathogens Genes e.g. B. efficient broadband resistances are built, their effect on the Site of infection is limited. Thus, the pathogen can be combated without damaging the unaffected plant parts guaranteed.  

The regulatory nucleic acid sequence according to the invention according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their fragment or homologously linked functional nucleic acids encode particularly preferably for gene products from the following list:
Structural proteins with repetitive peptide motifs Ser-Hyp ₄ , Gly-X or Val-Tyr-Lys-Pro-Pro. Structural proteins that contain repetitive peptide motifs with greater proportions of hydroxyproline (Hyp), proline (Pro) or glycine (Gly) can strengthen, change or repair the plant cell wall in the course of an infection and thus represent a barrier to the further penetration of the pathogen.

Hydrogen peroxide producing enzymes. Various proline-rich cell wall proteins are oxidatively linked to other proteins by H ₂ O ₂ after pathogen attacks and in this way strengthen the cell walls. Furthermore, aggressive oxygen derivatives in the context of the plant's hypersensitive immune response contribute to the death of the infected plant cells and help in the direct killing of the pathogen.

Lignin and callose producing enzymes. A frequently used defense strategy of the plant consists in the synthesis of polymeric cell wall components such as lignin or callose, which are incorporated into the Cell wall are installed and serve as a mechanical barrier against the pathogen. On An example of this class of substances is the "catalytic UE of callose synthase" (NCBI Acc. NO. AF085717).

Chitinases. Chitinases break down the carbohydrate polymer chitin while absorbing water. chitin is an essential scaffold z. B. many phytopathogenic fungi. An example of this Substance class is "Chitinase 2" (NCBI Acc. NO. AF241267).

β-1,3-glucanases. β-1,3-glucanases cleave β-1,3-glucan while taking up water. This too Polymer occurs as a scaffold in phytopathogenic fungi.

Antipathogenically active enzyme inhibitors. The cell wall-bound can be used here as an example Polygalacturonidase inhibiting protein (PGIP), for example from the kidney bean Phaseolus vulgaris, to be named. This inhibits the action of the harmful fungus Fusarium monoliform  produced enzyme α-1,4-D-polygalacturonidase, which the fungus used to dissolve the Bean cell walls used.

Thionins. Thionins are small basic and cysteine-rich proteins that have been predominantly found in Cereals have been detected. An antifungal effect of thionines is experimental proven and based on the ability of this class of proteins to destroy membranes.

Lectins with chitin-binding domains. Lectins are proteins with the ability to be highly specific bind to carbohydrates. An antifungal lectin with a chitin binding domain (NCBI Acc. NO. AAA34219) could be isolated from nettle rhizomes.

Ribosome Inactivating Proteins (RIPs). Ribosome inactivating proteins (see e.g. Leach et al., J. Biol. Chem. 266, 1564-1573, 1990) more inhibit protein synthesis Fungi and thus prevent their further growth. Plant's own ribosomes are due to the different structure not damaged.

PR proteins ("pathogenesis related"). The class of PR proteins (see e.g. Bol et al., Ann. Rev. Phytopathol. 28, 113-138, 1990) contains vegetable proteins found in the healthy Plant does not occur, but can be synthesized after pathogen attack. As examples can be mentioned here: PR-1 from Solanum tuberosum (NCBI Acc. NO. CAB58263), pathogenesis-related protein 4 from Hordeum vulgare (NCBI Acc. NO. T06171), Transcription factor Pti6 from Solanum tuberosum (NCBI Acc. NO. T07728), thaumatin similar PR proteins, e.g. B. from Hordeum vulgare (NCBI Acc. NO. CAB99485).

Enzymes for the synthesis of phytoalexins. Phytoalexins are plant secondary metabolites with strong antimicrobial efficacy that accumulates around the site of infection. These include, for example, phenol derivatives, isoflavonoids and sesquiterpenes, e.g. B. Chlorogenic acid, caffeic acid, scopoletin, medicarpin, glyceollin I, rishitin, gossypol, lubimin, Capsidiol, Orchinol, Pisatin, Momilacton, Resveratol, Safynol.

Enzymes for the synthesis of saponins. Saponins are steroid glycosides, the fungal membranes can destroy by binding to sterols.

R gene-gene products. R genes (resistance genes) code for proteins directly or indirectly  about other proteins with gene products from avr genes (avirulence genes) of the pathogen interact and to programmed cell death of infected plant cells at the Lead infection site, e.g. B. NCBI Acc. NO. BE039015 (downy mildew resistance protein rpp5), NCBI Acc. NO. AF122994 (RPM1 variant gene, NCBI Acc.NO.AF098962 (disease resistance protein RPP1-WsA gene).

avr gene products. avr gene products are encoded by the pathogen's avirulence genes and activate the in the course of an infection through contact with herbal R-gene products Defense against pathogens and programmed cell death (apoptosis) of plant cells on Of infection.

Enzymes for the synthesis of salicylic acid. If a plant is infected by a pathogen survived, it often developed resistance to a wide range of pathogens. This So-called "systemic acquired resistance (SAR) is generated by the signaling molecule salicylic acid mediates, first in the infection zone and later in different parts of the plant is formed. The metabolic pathway underlying salicylic acid synthesis starts from Phenylalanine from and is known to the person skilled in the art.

Enzymes for the synthesis of jasmonic acid. Jasmonic acid can create resistance for example against Phytophtora infestans can be used by the jasmonic acid is applied externally. In the organism, jasmonic acid is injured when the plants, e.g. B. after contact with the pathogen, produces and activates various factors relevant for the defense against pathogens Genes, e.g. B. proteinase inhibitors, thionins and plant defensin genes such. B. PDF1 (described in WO98 / 00023). Herbal defensins are a family of cysteine-rich basic proteins with structural similarity to antimicrobial active defensins, which in various insect species have been detected. That of jasmonic acid synthesis underlying metabolic pathway starts from linolenic acid and is known to the person skilled in the art.

In a particularly preferred embodiment of the method according to the invention, further increase the efficiency of pathogen defense by using several, i.e. H. two, three or many different gene promoter combinations, each consisting of one Regulatory nucleic acid sequence according to the invention according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their  Fragment or homologue and in each case a gene involved in the defense against pathogens or a Gene coding for toxic components according to the above list, in the plant Genome to be integrated. For example, a combination 1 consisting of a regulatory nucleic acid sequence according to the invention and an R gene, together with a combination 2 consisting of a regulatory according to the invention Nucleic acid sequence and an avr gene, are integrated into the plant genome, so that is strengthened by integrating both gene-promoter combinations Results in action against a pathogen.

Those with the regulatory nucleic acid sequence according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ 117 NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their fragment or homologue of functionally linked genes or other functional genes Nucleic acids can be endogenous or exogenous genomic DNA sections or cDNAs or their fragments or derivatives or synthetic or semi-synthetic nucleic acids his. Endogenous means that the nucleic acid sequence from the same organism comes in which it is integrated with the inventive method, for. Legs Nucleic acid sequence from Arabidopsis thaliana is in with the inventive method in Arabidopsis thaliana integrated. Exogenous means that the nucleic acid sequence consists of one other organism, e.g. B. a nucleic acid sequence from Arabidopsis thaliana with the inventive method in a crop, e.g. B. wheat, integrated. The functionally linked genes or other functional nucleic acid sequences compared to the naturally occurring nucleic acid sequences deletions, substitutions, Have additions, insertions and / or inversions.

Furthermore, the regulatory nucleic acid sequence according to the invention is also suitable for the Regulation of expression of other gene sequences. The promoter can be used in combination with any genes are present, both in a vector and in transgenic organisms.

Nucleic acid sequences with an identical physiological function to the nucleic acid sequences according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 can easily be obtained from other organisms with the help of special, to the state of Technique belonging to PCR, hybridization or screening methods are isolated.  

For example, the nucleic acid sequences listed above can be used as a probe for the Homology screening in DNA libraries, using hybridization to single-stranded Nucleic acids of a similar base sequence can be used. Furthermore, through the Knowledge of the base sequence of the nucleic acid sequences primer listed above are used to amplify PCR fragments coding for homologous Gene products from other organisms is possible.

The regulatory nucleic acid sequences according to the invention according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their Fragments or homologues can be of natural origin or have been produced artificially his. The functionally related genes or the other functional ones Nucleic acid sequences can both in the direction of the natural transcription Sense as well as reverse antisense orientation.

The regulatory nucleic acid sequence according to the invention according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ 117 NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their Fragments or homologs can be found in vectors, expression systems or plants, Plant tissues or plant cells or animal cells or microorganisms for Changes in the expression pattern of various gene products can be used. The Expression of the gene products can be compared to their natural expression be reinforced as well as reduced.

The present invention accordingly further relates to vectors comprising a regulatory Nucleic acid sequence according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ NO: 19, SEQ ID NO: 20, SEQ ID NO: 1, SEQ ID NO: 22 or SEQ ID NO: 23 or their fragment or homolog.

Suitable vectors for recording and transfer of the nucleic acid sequences and some of those used in commercial transformation and expression systems Transformation methods for the transfer of foreign genes (transformation) into the genome of plants have already been mentioned above.  

The present invention further relates to a method for producing a plant with altered gene expression, comprising the stable integration of a regulatory sequence according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or its fragment or homolog with the biological function of a Promotors and one functionally linked to this sequence for a gene product coding nucleic acid sequence in the genome of plant cells or plant tissues and regeneration of the plant cells or plant tissues obtained to plants.

Endogenous or exogenous nucleic acid sequences can be used for the methods according to the invention according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their fragments or homologs can be used. After the stable Integration of the transformed nucleic acid sequence then lies two or more WRKY33 Promoters in the genome so that several genes are regulated by these promoters. In In a preferred embodiment, the nucleic acid sequences are opposite to those of course occurring nucleic acid sequences deletions, substitutions, additions, insertions and / or inversions.

The method according to the invention can be applied to all plant organisms. In particular, the method according to the invention for the production of pathogen-resistant mono- and dicotyledonous plants can be used. Again, plants are particularly preferred the crops already listed above.

The invention further relates to transgenic plants with a stably integrated into the genome regulatory nucleic acid sequence according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their fragment or Homologous to and with the biological function of a promoter Nucleic acid sequence functionally linked coding for a gene product Nucleic acid sequence corresponding to the examples listed and described above for such coding nucleic acid sequences.

The present invention further relates to a transformed cell, in particular a  transformed plant cell or a transformed plant tissue in which the Nucleic acid sequence according to the invention according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their fragments or Homologues that are stably integrated. Furthermore, the present invention relates to a Nucleic acid sequence according to the invention according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 or their fragments or Homologous transformed plant cell or plant tissue, the or that can be regenerated into a fertile plant. In particular, the present invention relates to a Plant which can be obtained by the process according to the invention. Furthermore, the present invention seed obtained from plants grown after the Process according to the invention can be obtained. The invention further relates to that of Plants produced fruit, seeds, leaves, shoots and storage organs, e.g. B. fruit, berries, Grapes, cereals and potatoes.

The plants transformed with the nucleic acid sequence can be unmodified Wild type plants or plants obtained by breeding or modified plants z. B. be transgenic plants. The method according to the invention can also be used in plant tissues and in plant cells, e.g. B. in a cell culture or in expression systems to reinforce Pathogen resistance can be applied.

Since the pathogen defense based on WRKY proteins is not only specific but also specific Defense reactions causes, the inventive method for improving the apply general resistance to a variety of plant pathogens. In particular, this method according to the invention can also provide protection against the Plant diseases and pests described above (nematodes, fungi, bacteria, Viruses and other animal pathogens of the types already mentioned) used are, but is not limited to, these.

The invention is illustrated by the examples which now follow, but is not limited to these limited:  

example 1 Quantitative RT-PCR

For the identification of WRKY genes, which are used during the defense against Arabidopsis plants Peronospora parasitica, a quantitative RT-PCR approach was chosen and so the expression of specific WRKY genes checked.

First the pathogen Peronospora parasitica pv Cala was grown on Ler-1 (Arabidopsis thaliana var. Landsberg erecta) plants and the sporangiophores and spores were harvested by cutting off the leaves and cotyledons of the Ler-1 plants. To get the spores in solution, the leaves and cotyledons were shaken in sterile-filtered, double-distilled water. The spores were pelleted by centrifugation of 4000 × g at 10 ° C. for five minutes and taken up in new, double-distilled water. A spore solution of 2 × 10 ⁵ spores / ml was counted and set using a counting chamber under the microscope. The spores were then sprayed onto two-week-old plants grown under short day. Plants were harvested at approximately 1 to 2 hour intervals and frozen in liquid nitrogen. This plant material was used for RNA isolation. The Quiagen-RNA / DNA-Maxi-Kit was used for this RNA isolation and proceeded exactly according to the manufacturer's instructions. For the RT-PCR approach, RNA quantities between 10 pg and 2 µg were used. The concentration of the RNA was determined photometrically at 260 nm. In a first run, different amounts of RNA were used in the RT-PCR batches in order to determine the optimal concentration to be used for a particular WRKY-RNA. This ensured that the amount of cDNA obtained in the RT-PCR was still in the exponential and not already in the saturated range. The RT-PCR batches were all carried out using the one-step RT-PCR kit (Qiagen). The manufacturer's instructions were followed closely. The program in the thermal cycler (model PTC225 from MJResearch) was entered as follows: 30 min 50 ° C; 15 min 95 ° C; 35 cycles with 40 sec 94 ° C, 1 min 55 ° C or 65 ° C depending on the primer sequence (AtWK55-3 and AtWK55- 6: 65 ° C; all other primers: 55 ° C), 1 min 72 ° C ; followed by 10 min 72 ° C and then 4 ° C until the batches are removed. The following primers were used (sequence from 5 'to 3'):

The cDNA obtained was separated on a 0.8-1% agarose gel in TAE and photographed. Analysis of some Arabidopsis WRKY genes showed that during the Defense reaction of the Arabidopsis thaliana Col-0 plants against Peronospora parasitica pv. Cala2 can be induced. Other WRKY genes were not induced. The induced WRKY Genes were the following: WRKY30, WRKY33, WRKY41, WRKY46, WRKY54, WRKY 55, WRKY62, WRKY63, WRKY64, WRKY67, WRKY70.

Example 2 Northern analysis

For further detection of the induction of WRKY33 by the infestation of Peronospora parasitica, Northern analyzes were carried out. To do this, RNA became two weeks old Arabidopsis plants sprayed with Peronospora parasitica pv.Cala (ecotype Col-0) isolated. The Quiagen-RNA / DNA-Maxi-Kit was used for this RNA isolation and exactly proceed according to the manufacturer's instructions. 20 µg RNA per lane were placed on a formamide Agarose gel applied and applied with a voltage of 1 volt per cm electrode gap Electrophoretically separated at night. The RNA was then placed on a PALL-B membrane (Pall Corp., New York, USA) blotted, also with the manufacturer's information were adhered to exactly. 20xSSC (175.3 g / l NaCl, 88.2 g / l  Sodium citrate, pH 7.0) and blotted capillary for 24 hours. The RNA was afterwards heat-fixed to the membrane by baking the membrane at 80 ° C for 3 hours. In order to show the expression of the WRKY33 gene, the membrane was covered with a WRKY33 specific probe hybridized. For this purpose, a 350 bp fragment was PCR amplified and this after elution with the QuiaexII gel manufactured by Quiagen Extraction kit (method according to the manufacturer's instructions) marked with radioactive P32-dCTP. The Ready-To-G ™ kit from Amersham Pharmacia Biotech was used for the labeling reaction Inc. used. Hybridization took place overnight in 12% dextran sulfate buffer (1 M NaCl; 1% SDS; 12% dextran sulfate) at 65 ° C and became stringent with 2xSSC / 0.5% SSC at 62 ° C washed. It was then determined by means of autoradiography whether induction of the Expression of WRKY33 in those sprayed with the avirulent Peronospora strain Cala2 Arabidopsis plants existed. A clear signal in the case of Peronospora parasitica pv Plants sprayed with Cala2 were detectable 2 hours after contact with the pathogen. Was against no signal recognizable in Arabidopsis plants which were only sprayed with water. from that follows that WRKY33 in the defense reaction of the Col-0 plants against Peronospora parasitica Cala2 is induced.

Example 3 Promoter-GUS constructs

In order to determine whether other pathogens also influence WRKY gene expression, promoter-GUS constructs were produced and stably integrated into the genome of Arabidopsis plants. In this way it could be shown that very different pathogens, e.g. B. Peronospora, Pythium, Altemaria and Sclerotinia, which induce expression of WRKY33. In addition, it could be shown that the induction of this gene is locally limited and is located directly around the infection site. To determine which sequence is that of the promoter, the start of transcription was identified. 5'Race experiments were carried out to determine the transcription start point of the WRKY33 gene. The Boehringer 5'-3'RACE kit was used and the manufacturer's instructions followed. After the transcription start point had been determined, promoter regions of different lengths were amplified by means of PCR and gene-specific primers, as shown in FIG. 21.

The following were used as forward primers:

The recognition sequences for a restriction enzyme are underlined in each case. The following was used as reverse primer for all four fragments mentioned above:
5'-ccggggatccggttctgctattgtccattgtaag-3 '

The program in the thermal cycler (model PTC225 from MJResearch) was as follows entered: 150 sec at 94 ° C, 60 sec at 80 ° C (addition of Tfu polymerase, HotStart method), 38 cycles with 60 sec 60 ° C, 100 sec 71 ° C, 45 sec 94 ° C, followed by 60 sec 60 ° C and 90 sec 71 ° C.

The amplified promoter fragments were again by means of PCR using the Tfu polymerase (Stratagene) cloned in front of a GUS reporter gene, so that continuous translation is possible was. The constructs were in the pGPTV vector (Plant Mol Biol. 20 (6), 1195-1197 (1992)) brought and by means of agrobacterial transformation into the genome of Arabidopsis plants Ecotype Col-0 stably integrated. The transformants were identified using selection plates. The vector used contains a kanamycin resistance and thus enables the selection of Transformants on kanamycin-containing plates. These plants were made after selection Earth transplanted and grown. The seeds of these plants were used for the application of different pathogens used. For this purpose, the seeds were applied sterile on MS plates or attracted directly to earth. The pathogens were identified according to the following literature Applied: Pythium: Vijayan, P. et al.: A role for jasmonate in pathogen defense of Arabidopsis. Proc. Nat. Acad. Sci.USA 95, 7209-7214, 1998. Alternaria and Sclerotinia as in the following Publication for Rhizoctonia described: Broekaert et al. An automated quantitative assay for fungal growth inhibition FEMS Microbiology Letters 69, 55-60, 1990). With all tested Pathogens showed a clear and local induction of the WRKY33 gene.  

SEQUENCE LISTING

Claims (21)

1. Transgenic plant with improved resistance to pathogens, the improved resistance through increased expression of the nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11, or its homologue or derivative or fragment, or by changing the biological activity of the nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11, or their homologue or Derived derivative or fragment encoded gene product. 2. Transgenic plant according to claim 1 with increased expression of the nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11, or its homolog or derivative or fragment, the nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11, or its homologue or derivative or fragment and one with this (these) Nucleic acid sequence (s) functionally linked regulatory nucleic acid sequence is (are) also stable integrated in the genome. 3. A transgenic plant according to claim 2, comprising at least two copies of one of the Nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11, or their homolog or derivative or fragment. 4. Transgenic plant according to claim 2 or 3, wherein the regulatory nucleic acid sequence is selected from the group of promoters CaMV 35S promoter, ubiquitin promoter, PRPI promoter, phaseolin promoter, isoflavone reductase promoter, ST-LSI promoter, promoter inducible by salicylic acid, promoter inducible by benzenesulfonamide, promoter inducible by tetracycline, promoter inducible by abscisic acid, ethanol or cyclohexanone inducible promoter, promoter according to one of the  Claims 10 to 12, or their fragment or homolog with the biological function a promoter, or a seed-specific tobacco promoter. 5. Transgenic plant according to claim 1 with increased expression of the nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11, or its homolog or derivative, the endogenous promoter being the Nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11, or their homolog or derivative, by modification or Addition of functional components is modified. 6. Transgenic plant according to claim 1 with increased expression of the nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11, or its homolog or derivative, the endogenous promoter being the Nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11, or their homolog or derivative, is replaced by a strong promoter, in particular by a CaMV 35S promoter from the cauliflower Mosaic virus or the ubiquitin promoter from corn. 7. The transgenic plant of claim 1, wherein the changed biological activity of the the nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID Encoded NO: 10 and / or SEQ ID NO: 11 or their homolog or derivative or fragment Gene product by modification of functional protein domains, in particular of Receptor binding, phosphorylation and / or DNA binding domains in particular WRKY DNA binding domain is highlighted. 8. Transgenic plant according to claim 7, wherein the modifications deletions, additions or Substitutions of amino acid residues include. 9. Nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,  SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, or their homolog or derivative or fragment. 10. Nucleic acid sequence according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23. 11. Fragment or derivative of the nucleic acid sequence according to claim 10 or one Nucleic acid sequence that matches the nucleic acid sequence according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ 117 NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 hybridizes and has the biological activity of a promoter. 12. The nucleic acid sequence according to claim 11, wherein the hybridizing nucleic acid sequence under stringent conditions with the nucleic acid sequence according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 hybridized. 13. Transgenic plant with at least one stable after its transformation into the genome integrated regulatory nucleic acid sequence according to one of claims 10 to 12 and one functionally linked to this nucleic acid sequence for a gene product coding nucleic acid sequence. 14. The transgenic plant according to claim 13, wherein the nucleic acid coding for a gene product is selected from the nucleic acids coding for structural proteins with repetitive peptide motifs Ser-Hyp ₄ , Gly-X or Val-Tyr-Lys-Pro-Pro, H ₂ O ₂ -producing enzymes, lignin and callose producing enzymes, chitinases, β-1,3-glucanases, antipathogenically active enzyme inhibitors, thionines, lectins with chitin-binding domains, ribosome-inactivating proteins, PR proteins, enzymes for the synthesis of phytoalexins, enzymes for the synthesis of saponins, R gene products, avr gene products, enzymes for the synthesis of salicylic acid. 15. Transgenic plant according to claim 13 or 14, wherein two or more different  Combinations of regulatory nucleic acid sequences and for a gene product coding nucleic acid sequences are stably integrated into the genome. 16. A method for producing a transgenic plant according to any one of claims 1 to 6 or 13 to 1 p. Comprising the stable integration of a regulatory nucleic acid sequence, in particular a nucleic acid sequence according to any one of claims 10 to 12, and if appropriate, a functionally linked to this nucleic acid sequence for a Nucleic acid sequence coding for the gene product, in particular the nucleic acid sequence Claim 9, in the genome of plant cells or plant tissues and regeneration of obtained plant cells or plant tissues to plants. 17. Transformed plant cell or transformed plant tissue with at least one after their transformation, the regulatory is stable integrated into the genome Nucleic acid sequence, in particular a nucleic acid sequence according to one of the claims 10 to 12 and optionally a functionally linked to this nucleic acid sequence for a gene product coding nucleic acid sequence, in particular the Nucleic acid sequence according to claim 9. 18. Plant cell or plant tissue according to claim 17, regenerable to a fertile Plant. 19. Seed obtained from plants according to one of claims 1 to 8 or 13 to 15. 20. Vector comprising a nucleic acid sequence according to one of claims 9 to 12. 21. A method for producing transgenic plants using the vector according to Claim 20.