DE10232773A1 - Proteins of the ubiquitin proteosome metabolic pathway, useful as targets for identifying fungicides for use in plant protection, also new nucleic acids, proteins and fungicides - Google Patents

Abstract

Use of proteins (I) of the ubiquitin proteosome metabolic pathway as targets for fungicides. (I) are encoded by: (a) any of 9 sequences (X; 444-1266 bp), reproduced; (b) sequences (Y) derived by reverse translation, within the degeneracy of the genetic code, from 9 protein sequences (Z), reproduced; or (c) functional equivalents of (a) or (b), having at least 60% identity. Independent claims are also included for the following: (1) new nucleic acid sequences (IIa) that are (X), (Y) and functional equivalents having 65-86% identity with (X) or 71-98% identity with (Y), with the precise minimal degree of identity being specified for each sequence, individually; (2) polypeptides (Ia) encoded by (IIa); (3) method for detecting functional analogs of (X) by preparing a probe and then screening a genomic or cDNA library from the appropriate species, or by computer-based searching for analogs in electronic databases; (4) method for identifying mutations in (I); (5) expression cassette (EC) comprising control sequences linked to (IIa) and/or additional functional elements; (6) vector that contains EC; (7) non-human transgenic organism (bacterium, yeast, fungus or animal or plant cell) that contains (IIa), EC or the vector of (6); (8) method for identifying fungicides; (9) carrier for use in the new methods comprising (IIa), vectors of (6), hosts of (7) and (Ia); and (10) fungicides identified by method (8).

Description

The present invention relates to the provision of proteins of the ubiquitin proteasome pathway as targets for Fungicides, the provision of new nucleic acid sequences, functional equivalents the above-mentioned nucleic acid sequences and the use of the gene products of the aforementioned nucleic acid sequences as new targets for Fungicides in a method of identifying antifungal Compounds that are involved in ubiquitination and subsequent Inhibit protolysis in the proteins involved in the proteasome. Furthermore The invention relates to the use of these compounds identified via the Processes mentioned above as fungicides.

The basic principle of identifying fungicides by inhibiting a defined target is known (e.g. US 5.187071 . WO 98/33925 . WO 00/77185 ). In general, there is a great need to detect enzymes that could represent new targets for fungicides. In addition to the problem of resistance problems, the reasons for this are the constant effort to identify new fungicidal active ingredients which are characterized by the broadest possible range of action, ecological and toxicological safety and low application rates.

The detection of new targets is in practice with large Difficulties associated with the inhibition of an enzyme, the component of a metabolic pathway is common growth or infectivity of the pathogenic Fungus no longer affected. This may be because the pathogenic fungus switches to alternative metabolic pathways, the Existence is not known or that the inhibited enzyme is not limiting for is the metabolic pathway. The target suitability of a gene product can be therefore do not predict even with knowledge of gene function.

Object of the present invention therefore consists in identifying fungicidal targets and procedures provide which to identify connections with fungicidal activity are suitable.

The task was solved by the provision of nucleic acid sequences coding for Proteins of the Ubiqutin Proteasome pathway, namely the Ubiquitin conjugating enzymes E2, as well as catalytic proteins and regulatory subunit of the 26s proteasome as new targets for fungicides and by providing nucleic acid sequences coding for the above called polypeptides. Proteins of the catalytic subunit of 26s proteasomes are the proteins of the 19s proteasome. Proteins of regulatory Subunits of the 26s proteasome are proteins of the so-called 20s proteasome.

• a) einer Nukleinsäuresequenz mit der in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 oder SEQ ID NO:17 dargestellten Sequenz; oder
• b) einer Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes aus den durch Rückübersetzung der in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 oder SEQ ID NO:18 dargestellten Aminosäuresequenzen ableiten läßt; oder
• c) funktionelle Äquivalente der SEQ ID NO:1 mit einer Identität von mindestens 60% zu der SEQ ID NO:1, der SEQ ID NO:3 mit einer Identität von mindestens 60% zu der SEQ ID NO:3, der SEQ ID NO:5 mit einer Identität von mindestens 60% zu der SEQ ID NO:5, der SEQ ID NO:7 mit einer Identität von mindestens 60% zu der SEQ ID NO:7, der SEQ ID NO:9 mit einer Identität von mindestens 60% zu der SEQ ID NO:9, der SEQ ID NO:11 mit einer Identität von mindestens 60% zu der SEQ ID NO:11, der SEQ ID NO:13 mit einer Identität von mindestens 60% zu der SEQ ID NO:13, der SEQ ID NO:15 mit einer Identität von mindestens 60% zu der SEQ ID NO:15 oder der SEQ ID NO:17 mit einer Identität von mindestens 60% zu der SEQ ID NO:17;
• d) eine Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes durch Rückübersetzung der Aminosäuresequenz eines funktionellen Äquivalents der SEQ ID NO:2, das eine Identitiät mit der SEQ ID NO:2 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:4, das eine Identitiät mit der SEQ ID NO:4 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:6, das eine Identitiät mit der SEQ ID NO:6 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:8, das eine Identitiät mit der SEQ ID NO:8 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:10, das eine Identitiät mit der SEQ ID NO:10 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:12, das eine Identitiät mit der SEQ ID NO:12 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:14, das eine Identitiät mit der SEQ ID NO:14 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:16, das eine Identitiät mit der SEQ ID NO:16 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:18, das eine Identitiät mit der SEQ ID NO:18 von mindestens 60% aufweist,

ableiten läßt.The present invention therefore relates to the use of proteins of the Ubiqutin Proteasome pathway encoded by a nucleic acid sequence comprising

• a) a nucleic acid sequence with the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 sequence shown; or
• b) a nucleic acid sequence which, on the basis of the degenerate genetic code, results from the back-translation of the sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18 derived amino acid sequences; or
• c) functional equivalents of SEQ ID NO: 1 with an identity of at least 60% to SEQ ID NO: 1, SEQ ID NO: 3 with an identity of at least 60% to SEQ ID NO: 3, SEQ ID NO : 5 with an identity of at least 60% to SEQ ID NO: 5, SEQ ID NO: 7 with an identity of at least 60% to SEQ ID NO: 7, SEQ ID NO: 9 with an identity of at least 60 % to SEQ ID NO: 9, SEQ ID NO: 11 with an identity of at least 60% to SEQ ID NO: 11, SEQ ID NO: 13 with an identity of at least 60% to SEQ ID NO: 13 , SEQ ID NO: 15 with an identity of at least 60% to SEQ ID NO: 15 or SEQ ID NO: 17 with an identity of at least 60% to SEQ ID NO: 17;
• d) a nucleic acid sequence which, owing to the degenerate genetic code, is translated back by the amino acid sequence of a functional equivalent of SEQ ID NO: 2, which has an identity with SEQ ID NO: 2 of at least 60%, of a functional equivalent of SEQ ID NO: 4, which has an identity with SEQ ID NO: 4 of at least 60%, a functional equivalent of SEQ ID NO: 6, which has an identity with SEQ ID NO: 6 of at least 60%, a functional equivalent of SEQ ID NO: 8, which has an identity with SEQ ID NO: 8 of at least 60%, a functional equivalent of SEQ ID NO: 10, which has an identity with SEQ ID NO: 10 of at least 60%, a functional equivalent of SEQ ID NO: 12, which has an identity with SEQ ID NO: 12 of at least 60%, a functional equivalent of SEQ ID NO: 14, which has an identity with SEQ ID NO: 14 of at least 60%, a functional equivalent eq equivalent of SEQ ID NO: 16, which has an identity with SEQ ID NO: 16 of at least 60%, a functional equivalent of SEQ ID NO: 18, which has an identity with SEQ ID NO: 18 of at least 60%,

can be derived.

as targets for fungicides.

At this point, some are now of the terms used in the description. Encoding proteins for a Ubiquitin conjugating enzyme E2 and proteins of the 26s proteasome are also given below with the term "protein (s) of the ubiquitin proteasome pathway".

"Affinity Tag": denotes an Peptide or polypeptide, the coding nucleic acid sequence with which encode a protein of the ubiquitin proteasome pathway nucleic acid sequence directly or via a linker using common cloning techniques can be merged. The affinity tag is for isolation, Enrichment and / or targeted purification of the recombinant target protein using affinity chromatography from total cell extracts. The linker mentioned above can be beneficial a protease interface (e.g. for Thrombin or factor Xa) contain, whereby the affinity tag at Can be split off from the target protein as required. Examples of common affinity tags are these "His-Tag" e.g. von Quiagen, Hilden, "Strep-Tag", the Myc-Tag "(Invitrogen, Carlsberg), the domain consisting of a chitin-binding domain and an intein New England Biolabs Day, the Maltose Binding Protein (pMal) from New England Biolabs and the so-called CBD tag from Novagen. The affinity day can be on 5 'or 3 'end of the coding nucleic acid sequence with the for the sequence coding for the target protein may be appropriate.

"Enzymatic activity / Activity test ": The term enzymatic activity describes the ability on the one hand of an enzyme to convert a substrate into a product. The enzymatic activity In a so-called activity test, the increase in the product, the removal of the substrate (or starting material) or the removal of one specific cofactor or over a combination of at least two of the above parameters dependent on within a defined period of time. On the other hand describes the term activity, that by the presence of the protein of the ubiquitin proteasome pathway in organisms how yeast and mushrooms are taught to grow and survive. Becomes the activity of the protein involved in ubiquitination is inhibited reduced growth or infectivity or growth arrest, the loss of infectivity or dying of the organism.

"Expression cassette": An expression cassette contains a nucleic acid sequence according to the invention functionally linked with at least one genetic control element such as a promoter and advantageously with a further control element such as a terminator. The nucleic acid sequence the expression cassette can, for example, be a genomic or a complementary DNA sequence or an RNA sequence as well as semi or fully synthetic Analogues of it. These sequences can be in linear or circular form, extra-chromosomal or integrated into the genome. The corresponding nucleic acid sequences can synthetically manufactured or natural can be obtained or a mixture of synthetic and natural Contain DNA components, as well as from various heterologous Genetic sections of different organisms exist.

Also artificial nucleic acid sequences are suitable as long as they express the protein of Ubiquitin proteasome pathway in a cell or a cell Enable organism. For example synthetic nucleotide sequences are generated which are related to the Codon usage optimized by the organisms to be transformed were.

All of the nucleotide sequences mentioned above are known per se by chemical synthesis from the Nucleotide building blocks such as by fragment condensation single overlapping, complementary Nucleotide building blocks of the double helix can be produced. Chemical synthesis of oligonucleotides can, for example, in a known manner the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). When preparing an expression cassette can Different DNA fragments are manipulated to form a nucleotide sequence is obtained with correct reading direction and correct reading frame. The connection of the nucleic acid fragments among themselves takes place over general cloning techniques such as those used in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1994) are described.

"Functional Link ": Under a functional or operational link one understands the sequential arrangement of regulatory sequences or genetic control elements such that each of the regulatory sequences or each of the genetic control elements their function in the Expression of the coding sequence can meet the intended purpose.

"Functional equivalents" describe here Nucleic acid sequences, which under standard conditions with that for the ubiquitination involved protein coding nucleic acid sequence or parts of the for protein of the nucleic acid sequence encoding the ubiquitin proteasome pathway hybridize and empowered are the expression of an active protein of the ubiquitin proteasome Cause metabolic pathway in a cell or an organism.

For hybridization, it is advantageous to use short oligonucleotides with a length of about 10-50 bp, preferably 15-40 bp [lengths ok?], For example the conserved or other areas, which are compared can be determined with other related genes in a manner known to those skilled in the art. However, longer fragments of the nucleic acids according to the invention with a length of 100-500 bp or the complete sequences can also be used for the hybridization. These standard conditions vary depending on the nucleic acid used: oligonucleotide, longer fragment or complete sequence or depending on the type of nucleic acid DNA or RNA used for the hybridization. For example, the melting temperatures for DNA: DNA hybrids are approx. 10 ° C lower than those of DNA: RNA hybrids of the same length.

Under standard hybridization conditions for example, depending on the nucleic acid, temperatures are between 42 and 58 ° C in an aqueous buffer solution a concentration between 0.1 to 5 x SSC (1 x SSC = 0.15 M NaCl, 15 mM Sodium citrate, pH 7.2) or additional in the presence of 50% formamide such as 42 ° C in 5 × SSC, 50% To understand formamide. The hybridization conditions advantageously lie for DNA: DNA hybrids at 0.1 × SSC and temperatures between about 20 ° C up to 65 ° C, preferably between about 30 ° C. up to 45 ° C. For DNA: RNA hybrids the hybridization conditions are advantageously 0.1 × SSC and Temperatures between about 30 ° C up to 65 ° C, preferably between about 45 ° C up to 55 ° C. These specified temperatures for the hybridization are exemplary calculated melting temperature values for one Nucleic acid with a length of approximately 100 nucleotides and a G + C content of 50% in the absence of Formamide. The experimental conditions for DNA hybridization are in relevant textbooks genetics such as Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be described according to formulas known to the person skilled in the art for example dependent of length of nucleic acids, the type of hybrid or the G + C content. Further information For hybridization, the specialist can find the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

Under a functional equivalent one also understands in particular also natural or artificial Mutations of the corresponding nucleic acid sequences of the nucleic acid sequences according to the invention encoded protein and their homologues from other organisms.

Thus, for example, too such nucleotide sequences are encompassed by the present invention, which is obtained by modifying the nucleic acid sequence at the ubiquitination protein involved. Exemplary such modifications by techniques familiar to the person skilled in the art, such as "site directed mutagenesis", "error prone PCR", "DNA shuffling" (Nature 370, 1994, pp.389-391) or "Staggered Extension Process "(Nature Biotechnol. 16, 1998, pp.258-261). Aim of such Modification can e.g. the insertion further restriction enzyme interfaces, the removal of DNA for shortening the sequence, the exchange of nucleotides for codon optimization or adding other sequences. Proteins that have modified nucleic acid sequences must be encoded despite a different nucleic acid sequence still the ones you want Have functions.

The concept of the functional equivalent can also affect that through the appropriate nucleic acid sequence encoded amino acid sequence Respectively. In this case, the term describes the functional equivalent a protein whose amino acid sequence with the for the protein involved in ubiquitination to a certain level Percentage is identical or homologous.

Functional equivalents thus include naturally occurring ones Variants of the sequences described here as well as artificial e.g. nucleic acid sequences obtained by chemical synthesis and adapted to codon use or the amino acid sequences derived therefrom.

"Genetic Control sequence ": The concept of genetic control sequences should be seen as equivalent to that Term "regulatory Sequence "describes the sequences that have an impact on transcription and where appropriate Translation of the nucleic acids according to the invention into have prokaryotic or eukaryotic organisms. Examples are for this are promoters, terminators or so-called "enhancer" sequences. In addition to these control sequences or instead of these sequences can the natural regulation of these sequences be present before the actual structural genes and if necessary have been genetically modified so that natural regulation switched off and the expression of the target gene modified, so elevated or has been humiliated. The control sequence is selected depending on the Host organism or parent organism. Genetic control sequences also include the 5 'untranslated Region, introns or the 3 'non-coding region of genes. As control sequences are still to be understood as those that have a homologous recombination enable insertion into the genome of a host organism or allow removal from the genome.

"Homology" or "identity" between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid sequence / polypeptide sequence over the respective total sequence length, which can be determined by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG ), Madison, USA) using the following parameters:
Gap Weight: 50 Length Weight: 3
Average Match: 10 Average Mismatch: –9
Instead of the term homologous or homology, the term identity is used synonymously below.

"Mutations" include substitutions (= Replacements), additions (addition), deletions (deletion), Inversion (changes) or insertions one or more nucleotide residues, whereby the corresponding amino acid sequence the target protein by substitution, insertion or deletion of a or more amino acids change can.

"Knock-out transformants" denotes individual cultures of a transgenic organism in which a specific Gene was specifically inactivated.

"Natural genetic Environment "means the natural chromosomal locus in the organism of origin. In the case of a genomic Library is the natural one genetic environment of the nucleic acid sequence preferably at least partially preserved. The surrounding area flanked the nucleic acid sequence at least on 5'- or 3 'side and has a sequence length of at least 50 bp, preferably at least 100 bp, particularly preferred at least 500 bp, very particularly preferably at least 1000 bp, most preferably at least 5000 bp.

"Response time" means the time which one for the implementation an activity test until a significant statement about an activity is received and depends on both of the specific activity the protein used in the test and the method used and the sensitivity of the devices used. The expert is the Determination of the response times known. At on photometric Method based procedures for the identification of substances with fungicidal activity the reaction times generally between> 0 to 360 minutes.

"Recombinant DNA "describes a combination of DNA sequences can be produced by recombinant DNA technology.

"Recombinant DNA technology ': generally known techniques for fusing DNA sequences (e.g. described in Sambrook et al., 1989, Cold Spring Habour, NY, Cold Spring Habour Laboratory Press).

Ensure "origins of replication" the multiplication of the expression cassettes according to the invention or vectors in microorganisms and yeasts, e.g. B. the pBR322 on , ColE1 or the P15A on in E. coli (Sambrook et al .: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and the ARS1 on in yeast (Nucleic Acids Research, 2000, 28 (10): 2060-2068).

"Reporter genes" code for easy quantifiable proteins. about Growth, fluorescence, chemo, bioluminescence or resistance assay or about a photometric measurement (intrinsic color) or enzyme activity can be done using of these genes an assessment of the transformation efficiency or the Expression location or time can be made. Most notably reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol.

1999; 13 (1): 29-44) like "green fluorescence Protein "(GFP) (Gerdes HH and Kaether C, FEBS Lett. 1996; 389 (1): 44-47; Chui WL et a1., Curr Biol 1996, 6: 325-330; Leffel SM et al., Biotechniques. 23 (5): 912-8, 1997), chloramphenicol acetyl transferase, a luciferase (Giacomin, Plant Sci 1996, 116: 59-72; Scikantha, J Bact 1996, 178: 121; Millar et al., Plant Mol Biol Rep 1992 10: 324-414), and luciferase genes generally the β-galactosidase, or the β-glucuronidase (Jefferson et al., EMBO J. 1987, 6, 3901-3907) or the Uraa gene.

"Selection markers" confer resistance to antibiotics or other toxic compounds: examples include the neomycin phosphotransferase gene, which is resistant to the aminoglycoside antibiotics neomycin (G 418), kanamycin, paromycin (Deshayes A et al., EMBO J. 4 (1985) 2731-2737), the sul gene coding for a mutated dihydropteroate synthase (Guerineau F et al., Plant Mol Biol. 1990; 15 (1): 127-136), the hygromycin B phosphotransferase gene (Gen Bank Accession NO: K 01193) and the shble resistance gene, which is resistant to bleomycin antibiotics such as. Zeocin gives. Further examples of selection marker genes are genes which are resistant to 2-deoxyglucose-6-phosphate ( WO 98/45456 ) or phosphinotricin etc. or those which confer antimetabolite resistance, for example the dhfr gene (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994) 142-149). Also suitable are genes such as trpB or hisD (Hartman SC and Mulligan RC, Proc Natl Acad Sci US A. 85 (1988) 8047-8051). The gene of mannose phosphate isomerase ( WO 94/20627 ), the ODC (ornithine decarboxylase) gene (McConlogue, 1987 in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, ed.) or the deaminase from Aspergillus terreus (Tamura K et al., Biosci Biotechnol Biochem. 59 (1995) 2336 to 2338).

"Significant Decrease ": related on the activity of about a nucleic acid sequence according to the invention encoded polypeptide is a decrease in activity with a test compound added polypeptide compared to the activity of the test compound incubated meant the outside a measurement error lies.

"Target / Target Protein ": a on Ubiquitin proteasome pathway involved protein, which can be an enzyme in the classical sense or e.g. a structural protein, one for Development processes relevant protein, transport proteins, regulatory Subunits that regulate an enzyme complex substrate or activity to lend. It is common to all targets or action sites that the functional presence of the target protein makes it essential for survival or the normal development, growth and / or infectivity of the phytopathogenic Organism.

"Transformation" describes you Introduction process heterologous DNA in a pro- or eukaryotic cell. With a transformed cell is not just the product of the transformation process described per se, but also all transgenic descendants of the transgenic organism produced by the transformation '.

"Transgene": Relating to one Nucleic acid sequence an expression cassette or a vector containing a nucleic acid sequence according to the invention or an organism transformed with a nucleic acid sequence according to the invention, Expression cassette or vector describes the expression transgenic all such constructions made by genetic engineering methods, in which either the nucleic acid sequence of the target protein or one with the nucleic acid sequence of the target protein functionally linked genetic control sequence or a combination of the above is not natural genetic environment or by genetic engineering methods were modified. The modification can here, for example, via mutation one or more nucleotide residues of the corresponding nucleic acid sequence can be achieved.

For the targeted degradation of numerous short-lived proteins in eukaryotes the ubiquitin proteasome pathway is responsible. The proteins are first of all labeled with one or more ubiquitin molecules. This so-called Ubiquitination is a complex, multi-step process that involves Ubiquitin activation by the ubiquitin activating enzyme E1 and the ubiquitin conjugation over includes the ubiquitin-conjugating enzyme E2. The actual transfer of the ubiquitin-conjugating enzyme E2 can be done in two ways. Firstly, the ubiquitin can be obtained directly by condensation of the terminal Carboxyl group of the ubiquitin residue via an acid amide bond with the lysine residue of the target protein become. Alternatively, ubiquitination via the ubiquitin protein ligase E3 are made that have a specific complex with the target protein and forms the E2-ubiquitin and transfers the ubiquitin residue to the target protein (black S.E. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 560-64).

The proteins labeled with ubiquitin are then over at least two routes to proteolytic degradation. Either there is proteolytic degradation in the cytosol by proteasomes or transmembrane proteins are internalized in response to the signal and the degradation takes place lysosomally instead of. The non-lysosomal proteolytic degradation of the ubiquitin-labeled Proteins occur in an ATP-dependent reaction in the 26s proteasome. This complex consists of two protein aggregates, the 19s and the 20s particle together. The central catalytic unit is the 20s proteasome (Dick, T.P. et al. (1998) J. Biol. Chem. 273 (40), 25637-46). The regulation takes place through the 19s subunit. It is assumed that the substrate is recognized and unfolded there is then passed on to the catalytic subunit (Glickman M.H. et al. (1998) Mol. Cell. Biol., 3149-62).

Nucleic acid sequences coding for an ubiquitin-conjugating Enzyme E2 are from Schizosaccharomyces pombe (Gen Bank Acc.No Q9PR4; identity with SEQ ID NO: 1 = 72.9%, identity with SEQ ID NO: 2 = 79.01%) and Colletotrichum gloeosporioides (Gen Bank Acc.No Q74196; identity with the SEQ ID NO: 3 = 85.5%, identity with SEQ ID NO: 4 = 97.3%).

Nucleic acid sequences coding for the 20s Proteasome subunit are from S. cerevisiae (Gen Bank Acc.No PSB6 YEAST; identity with SEQ ID NO: 5 = 72.4%, identity with SEQ ID NO: 6 = 72.2%, gene Bank Acc.No PSB5 YEAST; identity with SEQ ID NO: 7 = 64.2%, identity with SEQ ID NO: 8 = 73.9%, Gen Bank Acc.No PSA2 YEAST; Identity with SEQ ID NO: 11 = 64.1%, identity with SEQ ID NO: 12 = 72.8%), Neurospora crassa (Gen Bank Acc.No PSB2 NEUCR; identity with SEQ ID NO: 9 = 72.8%, identity with SEQ ID NO: 10 = 88.0%) and Schizosaccharomyces pombe (Gen Bank Acc.No PSA4-SCHPO; identity with the SEQ ID NO: 13 = 68.3%, identity with SEQ ID NO: 14 = 69.6%).

Nucleic acid sequences coding for the regulatory Subunits of the 26s proteasome are from Aspergillus nidulans (Gen Bank Acc.No PRS6 ASPNG; identity with SEQ ID NO: 15 = 77.3%, identity with SEQ ID NO: 16 = 89.3%) and S. cerevisiae (Gen Bank Acc.No SUG2 YEAST; identity with the SEQ ID NO: 17 = 67.2%, identity with SEQ ID NO: 18 = 71.9%).

Surprisingly components of the ubiquitin proteasome pathway were found from phytopathogenic filamentous Mushrooms are suitable as targets.

• a) einer Nukleinsäuresequenz mit der in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 oder SEQ ID NO:17 dargestellten Sequenz;
• b) einer Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes aus den durch Rückübersetzung der in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 oder SEQ ID NO:18 dargestellten Aminosäuresequenzen ableiten läßt;
• c) funktionelle Äquivalente der SEQ ID NO:1 mit einer Identität von mindestens 60% zu der SEQ ID NO:1, der SEQ ID N0:3 mit einer Identität von mindestens 60% zu der SEQ ID N0:3, der SEQ ID NO:5 mit einer Identität von mindestens 60% zu der SEQ ID NO:5, der SEQ ID NO:7 mit einer Identität von mindestens 60% zu der SEQ ID NO:7, der SEQ ID NO:9 mit einer Identität von mindestens 60% zu der SEQ ID NO:9, der SEQ ID NO:11 mit einer Identität von mindestens 60% zu der SEQ ID NO:11, der SEQ ID NO:13 mit einer Identität von mindestens 60% zu der SEQ ID NO:13, der SEQ ID NO:15 mit einer Identität von mindestens 60% zu der SEQ ID NO:15 oder der SEQ ID NO:17 mit einer Identität von mindestens 60% zu der SEQ ID NO:17; oder
• d) eine Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes durch Rückübersetzung der Aminosäuresequenz eines funktionellen Äquivalents der SEQ ID NO:2, das eine Identitiät mit der SEQ ID NO:2 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:4, das eine Identitiät mit der SEQ ID NO:4 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:6, das eine Identitiät mit der SEQ ID NO:6 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:8, das eine Identitiät mit der SEQ ID NO:8 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:10, das eine Identitiät mit der SEQ ID NO:10 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:12, das eine Identitiät mit der SEQ ID NO:12 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:14, das eine Identitiät mit der SEQ ID NO:14 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:16, das eine Identitiät mit der SEQ ID NO:16 von mindestens 60% aufweist, eines funktionellen Äquivalents der SEQ ID NO:18, das eine Identitiät mit der SEQ ID NO:18 von mindestens 60% aufweist, ableiten läßt.

The present invention therefore relates to the use of at least one protein of the ubiquitin proteasome pathway encoded by a nucleic acid sequence selected from the group consisting of

• a) a nucleic acid sequence with the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 sequence shown;
• b) a nucleic acid sequence which, on the basis of the degenerate genetic code, results from the back-translation of the sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18 derived amino acid sequences;
• c) functional equivalents of SEQ ID NO: 1 with an identity of at least 60% to SEQ ID NO: 1, SEQ ID N0: 3 with an identity of at least 60% to SEQ ID N0: 3, SEQ ID NO : 5 with an identity of at least 60% to SEQ ID NO: 5, SEQ ID NO: 7 with an identity of at least 60% to SEQ ID NO: 7, SEQ ID NO: 9 with an identity of at least 60 % to SEQ ID NO: 9, SEQ ID NO: 11 with an identity of at least 60% to SEQ ID NO: 11, SEQ ID NO: 13 with an identity of at least 60% to SEQ ID NO: 13 , SEQ ID NO: 15 with an identity of at least 60% to SEQ ID NO: 15 or SEQ ID NO: 17 with an identity of at least 60% to SEQ ID NO: 17; or
• d) a nucleic acid sequence which, owing to the degenerate genetic code, is translated back by the amino acid sequence of a functional equivalent of SEQ ID NO: 2, which has an identity with SEQ ID NO: 2 of at least 60%, of a functional equivalent of SEQ ID NO: 4, which has an identity with SEQ ID NO: 4 of at least 60%, a functional equivalent of SEQ ID NO: 6, which has an identity with SEQ ID NO: 6 of at least 60%, a functional equivalent of SEQ ID NO: 8, which has an identity with SEQ ID NO: 8 of at least 60%, a functional equivalent of SEQ ID NO: 10, which has an identity with SEQ ID NO: 10 of at least 60%, a functional equivalent of SEQ ID NO: 12, which has an identity with SEQ ID NO: 12 of at least 60%, a functional equivalent of SEQ ID NO: 14, which has an identity with SEQ ID NO: 14 of at least 60%, a functional equivalent eq equivalent of SEQ ID NO: 16, which has an identity with SEQ ID NO: 16 of at least 60%, a functional equivalent of SEQ ID NO: 18, which has an identity with SEQ ID NO: 18 of at least 60%, can be derived.

as a target for fungicides. The functional equivalents according to c) or d) are characterized by essentially the same functionality, i.e. they have the physiological function of a protein of ubiquitin Proteasome pathway.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 1 have an identity with SEQ ID No: 1 of at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% preferably at least 69%, 70%, 71%, 72%, 73%, 70%, 71%, 72%, 73%, 70%, 71%, 72%, 73% preferably at least 74%, 75%, 76%, 77%, 78%, 79%, 80% particularly preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% are particularly preferred at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 3 have an identity with SEQ ID No: 3 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% particularly preferably at least 86%, 87%, 88%, 89%, 90% very particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID N0: 5 show an identity with SEQ ID No: 5 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% are particularly preferred at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% whole particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 7 have an identity with SEQ ID No: 7 of at least 60%, 61%, 62%, 63%, 64% preferably at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73% preferred at least 74%, 75%, 76%, 77%, 78%, 79%, 80% are particularly preferred at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% whole particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 9 show an identity with SEQ ID No: 9 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% preferably at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% particularly preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% very special preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID: 11 have an identity with SEQ ID No: 11 of at least 60%, 61%, 62%, 63%, 64% preferably at least 65%, 66%, 67%, 68% preferably at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% particularly preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% very special preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID: 13 demonstrate an identity with SEQ ID No: 13 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% adds at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% particularly preferably at least 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90% very particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ,

Functional equivalents of the nucleic acid sequences SEQ ID: 15 have an identity with SEQ ID No: 15 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, particularly preferably at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% whole particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID: 17 have an identity with SEQ ID No: 17 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67% preferably at least 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% especially preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% very particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID: 2 have an identity with SEQ ID No: 2 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% are particularly preferred at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% very particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID: 4 have an identity with SEQ ID No: 4 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% are particularly preferred at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% very particularly preferably at least 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID: 6 show an identity with SEQ ID No: 6 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% preferred at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% particularly preferred at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% whole particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID: 8 have an identity with SEQ ID No: 8 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% preferably at least 75%, 76%, 77%, 78%, 79%, 80% particularly preferred at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% whole particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID: 10 have an identity with SEQ ID No: 10 of at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% preferably at least 69%, 70%, 71%, 72%, 73%, 70%, 71%, 72%, 73%, 70%, 71%, 72%, 73% preferably at least 74%, 75%, 76%, 77%, 78%, 79%, 80% particularly preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, very particularly preferably at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 12 have an identity with SEQ ID No: 12 of at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67% preferably at least 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% preferably at least 79%, 80%, 81%, 82%, 83%, 84%, 85% particularly preferably at least 86%, 87%, 88%, 89%, 90% entirely particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID: 14 have an identity with SEQ ID No: 14 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73% preferably at least 74%, 75%, 76%, 77%, 78%, 79%, 80% especially preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% very particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID: 16 have an identity with SEQ ID No: 16 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% particularly preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% are particularly preferred at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID: 18 have an identity with SEQ ID No: 18 of at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% preferred at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% particularly preferred at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% whole particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

The nucleic acid sequences mentioned above preferably originate from a pyhtopathogenic filamentous fungus, preferably from a filamentous, phytopathogenic fungus of the genus Alternaria, Po dosphaera, Sclerotinia, Physalospora, Botrytis, Corynespora; Colletotrichum; Diplocarpon; Elsinoe; Diaporthe; Sphaerotheca; Cinula, Cercospora; Erysiphe; Sphaerotheca; Leveillula; Mycosphaerella; Phyllactinia; Gloesporium; Gymnosporangium, Leptotthrydium, Podosphaera; Gloedes; Cladosporium; Phomopsis; Phytopora; Phytophthora; Erysiphe; Fusarium; Verticillium; Glomerella; Drechslera; Bipolaris; Personospora; Phaeoisariopsis; Spaceloma; Pseudocercosporella; Pseudoperonospora; Puccinia; Typhula; Pyricularia; Rhizoctonia; Stachosporium; Uncinula; Ustilago; Gaeumannomyces or Fusarium, particularly preferably from a phytopathogenic filamentous fungus selected from the genera and species Alternaria, Podosphaera, Sclerotinia, Physalospora eg Physalospora canker, Botrytis eg Botrytis cinerea, Corynespora eg Corynespora melonis; Colletotrichum; Diplocarpon eg Diplocarpon rosae; Elsinoe, for example Elsinoe fawcetti, Diaporthe, for example, Diaporthe citri; Sphaerotheca; Cinula, e.g. Cinula neccata, Cercospora; Erysiphe, for example Erysiphe cichoracearum and Erysiphe graminis; Sphaerotheca eg Sphaerotheca fuliginea; Leveillula eg Leveillula taurica; Mycosphaerella; Phyllactinia, for example Phyllactinia kakicola; Gloesporium eg Gloesporium kaki; Gymnosporangium eg Gymnosporangium yamadae, Leptotthrydium eg Leptotthrydium pomi, Podosphaera eg Podosphaera leucotricha; Gloedes eg Gloedes pomigena; Cladosporium, for example Cladosporium carpophilum; Phomopsis; Phytopora; Phytophthora eg Phytophthora infestans; Verticillium; Glomerella, for example Glomerella cingulata; Drechslera; Bipolaris; Personospora; Phaeoisariopsis, for example Phaeoisariopsis vitis; Spaceloma, for example Spaceloma ampelina; Pseudocercosporella, for example Pseudocercosporella herpotrichoides; Pseudoperonospora; Puccinia; Typhula; Pyricularia, for example Pyricularia oryzae; Rhizoctonia; Stachosporium eg Stachosporium nodorum; Uncinula eg Uncinula necator; Ustilago; Gaeumannomyces species, e.g. Gaeumannomyces graminis and Fu sarium-species, e.g. Fusarium dimerium, Fusarium merismoides, Fusarium lateritium, Fusarium decemcellulare, Fusarium poae, Fusarium tricinctum, Fusarium sporotrichioides, Fusarium chlamydosiumusumumililararumarifarium fifarium fifarium, Fusarium Fusarium oxysporum, Fusarium solani, Fusarium culmorum, Fusarium sambucinum, Fusarium crookwellense, Fusarium avenaceum ssp. avenaceum, Fusarium avenaceum ssp. Ay values, Fusarium avenaceum ssp. nurragi, Fusarium hetrosporum, Fusarium acuminatum ssp. acuminatum, Fusarium acuminatum ssp. armeniacum, Fusarium longipes, Fusarium compactum, Fusarium equiseti, Fusarium scripi, Fusarium polyphialidicum, Fusarium semitectum and Fusarium beomiforme and Fusarium graminearum, very particularly preferably from a phytopathogenic filamentous fungus selected from the genus Fusariumususususususususususususususususususususususususususus), Fusarium graminearium, very particularly preferably from a phytopathogenic filamentous fungus selected from the genus Fusariumusususususususususususususususususususususususususususus) Fusarium decemcellulare, poae Fusarium, tricinctum, Fusarium sporotrichioides, Fusarium moniliforme chlamydosporum, Fusarium, Fusarium proliferatum anthophilum, Fusarium subglutinans, Fusarium nygamai, Fusarium oxysporum, Fusarium solani, Fusarium culmorum sambucinum, Fusarium, Fusarium crookwellense, Fusarium avenaceum ssp. avenaceum, Fusarium avenaceum ssp. Ay values, Fusarium avenaceum ssp. nurragi, Fusarium hetrosporum, Fusarium acuminatum ssp. acuminatum, Fusarium acuminatum ssp. armeniacum, Fusarium longipes, Fusarium compactum, Fusarium equiseti, Fusarium scripi, Fusarium polyphialidicum, Fusarium semitectum and Fusarium beomiforme are preferred, with the fungus Fusarium graminearum being particularly preferred from the genus Fusarium.

• a) eine Nukleinsäuresequenz mit der in SEQ ID :1, SEQ ID :3, SEQ ID :5, SEQ ID :7, SEQ ID :9, SEQ ID :11, SEQ ID :13, SEQ ID :15 oder SEQ ID :17 dargestellten Sequenz; oder
• b) eine Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes aus den durch Rückübersetzung der in SEQ ID NO:2, SEQ ID NO:4, SEQ ID :6, SEQ ID :8, SEQ ID :10, SEQ ID :12, SEQ ID NO:14, SEQ ID NO:16 oder SEQ ID NO:18 dargestellten Aminosäuresequenzen ableiten läßt;
• c) funktionelle Äquivalente der SEQ ID NO:1 mit einer Identität von mindestens 74% zu der SEQ ID NO:1, der SEQ ID NO:3 mit einer Identität von mindestens 86% zu der SEQ ID NO:3, der SEQ ID NO:5 mit einer Identität von mindestens 73% zu der SEQ ID NO:5, der SEQ ID NO:7 mit einer Identität von mindestens 65% zu der SEQ ID NO:7, der SEQ ID NO:9 mit einer Identität von mindestens 73% zu der SEQ ID NO:9, der SEQ ID NO:11 mit einer Identität von mindestens 65% zu der SEQ ID NO:11, der SEQ ID NO:13 mit einer Identität von mindestens 69% zu der SEQ ID NO:13, der SEQ ID NO:15 mit einer Identität von mindestens 78% zu der SEQ 2D NO:15 oder der SEQ ID NO:17 mit einer Identität von mindestens 68% zu der SEQ ID NO:17; oder
• d) eine Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes durch Rückübersetzung der Aminosäuresequenz eines funktionellen Äquivalents der SEQ ID NO:2, das eine Identitiät mit der SEQ ID NO:2 von mindestens 80% aufweist, eines funktionellen Äquivalents der SEQ ID NO:4, das eine Identitiät mit der SEQ ID NO:4 von mindestens 98% aufweist, eines funktionellen Äquivalents der SEQ ID NO:6, das eine Identitiät mit der SEQ ID NO:6 von mindestens 73% aufweist, eines funktionellen Äquivalents der SEQ ID NO:8, das eine Identitiät mit der SEQ ID NO:8 von mindestens 75% aufweist, eines funktionellen Äquivalents der SEQ ID NO:10, das eine Identitiät mit der SEQ ID NO:10 von mindestens 89% aufweist, eines funktionellen Äquivalents der SEQ ID NO:12, das eine Identitiät mit der SEQ ID NO:12 von mindestens 74% aufweist, eines funktionellen Äquivalents der SEQ ID NO:14, das eine Identitiät mit der SEQ ID NO:14 von mindestens 71% aufweist, eines funktionellen Äquivalents der SEQ ID N0:16, das eine Identitiät mit der SEQ ID NO:16 von mindestens 90% aufweist, eines funktionellen Äquivalents der SEQ ID NO:18, das eine Identitiät mit der SEQ ID NO:18 von mindestens 73% aufweist, ableiten läßt.

The present invention further claims nucleic acid sequences which code for a protein of the ubiquitin proteasome pathway for use as a target for fungicides, comprising:

• a) a nucleic acid sequence with the SEQ ID: 1, SEQ ID: 3, SEQ ID: 5, SEQ ID: 7, SEQ ID: 9, SEQ ID: 11, SEQ ID: 13, SEQ ID: 15 or SEQ ID: 17 sequence shown; or
• b) a nucleic acid sequence which, on the basis of the degenerate genetic code, is obtained from the back-translation of the sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID: 6, SEQ ID: 8, SEQ ID: 10, SEQ ID: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18 derived amino acid sequences;
• c) functional equivalents of SEQ ID NO: 1 with an identity of at least 74% to SEQ ID NO: 1, SEQ ID NO: 3 with an identity of at least 86% to SEQ ID NO: 3, SEQ ID NO : 5 with an identity of at least 73% to SEQ ID NO: 5, SEQ ID NO: 7 with an identity of at least 65% to SEQ ID NO: 7, SEQ ID NO: 9 with an identity of at least 73 % to SEQ ID NO: 9, SEQ ID NO: 11 with an identity of at least 65% to SEQ ID NO: 11, SEQ ID NO: 13 with an identity of at least 69% to SEQ ID NO: 13 , SEQ ID NO: 15 with an identity of at least 78% to SEQ 2D NO: 15 or SEQ ID NO: 17 with an identity of at least 68% to SEQ ID NO: 17; or
• d) a nucleic acid sequence which, owing to the degenerate genetic code, is converted back by the amino acid sequence of a functional equivalent of SEQ ID NO: 2, which has an identity with SEQ ID NO: 2 of at least 80%, of a functional equivalent of SEQ ID NO: 4, which has an identity with SEQ ID NO: 4 of at least 98%, a functional equivalent of SEQ ID NO: 6, which has an identity with SEQ ID NO: 6 of at least 73%, a functional equivalent of SEQ ID NO: 8, which has an identity with SEQ ID NO: 8 of at least 75%, a functional equivalent of SEQ ID NO: 10, which has an identity with SEQ ID NO: 10 of at least 89%, a functional equivalent of SEQ ID NO: 12, which has an identity with the SEQ ID NO: 12 from has at least 74%, a functional equivalent of SEQ ID NO: 14, which has an identity with SEQ ID NO: 14 of at least 71%, a functional equivalent of SEQ ID N0: 16, which has an identity with SEQ ID NO: 14 16 of at least 90%, a functional equivalent of SEQ ID NO: 18, which has an identity with SEQ ID NO: 18 of at least 73%, can be derived.

The functional equivalents according to c) or d) are characterized by essentially the same functionality, i.e. they have the physiological function of a protein of ubiquitin Proteasome pathway.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 1 have an identity with SEQ ID No: 1 of at least 74%, preferably at least 75%, 76%, 77% preferably at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, particularly preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% very particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 3 have an identity with SEQ ID No: 1 of at least 86%, preferably at least 87%, 88% preferably at least 89%, 90%, 91%, 92%, 93% particularly preferred at least 94%, 95%, 96% very particularly preferably at least 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 5 show an identity with SEQ ID No: 5 of at least 73%, preferably at least 74% 75%, 76%, 77% preferably at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, particularly preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% very particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 7 have an identity with SEQ ID No: 7 of at least 65%, preferably at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73% preferably at least 74%, 75%, 76%, 77%, 78%, 79%, 80% particularly preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% very particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 9 show an identity with SEQ ID No: 9 of at least 73%, preferably at least 74% 75%, 76%, 77% preferably at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, particularly preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% very particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 11 have an identity with SEQ ID No: 11 of at least 73%, preferably at least 74% 75%, 76%, 77% preferably at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, particularly preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% very particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 13 have an identity with SEQ ID No: 13 of at least 69%, preferably at least 70%, 71%, 72%, 73%, 74,% 75%, 76%, 77% preferably at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, particularly preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% very particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ 2D NO: 15 show an identity with SEQ ID No: 15 of at least 78%, preferably at least 79%, 80%, 81%, 82% preferably at least 83%, 84%, 85%, 86%, 87% especially preferably at least 88%, 89%, 90%, 91%, 92% very particularly preferred at least 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 17 have an identity with SEQ ID No: 17 of at least 68%, preferably at least 69%, 70%, 71%, 72%, 73%, 74, 75%, 76%, 77% preferably at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, particularly preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% very particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 2 have an identity with SEQ ID No: 2 of at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87% preferably at least 88%, 89%, 90%, 91%, 92%, 93% particularly preferably at least 94%, 95%, 96% entirely particularly preferably at least 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 4 have an identity with SEQ ID No: 4 of at least 98%, preferably at least 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 6 show an identity with SEQ ID No: 6 of at least 73%, preferably at least 74% 75%, 76%, 77% preferably at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, particularly preferred at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% very particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 8 have an identity with SEQ ID No: 8 of at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, preferably at least 81%, 82%, 83%, 84%, particularly preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% entirely particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 10 have an identity with SEQ ID No: 10 of at least 89%, preferably at least 90%, 91% preferably at least 92%, 93%, 94% particularly preferred at least 95%, 96%, 97% very particularly preferably at least 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 12 have an identity with SEQ ID No: 12 of at least 74%, preferably at least 75%, 76%, 77%, 78%, 79%, 80% preferably at least 81%, 82%, 83%, 84%, particularly preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% very particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 14 have an identity with SEQ ID No: 14 of at least 71%, preferably at least 72%, 73%, 74,% 75%, 76%, 77%, 78% preferably at least 79%, 80%, 81%, 82%, 83%, 84%, particularly preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% very particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 16 have an identity with SEQ ID No: 16 of at least 90%, preferably at least 91%, 92%, 93%, 94% preferably at least 95%, 96% particularly preferred at least 97%, 98% very particularly preferably at least 99%.

Functional equivalents of the nucleic acid sequences SEQ ID NO: 18 have an identity with SEQ ID No: 18 of at least 73%, preferably at least 74% 75%, 76%, 77% preferably at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, particularly preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% very particularly preferably at least 93%, 94%, 95%, 96%, 97%, 98%, 99% on.

The SEQ ID NO: 1 and its functional equivalents (e.g. with an identity of at least 60% or 74% to SEQ ID NO: 1) and SEQ ID NO: 3 as well as their functional equivalents (e.g. with an identity of at least 60% or 86% to SEQ ID NO: 3) code for a polypeptide with the biological activity an E2 (hereinafter referred to as "E2").

The SEQ ID NO: 5 and its functional equivalents like (e.g. with an identity of at least 60% or 73% to SEQ ID NO: 5), SEQ ID NO: 7 as well their functional equivalents (e.g. with an identity of at least 60% or 65% to SEQ ID NO: 7), SEQ ID NO: 9 as well as their functional equivalents (e.g. with an identity of at least 60% or 73% to SEQ ID NO: 9), SEQ ID NO: 11 as well as their functional equivalents (e.g. with an identity of at least 60% or 65% to SEQ ID NO: 11), SEQ ID NO: 13 as well their functional equivalents (e.g. with an identity of at least 60% or 69% to SEQ ID NO: 13) code for polypeptides the 20s proteasome (hereinafter referred to as "20s"), their presence for the functionality of the 20s proteasome is essential. In the 20s proteasome are which are the catalytic protease centers, i.e. the proteolysis takes place held in this UE.

The SEQ ID NO: 15 and its functional equivalents like (e.g. with an identity of at least 60% or 78% to SEQ ID NO: 37), SEQ ID NO: 17 as well as their functional equivalents (e.g. with an identity of at least 60% or 68% to SEQ ID NO: 39) code for polypeptides of the 19s (hereinafter referred to as "19s") (hab I really understood that ??) proteasome, its presence for the functionality of the 26s Proteasomes is essential. The 19s proteasome is for binding of the ubiquitinated substrate proteins on the proteasome.

The nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ 2D NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and those defined above functional equivalents of the above nucleic acid sequences are as follows also referred to with the term "nucleic acid sequences according to the invention". The by the nucleic acid sequences according to the invention encoded to proteins of the ubiquitin proteasome metabolism hereinafter referred to as "UPS proteins".

The nucleic acid sequences according to the invention can for the Manufacture of hybridization probes are used, via which functional equivalents of the nucleic acid sequences according to the invention isolated can be. The manufacture of these probes and the conduct of the experiments are known. she can for example about the targeted manufacture of radioactive or non-radioactive probes by means of PCR and the use of appropriately labeled oligonucleotides followed by Hybridization experiments. The technologies required for this are described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989). The corresponding probes can still using standard technologies (Lit. SDM or random mutagenesis) be modified so that they can be used for other purposes can be e.g. as a probe that is specific to mRNA and the corresponding coding sequences hybridized for analysis of the corresponding Sequences in other organisms. The probe can e.g. B. for screening in a genomic or cDNA library of the corresponding fungus or in a computer research for analog sequences in electronic Databases used.

Further applications of the probes described above are the analysis of possibly changed expression profiles of the nucleic acid sequences according to the invention in various fungi, preferably phytopathogenic fungi especially in connection with certain factors such as increased resistance to fungicides, the detection of the fungus in plant material and the detection of emerging resistances. The term phytopathogenic fungi has already been defined above.

Increasing resistance to one Fungicide targeting a UPS protein is often based on Mutation, e.g. the exchange of amino acids caused by a Mutation in the nucleic acid sequence, on for the substrate specificity essential places such as in the area of the active center or elsewhere in the protein, which is the binding of the substrate influence. Due to the changes described above can bind the inhibitor acting as a fungicide to the UPS Protein can be difficult or even prevented, so that a restricted or no fungicidal activity was observed in the corresponding crops is.

Because the changes that occur here often comprising only a few base pairs, those described above can be used Probes based on the nucleic acid sequences according to the invention or a functional equivalent as described above as described above for the detection of mutant nucleic acid sequences according to the invention in completely or partially resistant phytopathogenic fungi can be used.

After isolation of the corresponding Gene or gene segment of the UPS protein from the corresponding resistant Mushroom using the above mentioned Probes followed by sequencing and comparison with the corresponding one Wild-type nucleic acid sequence stand for analytics basically has two methods:

• 1. According to one preferred embodiment includes the proof according to step ii and variant 3 of the inventive method comprising the following steps: Using common Methods two primer pairs are constructed, the first complementary to that Wild type sequence is the second complementary to that mutated accordingly Sequence. about PCR can now quantitatively and qualitatively identify the mutant species be detected.
• 2. According to one preferred embodiment includes the proof according to step ii and variant 3 of the inventive method comprising the following steps: By changing the base sequence, restriction enzyme interfaces can arise or disappear. The corresponding area is created using flanking primer amplified and then with the corresponding Restriction enzymes are digested and / or sequenced, thereby increasing the presence the corresponding mutation can be detected.

Another object of the invention are containing expression cassettes

• a) eine Nukleinsäuresequenz mit der in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 oder SEQ ID NO:17 dargestellten Sequenz; oder
• b) eine Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes aus den durch Rückübersetzung der in SEQ ID N?:2, SEQ ID N?:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 oder SEQ ID NO:18 dargestellten Aminosäuresequenzen ableiten läßt; oder
• c) funktionelle Äquivalente der SEQ ID NO:1 mit einer Identität von mindestens 74% zu der SEQ ID NO:1, der SEQ ID NO:3 mit einer Identität von mindestens 86% zu der SEQ ID NO:3, der SEQ ID NO:5 mit einer Identität von mindestens 73% zu der SEQ ID NO:5, der SEQ ID NO:7 mit einer Identität von mindestens 65% zu der SEQ ID NO:7, der SEQ ID NO:9 mit einer Identität von mindestens 73% zu der SEQ ID NO:9, der SEQ ID NO:11 mit einer Identität von mindestens 65% zu der SEQ ID NO:11, der SEQ ID NO:13 mit einer Identität von mindestens 69% zu der SEQ ID NO:13, der SEQ ID NO:15 mit einer Identität von mindestens 78% zu der SEQ ID NO:15 oder der SEQ ID NO:17 mit einer Identität von mindestens 68% zu der SEQ ID NO:17;
• d) eine Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes durch Rückübersetzung der Aminosäuresequenz eines funktionellen Äquivalents der SEQ ID NO:2, das eine Identitiät mit der SEQ ID NO:2 von mindestens 80% aufweist, eines funktionellen Äquivalents der SEQ ID NO:4, das eine Identitiät mit der SEQ ID NO:4 von mindestens 98% aufweist, eines funktionellen Äquivalents der SEQ ID NO:6, das eine Identitiät mit der SEQ ID NO:6 von mindestens 73% aufweist, eines funktionellen Äquivalents der SEQ 2D NO:8, das eine Identitiät mit der SEQ ID NO:8 von mindestens 75% aufweist, eines funktionellen Äquivalents der SEQ ID NO:10, das eine Identitiät mit der SEQ ID NO:10 von mindestens 89% aufweist, eines funktionellen Äquivalents der SEQ ID NO:12, das eine Identitiät mit der SEQ ID NO:12 von mindestens 74% aufweist, eines funktionellen Äquivalents der SEQ ID NO:14, das eine Identitiät mit der SEQ ID NO:14 von mindestens 71% aufweist, eines funktionellen Äquivalents der SEQ ID NO:16, das eine Identitiät mit der SEQ ID NO:16 von mindestens 90% aufweist, eines funktionellen Äquivalents der SEQ ID NO:18, das eine Identitiät mit der SEQ ID NO:18 von mindestens 73% aufweist, ableiten läßt;
• b) zusätzliche Funktionselemente; oder
• c) eine Kombination aus a) und b).

sowie die Verwendung von Expressionskassetten enthaltend

• a) genetische Kontrollsequenzen in funktioneller Verknüpfung mit einer erfindungsgemäße Nukleinsäuresequenz
• b) zusätzliche Funktionselemente; oder c) eine Kombination aus a) und b) in "in vitro" oder "in vivo" Testsystemen.

a) comprising genetic control sequences in functional linkage with a nucleic acid sequence

• a) a nucleic acid sequence with that in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 sequence shown; or
• b) a nucleic acid sequence which, on the basis of the degenerate genetic code, is obtained from the back-translation of the sequences in SEQ ID N: 2, SEQ ID N: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18 derived amino acid sequences; or
• c) functional equivalents of SEQ ID NO: 1 with an identity of at least 74% to SEQ ID NO: 1, SEQ ID NO: 3 with an identity of at least 86% to SEQ ID NO: 3, SEQ ID NO : 5 with an identity of at least 73% to SEQ ID NO: 5, SEQ ID NO: 7 with an identity of at least 65% to SEQ ID NO: 7, SEQ ID NO: 9 with an identity of at least 73 % to SEQ ID NO: 9, SEQ ID NO: 11 with an identity of at least 65% to SEQ ID NO: 11, SEQ ID NO: 13 with an identity of at least 69% to SEQ ID NO: 13 , SEQ ID NO: 15 with an identity of at least 78% with SEQ ID NO: 15 or SEQ ID NO: 17 with an identity of at least 68% with SEQ ID NO: 17;
• d) a nucleic acid sequence which, owing to the degenerate genetic code, is converted back by the amino acid sequence of a functional equivalent of SEQ ID NO: 2, which has an identity with SEQ ID NO: 2 of at least 80%, of a functional equivalent of SEQ ID NO: 4, which has an identity with SEQ ID NO: 4 of at least 98%, a functional equivalent of SEQ ID NO: 6, which has an identity with SEQ ID NO: 6 of at least 73%, a functional equivalent of SEQ 2D NO: 8, which has an identity with SEQ ID NO: 8 of at least 75%, a functional equivalent of SEQ ID NO: 10, which has an identity with SEQ ID NO: 10 of at least 89%, a functional equivalent of SEQ ID NO: 12, which has an identity with SEQ ID NO: 12 of at least 74%, a functional equivalent of SEQ ID NO: 14, which has an identity with SEQ ID NO: 14 of at least 71%, a functional equivalent eq equivalents of SEQ ID NO: 16, which have an identity with SEQ ID NO: 16 of at least 90%, a functional equivalent of SEQ ID NO: 18, which has an identity with SEQ ID NO: 18 of at least 73%, can be derived;
• b) additional functional elements; or
• c) a combination of a) and b).

and containing the use of expression cassettes

• a) genetic control sequences in functional linkage with a nucleic acid sequence according to the invention
• b) additional functional elements; or c) a combination of a) and b) in "in vitro" or "in vivo" test systems.

Another object is the use of the above-mentioned embodiments the expression cassettes (hereinafter referred to as "expression cassettes according to the invention") for Expression of a UPS protein for in vitro or in vivo test systems. The expression cassettes described above are hereinafter referred to as expression cassettes according to the invention.

According to a preferred embodiment comprises an expression cassette according to the invention at the 5 'end of the coding Sequence a promoter and at the 3 'end Transcription termination signal and possibly further genetic Control sequences, which with the coding in between Sequence for the gene of the UPS protein functionally linked are.

Equivalents are also according to the invention of the expression cassettes described above, for example by a combination of the individual nucleic acid sequences on a polynucleotide (Multiple constructs), on several polynucleotides in one cell (Co-transformation) or by sequential transformation can come ,

Beneficial genetic control sequences for the Expression cassettes according to the invention or vectors contained therein are, for example, promoters such as cos, tac, trp, tet, lpp, lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, λ-PR or in the λ-PL promoter, which for Expression of the UPS proteins used in gram-negative bacterial strains can be.

Further advantageous genetic control sequences are for example in the promoters amy and SPO2, which are used for expression the SSP can be used in gram-positive bacterial strains, as well in the yeast or fungal promoters AUG1, GPD-1, PX6, TEF, CUP1, PGK, GAP1, TPI, PH05, AOX1, GAL10 / CYC1, CYC1, OliC, ADH, TDH, Kex2, MFa or NMT or combinations of the above promoters (Degryse et al., Yeast 1995 Jun 15; 11 (7): 629-40; Romanos et a1. Yeast 1992 Jun. 8 (6): 423-88; Benito et a1. Eur. J. Plant Pathol. 104: 207-220 (1998); Cregg et a1. Biotechnology (NY) 1993 Aug; 11 (8): 905-10; luo X., Gene 1995 Sep 22; 163 (1): 127-31; Nacken et al., Gene 1996 Oct. 10; 175 (1-2): 253-60; Turgeon et al., Mol Cell Biol 1987 Sep; 7 (9): 3297-305) or the transcription terminators NMT, Gcy1, TrpC, AOX1, nos, the PGK or CYC1 (Degryse et al., Yeast 1995 Jun 15; 11 (7): 629-40; Brunelli et a1. Yeast 1993 Dec9 (12): 1309-18; Frisch et al., Plant Mol. Biol. 27 (2), 405-409 (1995); Scorer et al., Biotechnology (N.Y.) 12 (2), 181-184 (1994), Genbank acc. number Z46232; Zhao et al. Genbank acc number: AF049064; Punt et al., (1987) Gene 56 (1), 117-124) used to express SSP in yeast strains can be.

As for expression in insect cells suitable genetic control elements are the polyhedrin promoter and the p10 promoter (Luckow, V.A. and Summers, M.D. (1988) Bio / Techn. 6, 47-55) and optionally also suitable ones known to the person skilled in the art Terminators.

Beneficial genetic control sequences for expression of the UPS proteins in cell culture are in addition to polyadenylation sequences for example eukaryotic promoters of viral origin such as e.g. Promoters of polyoma, adenovirus 2, cytomegalovirus, HIV thymidine kinase or Simian Virus 40 and, if appropriate, also suitable ones to the person skilled in the art well-known terminators.

Under additional functional elements b) are exemplary but not restrictive reporter genes, origins of replication, selection markers and so-called affinity tags, fused with the nucleic acid sequence according to the invention containing a protease interface either directly or by means of a linker to understand. Are also particularly preferred as additional ones Functional sequences that target the vacuole Mitochondrium, the peroxisome, the endoplasmic reticulum (ER) or a lack of corresponding operative sequences Ensure that it remains in the compartment of formation, the cytosol (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423).

According to the invention are also vectors that at least one copy of the nucleic acid sequences according to the invention and / or the expression cassettes according to the invention contain.

Among vectors are plasmids also all other vectors known to the person skilled in the art, such as, for example Phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA understand. These vectors can replicated autonomously in the host organism or replicated chromosomally chromosomal replication is preferred.

In a further embodiment of the vector, the nucleic acid construct according to the invention can also advantageously be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination. This linear DNA can be from a linearized plasmid or only from the nucleic acid construct as a vector or the one used Nucleic acid sequences exist.

More prokaryotic and eukaryotic Expression systems are mentioned in chapters 16 and 17 in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Other advantageous vectors are found in Hellens et al. (Trends in plant science, 5, 2000).

The expression cassette according to the invention as well derived vectors can for the transformation of bacteria, cyanobacteria, yeasts, filamentous fungi and algae and eukaryotic, non-human cells (e.g. insect cells) are used with the aim of recombinant production of the UPS proteins, whereby the production of a suitable expression cassette the organism in which the gene is to be expressed, directed.

In a further advantageous embodiment can those in the method according to the invention nucleic acid sequences used can also be introduced into an organism alone.

Should be next to the nucleic acid sequences more genes can be introduced into the organism, so everyone can together in a single vector or each gene in each a vector can be introduced into the organism, the various Vectors can be introduced simultaneously or successively.

The introduction of the nucleic acid (s) according to the invention, the expression cassette or the vector into the corresponding organisms (Transformation) in principle by all methods known to the person skilled in the art respectively.

For the transformation of microorganisms can be carried out by a person skilled in the art Methods the textbooks by Sambrook, J. et al.

(1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, by F.M. Ausubel et al. (1994) Current protocols in molecular biology, John Wiley and Sons, by D.M. Glover et al., DNA Cloning Vo1.1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Habor Laboratory Press or Guthrie et al. Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994, Take Academic Press.

In the transformation of filamentous mushrooms The following methods are available: On the one hand, the production of Protoplasts and transformation using PEG (would remain et al. (1997) Mycol. Res. 101 (7): 971-877; Proctor et al. (1997) Microbiol. 143, 2538-2591), on the other hand the transformation under to help Agrobacterium tumefaciens (de Groot et al. (1998) Nat. Biotech. 16, 839-842).

The one with transformation of the above-described embodiments an expression cassette or a vector produced transgenic Organisms containing as nucleic acid sequence SEQ ID NO: 21, SEQ according to the invention ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 39 as well as the functional equivalents defined above the above-mentioned nucleic acid sequences and the means Expression available from the above-mentioned transgenic organism recombinant UPS proteins are the subject of the present invention. Transgenic organisms containing a nucleic acid sequence according to the invention are also used as organisms according to the invention designated.

Organisms for recombinant expression In addition to bacteria, yeasts, mosses, algae and fungi, they are also eukaryotic Cell lines, preferably bacteria, yeast and fungi.

Inside the bacteria are bacteria the genus Escherichia, Erwinia, Flavobacterium, Alcaligenes or Cyanobacteria, for example, of the genus Synechocystis or Anabena prefers.

Genes are preferred yeasts Saccharomyces, Schizosaccheromyces or Pichia.

Preferred mushrooms are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria, Mortierella, Saprolegnia, Pythium, or others in Indian Chem Engr. Section B. Vol 37, No 1.2 (1995) mushrooms.

In principle, are as host organisms transgenic animals are also suitable, for example C. elegans.

Use is also preferred of expression systems and vectors that are publicly available or commercially available are.

For use in E. coli bacteria are the typical advantageous, commercially available Fusion and Expression Vectors pGEX [Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67: 31-40], pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which Contains glutathione S-transferase (GST), maltose binding protein, or Protein A, the pTrc vectors (Amann et al., (1988) Gene 69: 301-315) the "pKK233-2" from CLONTECH, Palo Alto, CA and the "pET" and "pBAD" vector series from Stratagene to call La Jolla.

Further advantageous vectors for use in yeast are pYep-Sec1 (Baldari, et al., (1987) Embo J. 6: 229-234), pMFa (Kurfan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 ( Schultz et al., (1987) Gene 54: 113-123), and pYES derivatives, pGAPZ derivatives, pPICZ derivatives and the vectors of the "Pichia Expression Kit" (Invitrogen Corporation, San Diego, CA). Vectors for use in filamentous fungi are described in: van den Hondel, CAMJJ & Punt, PJ (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, JF Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge.

Alternatively, insect cell expression vectors can also be used advantageously are used e.g. For expression in Sf9, Sf21 or His cells, which via recombinant Baculoviruses get infected. These are e.g. the vectors of the pAc Series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39). Farther the Baculovirus expression systems "MaxBac 2.0 Kit" and "Insect Select System" from Invitrogen may be mentioned, Calsbald or "BacPAK Baculovirus Expression System "from CLONTECH, Palo Alto, CA. Alternatively, insect cell expression vectors can also be used advantageously are used e.g. For expression in Sf9, Sf21 or Hi5 cells, which via recombinant Baculoviruses get infected. These are e.g. the vectors of the pAc Series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39). Farther the Baculovirus expression systems "MaxBac 2.0 Kit" and "Insect Select System" from Invitrogen may be mentioned, Calsbald or "BacPAK Baculovirus Expression System "from CLONTECH, Palo Alto, CA. The handling of insect cells in cell culture as well their infection for the expression of proteins are known to the person skilled in the art and can in analogy to known methods (Luckow and Summers, Bio / Tech. 6, 1988, pp.47-55; Glover and Hames (eds) in DNA cloning 2, A practical Approach, Expression Systems, Second Edition, Oxford University Press, 1995, 205-244).

Furthermore, can be advantageous for gene expression Plant cells or algae cells can be used. Examples of plant expression vectors can be found in Becker, D., et al. (1992) "New plant binary vectors with selectable markers located proximal to the left border ", Plant Mol. Biol. 20: 1195-1197 or in Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation ", Nucl. Acid. Res. 12: 8711-8721.

Furthermore, the nucleic acid sequences according to the invention in mammalian cells be expressed. example for corresponding expression vectors are called pCDM8 and pMT2PC in: Seed, B. (1987) Nature 329: 840 or Kaufuman et a1. (1987) EMBO J. 6: 187-195). Promoters to be used are preferably viral Origin such as Promoters of polyoma, adenovirus 2, cytomegalovirus or Simian Virus 40. Other prokaryotic and eukaryotic expression systems are mentioned in Chapters 16 and 17 in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Further advantageous vectors are in Hellens et al. (Trends in plant science, 5, 2000).

All, Embodiments described above of the transgenic organisms are called "transgenic invention Organism "summarized.

Another subject of the present Invention is the use of nucleic or amino acid sequences a UPS protein in a connection identification process with a fun gicidal effect. All Procedure for the identification of inhibitors with fungicidal activity in the following as the method according to the invention designated.

• i. Inkontaktbringen eines UPS Proteins kodiert durch eine erfindungsgemäße Nukleinsäursequenz mit einer oder mehreren Testsubstanzen unter Bedingungen, die die Bindung der Testsubstanz en) an das Nukleinsäuremolekül oder das Polypeptid, das über das vorstehend genannte Nukleinsäuremolekül kodiert wird, erlauben; und
• ii. Nachweis, ob die Testsubstanz an das UPS Protein aus i) bindet ; oder
• iii.Nachweis, ob die Testsubstanzen die Aktivität des UPS Proteins aus i) reduzieren oder blockieren; oder
• iv. Nachweis, ob die Testsubstanzen die Transkription, Translation oder Expression des UPS aus i) reduzieren oder blockieren.

A preferred embodiment of the method according to the invention comprises the following steps:

• i. Bringing a UPS protein encoded by a nucleic acid sequence according to the invention into contact with one or more test substances under conditions which allow the binding of the test substance (s) to the nucleic acid molecule or the polypeptide which is coded via the aforementioned nucleic acid molecule; and
• ii. Evidence of whether the test substance binds to the UPS protein from i); or
• iii.Proof whether the test substances reduce or block the activity of the UPS protein from i); or
• iv. Evidence as to whether the test substances reduce or block the transcription, translation or expression of the UPS from i).

Evidence according to step ii of the above The procedure can be based on techniques that interact point between protein and ligand, detect, take place. in this connection either the test compound or the enzyme can be a detectable Include markers such as a fluorescent, radioisotope, chemiluminescent or enzymatic labeling. Examples of enzymatic Labels are Horseraddish Peroxidase, Alkaline Phosphatase or lucifierase. The subsequent one Detection depends on the marking and is a specialist known.

• 1. Gemäß einer bevorzugten Ausführungsform umfasst der Nachweis gemäß Schritt ii und Variante 3 des erfindungsgemäße Verfahren umfassend die folgenden Schritte: Über Fluoreszenz Korrelations Spektroskopie (FCS) (Proc. Natl. Acad. Sci. USA (1994) 11753-11575) läßt sich die durchschnittliche Diffusionsrate eines Fluoreszenzmoleküls in Abhängigkeit zur Masse in einem kleinen Probenvolumen bestimmen. Durch Messen der Massenänderung bzw. der daraus resultierenden veränderten Diffunsionsrate einer chemischen Verbindung beim Binden an das erf.-Protein läßt sich FCS zur Bestimmung von Protein-Inhibitor-Wechselwirkungen einsetzten. Ein erfindungsgemäßes Verfahren kann direkt zur Messung der Bindung einer durch ein Fluoreszenzmolekül markierten Testerbindung aufgebaut werden. Alternativ kann das erfindungsgemäße Verfahren so konzipiert sein, dass eine durch ein Fluoreszenzmolekül markierte chemische Referenzverbindung durch zu testende weitere chemische Verbindungen verdrängt wird ("Verdrändungsassay"). Die so identifizierten Verbindugnen können als Inhibitoren geeignet sein.
• 2. Gemäß einer bevorzugten Ausführungsform umfasst der Nachweis gemäß Schritt ii und Variante 3 des erfindungsgemäße Verfahren umfassend die folgenden Schritte: Fluoreszenzpolarisation nutzt die Eigenschaft eines mit polarisiertem Licht angeregten ruhenden Fluorophors ebenfalls wieder polarisiertes Licht zu emittieren. Kann der Fluorophor allerdings während des angeregten Zustands rotieren, so geht die Polarisation des emittierten Fluoreszenzlichts mehr oder weniger verloren. Bei sonst gleichen Bedingungen (z.B. Temperatur, Viskosität, Lösungsmittel) ist die Rotation eine Funktion der Molekülgröße, womit man über das Messsignal eine Aussage über die Größe des am Fluorophor gebundenen Rests treffen kann (Methods in Enzymology 246 (1995), pp. 283-300). Ein erfindungsgemäßes Verfahren kann direkt zur Messung der Bindung einer durch ein Fluoreszenzmolekül markierten Testerbindung an das Erf.-Protein aufgebaut werden. Alternativ kann das erfindungsgemäße Verfahren auch in Form des unter 1 beschriebenen "Verdrängungsassays" konzipiert sein. Die so identifizierten Verbindugnen können als Inhibitoren geeignet sein.
• 3. Gemäß einer bevorzugten Ausführungsform umfasst der Nachweis gemäß Schritt ii und Variante 3 des erfindungsgemäße Verfahren umfassend die folgenden Schritte: Fluoreszenz-Resonanz Energie Tranfer" (FRET) basiert auf der strahlungslosen Energieübertragung zwischen zwei räumlich benachbarten Fluoreszenzmolekülen unter geeigneten Bedingungen. Eine Voraussetzung ist die Überlappung des Emissionsspektrums des Donormoleküls mit dem Anregungsspektrum des Akzeptormoleküls. Durch Fluoreszenzmarkierung des UPS Proteins und den Testverbindungen kann mittels FRET die Bindung gemessen werden (Cytometry 34, 1998, pp. 159-179). Alternativ kann das erfindungsgemäße Verfahren auch in Form des unter 1 beschriebenen "Verdrängungsassays" konzipiert sein. Eine besonders geeignete Ausführungsform der FRET Technologie ist die "Homogenous Time Resolved Fluorescence" (HTRF), wie sie von Packard BioScience vertrieben wird. Die so identifizierten Verbindugnen können als Inhibitoren geeignet sein.
• 4. Gemäß einer bevorzugten Ausführungsform umfasst der Nachweis gemäß Schritt ii und Variante 3 des erfindungsgemäße Verfahren umfassend die folgenden Schritte: Surface Enhanced-Laser Desorption/Ionisation (SELDI) in Kombination mit einem Time of Flight Massenspektrometer (MALDI-TOF) ermöglicht die schnelle Analyse von Molekülen auf einem Träger und kann zur Analyse von Protein-Ligand Wechselwirkungen verwendet werden (Worral et al., (1998) Anal. Biochem. 70:750-756). In einer bevorzugten Ausführungsform immobilisiert man nun das UPS Protein auf einem geeigneten Träger und inkubiert diesen mit der zu untersuchenden chemischen Verbindung. Nach einem oder mehreren geeigneten Waschschritten kann die an das UPS Protein zusätzlich gebundenen Moleküle der chemischen Verbindung mittels der oben erwähnten Methodik detektieren und somit Inhibitoren selektieren. Die so identifizierten Verbindungen können als Inhibitoren geeignet sein.
• 5. Gemäß einer bevorzugten Ausführungsform umfasst der Nachweis gemäß Schritt ii und Variante 3 des erfindungsgemäße Verfahren umfassend die folgenden Schritte: Die Messung von Oberflächen Plasmonenresonanz basiert auf der Änderung des Brechnungsindexes an einer Oberfläche beim Binden einer chemischen Verbindung an ein auf besagter Oberfläche immobilisierten Protein. Da die Änderung des Brechungsindex für eine definierte Änderung der Massenkonzentration an der Oberfläche quasi für alle Proteine und Polypeptide identisch ist, kann diese Methode prinzipiell auf jedes Protein angewendet werden (Lindberg et al. Sensor Actuators 4 (1983) 299-304; Malmquist Nature 361 (1993) 186-187). Die Messung kann beipielsweise mit Hilfe der von Biacore (Freiburg) vertriebenen auf Oberflächen-Plasmonenresonanz basierenden Analyseautomaten in einem Durchsatz von derzeit bis zu 384 Proben pro Tag durchgeführt werden. Ein erfindungsgemäßes Verfahren kann direkt zur Messung der Bindung der Testverbindung an das UPS Protein aufgebaut werden. Alternativ kann das erfindungsgemäße Verfahren auch in Form des unter 1 beschriebenen "Verdrängungsassays" konzipiert sein. Die so identifizierten Verbindungen können als Inhibitoren geeignet sein.

In this context, five preferred embodiments are to be mentioned in particular, which in connection with the present invention are also suitable for high-throughput methods (HTS):

• 1. According to a preferred embodiment, the detection according to step ii and variant 3 of the method according to the invention comprises the following steps: The fluorescence correlation spectroscopy (FCS) (Proc. Natl. Acad. Sci. USA (1994) 11753-11575) can be used determine the average diffusion rate of a fluorescence molecule as a function of mass in a small sample volume. By measuring the change in mass or the resulting change in diffusion rate of a chemical compound when binding to the invented protein, FCS can be used to determine protein-inhibitor interactions. A method according to the invention can be used directly for measuring the binding a tester bond marked by a fluorescent molecule. Alternatively, the method according to the invention can be designed in such a way that a chemical reference compound marked by a fluorescence molecule is displaced by further chemical compounds to be tested ("displacement assay"). The compounds identified in this way can be suitable as inhibitors.
• 2. According to a preferred embodiment, the detection according to step ii and variant 3 of the method according to the invention comprises the following steps: Fluorescence polarization uses the property of a resting fluorophore excited with polarized light also to emit polarized light again. However, if the fluorophore can rotate during the excited state, the polarization of the emitted fluorescent light is more or less lost. Under otherwise identical conditions (eg temperature, viscosity, solvent) the rotation is a function of the molecular size, with which one can make a statement about the size of the residue bound to the fluorophore via the measurement signal (Methods in Enzymology 246 (1995), pp. 283- 300). A method according to the invention can be set up directly for measuring the binding of a tester bond marked by a fluorescent molecule to the Erf. protein. Alternatively, the method according to the invention can also be designed in the form of the "displacement assay" described under 1. The compounds identified in this way can be suitable as inhibitors.
• 3. According to a preferred embodiment, the detection according to step ii and variant 3 of the method according to the invention comprises the following steps: fluorescence resonance energy transfer "(FRET) is based on the radiation-free energy transfer between two spatially adjacent fluorescence molecules under suitable conditions. A prerequisite is Overlap of the emission spectrum of the donor molecule with the excitation spectrum of the acceptor molecule The binding can be measured by means of FRET by fluorescent labeling of the UPS protein and the test compounds (Cytometry 34, 1998, pp. 159-179). Alternatively, the method according to the invention can also be used in the form of 1 A particularly suitable embodiment of the FRET technology is the "Homogeneous Time Resolved Fluorescence" (HTRF), as sold by Packard BioScience. The compounds identified in this way can be used as inhibitors on.
• 4. According to a preferred embodiment, the detection according to step ii and variant 3 of the method according to the invention comprises the following steps: Surface enhanced laser desorption / ionization (SELDI) in combination with a time of flight mass spectrometer (MALDI-TOF) enables rapid analysis of molecules on a support and can be used for the analysis of protein-ligand interactions (Worral et al., (1998) Anal. Biochem. 70: 750-756). In a preferred embodiment, the UPS protein is now immobilized on a suitable carrier and incubated with the chemical compound to be investigated. After one or more suitable washing steps, the molecules of the chemical compound additionally bound to the UPS protein can be detected using the above-mentioned methodology and thus select inhibitors. The compounds identified in this way can be suitable as inhibitors.
• 5. According to a preferred embodiment, the detection according to step ii and variant 3 of the method according to the invention comprises the following steps: The measurement of surface plasmon resonance is based on the change in the refractive index on a surface when a chemical compound binds to a protein immobilized on said surface. Since the change in the refractive index for a defined change in the mass concentration at the surface is virtually identical for all proteins and polypeptides, this method can in principle be applied to any protein (Lindberg et al. Sensor Actuators 4 (1983) 299-304; Malmquist Nature 361 (1993) 186-187). The measurement can be carried out, for example, with the aid of the automated analyzers based on surface plasmon resonance sold by Biacore (Freiburg) in a throughput of currently up to 384 samples per day. A method according to the invention can be set up directly for measuring the binding of the test compound to the UPS protein. Alternatively, the method according to the invention can also be designed in the form of the "displacement assay" described under 1. The compounds identified in this way can be suitable as inhibitors.

All of which Substances identified above can then be identified in another embodiment of the method according to the invention be checked for their fungicidal activity.

There is also the possibility of clarifying the three-dimensional structure of the UPS protein using X-ray structure analysis to detect further potential fungicidal active ingredients by means of "molecular modeling". The production of for the X-ray structure analysis required Protein crystals as well as the corresponding measurements and subsequent evaluations of these measurements, the detection of a binding site in the protein as well the prediction of possible Inhibitor structures are known to the person skilled in the art. In principle, optimization is also about "Molecular Modeling" the over the active substances identified above are possible.

• a) entweder ein UPS Protein in einem Organismus exprimiert wird oder ein Organismus, der naturgemäß ein UPS Protein enthält, kultiviert wird;
• b) das UPS Protein aus Schritt a) im Zellaufschluss des transgenen bzw. nicht transgenen Organismus, in partiell gereinigter Form oder in zur Homogenität gereinigten Form mit einer Testverbindung in Kontakt gebracht wird; und
• c) eine Verbindung selektiert wird, welche die Aktivität des UPS Proteins reduziert oder blockiert, wobei die Aktivität des mit der Testverbindung inkubierten UPS Proteins mit der Aktitivtät eines nicht mit einer Testverbindung inkubierten UPS Proteins ermittelt wird.

A preferred embodiment of the method according to the invention, which is based on steps i) and iii), consists of the following steps:

• a) either a UPS protein is expressed in an organism or an organism that naturally contains a UPS protein is cultivated;
• b) the UPS protein from step a) is brought into contact with a test compound in the cell disruption of the transgenic or non-transgenic organism, in partially purified form or in a form purified to homogeneity; and
• c) a compound is selected which reduces or blocks the activity of the UPS protein, the activity of the UPS protein incubated with the test compound being determined with the activity of a UPS protein not incubated with a test compound.

Here, connections are made in step (c) selected that a significant decrease in the activity of the UPS Result in a reduction of at least 10 %, advantageously at least 20%, preferably at least 30%, particularly preferably by at least 50% and very particularly preferably by at least 70% or a 100% reduction (blocking) is achieved.

The solution containing the UPS protein can from the lysate of the original Organism or the transgenic organism. If necessary can partially or completely purify the UPS protein using common methods become. A general overview of common techniques for the purification of proteins are described, for example, in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1994); ISBN 0-87969-309-6. In the case of recombinant Dar a cleaning of the with a Affinity tag fused polypeptides over affinity chromatography respectively.

The UPS required for in vitro processes Protein can thus be called either by means of heterologous expression from a transgenic according to the invention Organism can be expressed. This methodology is particularly suitable for E2. Alternatively, the UPS can also come from an organism that contains a UPS protein contains, isolated from a mushroom or a yeast, for example. suitable Yeasts are in the genera Saccharomyces, Schizosaccheromyces or Pichia included. Suitable mushrooms are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria, Mortierella, Saprolegnia, Pythium, or other mushrooms, e.g. the phytopathogenic filamentous fungi mentioned at the beginning. For example, the 20s proteasome or 26s proteasome from one suitable organism such as S. cerevisiae e.g. according to Groll et al. (1997 Nature 386, 463-471) can be isolated.

Determining the activity of the UPS For example, protein can an enzymatic activity test take place, i.e. by incubation of the polypeptide according to the invention with a suitable substrate, the decrease in the substrate or the rise of the resulting product is tracked.

In a particularly preferred embodiment the turnover of the substrate is monitored photometrically.

• a) Beladung des E2 Proteins mit Ubiquitin;
• b) Markierung des Ubiquitins am E2 mit Allophycocyanin und eines geeigneten Akzeptorproteins mit Europium Chelat;
• c) Inkontaktbringung des markierten ubiquitinbeladenen E2 mit einer Testverbindung
• d) Reaktionsstart durch Zugabe des markierten Akzeptorproteins;
• e) Anregung der Europiummarkierung mit 340 nm;
• d) Messung der E2 Aktivität durch Detektion bei 665 nm.

A particularly preferred embodiment of this method for identifying inhibitors for E2 is based on a method described by Boisclair et al (2000, J. Biomol. Screen. 5 (5) 319-28) and comprises the following steps:

• a) loading the E2 protein with ubiquitin;
• b) labeling the ubiquitin on E2 with allophycocyanin and a suitable acceptor protein with europium chelate;
• c) contacting the labeled ubiquitin-laden E2 with a test compound
• d) start of the reaction by adding the labeled acceptor protein;
• e) excitation of the Europium marking with 340 nm;
• d) Measurement of the E2 activity by detection at 665 nm.

• a) Inkontaktbringen des aufgereinigten 26s oder 20s Proteasoms mit einer Testverbindung;
• b) Zugabe eines geeigneten Substrates mit Fluorogener Abgangsgruppe;
• c) Bestimmung der Fluoreszenz der Abgangsgruppe (nach ca. 1-5h);
• d) nach Inkubation (ca. 1-5h) Anregung bei einer geeigneten Wellenlänge;
• e) Messung der Aktivität durch Detektion bei einer geeigneten Wellenlänge.

Another particularly preferred embodiment of this method for identifying inhibitors for the 26s proteasome or the 20s subunit of the 26s proteasome is based on a method according to Dick et al. ((1998) J. Biol. Chem. 273 (40), 25637-46) which comprises the following steps:

• a) contacting the purified 26s or 20s proteasome with a test compound;
• b) adding a suitable substrate with fluorogenic leaving group;
• c) determination of the fluorescence of the leaving group (after approx. 1-5 h);
• d) after incubation (approx. 1-5h) excitation at a suitable wavelength;
• e) measurement of the activity by detection at a suitable wavelength.

Examples of suitable substrates with fluorogenic Leaving groups are benzyloxycarbonyl-Ala-Arg-Arg-7-amino-4-methylcoumarin (Z-GGL-AMC), succinyl-Leu-Leu-Val-Tyr-7-amino-4 methylcoumarin, Benzyloxycarbonyl-Gly-Gly-Leu-7-amino-4 methylcoumarin, benzyloxycarbonyl-Leu-Leu-Glu-beta-naphthylamide.

• i. der Herstellung eines transgenen Organismus enthaltend eine erfindungsgemäße Nukleinsäuresequenzen;
• ii. das Aufbringen einer Testsubstanz auf den transgenen Organismus nach Anspruch a) und auf einen nicht-transgenen Organismus der gleichen Art;
• iii.das Bestimmen des Wachstums, der Überlebensfähigkeit und/oder der Ifektiosistät des transgenen und des nicht transgenen Organismus nach der Aufbringung der Testsubstanz; und
• iv. der Selektion von Testsubstanzen, die ein vermindertes Wachstum, Überlebensfähigkeit und/oder Infektiosität des nichttransgenen Organismus bewirken verglichen mit dem Wachstum des transgenen Organmismus bewirken.

Suitable wavelengths for excitation and measurement are e.g. 380nm / 440 nm for AMC and 330nm / 410 nm for beta-NA A preferred embodiment of the method according to the invention which is based on based on steps i) and iv) consists of the following steps:

• i. the production of a transgenic organism containing a nucleic acid sequence according to the invention;
• ii. the application of a test substance to the transgenic organism according to claim a) and to a non-transgenic organism of the same type;
• iii. determining the growth, survivability and / or ifectivity of the transgenic and non-transgenic organisms after application of the test substance; and
• iv. the selection of test substances which bring about a reduced growth, viability and / or infectivity of the non-transgenic organism compared to the growth of the transgenic organism.

Here the growth difference is or the difference in infectivity in step c) for the selection of an inhibitor with a fungicidal action at least 10%, preferably 20%, preferably 30%, particularly preferred 40% and very particularly preferably 50%. Infectivity is determined only if the organism is a phytopathogenic fungus is.

The production of the transgenic organism can by transforming the organism with a nucleic acid sequence according to the invention, an expression cassette according to the invention or a vector containing a nucleic acid sequence according to the invention or an expression cassette according to the invention.

The transgenic cells or organisms are bacteria, yeasts, filamentous fungi or eukaryotic Cell lines, preferably a phytopathogenic filamentous fungus, the phytopathogenic filamentous fungi mentioned above are particularly preferred. These transgenic organisms or cells therefore have an increased tolerance against chemical compounds, which the polypeptide of the invention inhibit.

All of which Substances identified above can then be identified in another in vivo activity test be checked for their fungicidal activity. A possibility consists of testing the appropriate substance in an agar diffusion test like with teeth, H. 1965 Biology of antibiotics, Berlin, Springer Verlag described, to test. Carried out the test is carried out with a culture of a filamentous phytopathogenic fungus, preferably a culture of a phytopathogenic, the fungicidal Effect e.g. about limited growth can be determined. Here are under the term phytopathogenic Mushroom the ones mentioned at the beginning To understand species.

According to the method according to the invention can also several test compounds in a method according to the invention be used. If through a group of test compounds one Influencing the target takes place, then it is either possible to isolate individual test compounds directly or the group of Divide test compounds into different subgroups, e.g. if it is made up of a variety of different components so the number of different test compounds in the method according to the invention to reduce. The method according to the invention then again with the individual test connection or the corresponding one Subset of test compounds. Depending on the complexity of the sample can the steps described above are repeated several times, preferably until according to the method of the invention Subgroup identified only a small number of test compounds or comprises only one test connection.

The method according to the invention can be advantageous carried out in a HTS become.

HTS enables parallel testing a variety of different connections.

In HTS offers for the practical execution the use of straps to one or more of the nucleic acid molecules according to the invention, a or more of the nucleic acid sequence according to the invention containing vectors, one or more transgenic organisms, which at least one of the nucleic acid sequences according to the invention contain or one or more (poly) peptides encoded via the nucleic acid sequences according to the invention. The carrier used can be solid or liquid is preferably solid, particularly preferably a microtiter plate. The carriers mentioned above are also the subject of the present invention. According to the on the most widespread technology uses 96-well microtiter plates, which can usually contain volumes from 50 to 500μl. In addition to the microtiter plates are the other components of a HTS system to match 96-well microtiter plates like many instruments, materials, automatic Pipetting devices, robots, automated plate readers as well Plate washer commercially available.

In addition to the HTS methods based on microtiter plates, so-called "free format assays" or test systems which have no physical barriers between the samples can also be used, as described, for example, in Jayaickreme et al., Proc. Natl. Acad. Sci USA 19 (1994) 161418; Chelsky, "Strategies for Screening Combinatorial Libaries, First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 710, 1995); Salmon et al., Molecular Diversity 2 (1996), 5763 and US 5,976,813 ,

The invention further relates to compounds identified by the methods according to the invention. These compounds are referred to below as "selected compounds". You assign a Mon molecular weight of less than 1000 g / mol, advantageously less than 500 g / mol, preferably less than 400 g / mol, particularly preferably less than 300 g / mol. Compounds with a fungicidal activity have a Ki value of less than 1 mM, preferably less than 1 μM, particularly preferably less than 0.1 μM, very particularly preferably less than 0.01 μM.

Own the selected connections to fight phytopathogenic mushrooms. Examples of phytopathogenic fungi are the following genera and species: Alternaria species, Podosphaera species, Sclerotinia species, Physalospora canker on vegetables and fruits, Botrytis cinerea (Gray mold) on strawberries, vegetables, Ornamental plants and vines, Corynespora melonis on cucumbers, strawberries; Colletotrichum species on cucumbers; Diplocarpon rosae on roses; Elsinoe fawcetti and Diaporthe citri on citrus fruits; Sphaerotheca species Cucumbers, pumpkin family, strawberries and roses; Cinula neccata on cucumbers, Cercospora species on peanuts, sugar beets, egg plants and date plums; Erysiphe cichoracearum and Sphaerotheca fuliginea on pumpkin family, Leveillula taurica on allspice; Mycosphaerella species on apples and Japanese apricot; Phyllactinia kakicola, Gloesporium kaki, on Japanese apricot; Gymnosporangium yamadae, Leptotthrydium pomi, Podosphaera leucotricha and Gloedes pomigena on apples; Cladosporium carpophilum on pears and Japanese apricot; Phomopsis species on pears; Phytopora species on citrus fruits, potatoes, onions; Phytophthora infestans on potatoes and tomatoes, Erysiphe graminis (powdery mildew) on cereals, Fusarium and Verticillium species various plants, Glomerella cingulata on tea; Drechslera or Bipolaris species on cereals, Mycosphaerella species on bananas and peanuts, Plasmopara viticola on vines and grapefruits, Personospora species onions, spinach and chrysanthemums; Phaeoisariopsis vitis and Spaceloma ampelina on grapefruit; Pseudocercosporella herpotrichoides on wheat and barley, Pseudoperonospora species on hops and cucumbers, Puccinia species and Typhula species on cereals, Pyrenophora teres on barley, Pyricularia oryzae on rice, Rhizoctonia species on cotton, Rice and lawn, Stachosporium nodorum on wheat, Uncinula necator on vines, Ustilago species on cereals and sugar cane, Gaeumannomyces graminis on oats and beets as well as Venturia species (Scab) on apples and pears.

The selected connections can also in the form of their agriculturally useful salts. Under agriculturally useful salts come mainly from the salts of those cations or the acid addition salts of those acids into consideration, the cations or anions of which are fungicidal Effect of over the method according to the invention identified compounds with fungicidal activity not negative affect.

All of which Compounds identified above are provided, provided they chirality centers contain, both as pure enantiomers or diastereomers or as their mixtures and as a racemate the subject of the present Invention.

The selected compounds can be chemical be synthesized or microbiologically produced substances and e.g. in cell extracts of e.g. Plants, animals or microorganisms occur. The reaction mixture can be a cell-free extract or comprise a cell or cell culture. Suitable methods are known to the person skilled in the art and are e.g. generally described in Alberts, Molecular Biology the cell, 3rd Edition (1994), e.g. Chapter 17.

Possible Test connections can Expression libraries such as cDNA expression libraries, peptides, Proteins, nucleic acids, Antibody, small organic substances, hormones, PNAs or the like (Milner, Nature Medicin 1 (1995), 879-880; Hupp, Cell. 1995, 83: 237-245; Gibbs, Cell. 79 (1994), 193-198 and references cited therein).

Fungicidal agents which the selected Contain connections, fight phytopathogenic mushrooms very good, especially with high application rates. They work in crops such as wheat, rice, corn, soybeans and cotton against phytopathogenic fungi, without the crops worth mentioning to harm. This effect occurs especially at low application rates. If with the aid of the method according to the invention found fungicidal active ingredients as total or selective fungicides act depends among other things, the amount used, their selectivity and others Factors.

The substances can be used to combat the already mentioned above pathogenic fungi can be used.

Depending on the particular Application method can the selected compounds or compositions containing them advantageous for eliminating the phytopathogenic agents already mentioned at the beginning Mushrooms are used.

Another object of the invention a process for the preparation of the fungicides already mentioned above Composition, characterized in that selected compounds with for formulated the formulation of fungicides suitable aids.

The selected compounds can be formulated, for example, in the form of directly sprayable aqueous solutions, powders, suspensions, also high-strength aqueous, oily or other suspensions or suspoemulsions or dispersions, emulsifiable concentrates, emulsions, oil dispersions, pastes, dusts, sprinkling agents or granules and by spraying , Atomizing, dusting, scattering or pouring can be used. The application forms depend on the purposes and the nature of the selected compounds and should in any case be the finest possible distribution of the se ensure selected connections. The fungicidal composition contains a fungicidally effective amount of at least one selected compound and auxiliaries customary for the formulation of fungicidal compositions.

For the production of emulsions, pastes or watery or oily Formulations and dispersible concentrates (DC) can be used selected compounds dissolved in an oil or solvent or are dispersed, with further formulation auxiliaries for homogenization can be added. It can but also from a selected compound, optionally solvents or oil as well as optionally other aids existing liquid or solid concentrates are made that can be diluted with water are suitable. Emulsifiable concentrates (EC, EW), suspensions (SC), soluble concentrates (SL), dispersible concentrates (DC), pastes, pastilles wettable Powders or granules, the solid formulations being either soluble in water (soluble) or dispersible (wettable). Furthermore, corresponding Powder or granules or tablets still with a solid, the Abrasion or a premature drug release preventing coating ("coating").

In principle are under the term Assistants to understand the following substance classes: anti-foaming agents, Thickeners, wetting agents, adhesives, dispersants, emulsifiers, Bactericides and / or thixotrophic agents. The meaning of the above mentioned means is known to the expert.

SLs, EWs and ECs can be produced by simply mixing the corresponding ingredients, powder by mixing or grinding in special mill types (e.g. hammer mills). DC, SCs and SEs are usually produced by wet milling, it being possible to produce an SE from a SC by adding an organic phase which may contain further auxiliaries or selected compounds. The manufacture is known. Powders, materials for broadcasting and dusts can advantageously be prepared by mixing or grinding the active substances together with a solid carrier. Granules, for example coated granules, impregnated granules and homogeneous granules, can be prepared by binding the selected compounds to solid carriers. Further details of the production are known to the person skilled in the art, and e.g. listed in the following writings: US 3,060,084 , EP-A 707445 (for liquid concentrates), Browning, "Agglomeration", Chemical Engineering, Dec. 4, 1967, 147-48, Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New York, 1963, pages 8-57 and ff. WO 91/13546 , US 4,172,714, US 4,144,050, US 3,920,442, US 5,180,587, US 5,232,701, US 5,208,030, GB 2,095,558, US 3,299,566, Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961, Hance et al. , Weed Control Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989 and Mollet, H., Grubemann, A., Formulation technology, Wiley VCH Verlag GmbH, Weinheim (Federal Republic of Germany), 2001.

Inert liquid and / or solid carriers suitable for the formulations according to the invention are known to the expert in a variety, such as. liquid additives like mineral oil fractions from medium to high boiling point such as kerosene or diesel oil coal as well as oils of vegetable or animal origin, aliphatic, cyclic and aromatic hydrocarbons, e.g. Paraffin, tetrahydronaphthalene, alkylated Naphthalenes or their derivatives, alkylated benzenes or their derivatives, Alcohols such as methanol, ethanol, propanol, butanol, cyclohexanol, Ketones such as cyclohexanone or strongly polar solvents, e.g. Amines like N-methylpyrrolidone or water.

Solid carriers are for example Mineral soils like silicas, Silica gels, silicates, talc, kaolin, limestone, lime, chalk, bolus, Loess, clay, Dolomite, diatomaceous earth, calcium and magnesium sulfate, magnesium oxide, ground plastics, fertilizers, such as ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas and vegetable products such as flour, tree bark, wood and nutshell, Cellulose powder or other solid carriers.

Surfactants (surfactants) suitable for the formulations according to the invention are known to the expert in a variety, such as. Alkali-, Alkaline earth, ammonium salts of aromatic sulfonic acids, e.g. Lignin, phenol, naphthalene and dibutylnaphthalenesulfonic acid, as well of fatty acids, Alkyl and alkylaryl sulfonates, alkyl, lauryl ether and fatty alcohol sulfates, and salts of sulfated hexa-, hepta- and octadecanols and of Fatty alcohol glycol ether, condensation products of sulfonated naphthalene and its derivatives with formaldehyde, condensation products of naphthalene or the naphthalenesulfonic acids with Phenol and formaldehyde, polyoxyethylene octylphenol ether, ethoxylated Isooctyl, octyl or nonylphenol, alkylphenyl, tributylphenyl polyglycol ether, Alkylaryl polyether alcohols, isotridecyl alcohol, fatty alcohol ethylene oxide condensates, ethoxylated castor oil, Poly oxyethylene alkyl ether or polyoxypropylene alkyl ether, lauryl alcohol polyglycol ether acetate, Sorbitol esters, lignin sulfite liquors or methyl cellulose.

Application of the fungicidal compositions or the active ingredients can be curative, eradicative or protective.

The application rates of fungicidal active ingredient (= Substance and / or agent) depending on the target, Season, target plants and stage of growth 0.001 to 3.0, preferably 0.01 to 1.0 kg / ha.

The invention is by the now following examples, but is not limited to this.

The genetic engineering processes on which the following exemplary embodiments are based are briefly described below: cloning processes such as restriction cleavages, DNA isolation, Agaro se gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, sequence analysis of recombinant DNA as well as Southern and Western blots were carried out as in Sambrook et al ., Cold Spring Harbor Laboratory Press (1989) and Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1994); ISBN 0-87969-309-6.

The bacterial strains used below (E. coli TOP 10) were obtained from Invitrogen, Carlsberg, CA. As F. Graminearum wild type strain 8/1 can e.g. the DSM: 4527 can be used.

Furthermore, all chemicals used below, unless otherwise stated, were obtained in pA quality from Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Solutions were prepared with treated, pyrogen-free water, hereinafter referred to as H ₂ O, from a Milli-Q Water System water treatment plant (Millipore, Eschborn). Restriction enzymes, DNA-modifying enzymes and molecular biological kits were made by the companies AGS (Heidelberg), Amersham (Braunschweig), Biometra (Göttingen), Roche (Mannheim), Genomed (Bad Oeynnhausen), New England Biolabs (Schwalbach / Taunus), Novagen ( Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Promega (Madison, Wisconsin, USA), Pharmacia (Freiburg), Qiagen (Hilden) and Stratagene (Heidelberg). Unless otherwise stated, they were used according to the manufacturer's instructions.

All for genetic engineering experiments Media and buffer used were either via sterile filtration or heating sterilized in an autoclave.

Examples A total of three different cDNA libraries made from F. graminearum.

The mycelium, on the basis of which the first cDNA bank was produced, comes from the mycelium of a Fusarium graminearum strain, which on complete medium (1 g KH ₂ PO ₄ , 1 g KNO ₃ , 0.5 g MgSO ₄ × 7 H ₂ O, 0.5 g KCl, 20 g sorbitol ad 1000 ml H ₂ O) was attracted.

The mycelium, on the basis of which the second cDNA bank was produced, comes from mycelium of a Fusarium graminearum strain which is based on N-deficient medium (1 g KH ₂ PO ₄ , 0.018 KNO ₃ , 0.5 g MgSO ₄ × 7 H ₂ O, 0 , 5g KCL, 10g glucose, 10g sucrose ad 1000 ml H ₂ O) was attracted.

For the production of the third cDNA bank, conidia were used, which were incubated for three days by incubating spores of a Fusarium graminearum strain for three days on a medium which allowed the conidia to germinate (1 g NH ₄ H ₂ PO ₄ , 3 g K ₂ HPO ₄ , 0, 2g MgSO ₄ × 7 H ₂ O, 5g NaCl, 40g sucrose, 10g glycerol ad 1000 ml H2O) were obtained.

The RNA isolation was carried out with the pegGOLD RNAPure ^™ from Promega according to the manufacturer's instructions. The amount was quantified photometrically and the quality of the RNA was checked by gel electrophoresis.

The cDNA banks were produced the first strand synthesis after the "primer switch Method "(from Clontech, according to the manufacturer's instructions) with the help of an oligo-dT primer Included adapter with a NotI binding site. During the First strand synthesis became a second oligonucleotide primer at the 5 'end of the first strand cDNA introduced, which contained an AscI interface. The synthesis of the second cDNA strands were carried out with the adapter sequences as primer binding sites for the Amplification primers. The sequences were converted into the vector pUCminIV cloned and sequenced.

The 3 cDNA banks were separated sequenced. Following were the sequences obtained were processed using bioinformatic methods. After various processing, a clustering contigs created. The programs became clusters and annotations Harvesters and pedants used by Biomax, Martinsried. Contigs consisting of sequences that occurred in all three banks, were filtered out and are referred to below as "house keeping genes".

These "house keeping" genes thus obtained were used against the essential yeast genes of Mips (see e.g. ewes, H. W., D. Frishman, U.

Guldener, G. Mannhaupt, K. Mayer, M. Mokrejs, B. Morgenstern, M. Munsterkotter, S. Rudd, and B. Weil. MIPS: a database for genomes and protein sequences. Nucleic Acids Res 30: 31-34, 2002. blasted. (cutoff value 10-6). The nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17, which have been identified as house keeping genes and which are also essential for S. cerevisiae, are therefore essential for F. graminearum and represent targets for fungicides. SEQUENCE LISTING

Claims (21)

Use of a protein from the Ubiquitin Proteasome pathway encoded by a nucleic acid sequence full a) a nucleic acid sequence with that in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 sequence shown; or b) a nucleic acid sequence, resulting from the degenerate genetic code from the retranslation the one in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18 amino acid sequences shown can be derived; or c) functional equivalents the SEQ ID NO: 1 with an identity of at least 60% to the SEQ ID NO: 1, the SEQ ID NO: 3 with an identity of at least 60% to that SEQ ID NO: 3, the SEQ ID NO: 5 with an identity of at least 60% to that SEQ ID NO: 5, the SEQ ID NO: 7 with an identity of at least 60% to the SEQ ID NO: 7, the SEQ ID NO: 9 with an identity of at least 60% to that SEQ ID NO: 9, the SEQ ID NO: 11 with an identity of at least 60% to that SEQ ID NO: 11, the SEQ ID NO: 13 with an identity of at least 60% to that SEQ ID NO: 13, the SEQ ID NO: 15 with an identity of at least 60% to that SEQ ID NO: 15 or SEQ ID NO: 17 with an identity of at least 60% to SEQ ID NO: 17; d) a nucleic acid sequence that is due to of the degenerate genetic code by back-translating the amino acid sequence of a functional equivalent the SEQ ID NO: 2, which has an identity with the SEQ ID NO: 2 of at least 60%, a functional equivalent of SEQ ID NO: 4, that is an identity with SEQ ID NO: 4 of at least 60%, a functional equivalent the SEQ ID NO: 6, which has an identity with the SEQ ID NO: 6 of at least 60%, a functional equivalent of SEQ ID NO: 8, that is an identity with SEQ ID NO: 8 of at least 60%, a functional equivalent the SEQ ID NO: 10, which has an identity with the SEQ ID NO: 10 of has at least 60%, a functional equivalent of SEQ ID NO: 12, that is an identity with SEQ ID NO: 12 of at least 60%, a functional equivalent the SEQ ID NO: 14, which has an identity with the SEQ ID NO: 14 from has at least 60%, a functional equivalent of SEQ ID NO: 16, that is an identity with SEQ ID NO: 16 of at least 60%, a functional equivalent the SEQ ID NO: 18, which has an identity with the SEQ ID NO: 18 from has at least 60%, can be derived. as well as by the above called nucleic acid sequences encoded proteins as targets for Fungicides. Nucleic acid sequences encoding a protein of the ubiquitin proteasome metabolic pathway for use as a target for fungicides, comprising: a) a nucleic acid sequence with the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17; or b) a nucleic acid sequence which, on the basis of the degenerate genetic code, is obtained from the back-translation of the sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18 derived amino acid sequences; or c) functional equivalents of SEQ ID NO: 1 with an identity of at least 74% to SEQ ID NO: 1, SEQ ID NO: 3 with an identity of at least 86% to SEQ ID NO: 3, SEQ ID NO: 5 with an identity of at least 73% to SEQ ID NO: 5, SEQ ID NO: 7 with an identity of at least 65% to SEQ ID NO: 7, SEQ ID NO: 9 with an identity of at least 73% to SEQ ID NO: 9, SEQ ID NO: 11 with an identity of at least 65% to SEQ ID NO: 11, SEQ ID NO: 13 with an identity of at least 69% to SEQ ID NO: 13, SEQ ID NO: 15 with an identity of at least 78% to SEQ ID NO: 15 or SEQ ID NO: 17 with an identity of at least 68% to SEQ ID NO: 17; d) a nucleic acid sequence which, owing to the degenerate genetic code, is converted back by the amino acid sequence of a functional equivalent of SEQ ID NO: 2, which has an identity with SEQ ID NO: 2 of at least 80%, of a functional equivalent of SEQ ID NO: 4, which has an identity with SEQ ID NO: 4 of at least 98%, a functional equivalent of SEQ ID NO: 6, which has an identity with SEQ ID NO: 6 of at least 73%, a functional equivalent of SEQ ID NO: 8, which has an identity with SEQ ID NO: 8 of at least 75%, a functional equivalent of SEQ ID NO: 10, which has an identity with SEQ ID NO: 10 of at least 89%, a functional equivalent of SEQ ID NO: 12, which has an identity with SEQ ID NO: 12 of at least 74%, a functional equivalent of SEQ ID NO: 14, which has an identity with SEQ ID NO: 14 of at least 71%, a functional equivalent eq uivalents of SEQ ID NO: 16, which has an identity with SEQ ID NO: 16 of at least 90%, a functional equivalent of SEQ ID NO: 18, which has an identity with SEQ ID NO: 18 of at least 73%, can be derived. nucleic acid sequence according to claim 2, which comes from a fungus of the genus Fusarium. nucleic acid sequence according to claim 2 or 3, which is derived from Fusarium graminearum. Polypeptide encoded by the nucleic acid molecule according to one of claims 2 to 4th Method for the detection of functional analogues of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 by making a probe followed by subsequent Search a genomic or cDNA bank of the corresponding Species or a computer search for analog sequences in electronic databases. Method of identifying mutations in one for a protein of the nucleic acid sequence encoding the ubiquitin proteasome pathway consisting of i. the production of on a nucleic acid sequence according to the claims 2 to 4 based oligonucleotides containing the mutation with subsequent PCR; or ii. based on the production of oligonucleotides a nucleic acid sequence according to the claims 2 to 4, the region flanking the mutation using PCR is amplified, followed by restriction digestion and / or Sequencing of the PCR product produced. Containing expression cassette a) genetic control sequences in functional connection with the over Expectations 2 to 4 defined nucleic acid sequence; or b) additional Functional elements; or c) a combination of a) and b). Vector containing an expression cassette according to claim B. Non-human transgenic organism containing at least one nucleic acid sequence according to claim 2 to 4, an expression cassette according to claim 8 or a vector according to claim 9 selected from Bacteria, yeasts, fungi, animal or plant cells. Use of a protein from the Ubiquitin Proteasome pathway encoded by a nucleic acid sequence full a) a nucleic acid sequence according to claims 2 to 4; b) functional equivalents the SEQ ID NO: 1 with an identity of at least 60% to the SEQ ID NO: 1, the SEQ ID NO: 3 with an identity of at least 60% to that SEQ ID NO: 3, the SEQ ID NO: 5 with an identity of at least 60% to that SEQ ID NO: 5, the SEQ ID NO: 7 with an identity of at least 60% to the SEQ ID NO: 7, the SEQ ID NO: 9 with an identity of at least 60% to that SEQ ID NO: 9, the SEQ ID NO: 11 with an identity of at least 60% to that SEQ ID NO: 11, the SEQ ID NO: 13 with an identity of at least 60% to that SEQ ID NO: 13, the SEQ ID NO: 15 with an identity of at least 60% to that SEQ ID NO: 15 or SEQ ID NO: 17 with an identity of at least 60% to SEQ ID NO: 17; c) a nucleic acid sequence that is due to of the degenerate genetic code by back-translating the amino acid sequence of a functional equivalent the SEQ ID N0: 2, which is an identity with the SEQ ID NO: 2 of at least one 60%, a functional equivalent of SEQ ID NO: 4, an identity with which has SEQ ID NO: 4 of at least 60%, a functional equivalent the SEQ ID NO: 6, which has an identity with the SEQ ID NO: 6 of at least 60%, a functional equivalent of SEQ ID NO: 8, that is an identity with SEQ ID NO: 8 of at least 60%, a functional equivalent of SEQ ID NO: 10, which is an identity with SEQ ID NO: 10 of at least 60%, a functional equivalent the SEQ ID NO: 12, which has an identity with the SEQ ID NO: 12 of has at least 60%, a functional equivalent of SEQ ID NO: 14, that is an identity with SEQ ID NO: 14 of at least 60%, a functional equivalent the SEQ ID NO: 16, which has an identity with the SEQ ID NO: 16 of has at least 60%, a functional equivalent of SEQ ID NO: 18, that is an identity with SEQ ID NO: 18 of at least 60%. such as of proteins encoded by the aforementioned nucleic acid sequences as targets for Fungicides. in a procedure for the identification of substances with fungicidal effect. Method of identifying substances with fungicidal Effect comprising the following steps: i. contacting Protein encoded by the ubiquitin proteasome pathway a nucleic acid sequence full a) a nucleic acid sequence according to claims 2 to 4; b) functional equivalents the SEQ ID NO: 1 with an identity of at least 60% to the SEQ ID NO: 1, the SEQ ID NO: 3 with an identity of at least 60% to that SEQ ID NO: 3, the SEQ ID NO: 5 with an identity of at least 60% to that SEQ ID NO: 5, the SEQ ID NO: 7 with an identity of at least 60% to the SEQ ID NO: 7, the SEQ ID NO: 9 with an identity of at least 60% to that SEQ ID NO: 9, the SEQ ID NO: 11 with an identity of at least 60% to that SEQ ID NO: 11, the SEQ ID NO: 13 with an identity of at least 60% to that SEQ ID NO: 13, the SEQ ID NO: 15 with an identity of at least 60% to that SEQ ID NO: 15 or SEQ ID NO: 17 with an identity of at least 60% to SEQ ID NO: 17; or c) a nucleic acid sequence, which are due to the degenerate genetic code through back translation the amino acid sequence of a functional equivalent the SEQ ID NO: 2, which has an identity with the SEQ ID NO: 2 of at least 60%, a functional equivalent of SEQ 2D NO: 4, that is an identity with SEQ ID NO: 4 of at least 60%, a functional equivalent the SEQ ID NO: 6, which has an identity with the SEQ ID NO: 6 of at least 60%, a functional equivalent of SEQ ID NO: 8, that is an identity with the SEQ 2D NO: 8 of at least 60%, a functional equivalent the SEQ ID NO: 10, which has an identity with the SEQ ID NO: 10 of has at least 60%, a functional equivalent of SEQ ID NO: 12, that is an identity with SEQ ID NO: 12 of at least 60%, a functional equivalent the SEQ ID NO: 14, which has an identity with the SEQ ID NO: 14 from has at least 60%, a functional equivalent of SEQ ID NO: 16, that is an identity with SEQ ID NO: 16 of at least 60%, a functional equivalent the SEQ ID NO: 18, which has an identity with the SEQ ID NO: 18 from has at least 60%, can be derived. with one or more Test substances under conditions that affect the binding of the test substance (s) to the nucleic acid molecule or the Polypeptide encoding the above nucleic acid molecule will allow; and ii. Evidence that the test connection is on the polypeptide from i) binds. iii.Proof of whether the test compound the activity of the polypeptide from i) reduced or blocked; or iv. Proof, whether the test compound is transcription, translation or expression the nucleic acid from i) reduced or blocked. A method according to claim 12, characterized in that i. either a protein of the ubiquitin proteasome pathway, encoded by a nucleic acid sequence comprising a) a nucleic acid sequence according to claims 2 to 4; b) functional equivalents of SEQ ID NO: 1 with an identity of at least 60% to SEQ ID NO: 1, SEQ ID NO: 3 with an identity of at least 60% to SEQ ID NO: 3, SEQ ID NO : 5 with an identity of at least 60% to SEQ ID NO: 5, SEQ ID NO: 7 with an identity of at least 60% to SEQ ID NO: 7, SEQ ID NO: 9 with an identity of at least 60 % to SEQ ID NO: 9, SEQ ID NO: 11 with an identity of at least 60% to SEQ ID NO: 11, SEQ ID NO: 13 with an identity of at least 60% to SEQ ID NO: 13 , SEQ ID NO: 15 with an identity of at least 60% to SEQ ID NO: 15 or SEQ ID NO: 17 with an identity of at least 60% to SEQ ID NO: 17; or c) a nucleic acid sequence which, owing to the degenerate genetic code, is translated back by the amino acid sequence of a functional equivalent of SEQ ID NO: 2, which has an identity with SEQ ID NO: 2 of at least 60%, of a functional equivalent of SEQ ID NO : 4, which has an identity with SEQ ID NO: 4 of at least 60%, a functional equivalent of SEQ ID NO: 6, which has an identity with SEQ ID NO: 6 of at least 60%, a functional equivalent of SEQ ID NO: 8, which has an identity with SEQ ID NO: 8 of at least 60%, a functional equivalent of SEQ ID NO: 10, which has an identity with SEQ ID NO: 10 of at least 60%, a functional equivalent SEQ ID NO: 12, which has an identity with SEQ ID NO: 12 of at least 60%, a functional equivalent of SEQ ID NO: 14, which has an identity with SEQ ID NO: 14 of at least 60%, one functional n equivalent of SEQ ID NO: 16, which has an identity with SEQ ID NO: 16 of at least 60%, a functional equivalent of SEQ ID NO: 18, which has an identity with SEQ ID NO: 18 of at least 60% , can be derived. is expressed in a transgenic organism or an organism which naturally contains a protein of the ubiquitin proteasome pathway, encoded by one of the nucleic acid sequences mentioned above, is cultivated; ii. the protein of the ubiquitin proteasome pathway from step i) is brought into contact with a test compound in the cell disruption of the transgenic or non-transgenic organism, in partially purified form or in a form purified to homogeneity; and iii. a compound is selected which reduces or blocks the activity of the protein of the ubiquitin proteasome metabolic pathway, the activity of the protein incubated with the test compound being determined with the activity of a protein not incubated with a test compound. A method according to claim 12, characterized in that it includes the following steps: i. Cultivation of a transgenic Organism according to claim 10 or a transgenic organism containing one nucleic acid sequence full a) a nucleic acid sequence according to claims 2 to 4; b) functional equivalents the SEQ ID NO: 1 with an identity of at least 60% to the SEQ ID NO: 1, the SEQ ID NO: 3 with an identity of at least 60% to that SEQ ID NO: 3, the SEQ ID NO: 5 with an identity of at least 60% to that SEQ ID NO: 5, the SEQ ID NO: 7 with an identity of at least 60% to the SEQ ID NO: 7, the SEQ ID NO: 9 with an identity of at least 60% to that SEQ ID NO: 9, the SEQ ID NO: 11 with an identity of at least 60% to that SEQ ID NO: 11, the SEQ ID NO: 13 with an identity of at least 60% to that SEQ ID NO: 13, the SEQ ID NO: 15 with an identity of at least 60% to that SEQ ID NO: 15 or SEQ ID NO: 17 with an identity of at least 60% to SEQ ID NO: 17; or c) a nucleic acid sequence, which are due to the degenerate genetic code through back translation the amino acid sequence of a functional equivalent the SEQ ID NO: 2, which has an identity with the SEQ ID NO: 2 of at least 60%, a functional equivalent of SEQ ID NO: 4, that is an identity with SEQ ID NO: 4 of at least 60%, a functional equivalent the SEQ ID NO: 6, which has an identity with the SEQ ID NO: 6 of at least 60%, a functional equivalent of SEQ ID NO: 8, that is an identity with SEQ ID NO: 8 of at least 60%, a functional equivalent the SEQ ID NO: 10, which has an identity with the SEQ ID NO: 10 of has at least 60%, a functional equivalent of SEQ ID NO: 12, that is an identity with SEQ ID NO: 12 of at least 60%, a functional equivalent the SEQ ID NO: 14, which has an identity with the SEQ ID NO: 14 from has at least 60%, a functional equivalent of SEQ ID NO: 16, that is an identity with SEQ ID NO: 16 of at least 60%, a functional equivalent the SEQ ID NO: 18, which has an identity with the SEQ ID NO: 18 from has at least 60%, can be derived; ii. the applying a test substance for the transgenic organism according to claim a) and in a non-transgenic organism of the same kind; iii.das Determine growth, survivability and / or the infectivity of the transgenic and the non-transgenic organism after application of the test substance; and iv. the selection of test substances that show a reduced Growth, survivability and / or infectivity of the non-transgenic organism compared to the growth of the transgenic organism. Process according to claims 14, characterized in that it is carried out with a mushroom. Process according to claims 15, characterized in that it has a filamentous, phytopathogenic Mushroom performed becomes. Method according to one of claims 12 to 16, characterized in that that identify the substances in a high-throughput screening carried out becomes. carrier for use in one of the methods according to claims 1 to 19, the one or several of the nucleic acid molecules after one of the claims 2 to 4, one or more vectors according to claim 9, one or several hosts according to claim 10 or one or more (poly) peptides according to claim 5. Compounds with fungicidal activity identified via one the method according to the claims 12 to 17. Process for the preparation of a fungicidal composition, characterized in that a fungicidal active ingredient over a the method according to the claims 12 to 17 identified, and this active ingredient with for the formulation formulated with fungicides. Control procedures of harmful fungi, characterized in that the fungi or those to be protected from fungal attack Materials, plants, soil or seeds with an effective amount a compound according to claim 19 or a composition by a method according to claim 20 treated.