WO2005098030A2 - Method for the identification of fungicidally-effective compounds based on thioredoxin reductases - Google Patents

Abstract

The invention relates to a method for the identification of fungicides, the use of fungal thioredoxin reductases for the identification of fungicides and the use of thioredoxin reductase inhibitors as fungicides.

Description

Method for identifying fungicidally active compounds based on thioredoxin reductases

The invention relates to a method for identifying fungicides, the use of fungal thioredoxin reductase to identify fungicides, and the use of inhibitors of thioredoxin reductase as fungicides.

Unwanted fungal growth, which causes considerable damage in agriculture every year, can be controlled through the use of fungicides. The demands on fungicides have steadily increased in terms of their effectiveness, costs and, above all, their environmental compatibility. There is therefore a need for new substances or classes of substances that can be developed into powerful and environmentally compatible new fungicides. In general, it is common to search for such new lead structures in greenhouse tests. However, such tests are labor intensive and expensive. The number of substances that can be tested in the greenhouse is limited accordingly. An alternative to such tests is the use of high-throughput screening (HTS). A large number of individual substances are tested for their effects on cells, individual gene products or genes in an automated process. If an effect is proven for certain substances, these can be examined in conventional screening methods and, if necessary, further developed.

Favorable targets for fungicides are often sought in essential biosynthetic pathways. Ideal fungicides continue to be substances that inhibit gene products that are crucial

Of importance in the expression of the pathogenicity of a fungus.

The object of the present invention was therefore to identify a suitable new point of attack for potential fungicidal active substances and to make them accessible, and a method for _. To make available, which enables the identification of modulators of this point of attack, which can then be used as fungicides.

Thioredoxin reductase (EC 1.8.1.9, formerly EC 1.6.4.5), also known as NADP-thioredoxin reductase, NADPH-thioredoxin reductase or thioredoxin: NADP ⁺ -oxidoreductase, catalyzes the reaction of NADP ⁺ and thioredoxin to thio-edoxin-disulfide NADPH + ϊ (Figure 1). The reaction of thioredoxin reductase is an essential step in the recycling of the oxidized form of thioredoxin (disulfide form), which provides the cell with reduced thioredoxin. The cytoplasmic thioredoxin reductase - hereinafter also referred to as TRR - belongs to class II of pyridine nucleotide disulfide oxido eductases. The enzyme recycles the oxidized form of thioredoxin and provides the cell with reduced thioredoxin. TRR is therefore important for maintaining redox homeostasis in the cell. Thioredoxins themselves are small, highly conserved oxidoreductases that contain two conserved cysteine residues in the active center. They were originally identified as electron donors for the enzyme ribonucleotide reductase, but are also necessary for a large number of metabolic enzymes that form disulfide bonds during their catalytic cycles, such as PAPS reductase. Thioredoxins are best characterized as antioxidants against reactive oxygen species, but their functions also include protection against reductive stress. It is believed that they also play a role in a variety of other cellular processes, such as protein folding and its regulation, the repair of oxidatively damaged proteins and in the sulfur metabolism.

Bacterial and fungal thioredoxin reductases are flavoenzymes and exist as homodimers. They contain a FAD cofactor and a pair of redox-active Cys groups

Transfer of the redox equivalents from the FAD to the substrate are involved.

Genes for the thioredoxin reductase were cloned from various organisms, including from different fungi such as Saccharomyces cerevisiae (Swissprot Accession No .: P38816), Schizosaccharomyces pom.be (Swissprot Accession No .: Q92375), Neurospora crassa (Swissprot Accession No. P51978) , Pneumocystis carinii (Swissprot Accession No .: Q7Z7S3), Pneumocystis jiroveci (Swissprot Accession No .: Q8J0U0) or Penicillium chrysogenum (Swissprot Accession No .: P43496). The sequence similarities are significant within the eukaryotic classes. In contrast, thioredoxin reductases from phytopathogenic fungi have so far not become known.

The object of the present invention was to identify new points of attack by fungicides in fungi, in particular in phytopathogenic fungi, and to make methods accessible in which inhibitors of such a point of attack or polypeptide can be identified and tested for their fungicidal properties. The object was achieved by isolating the nucleic acid coding for thioredoxin reductase from a phytopathogenic fungus, the polypeptide encoded therefrom, and a method using which

Inhibitors of this enzyme can be determined. The inhibitors identified with this method can be used against fungi in vivo. Description of the pictures

Figure 1: Schematic representation of the reduction of thioredoxin in its disulfide form to thioredoxin with free SH groups catalyzed by the thioredoxin reductase. In this reaction, NADP + H ^{+ is converted} to NADP ⁺ .

Figure 2: Sequence alignment of the amino acid sequence of thioredoxin reductases from various fungi. The homologous areas between the thioredoxin reductases are shown. In the sequences continue to the Prosite motif amino acids were appropriate for thioredoxin reductases ^'' marked. Yeastl and Yeast2: Saccharomyces cerevisiae. PNEUCA: Pneumocycstis carinii. PNEUJI: Pneumocystis jiroveci. POMBE: Saccharomyces pombe. NEUCR: Neurospora crassa. PENCH: Penicillium chrysogenum. Ustilago: Ustilago maydis.

Figure 3: SDS gel. The expression and purification of TRR1 from U. maydis was monitored using SDS gels (A) to (C). Lane 1: non-induced E. coli cells. Lane 2: is.co/z cells after induction. Lane 3: Precipitation after cell disruption. Lane 4: supernatant after cell disruption. Lane 5: wash fraction from the Ni-NTA column. Lane 6: Elution fraction after the Ni-NTA column. Lane 7: fraction after the PD-10 column. M: 10 kDa protein standard.

Figure 4: SDS gel. The heterologous expression and purification of thioredoxin, the substrate of thioredoxin reductase, was monitored using SDS gels (A) to (D). Lane 1: uninduced E. co / z ^' cells. Lane 2: E. co / z cells after induction. Lane 3: Precipitation after cell disruption. Lane 4: supernatant after cell disruption. Lane 5: wash fraction from the Ni-NTA column. Lane 6: Elution fraction from the Ni-NTA column. Lane 7: fraction after the PD-10 column. M: 10 kDa protein standard. Gel (D) shows samples of the individual thioredoxin purifications.

Figure 5: Graphical representation of the kinetics of the NADPH decrease in an activity test for the thioredoxin reductase. The enzymatic activity of thioredoxin reductase (TRR1) is determined based on the consumption of NADPH. For this purpose, the decrease in NADPH was investigated in 16 batches based on the change in fluorescence (excitation at 360 nm, emission at 465 nm) as a function of time (■). The same preparation conditions were used as a control in the absence of the enzyme (♦). The kinetics show one due to the enymatic activity of the TRR, almost linear decrease in the fluorescence yield within 35-40 min, which then changes into a plateau area.

Figure 6: Lineweaver-Burk plot for determining the K value of thioredoxin (A) and NADPH (B). The measured values are presented according to Lineweaver and Burk, ie 1 / V ₀ = 1 / V _max + 1 / S x (K _M / V _max ), where V _{0 is} the initial reaction rate, V _max the maximum achievable conversion rate and S the substrate concentration is. V _nιax and K _M can then be read as the abscissa and ordinate _sections 1 / V _max and 1 / K _M, respectively. The K _M value for E. co / z ^' thioredoxin is 5 μM, the K _M value for NADPH is between 20 and 40 μM.

Figure 7: Graphical representation of the decrease in the activity of thioredoxin reductase (TRR1) in the presence of an inhibitor. The activity of TRR1 was measured by determining the NADPH concentration over time. A smaller decrease in the NADPH concentration indicates a lower or inhibited enzymatic reaction of the TRR1. The measurements were carried out in the presence of different concentrations of n-ethylmaleimide. The concentrations used are given in the legend. O.I. stands for the reaction in the absence of the inhibitor, i.e. the uninhibited reaction (negative control).

SEQ ID NO: 1 nucleic acid sequence coding for the thioredoxin reductase from Ustilago maydis.

SEQ ID NO: 2 amino acid sequence of Uiorago maydis thioredoxin reductase.

definitions

The term "homology" or "identity" is to be understood as the number of matching amino acids (identity) with other proteins, expressed in percent. The identity is preferably determined by comparing a given sequence to other proteins with the aid of computer programs. If sequences which are compared with one another have different lengths, the identity is to be determined in such a way that the number of amino acids which the shorter sequence has in common with the longer sequence determines the percentage of identity. By default, the identity can be established by means of known computer programs available to the public, e.g. ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). ClustalW is e.g. made publicly available by Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can also be accessed from various websites, including at the IGBMC (Institut de Genetique et de Biologie Moleculaire et Cellulaire, BP163, 67404 Illkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and at the EBI (ftp: //ftp.ebi .ac.uk / pub / software /) and from all mirrored websites of the EBI (European Bioinfo matics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, K). If the ClustalW computer program version 1.8 is used to establish the identity between e.g. To determine a given reference protein and other proteins, set the following parameters: KTUPLE = 1, TOPDIAG = 5, WINDOW = 5, PAIRGAP = 3, GAPOPEN = 10, GAPEXTEND = 0.05, GAPDIST = 8, MAXDIV = 40, MATRIX = GONNET, ENDGAPS (OFF), NOPGAP, NOHGAP. One way to find similar sequences is to perform sequence database searches. Here, one or more sequences are specified as a so-called query. This query sequence is then compared using statistical computer programs with sequences that are contained in the selected databases. Such database queries ("blast searches") are known to the person skilled in the art and can be carried out at various providers. If such a database query e.g. carried out at the NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/), the standard settings that are specified for the respective comparison request should be used. For protein sequence comparisons ("blastp") these are the following settings: Limit entrez = not activated; Filter = low complexity activated; Expect value = 10; word size = 3; Matrix = BLOSUM62; Gap costs: Existence = 11, Extension = 1. As a result of such a query, among others

, Parameters also show the proportion of identity between the query sequence and the similar sequences found in the databases. A protein according to the invention should therefore be understood in connection with the present invention to mean those proteins which when using at least one of the methods described above for identity determination have an identity of at least 70%, preferably of at least 75%, particularly preferably of at least 80%, more preferably of at least 85%, and in particular of at least 90%.

The term "complete thioredoxin reductase" as used herein describes one

Thioredoxin reductase, which is encoded by a complete coding region of a transcription unit, comprising an ATG start codon and comprising all information-bearing exon regions of the gene coding for a thioredoxin reductase present in the organism of origin, as well as the signals necessary for correct termination of the transcription.

The term "biological activity of a thioredoxin reductase" as used in the present context refers to the ability of a polypeptide to effect the reaction described above, ie the reduction of thioredoxin in its disulfide form to thioredoxin with free SH groups using NADPH + H ^{1 "} (Figure 1). Two cysteine residues and the amino acids surrounding these cysteine residues are characteristic of thioredoxin reductases (Figure 2).

The term "active fragment" as used herein no longer describes complete nucleic acids coding for thioredoxin reductase, but which still code for polypeptides with the biological activity of a thioredoxin reductase and which can catalyze a reaction characteristic of the thioredoxin reductase as described above , Such fragments are shorter than the complete nucleic acids encoding thioredoxin reductase described above. Nucleic acids may have been removed both at the 3 'and / or 5' ends of the sequence, but parts of the sequence can also be deleted, i.e. have been removed that do not significantly affect the biological activity of the thioredoxin reductase. A lower or possibly also an increased activity, which, however, still allows the characterization or use of the resulting thioredoxin reductase fragment, is understood to be sufficient in the sense of the expression used here. The expression "active fragment" can also refer to the amino acid sequence of thioredoxin reductase and then applies analogously to the above explanations for those polypeptides which, in comparison to the complete sequence defined above, no longer contain certain parts, the biological activity of the enzyme, however, not being decisive is impaired. The fragments can have different lengths.

The terms "thioredoxin reductase inhibition test" or "inhibition test" as used herein refer to a method or a test which allows the inhibition of the enzymatic activity of a polypeptide with the activity of a thioredoxin reductase recognize one or more chemical compounds (candidate compound (s)), whereby the chemical compound can be identified as an inhibitor of thioredoxin reductase.

The term "gene" as used herein is the term for a portion of the genome of a cell that is responsible for the synthesis of a polypeptide chain.

The term "fungicide" or "fungicidal" as used herein refers to chemical compounds which are suitable for combating fungi which are pathogenic to humans, animals and plants, in particular fungi which are pathogenic to plants. Such phytopathogenic fungi are mentioned below, the list is not exhaustive:

Plasmodiophoromycetes, Oomycetes, Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes, e.g.

Pythium species, such as, for example, Pythium ultimum, Phytophthora species, such as, for example, Phytophthora infestans, Pseudoperonospora species, such as, for example, Pseudoperonospora humuli or Pseudoperonospora cubensis, Plasmopara species, such as, for example, Plasmopara viticola, Bremia species, such as, for example, Bremia lactucae, Peronospor Species, such as, for example, Peronospora pisi or P. brassicae, Erysiphe species, such as, for example, Erysiphe graminis,

Sphaerotheca species, such as, for example, Sphaerotheca fuliginea, Podosphaera species, such as, for example, Podosphaera leucotricha, Venturia species, such as, for example, Venturia inaequalis, Pyrenophora species, such as, for example, Pyrenophora teres or P. graminea (conidial form: Drechslera, Syn: Helminthosobol Species, such as, for example, Cochliobolus sativus (conidial form: Drechslera, Syn: Helminthosporium), Uromyces species, such as

Uromyces appendiculatus, Puccinia species, such as, for example, Puccinia recondita, Sclerotinia species, such as, for example, Sclerotinia sclerotiorum, Tilletia species, such as, for example, Tilletia caries; Ustilago species, such as, for example, Ustilago nuda or Ustilago avenae, Pellicularia species, such as, for example, Pellicularia sasakii, Pyricularia species, such as, for example, Pyricularia oryzae, Fusarium species, such as, for example - Fusaήum culmorum, Botrytis species, Septoria species, such as, for example Septoria nodorum, Leptosphaeria species such as Leptosphaeria nodorum, Cercospora species such as Cercospora canescens, Alternaria species such as Alternaria brassicae or Pseudocercosporella species such as Pseudocercosporella herpotrichoides.

Of particular interest are e.g. also Magnaporthe grisea, Cochliobulus heterostrophus,

Nectria hematococcus and Phytophtora species. Fungicidal active ingredients which are found with the aid of the thioredoxin reductases according to the invention from plant-pathogenic fungi can also interact with thioredoxin reductases from human-pathogenic fungus species, the interaction with the different thioredoxin reductases occurring in these fungi not always having to be equally strong ,

The present invention therefore also relates to the use of inhibitors of

Thioredoxin reductase for the manufacture of agents for the treatment of diseases caused by human pathogenic fungi.

The following human pathogenic fungi are of particular interest, which can cause the following clinical pictures:

Dermatophytes, e.g. Trichophyton spec, Microsporum spec, Epidermophyton floccosum or

Keratomyces ajelloi, e.g. Cause foot mycoses (tinea pedis),

Yeasts such as Candida albicans, which causes thrush esophagitis and dermatitis, Candida glabrata, Candida krusei or Cryptococcus neoformans, which e.g. can cause pulmonary cryptococcosis and also torulose,

Mold, such as Aspergiltus fumigatus, A. flavus, A. niger, e.g. bronchopulmonary

Cause aspergillosis or fungal sepsis, Mucor spec, Absidia spec, or Rhizopus spec. Cause zygomycoses (intravascular mycoses), Rhinosporidium seeberi, which e.g. chronic granulomatous pharyngitis and tracheitis, Madurella myzetomatis, which e.g. causes subcutaneous mycetomas, Histoplasma capsulatum, which e.g. reticuloendothelial cytomycosis and Darling disease, Coccidioides immitis, which causes Z: B. pulmonary coccidioidomycosis and sepsis, Paracoccidioides brasiliensis, which e.g. Brazilian blastomycosis, Blastomyces dermatitidis, which e.g. Gilchrist's disease and North American blastomycosis, Loboa loboi, which e.g. Keloid blastomycosis and Lobo's disease, and Sporothrix schenckii, e.g. Sporotrichosis (granulomatous skin mycosis).

Fungicidal active ingredients which are found with the aid of a thioredoxin reductase obtained from a particular fungus, here from Ustilago maydis, can therefore also ^" interact with thioredoxin reductases from other numerous fungal species, especially with phytopathogenic fungi, the interaction with the different ones in these Thioredoxin reductases occurring in fungi do not always have to be of the same strength, which explains, among other things, the observed selectivity of the substances active on this enzyme.

The term "homologous promoter" as used in the present context refers to a promoter which controls the expression of the gene in question in the original organism. The The term "heterologous promoter" as used in the present context refers to a promoter which has different properties than the promoter which controls the expression of the gene in question in the original organism.

The term “competitor” as used in the present context refers to the property of the compounds to compete with other, if necessary, identifiable compounds in order to compete for binding to the thioredoxin reductase and to displace or be displaced by the enzyme.

The term "inhibitor" or "specific inhibitor" as used herein denotes a substance that directly inhibits an enzymatic activity of the thioredoxin reductase. A ^• Such an inhibitor preferably is "specific", ie it inhibits specifically the thioredoxin reductase activity at a concentration which is lower than the cause concentration of an inhibitor that is required to another, so that unbonded effect. The concentration is preferably two times lower, particularly preferably five times lower and very particularly preferably at least ten times or 20 times lower than the concentration of a compound which is required to produce an unspecific effect.

The term "modulator" as used herein represents a generic term for inhibitors and activators. Modulators can be small organic chemical molecules, peptides or antibodies which bind to the polypeptides according to the invention or which influence their activity. Furthermore, modulators can be small organic chemical molecules, peptides or antibodies which bind to a molecule which in turn binds to the polypeptides according to the invention and thereby influences their biological activity. Modulators can be natural substrates and ligands, or structural or functional mimetics thereof. The term "modulator" as used herein is preferably, however, those molecules which do not represent the natural substrates or ligands.

Description of the invention

In the present invention for the first time the complete sequence of a thioredoxin reductase from the plant pathogenic fungus Ustilago maydis is made available, which further research of thioredoxin reductases in particular from plant pathogenic fungi and thus the development of a new target protein for the identification of new fungicidal

Active ingredients enables.

Despite the extensive research on thioredoxin reductase, it was previously unknown that thioredoxin reductase in fungi can be a target protein (a so-called "target") of fungicidally active substances. This shows for the first time in the present invention that thioredoxin reductase is an enzyme that is particularly important for fungi and is therefore particularly suitable for use as a target protein in the search for further and improved fungicidally active compounds.

So far, inhibitors of thioredoxin reductase with a fungicidal activity have not been described. Various publications refer to the special role of thioredoxin reductase in the metabolism of organisms such as of the yeast

Saccharomyces cerevisiae. In yeast, two genes are annotated as thioredoxin reductases, whereby TRR1 is localized cytoplasmic and TRR2 is suspected mitochondrial. Deletion mutants of TRR2 are viable, hypersensitive to hydrogen peroxide, show temperature-sensitive growth and are auxotrophic for methionine (Machado et al., 1997; Pearson and Merril, 1998), while deletion mutants of the cytoplasmic TRR1 are not viable

(Giaever et al, 2002). In another study, the trri null mutant was fatal, but showed only very slow growth, trrl mutations were also sensitive to hydrogen peroxide. Mutations in the thioredoxin reductase gene from Neurospora crassa cause cysteine auxotrophy (K. Onai and H. Nakashima, 1997). However, none of the publications comment on the question whether the enzyme thioredoxin reductase is by

Active substances influenced, e.g. can be inhibited, and whether the control of fungi, in particular phytopathogenic fungi, is possible in vivo with an active ingredient which modulates the thioredoxin reductase. The thioredoxin reductase has not yet been described as a target protein for fungicides. There are no known active ingredients which have a fungicidal action and whose place of action is thioredoxin reductase.

In the context of the present invention it could be shown that the thioredoxin reductase is surprisingly a target or "target" for fungicidal active substances in phytopathogenic fungi, the inhibition of the thioredoxin reductase thus leading to damage or death of the fungus. So in the plant pathogenic fungus Ustilago maydis one for one Gene coding for thioredoxin reductase identified (um 140_5). Knock-out of this gene was found to be lethal (Example 1). In further experiments, which were directed towards the accessibility of the thioredoxin reductase for active substances in vitro and also in vivo, it could be shown that the thioredoxin reductase can be used in suitable test methods for identifying modulators or inhibitors of its enzymatic activity, which can be used in a

A series of theoretically interesting targets cannot be taken for granted.

In the context of the present invention, a method was finally developed which is suitable for determining the activity of the thioredoxin reductase and the inhibition of this activity by a chemical compound in a so-called inhibition test, in order in this way to inhibit the enzyme, e.g. in HTS and UHTS processes to identify and their fungicidal

To determine properties.

Finally, it was also found in the context of the present invention that thioredoxin reductase can be inhibited in vivo by active substances and that a fungal organism treated with these active substances can be damaged and killed by treatment with these active substances. The inhibitors of a fungal thioredoxin reductase can therefore be used as fungicides in crop protection or as antifungals in pharmaceutical indications. In the present invention it is shown, for example, that the inhibition of the thioredoxin reductase with a substance identified in a method according to the invention leads to the death of the treated fungi in synthetic media or on the plant.

Thioredoxin reductases can be obtained from various phytopathogenic or also from human or animal pathogenic fungi, e.g. from fungi such as the plant pathogenic fungus U maydis. For the production of the thioredoxin reductase from fungi, the gene can e.g. recombinantly expressed in Escherichia coli and an enzyme preparation is produced from E. coli cells (Example 2). Thioredoxin reductases from phytopathogenic fungi are preferably used to identify fungicides which can be used in crop protection. If the goal is to identify fungicides or antimycotics that are to be used in pharmaceutical indications, the use of thioredoxin reductases from human or animal pathogenic fungi is recommended.

Thus, for the expression of the U. maydis polypeptide TRR1 encoded by trrl, the associated ORF was amplified by means of PCR using selected primers using methods known to the person skilled in the art. The corresponding DNA was cloned into the expression vector pTrcHis2-TOPO, so that the TRR protein is expressed starting from the plasmid ptrcTRRlO with a C-terminal His ₆ tag (Example 2). To express the TRR1, the plasmid ptrcTRR10 was transformed into E. coli BL21 (DE3). The trrl gene from Ustilago maydis has a size of 1053 bp, es does not contain hitron. UM 140_5 (trrl) codes for a polypeptide of 351 amino acids in length with a molecular weight of 39000 daltons (Figure 3).

The present invention thus also provides a complete genomic sequence of a phytopathogenic fungus coding for a thioredoxin reductase, and its use or the use of the polypeptide encoded thereby for the identification of

Inhibitors of the enzyme are described.

The present invention therefore also relates to the nucleic acid from the fungus Ustilago maydis which codes for a polypeptide with the enzymatic function of a thioredoxin reductase.

Thioredoxin reductases share homologous areas. A conserved region containing two cysteines, which are involved in the redox-active disulfide bridge, is typical for thioredoxin reductases. The sequences surrounding these two cysteines are also characteristic of thioredoxin reductases.

This cysteine and its sequence environment is a characteristic of the sequence for thioredoxin reductases. Such a motif can be identified by a suitable search in the PROSITE database (Hofmann et al., 1999),

C-x (2) -C-D- [GA] -x (2,4) - [FY] -x (4) - [LIVM] -x- [LIVM] (2) -G (3) - [DNj,

the two cysteines forming the disulfide bridge.

PROSITE enables polypeptides to be assigned a function and thus to recognize thioredoxin reductases as such.

The "one-letter code" is used to display the Prosite motif. The symbol "x" stands for a position where every amino acid is accepted. A variable position at which various specific amino acids are accepted is shown in square brackets "[...]", whereby the possible amino acids at this position are listed. Amino acids that are not accepted at a certain position, however, are enclosed in curly brackets "{...}". A dash "-" separates the individual elements or positions of the motif. Repeats a certain position, e.g. "x", several times in succession, this can be represented by specifying the number of repetitions in a subsequent bracket, e.g. "x (3)" which stands for "x-x-x".

A Prosite motif ultimately represents the components of a consensus sequence, as well as the distances between the amino acids involved and is therefore typical for a specific enzyme great. On the basis of the nucleic acids according to the invention, this motif can be used to identify or assign further polypeptides from phytopathogenic fungi which belong to the same class as the polypeptide according to the invention and can therefore also be used in the manner according to the invention.

In the case of thioredoxin reductase from U. maydis, this motive is also present, e.g. in S. cerevisiae, S. pombe, P. carinii, P. jiroveci, P. chrysogenum or N. crassa (see Figure 2).

The above-mentioned Prosite motif or the specific consensus sequence are typical of the polypeptides according to the invention, which can be structurally defined on the basis of these consensus sequences and are therefore also clearly identifiable.

The present invention therefore also relates to polypeptides from plant pathogens

Mushrooms with the biological activity of a thioredoxin reductase which has the above-mentioned prostite motif Cx (2) -CD- [GA] -x (2.4) - [FY] -x (4) - [LIVM] -x- [LIVM ] (2) -G (3) - [DΝ] (see also Figure 2).

Those polypeptides according to the invention with the biological activity of a thioredoxin reductase which have the motif C-AN-C-D-G-AN-P-I-F-R-Ν-K-P-L-xN- are particularly preferred.

[VI] -G (3) -D include.

Because of the homologies that exist with species-specific nucleic acids coding for thioredoxin reductases, thioredoxin reductases from other phytopathogenic fungi can also be identified and used to achieve the above object, ie they can also be used to identify inhibitors of a thioredoxin reductase, which can in turn be used as fungicides in crop protection. However, it is also conceivable to use another fungus which is not pathogenic to the plant, or its thioredoxin reductase or the sequence coding therefor, in order to identify fungicidal inhibitors of thioredoxin reductase. Based on the sequence given here according to SEQ ID NO: 1 and any primers derived therefrom and, if appropriate, with the aid of the homologous regions (Figure 2) and the prosite motif shown above or the preferred motif as described above, it is possible for the person skilled in the art, for example by To obtain and identify PCR for further nucleic acids coding for thioredoxin reductases from other (phytopathogenic) fungi. Such nucleic acids and their use in methods for identifying fungicidal active substances are considered to be encompassed by the present invention. With the aid of the nucleic acid sequence according to the invention and sequences obtained from other phytopathogenic fungi according to the methods described above, further nucleic acid sequences coding for a thioredoxin reductase from other fungi can be identified.

The present invention therefore relates to nucleic acids from phytopathogenic fungi which code for a polypeptide with the enzymatic activity of a thioredoxin reductase, in particular polypeptides which comprise the motifs described above.

The present invention preferably relates to nucleic acids from the phytopathogenic fungal species mentioned above in the definitions section, which code for a polypeptide with the enzymatic activity of a thioredoxin reductase.

The present invention particularly preferably relates to the nucleic acid coding for the Ustilago maydis thioredoxin reductase with the SEQ ID NO: 1 and the nucleic acids coding for the polypeptides according to SEQ ID NO: 2 or active fragments thereof.

The nucleic acids according to the invention are in particular single-stranded or double-stranded deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). preferred

Embodiments are fragments of genomic DNA and cDNAs.

The nucleic acids according to the invention particularly preferably comprise a sequence of phytopathogenic fungi coding for a polypeptide with the enzymatic activity of a thioredoxin reductase selected from

a) a sequence according to SEQ ID NO: 1,

b) sequences which code for a polypeptide which comprises the amino acid sequence according to SEQ ID NO: 2,

c) sequences which code for a polypeptide with the enzymatic activity of a thioredoxin reductase which has the motif Cx (2) -CD- [GA] -x (2,4) - [FY] -x (4) - [LIVM] -x- [LIVM] (2) -G (3) - [DN] and preferably the motif CAVCDGAVPIFRNKPLx- V- [VI] -G (3) -D,

d) sequences which hybridize to the sequences defined under a) and b) at a hybridization temperature of 42-65 ° C., and e) sequences which have at least 80%, preferably at least 85% and particularly preferably at least 90% identity with the sequences defined under a) and b).

As already stated above, the present invention is not only limited to the thioredoxin reductase from Ustilago maydis. Polypeptides with the activity of a thioredoxin reductase can also be obtained from other fungi, preferably from phytopathogenic fungi, in an analogous manner to those skilled in the art. can be used in a method according to the invention. However, the thioredoxin reductase from Ustilago maydis is preferably used.

The present invention furthermore relates to DNA constructs which comprise a nucleic acid according to the invention and a homologous or heterologous promoter.

The choice of heterologous promoters depends on whether pro- or eukaryotic cells or cell-free systems are used for expression. Examples of heterologous promoters are the 35S promoter of the cauliflower mosaic virus for plant cells, the alcohol dehydrogenase promoter for yeast cells, the T3, T7 or SP6 promoters for prokaryotic cells or cell-free systems.

Fungal expression systems such as e.g. the Pichia pastoris system can be used, the transcription here being driven by the methanol-inducible AOX promoter.

The present invention further relates to vectors which are an inventive

Contain nucleic acid, a regulatory region according to the invention or a DNA construct according to the invention. All phages, plasmids, phagmids, phasmids, cosmids, YACs, BACs, artificial chromosomes or particles which are suitable for particle bombardment can be used as vectors.

Preferred vectors are e.g. the p4XXprom. Vector series (Mumberg et al, 1995) for yeast cells, pSPORT vectors (from Life Technologies) for bacterial cells, or the gateway vectors (from Life Technologies) for various expression systems in bacterial cells, plants, P. pastoris, S. cerevisiae or insect cells.

The present invention also relates to host cells which contain a nucleic acid according to the invention, a DNA construct according to the invention or a vector according to the invention. The term "host cell" as used in the present context refers to cells which do not naturally contain the nucleic acids according to the invention.

Suitable host cells are both prokaryotic cells, preferably E. coli, and eukaryotic cells, such as cells from Saccharomyces cerevisiae, Pichia pastoris, insects, plants, frog oocytes and cell lines from mammals.

The present invention furthermore relates to polypeptides with the biological activity of a thioredoxin reductase, which are encoded by the nucleic acids according to the invention.

The polypeptides according to the invention preferably comprise an amino acid sequence selected from phytopathogenic fungi

(a) the sequence according to SEQ ID NO: 2,

(b) sequences which have an at least 80%, preferably an at least 85%, particularly preferably a 90% and particularly preferably a 95% identity with the sequence defined under a),

(c) the sequences given under b) which contain the motif Cx (2) -CD- [GA] -x (2,4) - [FY] - x (4) - [LIVM] -x- [LIVM3 (2 ) -G (3) - [DN] and preferably include the motif CAVCDGAVPI-FRNKPLxV- [VI] -G (3) -D, and

(d) Fragments of the sequences given under a) to c), which have the same biological activity as the sequence defined under a).

The term "polypeptides" as used herein refers to both short amino acid chains, commonly referred to as peptides, oligopeptides, or oligomers, and longer amino acid chains, commonly referred to as proteins. It includes

Amino acid chains that can be modified either by natural processes, such as post-translational processing, or by chemical processes that are state of the art. Such

Modifications can occur at different locations and multiple times in a polypeptide, such as, for example, on the peptide backbone, on the amino acid side chain, on the amino and / or on

Carboxy terminus. They include, for example, acetylations, acylations, ADP ribosylations, amidations, covalent linkages with flavins, heme components, nucleotides or

Nucleotide derivatives, lipids or lipid derivatives or phosphatidylinositol, cyclizations,

Disulfide bridging, demethylation, cystine formation, formylation, gamma-carboxylation, glycosylation, hydroxylation, iodination, methylation, myristoyly rations, oxidations, proteolytic processing, phosphorylation, selenoy reactions and tRNA-mediated additions of amino acids.

The polypeptides according to the invention can be in the form of "mature" proteins or as parts of larger proteins, e.g. as fusion proteins. Furthermore, they can secreting or "leader" sequences, pro sequences, sequences that allow easy purification, such as multiple

Histidine residues, or additional stabilizing amino acids. The proteins according to the invention can also be present as they are naturally present in their organism of origin, from which they can be obtained directly, for example. Active fragments of a thioredoxin reductase can also be used in the method according to the invention, as long as they enable the determination of the enzymatic activity of the polypeptide or its inhibition by a candidate compound.

The polypeptides used in the methods according to the invention may have deletions or amino acid substitutions in comparison to the corresponding regions of naturally occurring thioredoxin reductases, as long as they at least still show the biological activity of a complete thioredoxin reductase. Conservative substitutions are preferred. Such conservative substitutions include variations in which one amino acid is replaced by another amino acid from the following group:

1. Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly;

2. Polar, negatively charged residues and their ide: Asp, Asn, Glu and Gin;

3. Polar, positively charged residues: His, Arg and Lys;

4. Large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and

5. Aromatic residues: Phe, Tyr and Trp.

A possible purification method for thioredoxin reductase is based on preparative electrophoresis, FPLC, HPLC (e.g. using gel filtration, reverse phase or slightly hydrophobic columns), gel filtration, differential precipitation, ion exchange chromatography or affinity chromatography (see Example 3).

A rapid method for isolating thioredoxin reductase, which is synthesized by host cells, begins with the expression of a fusion protein, whereby the fusion partner can be easily affinity-purified. The fusion partner can be, for example, an MBP or preferably also a histidine tag (cf. Example 3). The fusion protein can then turn on

Amylose resin or, if a histidine tag is present, for example on a Ni-NTA column getting cleaned. The fusion partner can be separated by partial proteolytic cleavage, for example on linkers between the fusion partner and the polypeptide to be purified according to the invention. The protein with a His tag can be removed from the column by lnidazole-containing buffers. The linker can be designed to include target amino acids, such as arginine and lysine residues, that define sites for trypsin cleavage. Standard cloning methods using oligonucleotides can be used to generate such linkers.

Further possible purification processes are based on preparative electrophoresis, FPLC, HPLC (e.g. using gel filtration, reverse phase or slightly hydrophobic columns), gel filtration, differential precipitation, ion exchange chromatography and affinity chromatography.

The terms "isolation or purification" as used in the present context mean that the polypeptides according to the invention are separated from other proteins or other macromolecules of the cell or the tissue. A composition containing the polypeptides according to the invention is preferred with regard to the protein content compared to a preparation from the

Host cells enriched at least 10 times and particularly preferably at least 100 times.

The polypeptides according to the invention can also be affinity-purified without a fusion partner using antibodies which bind to the polypeptides.

The method for producing polypeptides with the activity of a thioredoxin reductase, e.g. of the polypeptide TRR1 from U. maydis is characterized by

(a) cultivating a host cell containing at least one expressible nucleic acid sequence coding for a polypeptide from fungi with the biological activity of a thioredoxin reductase under conditions which ensure the expression of this nucleic acid, or

(b) expressing an expressible nucleic acid sequence coding for a polypeptide from fungi with the biological activity of a thioredoxin reductase in an in vitro system, and

(c) obtaining the polypeptide from the cell, the culture medium or the in vitro system. The cells obtained in this way, containing the polypeptide according to the invention or the purified polypeptide obtained in this way, are suitable for use in methods for identifying modulators or inhibitors of thioredoxin reductase.

The present invention also relates to the use of polypeptides from fungi, preferably from phytopathogenic fungi, which have at least one biological activity

Exercise thioredoxin reductase in methods of identifying fungicides, where the inhibitors of thioredoxin reductase can be used as fungicides. The thioredoxin reductase from Ustilago maydis is particularly preferably used.

Fungicidal active ingredients, which are found with the help of a thioredoxin reductase from a certain fungal species, can also interact with thioredoxin reductase from other fungal species, although the interaction with the different thioredoxin reductase occurring in these fungi does not always have to be equally strong. This explains, among other things, the selectivity of active substances. The use of the active ingredients found with a specific thioredoxin reductase as a fungicide in other fungi can be attributed to the fact that thioredoxin reductases from various fungal species are very close and in larger ones

Show areas of pronounced homology. Figure 2 clearly shows that such homology exists over considerable sequence sections between S. cerevisiae, N. crassa, S. pombe, P. carinii, P. jiroveci, P. chrysogenum and U. maydis and thus the effect of e.g. with the help of thioredoxin reductase from substances found in U maydis is not limited to U. maydis.

The present invention therefore also relates to a method for identifying fungicides by testing potential inhibitors or modulators of the enzymatic activity of thioredoxin reductase (candidate compound or test compound) in a thioredoxin reductase inhibition test.

Methods which are suitable for identifying modulators, in particular inhibitors or antagonists, of the polypeptides according to the invention are generally based on determining the activity or the biological functionality of the polypeptide. In principle, both whole-cell-based methods (in vivo methods) and methods based on the use of the polypeptide isolated from the cells, which can be present in purified or partially purified form or as a crude extract, are also possible. This cell-free z z vitro

Like in vivo methods, methods can be used on a laboratory scale, but preferably also in HTS or UHTS methods. Following the in vivo or in vitro identification of modulators of the polypeptide, tests can be carried out on fungal cultures in order to test the fungicidal activity of the compounds found. Many test systems that aim to test compounds and natural extracts are preferably geared towards high throughput numbers in order to maximize the number of substances tested in a given period of time. Test systems that are based on cell-free work require purified or semi-purified protein. They are suitable for a "first" test, which primarily aims to detect a possible influence of a substance on the target protein. Once such a first test has been carried out and one or more compounds, extracts etc. have been found, the effect of such compounds can be examined in a more targeted manner in the laboratory. In a first step, the inhibition or activation of the polypeptide according to the invention can be checked again in vitro in order to then test the effectiveness of the compound on the target organism, here one or more phytopathogenic fungi.

The compound can then optionally be used as a starting point for the further search and development of fungicidal compounds based on the original structure, but e.g. are optimized in terms of effectiveness, toxicity or selectivity.

To find modulators, e.g. incubating a synthetic reaction mix (e.g. products of in vitro transcription) or a cellular component such as a membrane, a compartment or any other preparation which contains the polypeptides according to the invention together with an optionally labeled substrate or ligand of the polypeptides in the presence and absence of a candidate molecule become. The maturity of the candidate molecule to inhibit the enzymatic activity of the polypeptides according to the invention is e.g. recognizable by a reduced binding of the optionally labeled ligand or by a reduced

Implementation of the optionally marked substrate. Molecules that inhibit the biological activity of the polypeptides according to the invention are good antagonists or inhibitors.

The detection of the biological activity of the polypeptides according to the invention can be improved by a so-called reporter system. Reporter systems in this regard include, but are not limited to, colorimetric or fluorometric detectable substrates contained in one

Product to be converted or a reporter gene that responds to changes in activity or, the expression of the polypeptides of the invention or other known binding tests.

Another example of a method with which modulators of the polypeptides according to the invention can be found is a displacement test, in which, under suitable conditions, the polypeptides according to the invention and a potential modulator with a molecule which is known to bind to the polypeptides according to the invention, such as a natural one Bring substrate or ligands or a substrate or ligand mimetic. The polypeptides according to the invention themselves can be labeled, for example fluorimetrically or colorimetrically, so that the number of polypeptides which are bound to a ligand is determined or who have participated in an implementation, can determine exactly. However, the binding can also be followed by means of the optionally labeled substrate, ligand or substrate analog. In this way, the effectiveness of an antagonist can be measured.

Effects such as cell toxicity are usually ignored in these in vitro systems. The test systems check both inhibitory or suppressive effects of the substances as well as stimulatory effects. The effectiveness of a substance can be checked using concentration-dependent test series. Control approaches without test substances or without enzyme can be used to evaluate the effects.

The host cells containing nucleic acids coding for a thiorexin reductase according to the invention and available on the basis of the present invention also enable the development of test systems based on cells for the identification of substances which modulate the activity of the polypeptides according to the invention.

The modulators to be identified are preferably small organic chemical compounds.

A method for identifying a compound that modulates the activity of a thioredoxin reductase from fungi and that can be used as a fungicide in crop protection is preferably that

a) a polypeptide according to the invention or a host cell containing this polypeptide is brought into contact with a chemical compound or with a mixture of chemical compounds under conditions which allow the interaction of a chemical compound with the polypeptide,

b) comparing the activity of the polypeptide according to the invention in the absence of a chemical compound with the activity of the polypeptide according to the invention in the presence of a chemical compound or a mixture of chemical compounds, ^" and

c) the chemical compound that specifically modulates the activity of the polypeptide according to the invention, and optionally

d) tests the fungicidal activity of the selected compound in vivo.

The compound which specifically inhibits the activity of the polypeptide according to the invention is particularly preferably determined. The term "activity" as used here refers to the biological activity of the polypeptide according to the invention. In a preferred embodiment, the fact is used that the thioredoxin reductase catalyzes the reduction of oxidized thioredoxin using NADPH. This reaction can be carried out, for example, in the presence of insulin, which can be used as a thioredoxin-regenerating system, since thioredoxin reductase reduces thioredoxin as disulfide reductase, converting insulin to oxidized insulin and thereby converting it back into the oxidized state. This oxidized thioredoxin then serves as the substrate for the thioredoxin reductase. NADPH is then consumed in the reaction of the thioredoxin reductase. The experimental approach can be shown schematically as follows:

Thioredoxin _ox + NADPH ► Thioredoxin _red + NADP + H ⁺

Thioredoxin _ox +

The measurement of the enzymatic activity of the thioredoxin reductase or the inhibition of this enzymatic activity by an inhibitor is then carried out on the basis of the decreasing NADPH concentration and can be carried out photospectrometrically by absorption or fluorescence measurement (absorption decrease at 340 nm or fluorescence decrease at 340 nm (emission at 465 nm)) can be determined (Figure 5). The lower or inhibited activity of the polypeptide according to the invention is tracked based on the photospectrometric determination of the degree of decrease in the NADPH concentration relative to a control batch. If the thioredoxin reductase is inhibited, there is no enzymatic conversion of the NADPH. The fluorescence intensity of the batch should correspond to that of the control batch, since there is no change in the NADPH concentration. An increase in the NADPH concentration can result if the enzymatic activity of the thioredoxin reductase is inhibited so strongly that the thioredoxin oxidized by the reaction with insulin is no longer reduced and thus accumulates.

Alternatively, the amount of NADPH e.g. even after the reaction has been completed by implementing

Resazurin to Resorußn in the presence of PMS (phenazine methosulfate) can be determined fluorimetrically (excitation 550 nm, emission 595 nm) compared to the amount used. In a chemical reaction, the electrons are first transferred from NADPH to PMS. The reduced PMS is then able to reduce resazurin to resorufin. The concentration of resurofin can then be detected fluorimetrically (excitation 550 nm, emission 595 nm). The amount of converted NADPH can then be determined by comparison with the concentrate of resorufin in the control batch. NADPH thus reduces non-fluorescent resazurin to intense red fluorescent resorufin. PMS serves as a redox mediator. The measurement can also be carried out in formats customary for HTS or UHTS assays, for example in microtiter plates in which, for example, a total volume of 5 to 50 μl per batch or per well is placed and the individual components are present in one of the final concentrations given above. The test compound, which potentially inhibits or activates the activity of the enzyme (candidate molecule), is introduced, for example, in a suitable concentration in test buffer containing the components necessary for the reaction. The polypeptide according to the invention is then added in the test buffer mentioned above and the reaction is started therewith. The mixture is then incubated, for example, for up to 30 minutes at a suitable temperature and, for example, the decrease in fluorescence (absorbance 340 nm; emission 465 nm) is measured.

A further measurement is carried out in a corresponding approach, but without adding one

Candidate molecule and without adding a polypeptide according to the invention (negative control). A further measurement is again carried out in the absence of a candidate molecule, but in the presence of the polypeptide according to the invention (positive control). Negative and positive control thus give the comparison values for the batches in the presence of a candidate molecule.

For optimal conditions for a procedure to identify inhibitors of thioredoxin

To determine reductase or to determine the activity of the polypeptides according to the invention, it may be advantageous to determine the respective K _M value of the polypeptide according to the invention used. This provides information about the preferred concentration of the substrate or substrates. In the case of the thioredoxin reductase from U maydis, a K _M of 5 μM for E. coli thioredoxin and a K _M of 20-40 μM for NADPH could be determined (Figure 6).

In this way, inhibitors of thioredoxin reductase could be identified with the method according to the invention.

It goes without saying that in addition to the methods mentioned for determining the enzymatic activity of a thioredoxin reductase or the inhibition of this activity and for identifying fungicides, other, e.g. already known methods or inhibition tests can be used as long as these methods allow the activity of a thioredoxin reductase to be determined and an inhibition of this activity to be recognized by a candidate compound.

Within the scope of the present invention it was also found that the inhibitors of a thioredoxin according to the invention identified with the aid of a method according to the invention

Reductase are suitable, optionally in a suitable formulation, to damage or kill fungi. For this purpose, for example, a solution of the active substance to be tested can be pipetted into the cavities of microtiter plates. After the solvent has evaporated, medium is added to each cavity. A suitable concentration of spores or mycelium of the fungus to be tested is added to the medium beforehand. The resulting concentrations of the active ingredient are, for example, 0J, 1, 10 and 100 ppm.

The plates are then incubated on a shaker at a temperature of approximately 22 ° C. until sufficient growth can be determined in the untreated control.

The evaluation is carried out photometrically at a wavelength of 620 nm. From the measurement data of the different concentrations, the dose of active substance can be determined, which leads to a 50% inhibition of fungal growth compared to the untreated control (ED ₅₀ ). The present invention therefore also relates to the use of modulators of thioredoxin reductase from fungi, preferably from phytopathogenic fungi, as fungicides. The present invention also relates to fungicides which have been identified using a method according to the invention.

Compounds which are identified with the aid of a method according to the invention and which have a fungicidal action due to the inhibition of the fungal thioredoxin reductase can thus be used for the preparation of fungicidal compositions.

The present invention also relates to the use of modulators of thioredoxin reductase from fungi, preferably from phytopathogenic fungi as fungicides, and to methods for combating fungal attack in plants by using an inhibitor of a

Thioredoxin reductase affects the fungus and / or the infected plant. These are preferably inhibitors of a thioredoxin reductase from a phytopathogenic fungus. The modulators are preferably small, organic-chemical molecules, but not inorganic molecules such as anions or cations. For the purposes of the present inventions, the term modulators also does not include any natural effectors of the enzymes which are used in the organism in question to regulate the activity of the enzyme, such as Metabolic products of the enzymes, cofactors of the enzymes, and no non-metabolizable analogues of the cofactors or educts of the enzyme.

Depending on their respective physical and / or chemical properties, the identified active ingredients can be converted into the usual formulations, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, very fine encapsulations in polymeric substances and in coating compositions for seeds, and ULV cold and warm mist formulations. These formulations are prepared in a known manner, for example by mixing the active ingredients with extenders, that is to say liquid solvents, pressurized liquefied gases and / or solid carriers, if appropriate using surface-active agents, that is to say emulsifiers and or dispersants and / or foam-generating agents. If water is used as an extender, organic solvents can, for example, also be used as auxiliary solvents. The following are essentially suitable as liquid solvents: aromatics, such as xylene, toluene or alkylnaphthalenes, chlorinated aromatics or chlorinated aliphatic hydrocarbons, such as chlorobenzenes, chlorethylenes or methylene chloride, aliphatic hydrocarbons, such as cyclohexane or paraffins, for example petroleum fractions, alcohols, such as butanol or glycol, and the like their ethers and esters, ketones, such as acetone, methyl ethyl ketone, methyl isobutyl ketone or

Cyclohexanone, strongly polar solvents such as dimethylformamide and dimethyl sulfoxide, as well as water. Liquefied gaseous extenders or carriers mean liquids which are gaseous at normal temperature and pressure, e.g. Aerosol propellants, such as halogenated hydrocarbons as well as butane, propane, nitrogen and carbon dioxide. As solid carriers, the following are possible: e.g. natural rock flours, such as kaolins, clays, talc,

Chalk, quartz, attapulgite, montmorillonite or diatomaceous earth and synthetic rock powder, such as highly disperse silica, aluminum oxide and silicates. The following are suitable as solid carriers for granules: e.g. broken and fractionated natural rocks such as calcite, marble, pumice, sepiolite, dolomite as well as synthetic granules from inorganic and organic flours as well as granules from organic material such as sawdust, coconut shells, corn cobs and tobacco stems. Possible emulsifiers and / or foaming agents are: e.g. non-ionic and anionic emulsifiers such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, e.g. Alkylaryl polyglycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates and protein hydrolyzates. Possible dispersants are: e.g. Lignin sulfite liquor and methyl cellulose.

Adhesives such as carboxymethyl cellulose, natural and synthetic powdery, granular or latex-shaped polymers such as gum arabic, polyvinyl alcohol, polyvinyl acetate, and natural phospholipids such as cephalins and lecithins, and synthetic phospholipids can be used in the formulations. Other additives can be mineral and vegetable oils.

Dyes such as inorganic pigments, e.g. Iron oxide, titanium oxide, ferrocyan blue and organic dyes such as alizarin, azo and metal phthalocyanine dyes and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc can be used.

The formulations generally contain between 0.1 and 95 percent by weight of active compound, preferably between 0.5 and 90%. The active compounds according to the invention can be used as such or in their formulations, also in a mixture with known fungicides, bactericides, acaricides, nematicides or insecticides, in order, for example, to broaden the spectrum of activity or to prevent the development of resistance; in many cases synergistic effects, ie the activity, are obtained the mixture is greater than the effectiveness of the individual components.

When using the compounds according to the invention as fungicides, the application rates can be varied within wide ranges depending on the application.

According to the invention, all plants and parts of plants can be treated. Plants are understood here to mean all plants and plant populations, such as desired and undesired wild plants or crop plants (including naturally occurring plants

Crop plants). Crop plants can be plants which can be obtained by conventional breeding and optimization methods or by biotechnological and genetic engineering methods or combinations of these methods, including the transgenic plants and including the plant varieties which can or cannot be protected by plant breeders' rights. Under plant parts, all above-ground and underground parts and organs of the

Plants such as sprout, leaf, flower and root are understood, with examples being leaves, needles, stems, stems, flowers, fruiting bodies, fruits and seeds as well as roots, tubers and rhizomes. The plant parts also include crops and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, offshoots and seeds.

The treatment of the plants and parts of plants with the active compounds according to the invention is carried out directly or by acting on their surroundings, living space or storage space using the customary treatment methods, e.g. by dipping, spraying, vaporizing, atomizing, scattering, spreading and, in the case of propagation material, in particular in the case of seeds, furthermore by coating in one or more layers.

The following examples illustrate various aspects of the present invention and are not to be interpreted as limitative. Examples

example 1

Production of trr knock-out mutants in U. maydis

Cultivation of U maydis

The strains were grown at 28 ° C on PD, YEPS or suitable minimal media (Holliday, 1974;

Tsukada et al., 1988). The formation of dikaryotic filaments was observed after dropping strains on PD plate media containing 1% charcoal (Holliday, 1974). Pathogenicity tests were performed as described (Gillessen et al., 1992). Overnight cultures of the strains were resuspended in a concentration of 4 × 10 ⁷ cells and young maize plants (Gaspe 'Flint) were injected or dropped onto them. For each strain, at least 25 plants were infected and tumors that developed were examined after 14-21 days.

Production of the knock-out cassette

Standard molecular biological methods were carried out according to Sambrook et al., 1989. To produce trr null mutants, the 5 'and 3' flanks of the trr gene were amplified by PCR. The UM518 strain was used as a template. For the 5 'edge

(1537bp) the primers LB2 with the sequence (5'-cacggcctgagtggccggctcggaatgatcgagctgagg-3 ') and pl5 with the sequence (5'-gatgacgcaggagggacacaagg-3') were used. The primers RB1 (5'-gtgggccatctaggccggctcatctcaggtaacagcgg-3 ') and pl3 (5'-ggcgcgacgatggcatttgaagg-3') were used for the 3'-flank (1654bp). The interfaces Sfi I (a) and Sfi I (b) were introduced with the primers LB2 and RB1. The amplicons were restricted with S / z I and ligated with the 1884 bp Sfi I fragment from the vector pBS (hygromycin B cassette). The 4742 bp trr knock-out cassette, which was used in the subsequent transformation (Kämper and.), Was amplified by PCR with the primers LB1 (5'-gacgggtcgattcggtcgcaagg-3 ') and RB2 (5'-ggctccgacggaaaacactttgg-3') Schreier, 2001).

Production of protoplasts by U maydis

50 ml of a culture in YEPS medium were grown at 28 ° C. to a cell density of approx. 5 × 10 7 / ml (OD 0.6 to 1.0) and then for 7 min. centrifuged at 2500g (Hereaus, 3500 rpm) in 50 ml Falkon tubes. The cell pellet was resuspended in 25 ml SCS buffer (20 mM Na citrate pH 5.8, 1.0 M sorbitol, (mix 20 mM Na citrate / 1.0 M sorbitol and 20 mM citric acid / 1.0 M sorbitol and adjust to pH 5.8 with a pH meter)) again 7 min. Centrifuge at 2500 g (3500 rpm) and the pellet in 2 ml SCS buffer, pH 5.8, with 2.5 mg / ml Novozym 234 resuspended. The protoplasting was carried out at room temperature and was microscopic every 5 min. tracked. The protoplasts were then mixed with 10 ml SCS buffer and at 1100 g (2300 rpm) for 10 min. centrifuged, the supernatant was discarded. The pellet was carefully resuspended in 10 ml SCS buffer and centrifuged again. The washing process with SCS buffer was repeated twice, the pellet was washed in 10 ml STC buffer. Finally, that was

Pellet resuspended in 500 μl cold STC buffer (10 M Tris / HCl pH 7.5, 1.0 M sorbitol, 100 mM CaC12) and kept on ice. Aliquots can be stored at -80 ° C for several months.

Transformation by U. maydis

The transformation of a diploid U. maydis strain was carried out according to Schulz et al., 1990. The isolation of genomic U. maydis DNA was carried out as described by Hoffmann and Winston 1987 or according to the protocol of the company Qiagen (DNeasy-Kit).

Max. Transfer 10 μl DNA (optimally 3-5 μg) into a 2 ml Eppendor tube, add 1 μl heparin (15 μg / μl) (SIGMA H3125) and then add 50 μl protoplasts and 10 min. incubated on ice. 500 ul 40% (w / w) PEG3350 (SIGMA P3640) in STC (sterile filtered) were added, mixed carefully with the protoplast suspension and 15 min. incubated on ice. The plating was carried out on gradient plates (lower agar: 10 ml of YEPS-1.5% agar-IM sorbitol with antibiotic; shortly before plating out, the lower agar layer was overlaid with 10 ml of YEPS-1.5% agar-IM sorbitol, which Spread protoplasts and incubate the plates for 3-4 days at 28 ° C.

The detection of homologous recombination in a genomic locus of trr was by means of

Standard methods (PCR or Southern analysis) of isolated genomic DNA were performed. It was shown that the trz'-knock-out cassette had been integrated into a genomic trrLokus and thus a wild-type copy of the trr gene was replaced, while the second copy was retained in this diploid strain. The heterozygous trr mutants created in this way were subsequently used in the pathogenicity test.

Spore analysis by U. maydis

Spores were isolated from the tumors generated in the pathogenicity test. The resulting sporidia were then isolated and examined phenotypically or genotypically. The phenotypic analysis was carried out using growth experiments on suitable full or minimal media (Holliday, 1974; Tsukada et al., 1988). It was shown that only 15 out of 48 sporidia analyzed grew under selective conditions. Of these, 7 strains were diploid. This was a first indication that the trr-null mutant phenotype was lethal. The genotypic Investigations were carried out by Southern or PCR-based analysis of the sporidia. It was found that no viable, haploid strain could be identified among the sporidia analyzed, in which only the trr gene was replaced by the trr knock-out cassette. In all cases where the trz 'knock-out cassette was homologously integrated into the trr genomic locus, an additional one was added

Example 2

Cloning, expression and purification of the TRR1 from IT. maydis

For the heterologous expression of the tπ gene, the gene was amplified with gene-specific oligonucleotides by means of PCR and cloned into the expression vector pTrcHis2-TOPO, so that the TRR protein is expressed starting from the plasmid ptrcTRRlO with a C-terminal His ₆ tag.

To express the TRR1, the plasmid ptrcTRR10 was transformed into E. coli BL21 (DE3). As a preculture, 5 ml of selection medium (dYT with 100 μg / ml ampicillin) were inoculated with a single colony and incubated at 37 ° C. overnight in a shaker. The main culture (dYT containing 100 ug / ml ampicillin) was inoculated 1:40 and incubated at 37 ° C with shaking to an OD ₆ oo of 0.8 the culture was induced by adding 1 mM IPTG (final concentration). After an incubation period of 4 h at 37 ° C, the cells were harvested and then frozen at ~ 20 ° C. The cells can be stored for several months at - 20 ° C without loss of activity. 2 g of cells were resuspended in 5 ml binding buffer (40 mM Tris / HCl pH 8.0, 100 mM NaCl), 600 ng lysozyme and 500, ng DNasel were added. The cell suspension was incubated with stirring on ice for 30 min. The digestion was carried out by 3

"Freeze and Thaw" cycles. After centrifugation at 11,000 rpm and 4 ° C. for 30 minutes, the supernatant was placed on a Ni-NTA column (volume: 2 ml), which was buffered with buffer A (20 mM Tris / HCl, pH 8.0 , 5 mM MgCl ₂ , 150 mM NaCl), the column was washed with 8 ml of buffer A, elution was carried out with 7 ml of buffer A + 100 mM hnidazole (Figure 1) .. The protein fraction was then analyzed using a NAP25 Column desalted and in storage buffer

(50 mM Tris / HCl, pH 7.5, 1 mM EDTA). From 500 l-E. co / i culture approximately 8.8 mg soluble TRR can be isolated. The protein solution was frozen at -80 ° C. The enzyme isolated in this way can be stored at -80 ° C for about 2 months without loss of activity.

Example 3

Cloning, expression and purification of E. coli thioredoxin (substrate)

To express thioredoxin in E. coli, the plasmid pEt32A (Novagen, see http://www.merckbiosciences.de/product/69015) was transformed into E. coli BL21 (DE3). As Pre-culture, 5 ml selection medium (dYT (double yeast tryptone) with 100 μg / ml ampicillin) was inoculated with a single colony and incubated at 37 ° C. overnight in a shaker. The main culture (dYT with 100 μg / ml ampicillin) was inoculated 1:40 and incubated at 37 ° C. with shaking. After reaching an OD ₆₀₀ of 0.8, the culture was induced by adding 1 mM IPTG (final concentration). After an incubation period of 4 h at 37 ° C, the cells were harvested and then frozen at -20 ° C. The cells can be stored for several months at -20 ° C without loss of activity. 6 g cells were resuspended in 94 ml binding buffer (40 mM Tris HCl pH 8.0, 100 mM NaCl) and lysozyme and DNasel were added. The cell suspension was incubated with stirring on ice for 30 min. The disruption was carried out by means of ultrasound in a rosette rim vessel with 8 cycles of 90 seconds and a break of 90 seconds each (cooled with ice water). After centrifugation at 13000 rpm (Eppendorf centrifuge) and 4 ° C for 30 minutes, the supernatant was placed on a Ni-NTA column (volume: 8 ml), which was buffered with buffer A (20 mM Tris / HCl, pH 8.0, 5 mM MgCl ₂ , 150 mM NaCl) had been equilibrated. The column was washed with 30 ml of buffer A, the elution was carried out with 30 ml of buffer A + 100 mM imidazole (Figure 4). The protein fraction was then desalted on a NAP25 column and in

Storage buffer (50 mM Tris / HCl, pH 8.0) transferred. Approx. 1. g soluble thioredoxin was isolated from 60 liters of an E. cσ / z culture.

The protein solution was frozen at -20 ° C. The enzyme isolated in this way can be stored at -20 ° C for several months without loss of activity.

Example 4

Determination of the ideal substrate concentrations (KM values for performing a thioredoxin reductase inhibition test

3J KM value determination for thioredoxin from E. coli

For the substrate thioredoxin, the K _M value was determined by means of the initial decrease in fluorescence at 360 nm / 465 nm. Due to the strong fluctuations in very small thioredoxin

Concentrations, the different quality of the thioredoxin preparations and the different background decrease in NADPH, slightly varying K _M values were obtained. The Lineweaver Burk plot (Figure 6 (A)) gives a K _M value between 0.75-5 μM. The measurements were carried out with 300 ng TRR. The amount of thioredoxin used was varied in order to obtain an optimal read-out. It showed up at about 1 μM

Thioredoxin (heterologously expressed in E. coli, see Example 2), which corresponds to approximately the lowest determined K _M value and approximately one sixth of the highest determined K _M value, a good relationship between read-out and sensitivity to inhibitors. 3.2 K value determination for NADPH

In order to obtain the highest possible read-out within a time window acceptable for the implementation of screening procedures, the test was carried out with different concentrations of NADPH. The consumption of NADPH is measured depending on the NADPH concentration used. The TRR1 concentration was 300 ng in a reaction volume of 50 μl. The application was carried out as a lineweaver burk plot (Figure 6 (B)). The K _M value of the thioredoxin reductase for NADPH from E. coli is 3-8 μM according to the literature. Since the NADPH fluorescence also serves as a measurement parameter, the K _M value determination was carried out in parallel by detecting the free SH groups of the thioredoxin formed with DTNB and the change in absorption was measured. A K _M value for NADPH of 16-40 μM resulted from the measurement with NADPH. A screening method or an inhibition test is therefore carried out, for example, with 30 μM NADPH in order to obtain an adequate read-out.

Example 5

^" Testing the functionality of the thioredoxin reductase inhibition test using a known inhibitor of thioredoxin reductase

The literature describes N-ethylmaleimide (M _r 125Jg / mol) as an inhibitor of thioredoxin reductase. In order to check whether inhibitors of thioredoxin reductase can be identified using the method according to the invention, the method was tested using this known inhibitor. The test was carried out in the presence of different concentrations of N-ethylmaleimide. It was shown that the NADPH fluorescence initially decreases very strongly in the presence of high concentrations of inhibitor. In addition, N-ethylmaleimide can also inhibit thioredoxin itself as an "SH group reagent". The measurement (Figure 7) shows that the inhibitory effect is concentration-dependent as desired, but only very high concentrations of N-ethylmaleimide lead to complete inhibition of the reaction If the inhibitor accepts the loss of part of the NADPH fluorescence, the inhibitor is suitable as a control inhibitor for this test. Example 6

Identification of modulators of thioredoxin reductase in 384-well MTP in an assay with a thioredoxin regenerating system

The negative control was pipetted into columns 1 and 2 of the MTP. This was composed of 5μl 5% (v / v) DMSO, 20μl substrate solution (18.75mM potassium phosphate buffer pH

7.4; 1, 25mM EDTA, 75μM NADPH) and 25μl enzyme solution without TRR (15mM potassium phosphate buffer pH 7.4; ImM EDTA, 256 μM insulin, 40nM / μl thioredoxin). The positive control was pipetted into columns 3 and 4. This consisted of 5μl 5% (v / v) DMSO, 20μl substrate solution (18.75mM potassium phosphate buffer pH 7.4; 1, 25mM EDTA, 75μM NADPH) and 25μl enzyme solution (15mM potassium phosphate buffer pH 7.4; ImM EDTA, 256 μM insulin,

40nM / ul thioredoxin and 4.8ng / ul TRR). Test substances in a concentration of 10 μM in DMSO were placed in the remaining columns, H ₂ 0 being used to dilute the substance. After adding 20 ml substrate solution (18J5mM potassium phosphate buffer pH 7.4; l, 25mM EDTA, 75 μM NADPH), 25 μl enzyme solution (15mM potassium phosphate buffer pH 7.4; ImM EDTA, 256 μM insulin, 40nM / μl thioredoxin and 4.8 ng / μl

TRR) added. There was an incubation at 30 ° C for 45 min. Then 50 μl of a resazurin PMS mix (300 mM sodium acetate pH 5.8, 1% (v / v) DMSO, 20 mM PMS, 10 μM resazurin) were added. The NADPH not reacted during the reaction reacts with resazurin in the presence of PMS to form fluorescent resorufin. The resorufin was detected by determining the decrease in the fluorescence intensity at 595 nm (excitation 550 nm) in a suitable SPECTRAFluor Plus reader from Tecan.

Example 7

Evidence of the fungicidal activity of the identified inhibitors of thioredoxin reductase

A methanolic solution of the active substance identified by means of a method according to the invention (Example 4), mixed with an emulsifier, is pipetted into the cavities of microtiter plates. After the solvent has evaporated, 200 μl of potato dextrose medium are added to each cavity. Suitable concentrations of spores or mycelia of the fungus to be tested are added to the medium beforehand.

The resulting concentrations of the active ingredient are e.g. 0J, 1, 10 and 100 ppm. The resulting concentration of the emulsifier is 300 ppm.

The plates are then incubated on a shaker at a temperature of 22 ° C. until sufficient growth can be determined in the untreated control. The evaluation photometrically at a wavelength of 620 nm. From the measurement of the different concentrations, the dose of active ingredient that results in a 50% inhibition of fungal growth compared to the untreated control (ED ₅ o) was calculated.

literature

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(2) Hoffman, C.S., and Winston, F. (1987): A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. In Gene, pp. 267-272.

(3) Hofmann, K. et al. (1999): The PROSITE database, is status in 1999. Nucl. Acid Res. 27, 215-219.

(4) Holliday, R. (1974): Ustilago maydis. In Handbook of Genetics, R.C. King, ed. (New York, Plenum), pp. 575-595.

(5) Kämper and Schreier (2001): Method for producing deletion mutants. WO 03/006663.

(6) Kaemper, J., and Schreier, P. (2003): A PCR method for the rapid construction of vectors for the generation of deletion mutants by gene knockout. In PCT Int. Appl. (WO, (Bayer Aktiengesellschaft, Germany).), Pp. 51 pp.

(7) Machado A.K. et al. (1997): Thioredoxin reductase-dependent inhibition of MCB cell cycle box activity in Saccharomyces cerevisiae. J. Biol. Chem. 272 (27), 17045-54.

(8) Mumberg, D. et al. (1995): Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119-122.

(9) Onai and Nakshima (1997): Mutation of the cys-9 gene, which encodes thioredoxin reductase, affects the circadian conidiation rhythm in Neurospora crassa. Genetics 146, 101-110.

(10) Pearson G. D. and Merrill G. F .: Deletion of the Saccharomyces cerevisiae TRR1 gene encoding thioredoxin reductase inhibits p53-dependent reporter gene expression. J. Biol. Chem. 273 (10), 5431-4.

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Claims

claims 1. A method of identifying fungicides, characterized in that one (a) contacting a fungal polypeptide with the enzymatic activity of a thioredoxin reductase with a chemical compound or a mixture of chemical compounds under conditions which allow the chemical compound to interact with the polypeptide, (b) comparing the activity of the thioredoxin reductase in the absence of a chemical compound with the activity of the thioredoxin reductase in the presence of a chemical compound or a mixture of chemical compounds, and (c) selects the chemical compound that specifically inhibits the thioredoxin reductase. 2. The method according to claim 1, characterized in that the activity of the thioredoxin reductase is determined on the basis of the decrease in the NADPH concentration. 3. The method according to claim 1 or 2, characterized in that an inhibition of the enzymatic activity of the thioredoxin reductase in the presence of a chemical compound is determined by means of a smaller decrease in the NADPH concentration compared to the enzymatic activity of the thioredoxin reductase in the absence of a chemical compound. 4. The method according to claim 1 or 2, characterized in that the activity of thioredoxin reductase is determined in the presence of insulin. 5. Verfaliren according to any one of claims 1 to 4, characterized in that in a further step the fungicidal activity of the identified compound is tested by bringing it into contact with a fungus. 6. The method according to any one of claims 1 to 5, characterized in that one uses a thioredoxin reductase from a phytopathogenic fungus. , 7. A method for combating fungal attack in plants, characterized in that an inhibitor of a fungal thioredoxin reductase is allowed to act on the fungus and / or its habitat. 8. The method according to claim 7, characterized in that the inhibitor is identified by a method according to one of claims 1 to 6. 9. nucleic acid coding for a polypeptide with the biological activity of a thioredoxin reductase, characterized in that it comprises a sequence from a phytopathogenic fungus which is selected from: a) a sequence according to SEQ ID NO: 1, b) sequences which code for a polypeptide which comprises the amino acid sequence according to SEQ ID NO: 2, c) sequences which code for a polypeptide which has the motif Cx (2) -CD- [GA] - x (2,4) - [FY] -x (4) - [LIVM] -x- [LIVM] (2) -G (3) - [DN], d) sequences which match the sequences defined under a) and b) at a hybridization temperature of 42- Hybridize at 65 ° C., and e) sequences which have at least 80%, preferably at least 85% and particularly preferably at least 90% identity with the sequences defined under a) and b). 10. DNA construct comprising a nucleic acid according to claim 9 and a heterologous promoter. 11. Vector comprising a nucleic acid according to claim 9 or a DNA construct according to claim 10.> 12. Vector according to claim 11, characterized in that the nucleic acid is functionally linked to regulatory sequences which ensure the expression of the nucleic acid in pro- or eukaryotic cells. 13. Host cell containing a nucleic acid. according to claim 9, a DNA construct according to claim 10 or a vector according to claim 11 or 12. 14. A polypeptide with the biological activity of a thioredoxin reductase, which is encoded by a nucleic acid according to claim 9. 15. Polypeptide with the biological activity of a thioredoxin reductase, characterized in that it comprises a sequence from a phytopathogenic fungus, which is selected from: a) the sequence according to SEQ ID N0: 2, b) sequences which have an at least 80%, preferably an at least 85%, particularly preferably a 90% and particularly preferably a 95% identity with the sequence defined under a), c) the sequences given under b) which contain the motif Cx (2) -CD- [GA] -x (2.4) - [FY] -x (4) - [LIVM] -x- [LIVM] (2 ) -G (3) - [DN] and preferably include the motif CAVC-DGAVPIFRNKPLxV- [VI] -G (3) -D.