WO2003012088A2 - Nucleic acid molecules encoding a glucosinolate sulfatase - Google Patents

Abstract

Nucleic acid molecules encoding glucosinolate sulfatase are described as well as the encoded protein. Moreover, vectors, host cells and methods for producing the encoded protein are described as well as methods for identifying antagonists/inhibitors of the glucosinolate sulfatase and methods for formulating these antagonists/inhibitors in an insecticidal composition.

Description

Nucleic acid molecules encoding a glucosinolate sulfatase

The present invention relates to nucleic acid molecules encoding a glucosinolate sulfatase. Moreover, this invention relates to the encoded protein, vectors, host cells and methods for producing said protein. It also relates to methods for identifying antagonists/inhibitors of the glucosinolate sulfatase and to methods for producing insecticidal compositions comprising such antagonists/inhibitors.

Plants are attacked by a broad array of herbivores and pathogens. In response, plants deploy an arsenal of defensive traits and have evolved a broad variety of defense mechanisms. These include preformed physical and chemical barriers, as well as inducible defenses. A well-studied example is the glucosinolate-myrosinase system (Fig. 1A), also referred to as "the mustard oil bomb" (Lϋthy and Matile, Biochem. Physiol. Pflanz. 179 (1984), 5; Bones and Rossiter, Physiol. Plant 97 (1996), 194). Cruciferous plants synthesize glucosinolates, a class of plant secondary compounds that share a core consisting of a β-thioglucose moiety and a sulfonated oxime, but differ by a variable side chain derived from one of several amino acids (Halkier, in Naturally Occurring Glycosides: Chemistry, Distribution and Biological Properties, R. Ikan, Ed. (John Wiley & Sons, New York (1999), 193-223)). More than 100 different glucosinolates have been identified (Rask, Plant Mol. Biol. 42 (2000), 93), and this diversity is believed to be of ecological importance (Kliebenstein et al., Plant Phys. 126 (2001), 811-825; Kroymann et al., in press). However, intact glucosinolates have limited biological activity. Their potency arises when plant tissue is damaged, and glucosinolates come into contact with myrosinase, a β- thioglucosidase. Myrosinase removes the β-glucose moiety from glucosinolates, leading to the formation of an unstable intermediate, and, finally, to a variety of toxic breakdown products (Fig. 1A). These compounds have diverse biological activity ranging from feeding deterrance for generalist insects to oviposition stimulation for specialists (Giamoustaris and Mithen, Ann. Appl. Biol. 126 (1995), 347). They may also be toxic to crucifer specialists (Li et al., J. Chem. Ecol. 26 (2000), 2401). However, the so-called „mustard oil bomb" is overcome by a crucifer specialist insect, Diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae). The host range of this world-wide distributed pest includes important crops such as cabbage, broccoli, cauliflower, collards, rapeseed, mustard, and Chinese cabbage. Pest management is difficult, as DBM rapidly developed resistance to many synthetic insecticides (Furlong and Wright, Pesticide Science 42 (1994), 315) and also to microbial Bacillus thuringiensis toxin sprays (Tabashnik et al., Proc. Natl. Acad. Sci. USA 94 (1997), 12780; Heckel et al., Proc. Natl. Acad. Sci. USA 96 (1999), 8373). It is also costly, exceeding U.S. $ 1 billion per year (Talekar, Annu. Rev. Entomol. 38 (1993), 275), in addition to the costs caused by crop yield loss. To utilize crucifers as host plants, DBM needs a mechanism to overcome the chemical defense posed by the "mustard oil bomb". Revelation of this mechanism may allow to devise strategies for a better DBM pest management.

Thus, the technical problem underlying the present invention is to provide means and methods allowing an efficient DBM pest management.

This problem is solved by the provision of the embodiments as characterized in the claims.

Accordingly, the present invention relates to a nucleic acid molecule encoding a glucosinolate sulfatase selected from the group consisting of

(a) nucleic acid molecules encoding a protein which comprises the amino acid sequence indicated in SEQ ID No: 2, 4 or 6;

(b) nucleic acid molecules comprising the nucleotide sequence of the coding region indicated in SEQ ID No: 1 , 3 or 5;

(c) nucleic acid molecules encoding a protein, the amino acid sequence of which has a homology of at least 40% to the amino acid sequence indicated in Seq ID No: 2, 4 or 6;

(d) nucleic acid molecules the complementary strand of which hybridizes to a nucleic acid molecule as defined in (a) or (b);

(e) nucleic acid molecules comprising a nucleotide sequence encoding an enzymatically active fragment of the protein which is encoded by any one of the nucleic acid molecules as defined in (a), (b), (c) or (d); and (f) nucleic acid molecules, the nucleotide sequence of which deviates because of the degeneracy of the genetic code from the sequence of the nucleic acid molecules as defined in any one of (a), (b), (c), (d) or (e).

Consequently, the present invention relates to nucleic acid molecules encoding a glucosinolate sulfatase, said molecules preferably encoding proteins comprising the amino acid sequence indicated in SEQ ID No: 2, 4 or 6.

A glucosinolate sulfatase is understood to mean an enzyme which can desulfate glucosinolates thereby forming desulfo-glucosinolates (see Figure 1). Glucosinolates are a class of plant secondary compounds that share a core consisting of a β-thioglucose moiety and a sulfonated oxime but differ by a variable side chain derived from one or several amino acids. They are generated through conversion of numerous amino acids (methionine, leucine, isoleucine, valine, tryptophan, tyrosine and phenylalanine) and their various carbon chain elongated analogues to the corresponding oxime by a variety of different enzymes (Dawson et al., J. Biol. Chem. 268 (1993), 27154-27159; Du et al., Proc. Natl. Acad. Sci. USA 92 (1995), 12505-12509). This oxime is then converted into the final glucosinolate through a thiohydroximate intermediate (Halkier and Du, Trends Plant Sci. 2 (1997), 425-431). In Brassicaceae, which include crop plants such as rapeseed, vegetables like cauliflower and cabbage, as well as Arabidopsis thaliana, the major glucosinolates are derived from chain elongated versions of methionine. The postulated pathway for the initial elongation of methionine (Chisholm and Wetter, Can. J. Biochem. 42 (1964), 1033-1040; Matsuo and Yamazaki, Chem. Pharm. Bull. 12 (1964), 1388-1389; Graser et al., Arch. Biochem. Biophys. 378 (2000), 411-419) involves transamination of methionine to form 2-oxo-4-methylthiobutanoic acid, condensation of 2-oxo-4-methylthiobutanoic acid with acetyl-CoA to generate 2-(2^'- methylthio)ethylmalic acid, isomerization of 2-(2^'-methylthio)ethylmalic acid to 3-(2^'- methylthio)ethylmalic acid, and oxidative carboxylation of 3-(2^'-methylthio)ethylmalic acid to form 2-oxo-5-methylthiopentanoic acid (Figure 6). The net result of these reactions is extension of the aliphatic side chain by one methylene group. Subsequent elongation cycles involving analogous reactions can further extend the side chain, each cycle resulting in the addition of one methylene group to the 2-oxo- acid. In Brassicaceae, chain elongated analogues of methionine with as many as 11 methylene groups have been reported (Halkier and Du, Trends Plant Sci. 2 (1997), 425-431). Chain elongation of other amino acids utilized for glucosinolate biosynthesis occurs analogously.

In the context of the present invention the term "glucosinolate" means any possible glucosinolate, i.e. any naturally occurring glucosinolate or chemically synthesized glucosinolate. It also covers modified forms of these glucosinolates. Preferably, the term refers to glucosinolates which are synthesized by plants of the order Capparales and more preferably by Brassicaceae plants.

The activity of a glucosinolate sulfatase can be tested as described, e.g., in Dodgson and Spencer (Methods of Biological Analysis 4 (1957), 211-255), wherein glucosinolate sulfatases are referred to as myrosulfatases or as described in the Examples. Preferably, it is determined as described hereinbelow. For example, it can be determined by incubating the protein preparation to be tested in an aqueous solution of glucosinolate under appropriate conditions, purifying the reaction products on an appropriate column (e.g. DEAE sephadex A-25) and analyzing the reaction products, e.g. by HPLC, for the presence of desulfo-glucosinolates. The occurrence of desulfo-glucosinolates indicates that the testes protein has glucosinolate sulfatase activity.

The invention in particular relates to nucleic acid molecules containing the nucleotide sequence of the coding region indicated under SEQ ID No: 1 , 3 or 5 or a part thereof or a corresponding ribonucleotide sequence.

Moreover, the present invention relates to nucleic acid molecules which encode a glucosinolate sulfatase and the complementary strand of which hybridizes with one of the above-described molecules.

The present invention also relates to nucleic acid molecules which encode a protein, which has a homology, that is to say a sequence identity, of at least 40%, preferably of at least 60%, more preferably of at least 70%, even more preferably of at least 80% and in particularly preferred of at least 90% to the entire amino acid sequence as indicated in SEQ ID No: 2, 4 or 6 the protein possessing the enzymatic or biological activity of a glucosinolate sulfatase. The present invention also relates to nucleic acid molecules, which encode a glucosinolate sulfatase and the sequence of which deviates from the nucleotide sequences of the above-described nucleic acid molecules due to the degeneracy of the genetic code.

The invention also relates to nucleic acid molecules comprising a nucleotide sequence which is complementary to the whole or a part of one of the above- mentioned sequences.

In the context of the present invention the term "hybridization" means hybridization under conventional hybridization conditions, preferably under stringent conditions, as for instance described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. In an especially preferred embodiment the term "hybridization" means that hybridization occurs under the following conditions: Hybridization buffer: 2 x SSC; 10 x Denhardt solution (Fikoll 400 + PEG +

BSA; ratio 1 :1:1); 0.1% SDS; 5 mM EDTA; 50 mM

Na₂HPO _;

250 μg/ml of herring sperm DNA; 50 μg/ml of tRNA; or

0.25 M of sodium phosphate buffer, pH 7.2;

1 mM EDTA

7% SDS Hybridization temperature T = 60°C Washing buffer: 2 x SSC; 0.1% SDS

Washing temperature T = 60°C.

Nucleic acid molecules which hybridize with the nucleic acid molecules of the invention can, in principle, encode glucosinolate sulfatase from any organism expressing such proteins. Preferably, they encode a glucosinolate sulfatase from an animal organism, more preferably from an insect, even more preferably from an insect belonging to the Lepidoptera, in particular to the Plutellidae, particularly preferred from a member of the genus Plutella. In a most preferred embodiment the nucleic acid molecule of the present invention encodes a protein from Plutella xylostella (Diamondback moth).

Nucleic acid molecules which hybridize with the molecules of the invention can for instance be isolated from genomic libraries or cDNA libraries. Alternatively, they can be prepared by genetic engineering or chemical synthesis.

Examples for such genomic sequences are the nucleotide sequences shown in SEQ

ID NOs: 7, 8 and 9, respectively. The exon/intron structure of these sequences is shown in the following Table and Figure 8:

Table 1

Such nucleic acid molecules may be identified and isolated by using the molecules of the invention or parts of these molecules or reverse complements of these molecules, for instance by hybridization according to standard methods (see for instance Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual, 2^nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Nucleic acid molecules comprising the same or substantially the same nucleotide sequence as indicated in SEQ ID NO: 1 , 3 or 5 for the coding region or parts thereof can, for instance, be used as hybridization probes. The fragments used as hybridization probes can also be synthetic fragments which are prepared by usual synthesis techniques, and the sequence of which is substantially identical with that of a nucleic acid molecule according to the invention. The molecules hybridizing with the nucleic acid molecules of the invention also comprise fragments, derivatives and allelic variants of the above-described nucleic acid molecules encoding a glucosinolate sulfatase. Herein, fragments are understood to mean parts of the nucleic acid molecules which are long enough to encode one of the described proteins, preferably showing the enzymatic or biological activity of a glucosinolate sulfatase. In this context, the term derivative means that the sequences of these molecules differ from the sequences of the above-described nucleic acid molecules in one or more positions and show a high degree of homology to these sequences. In this context, homology means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably of more than 80%, even more preferably of more than 81%, especially of more than 85% and particularly preferred of more than 90%. Deviations from the above-described nucleic acid molecules may have been produced, e.g., by deletion, substitution, insertion and/or recombination.

Preferably, the degree of homology is determined by comparing the respective sequence with the nucleotide sequence of the coding region of SEQ ID No: 1 , 3 or 5. When the sequences which are compared do not have the same length, the degree of homology preferably refers to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence. The degree of homology can be determined conventionally using known computer programs such as the DNA star program with the Clustal analysis. When using the Clustal analysis method to determine whether a particular nucleotide or amino acid sequence is, for instance, 80% identical to a reference sequence the settings are preferably as follows: Gap penalty 10, gap length penalty 10, ktuple 2, gap penalty 2, window 4 and diagonals saved 4. For amino acid sequences, PAM250 should used be as residue weight matrix.

Furthermore, homology means preferably that the encoded protein displays a sequence identity of at least 40%, more preferably of at least 60%, even more preferably of at least 70%, in particular of at least 75%, particularly preferred of at least 80%, especially of at least 90% and finally preferred of at least 95% to the amino acid sequence depicted under SEQ ID NO: 2, 4 or 6.

Preferably, sequences hybridizing to a nucleic acid molecule according to the invention comprise a region of homology of at least 90%, preferably of at least 93%, more preferably of at least 95%, still more preferably of at least 98% and particularly preferred of at least 99% identity to an above-described nucleic acid molecule, wherein this region of homology has a length of at least 500 nucleotides, more preferably of at least 600 nucleotides, even more preferably of at least 800 nucleotides and particularly preferred of at least 1000 nucleotides. Homology, moreover, means that there is a functional and/or structural equivalence between the corresponding nucleic acid molecules or proteins encoded thereby. Nucleic acid molecules which are homologous to the above-described molecules and represent derivatives of these molecules are normally variations of these molecules which represent modifications having the same biological function. They may be either naturally occurring variations, for instance sequences from other strains, varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. Furthermore, the variations may be synthetically produced sequences. The allelic variants may be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA techniques.

The proteins encoded by the different variants of the nucleic acid molecules of the invention possess certain characteristics they have in common. These include for instance enzymatic activity, molecular weight, immunological reactivity, conformation, etc., and physical properties, such as for instance the migration behavior in gel electrophoreses, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability, pH optimum, temperature optimum etc. A protein encoded by a nucleic acid molecule according to the invention is characterized by the feature that it has the enzymatic activity of a glucosinolate sulfatase.

Moreover, a polypeptide encoded by a nucleic acid molecule of the present invention is preferably characterized by the feature that it is an extracellular protein. This means that the protein comprises a targeting sequence for extracellular targeting. Preferably, the targeting sequence is located in the 19 N-terminal amino acid residues. The presence of such a targeting signal can, e.g., be determined by using the PSORTII program (Nakai and Horton, Trends Biochem. Sci. 24 (1999), 34). Furthermore, a polypeptide encoded by a nucleic acid molecule of the present invention preferably has a molecular weight of between 50 kDa and 70 kDa, more preferably of between 55 kDa and 65 kDa, even more preferably of between 60 kDa and 64 kDa and most preferably of about 62 kDa when calculated on the basis of the amino acid sequence.

The nucleic acid molecules of the invention can be DNA molecules, in particular genomic DNA (see, e.g., SEQ ID NOs: 7, 8 and 9) or cDNA (see, e.g., SEQ ID NOs: 1 , 3 and 5). Moreover, the nucleic acid molecules of the invention may be RNA molecules. The nucleic acid molecules of the invention can be obtained for instance from natural sources or may be produced synthetically or by recombinant techniques, such as PCR.

The present invention is based on the isolation of cDNA and genomic sequences from Plutella xylostella encoding a glucosinolate sulfatase which allows this crucifer plant specialist to overcome the co called "mustard oil bomb" by preventing the formation of toxic hydrolysis products arising from this plant defense system. The glucosinolate sulfatase desulfates the glucosinolates which renders them invisible to myrosinase and thereby prevents their conversion by this enzyme into toxic glucosinolate breakdown products.

SEQ ID NOs: 1, 3 and 5 show cDNA sequences obtained from a cDNA library of Plutelly xylostella. All three cDNA sequences comprise the complete coding sequence encoding glucosinolate sulfatase and short non-translated regions. In all three cDNAs the start codon lies at position 13 to 15 and the stop codon at position 1654 to 1656. SEQ ID NOs: 2, 4 and 6 show the corresponding amino acid sequences. The three cDNA sequences differ from each other by single nucleotide polymorphisms. Most of these polymorphisms do not alter the amino acid sequence since mostly the third position of the codons is affected.

SEQ ID NOs: 7, 8 and 9 show three genomic sequences of Plutella xylostella encoding glucosinolate sulfatase the exon/intron structure of which is indicated in Table 1 , supra.

The nucleic acid molecules of the invention now allow host cells to be prepared which produce recombinant enzymes having the activity of a glucosinolate sulfatase of high purity and/or in sufficient quantities. The only presently commercially available enzyme activity which corresponds to the glucosinolate sulfatase of Plutella xylostella is a crude protein extract form the Helix pomatia (Sigma). The provision of the nucleic acid molecules of the present invention now allows to provide a pure glucosinolate sulfatase. Such a protein is, e.g., extremely useful for identifying compounds which can be used as pesticides in order to control DBM infestation of plants as described below.

The provision of a recombinantly produced glucosinolate sulfatase allows to indicate a defined specific activity. Moreover, other contaminating enzyme activities, such as a glucuronidase in the sulfatase of Helix pomatia (Sigma) can be avoided. Furthermore, the preparation of sulfatase from Helix pomatia (snail) is problematic since the animals have to be collected or cultivated and have to be killed for the preparation of sulfatase.

The invention also relates to oligonucleotides specifically hybridizing to a nucleic acid molecule of the invention. Such oligonucleotides have a length of preferably at least 10, in particular at least 15, and particularly preferably of at least 50 nucleotides. They are characterized in that they specifically hybridize to the nucleic acid molecules of the invention, that is to say that they do not or only to a very minor extent hybridize to nucleic acid sequences encoding other proteins, in particular other glucosinolate sulfatases. The oligonucleotides of the invention can be used for instance as primers for amplification techniques such as the PCR reaction or as a hybridization probe to isolate related genes.

Moreover, the invention relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering, which contain the above-described nucleic acid molecules of the invention. In a preferred embodiment of the invention, the vectors of the invention are suitable for the transformation of fungal cells or cells of microorganisms. Preferably, such vectors permit the integration of the nucleic acid molecules of the invention, possibly together with flanking regulatory regions, into the genome of the transformed host cell. In another preferred embodiment, the nucleic acid molecules contained in the vectors are connected to regulatory elements ensuring transcription and synthesis of a translatable RNA in prokaryotic or eukaryotic cells. The expression of the nucleic acid molecules of the invention in prokaryotic or eukaryotic cells, for instance in Escherichia coli, Saccharomyces cerevisiae or insect cells, such as, e.g. Spodoptera frugiperda Sf9 cells, is interesting because it permits a more precise characterization of the enzymatic activities of the enzymes encoded by these molecules. Moreover, it is possible to express these enzymes in such prokaryotic or eukaryotic cells which are free from interfering enzymes. In addition, it is possible to insert different mutations into the nucleic acid molecules by methods commonly used in molecular biology (see for instance Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2^nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), leading to the synthesis of proteins possibly having modified biological properties. On the one hand it is possible in this connection to produce deletion mutants in which nucleic acid molecules are produced by progressive deletions from the 5' or 3' end of the coding DNA sequence, and said nucleic acid molecules lead to the synthesis of correspondingly shortened proteins.

On the other hand, the introduction of point mutations is also conceivable at positions at which a modification of the amino acid sequence for instance influences the enzyme activity or the control of the enzyme. In this manner, it is for instance possible to produce mutants which possess a modified stereo and regio selectivity or a modified K_m value or which are no longer subject to the control mechanisms normally existing in the cell and realized via an allosteric control or covalent modification.

Moreover, mutants possessing a modified substrate or product specificity can be prepared. Furthermore, it is possible to prepare mutants having a modified activity- temperature-profile.

Furthermore, in the case of expression in plants, the introduction of mutations into the nucleic acid molecules of the invention allows the gene expression rate and/or the activity of the proteins encoded by the nucleic acid molecules of the invention to be increased.

For genetic engineering in prokaryotic cells or eukaryotic cells, the nucleic acid molecules of the invention or parts of these molecules can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook et al., 1989, Molecular Cloning: A laboratory manual, 2^nd edition, Cold Spring Harbor Laboratory Press, NY, USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be connected to each other by applying adapters and linkers to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods.

Another embodiment of the invention relates to host cells, in particular prokaryotic or eukaryotic cells, transformed and/or genetically modified with an above-described nucleic acid molecule of the invention or with a vector of the invention, and to cells derived from such transformed cells and containing a nucleic acid molecule or vector of the invention. In a preferred embodiment the host cell is genetically modified in such a way that it contains a nucleic acid molecule stably integrated into the genome. More preferably the nucleic acid molecule can be expressed so as to lead to the production of a protein having the enzymatic activity of a glucosinolate sulfatase. Particularly preferred are eukaryotic host cells, in particular yeast cells or cells of animal origin. Preferred examples are insect cells and in particular insect cell lines, such as Sf9, in particular Spodoptera frugiperda Sf9, Sf21 or HighFive cell lines (Invitrogen). Other examples for suitable host cells are mammalian cells, in particular mammalian cell lines.

An overview of different expression systems is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1- 9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al., (Methods in Molecular Biology 75 (1997), 427-440). An overview of yeast expression systems is for instance given by Hensing et al. (Antonie van Leuwenhoek 67 (1995), 261-279), Bussineau et al. (Developments in Biological Standardization 83 (1994), 13-19), Gellissen et al. (Antonie van Leuwenhoek 62 (1992), 79-93, Fleer (Current Opinion in Biotechnology 3 (1992), 486-496), Vedvick (Current Opinion in Biotechnology 2 (1991), 742-745) and Buckholz (Bio/Technology 9 (1991), 1067-1072).

An overview of insect cell expression systems is for instance given by Hegedus et al. (Gene 201 (1998), 241-249). The expression of sequences in Sf9 cells is, e.g., described in detail in Li et al. (Protein Expression and Purificaton 21 (2001), 121-128) and in Jarvis et al. (Protein Expression and Purification 8 (1996), 191-203). Moreover, the examples of the present application describe the expression of the nucleic acid molecule of the invention in Spodoptera frugiperda Sf9 cells.

Expression vectors have been widely described in the literature. As a rule, they contain not only a selection marker gene and a replication-origin ensuring replication in the host selected, but also a bacterial or viral promoter, and in most cases a termination signal for transcription. An example for an insect cell expression vector is the commercially available vector plZTΛ/5-His (Invitrogen). Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a coding DNA sequence. The DNA sequence naturally controlling the transcription of the corresponding gene can be used as the promoter sequence, if it is active in the selected host organism. However, this sequence can also be exchanged for other promoter sequences. It is possible to use promoters ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) are sufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUN/5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez and Chamberlin (Eds), Promoters, Structure and Function; Praeger, New York, (1982), 462-481; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), Ip1 , rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of proteins. These promoters often lead to higher protein yields than do constitutive promoters. In order to obtain an optimum amount of protein, a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, a lac promoter is particularly suitable which can be induced by lactose or IPTG (=isopropyl-β-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature. Promoters for ensuring expression of a desired sequence in insect cells are known to the person skilled in the art and include, e.g., the ADH1 promoter, the polyhedrin (p10) promoter, the MT promoter and the Ac5 promoter (see England et al. (J. Biol. Chem. 265 (1990), 5086-5094); Hong et al. (J. Microbiol. Biotechnol. 11 (2001), 585-591); Pfeifer et al. (Gene 188 (1997), 183-190); Jervis and Kilburn (Cytotechnology 21 (1996), 217-223). A preferred promoter for expression in insect cells is the Orygia pseudotsugata multicapsid nucleopolyhedrosis virus immediate- early 2 (OplE2) promoter (Theilmann and Stewart (Virology 180 (1991), 492-508); Theilmann and Stewart (Virology 187 (1992), 84-96)).

The transformation of the host cell with a nucleic acid molecule or vector according to the invention can be carried out by standard methods, as for instance described in Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2^nd edition (1989) Cold Spring Harbor Press, New York; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990). The host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc.

Moreover, the invention relates to a protein and biologically active fragments thereof, which is encoded by a nucleic acid molecule according to the invention and to methods for its preparation, wherein a host cell according to the invention is cultured under conditions permitting the synthesis of the protein, and the protein is subsequently isolated from the cultured cells and/or the culture medium. Since the disclosed glucosinolate sulfatase is an extracellular enzyme, it is preferably recovered from the culture medium. The present invention also relates to the protein obtainable by such a method.

Furthermore, the present invention also relates to an antibody specifically recognizing a protein according to the invention. The antibody can be monoclonal or polyclonal and can be prepared according to methods well known in the art. The term "antibody" also comprises fragments of an antibody which still retain the binding specificity.

The present invention also relates to methods for identifying compounds which can act as antagonists/inhibitors of the polypeptide according to the invention comprising the steps of:

(a) contacting the polypeptide of the invention with a compound to be tested in the presence of a glucosinolate under appropriate conditions; and

(b) determining whether the compound reduces or abolishes the activity of the polypeptide to desulfate the glucosinolate.

As described above, the glucosinolate sulfatase activity of DBM enables this organism to desulfate glucosinolates which renders them invisible to myrosinase. This allows DBM to avoid the formation of toxic glucosinolate breakdown products. Thereby, DBM, so to say, disarmes the "mustard oil bomb". Consequently, the glucosinolate sulfatase of DBM constitutes an interesting target for DBM pest management since inhibitors of this enzyme should destroy the organisms mechanism to overcome the barrier that the cruciferous plants build up by synthesizing glucosinolates. Thus, the polypeptide of the present invention can be used in screening methods to identify compounds which inhibit the glucosinolate sulfatase and which can be used as pesticides or at least as lead structures for the design and synthesis of effective pesticides.

The polypeptide used in step (a) can be in any possible form which allows a determination of its activity. It may be in the form of a crude extract, e.g. a protein extract, from cells, tissue, organisms or host cells which express the protein. Thus, it may, e.g., be in the form of a protein extract from cells or tissue of an organism in which it is naturally expressed, for example, Plutella xylostella. It is possible, e.g., to use whole larvae of this organism or those tissues in which the protein is expressed, e.g. gut tissue or gut content . The protein may also be in the form of an extract from host cells/host cell cultures, which are transformed with a nucleic acid molecule of the invention. It is also possible to use the supernatant of such a host cell culture if the protein is secreted by the host cell. Preferably, the polypeptide used in the method according to the invention is a purified protein. "Purified" in this context means a protein which is at least 50% pure, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, particularly preferred at least 90% and most preferably 100%) pure.

The conditions in step (a) are preferably chosen in such a way that they ensure that the protein is active if the same conditions are used in the absence of the compound to be tested. The determination of these conditions is within the skill and the common general knowledge of the person skilled in the art.

The determination of the level of activity in step (b) of the method according to the invention can be carried out as described hereinabove or as described in the Examples. In order to determine whether the compound to be tested reduces or abolishes the enzymatic activity of the polypeptide of the invention, the reaction is preferably carried out as a control in parallel in an identical manner, however, in the absence of the compound to be tested.

The glucosinolate used in the method can be any possible glucosinolate which is accepted as a substrate by the polypeptide employed in the method. Preferably, it is a commercially available glucosinolate, such as, e.g. sinigrin (2-propenyl- glucosinolate (Aldrich)) or glucotropaeolin (benzyl glucosinolate, Calbiochem) or a purified intact glucosinolate, such as, e.g. glucoraphanin (4-methylsulfinylbutyl glucosinolate) or glucobrassicin (3-indoylmethyl glucosinolate) (Kliebenstein et al., Plant Cell 13 (2001), 684).

The compound to be tested in the method according to the invention may be any possible compound.

The term "compound" as used herein in particular describes any possible molecule which may be tested for its capability of inhibiting glucosinolate sulfatase activity. Preferably, the compound has a low toxicity for plant cells. It is possible to run a plurality of assay mixtures in parallel with different concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

Candidate compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate compounds preferably comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate compounds often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate compounds are also found among biomolecules including polypeptides, peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate compounds can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known insecticidal agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Preferably, the compound to be tested may be a compound which is already known or suspected to inhibit known sulfatases, such as, e.g., sulfit, derivatives thereof, compounds containing it or the substances mentioned in Skorey et al. (Protein Expression and Purification 15 (1999), 178-187) as potential inhibitors of sulfatases, such as phenol derivatives. These are in particular compounds which can form covalent complexes with vanadate, more preferably vanadate esters. An example for such a compound is N-acetyl-L-tyrosine ethyl ester (NATEE). Other examples are the compounds listed in Table 2 of Skorey et al. (loc. cit.) on page 184.

In a preferred embodiment of the method according to the invention, the compound to be tested is contacted with the polypeptide of the invention in the presence of vanadate and the compound is a compound which can form a covalent complex with vanadate as described above. A method which includes the addition of vanadate can basically be carried out as described in Skorey et al. (loc. cit.), in particular on page 181. Moreover, the present invention also relates to the use of the polypeptide of the invention, of a nucleic acid molecule encoding such a polypeptide or of cells, tissue, organisms or host cells expressing such a polypeptide for the identification of compounds which reduce or abolish the activity of the polypeptide to desulfate glucosinolate, i.e. antagonists/inhibitors of the polypeptide.

The term "reduce" in this context means a reduction of the glucosinolate sulfatase activity of at least 10%, preferably of at least 30%, more preferably of at least 50%, in particular of at least 70%, particularly preferred of at least 80% and most preferably of at least 90% when compared to the activity of the polypeptide in the absence of the antagonist/inhibitor.

The term "abolish" means a total loss of enzymatic activity, preferably in the sense that no enzymatic activity can be detected when the activity is determined as described in the Examples.

Moreover, the present invention relates to a method for the preparation of a composition comprising the steps (a) and (b) of the method as described above and furthermore the step of formulating the compound identified in step (b) as reducing or abolishing the glucosinolate sulfatase activity of the polypeptide as an active ingredient in a composition.

The composition prepared according to this method is preferably a pesticidal, more preferably an insecticidal composition designed for application to plants or parts of plants. Thus, the compound is preferably formulated into an insecticidal composition, e.g. by mixing it with ingredients normally used in this field.

The compound may, e.g., be formulated into dosage forms such as aerosols, granules, flowables, oil sprays, wettable powders, emulsions or emulsifiable concentrates, fumigants or poison baits. Such forms may be prepared by mixing the compound with an appropriate carrier, e.g. a liquid carrier, a gaseous carrier or a solid carrier and/or by adding, if necessary, surfactants or other auxiliaries generally used for the preparation of such formulations.

The content of the compound in the composition lies generally in the range form 0.1%) to 100%) (i.e. pure form without addition of other ingredients), preferably form 5% to 95%, more preferably from 10%) to 90%, from 20% to 80%, in particular from 30% to 70%, even more preferably from 40% to 60% and particularly preferred in the range of 50%.

Solid carriers which can be used when formulating the composition are, e.g., fine powder of granules of clay materials such as diatomaceous earth, synthetic hydrated silicon oxide, ceramics, quartz, sulfur, calcium carbonate, hydrated silica etc.

Liquid carries are, e.g., water, alcohols such as ethanol or methanol, aromatic hydrocarbons such as toluene, xylene, benzene, aliphatic hydrocarbons such as hexane, cyclohexane, gas oil or kerosine; ketones; esters such as butyl acetate or ethyl acetate; ethers; nitriles; acid amines; halogenated hydrocarbons or vegetable oils such as, e.g., soybean oil.

Gaseous carriers are, e.g., butan gas, dimethyl ether or carbon dioxide.

Surfactans which are often used in this field are, e.g., alkyl sufates, polyethylene glycol ethers, sugar alcohol derivatives, alkyl sulfonates etc. Auxiliaries used are, e.g., stabilizers, dispersing agents or fixing agents such as starch, cellulose derivatives, alginic acid, sugars, gelantin, or synthetic watersoluble polymers such as polyvinyl pyrrolidone. Stabilizers comprise, e.g., mineral oils, fatty acids and their esters and vegetable oils, as well as synthetic stabilizers.

The present invention also relates to a method for preventing infestation of plants by an organism, preferably an insect organism, which expresses a polypeptide according to the invention, in particular by larvae of such an organism, comprising the step of applying to the plants or parts of the plants a compound which can reduce or abolish the glucosinolate sulfatase activity of the polypeptide of the invention. Such a compound can be identified by a method according to the invention as described above. Most preferably, the organism belongs to the Lepidoptera, more preferably to the Plutellidae, even more preferably to the genus Plutella and most preferably to the species Plutella xylostella.

The compound can be applied to the plants or parts thereof in any form which is suitable to ensure that the organism will come into contact with it when trying to feed on the plant. Appropriate formulations are known to the person skilled in the art and have already been described above in connection with the method for formulating a composition. A suitable mode of application is, e.g., spraying of a solution or emulsion. Furthermore, the present invention also relates to a method for producing mutants of an organism, preferably an insect organism, which expresses a polypeptide according to the invention wherein said mutant shows a reduced or abolished activity of glucosinolate sulfatase comprising the steps of:

(a) subjecting the organism to a mutagenesis step;

(b) testing the mutants obtained according to step (a) for the expression and/or activity of the glucosinolate sulfatase according to the invention; and

(c) selecting those mutants in which the expression and/or activity of the glucosinolate sulfatase according to the invention is reduced or abolished.

The organism used in this method can be any organism which expresses a glucosinolate sulfatase according to the invention. Preferably, it is an insect organism, more preferably an insect belonging to the Lepidoptera, even more preferably an insect belonging to the Plutellidae, most perferably an insect of the genus Plutella, and particularly preferred an insect of the species Plutella xylostella. The mutagenesis of step (a) can be carried out according to methods known to the person skilled in the art, e.g., by chemical substances, such as ethylmethyl sulfonate (EMS) or by ionizing radiation.

The determination of the activity of the glucosinolate sulfatase according to step (b) of the described method can be carried out as described herein above or as described in the following Examples.

The expression level can be determined by methods well-known to the person skilled in the art, e.g. by determining the amount of corresponding RNA (e.g. by Northern Blot analysis) or the amount of protein (e.g. by Western Blot analysis). The terms "reduced" and "abolished" have preferably the meaning as described herein above. In a preferred embodiment the obtained mutants are moreover selected for temperature sensitivity, i.e. for mutants in which the effect of the mutation only occurs at higher temperatures. By this it would be possible that a mutation spreads in a DBM population before the corresponding effect of the mutation occurs at higher temperatures. The present invention also relates to a method for the production of a transgenic animal of an organism which expresses a polypeptide according to the invention, wherein said transgenic animal shows a reduced activity of a glucosinolate sulfatase according to the invention, comprising the step of genetically modifying such an organism with a nucleic acid molecule, the presence or expression of which in the cells of the organism reduces or abolishes the expression of the polypeptide of the present invention. The nucleic acid molecule used for the genetic modification of the organism may, e.g., be a nucleic acid molecule according to the invention or a fragment thereof, wherein said nucleic acid molecule or fragment thereof upon expression in said organism leads to the synthesis of an RNA molecule which inhibits or prevents the expression of an endogenous DNA encoding a glucosinolate sulfatase according to the invention. Examples of such RNA molecules are antisense RNA molecules, sense RNA molecules which effect a cosupression effect, ribozymes or RNAi molecules. The construction and expression of such molecules in order to achieve the desired effect is well-known to the person skilled in the art. E.g. an antisense RNA is characterized as being complementary to transcripts of a gene encoding a glucosinolate sulfatase according to the invention. Thereby, complementarity does not signify that the encoded RNA has to be 100% complementary. A low degree of complementarity may be sufficient as long as it is high enough to inhibit the expression of such a glucosinolate sulfatase upon expression of said RNA in cells of the organism. The transcribed RNA is preferably at least 90% and most preferably at least 95% complementary to the transcript of the nucleic acid molecule encoding the glucosinolate sulfatase. In order to cause an antisense effect during the transcription in the cells such RNA molecules have a length of at least 15 bp, preferably a length of more than 100 bp and most preferably a length or more than 500 bp, however, usually less than 5000 bp, preferably shorter than 2500 bp. Exemplary methods for achieving an antisense effect are for instance described by Mϋller-Rober (EMBO J. 11 (1992), 1229-1238), Landschϋtze (EMBO J. 14 (1995), 660-666), D'Aoust (Plant Cell 11 (1999), 2407-2418) and Keller (Plant J. 19 (1999), 131-141) and are herewith incorporated in the description of the present invention. Likewise, an antisense effect may also be achieved by applying a triple-helix approach, whereby a nucleic acid molecule complementary to a region of the gene, encoding the relevant polypeptide, designed according to the principles for instance laid down in Lee (Nucl. Acids Res. 6 (1979), 3073); Cooney (Science 241 (1998), 456) or Dervan (Science 251 (1991), 1360) may inhibit its transcription.

A similar effect as with antisense techniques can be achieved by producing transgenic animals expressing suitable constructs in order to mediate an RNA interference (RNAi) effect. Thereby, the formation of double-stranded RNA leads to an inhibition of gene expression in a sequence-specific fashion. More specifically, in RNAi constructs, a sense portion comprising the coding region of the gene to be inactivated (or a part thereof, with or without non-translated region) is followed by a corresponding antisense sequence portion. Between both portions, an intron not necessarily originating from the same gene may be inserted. After transcription, RNAi constructs form typical hairpin structures. In accordance with the method of the present invention, the RNAi technique may be carried out as described by Smith (Nature 407 (2000), 319-320) or Marx (Science 288 (2000), 1370-1372).

As mentioned above, also DNA molecules can be employed which, during expression in cells, lead to the synthesis of an RNA which reduces the expression of the gene encoding the glucosinolate sulfatase in the cells due to a co-suppression effect. The principle of co-suppression as well as the production of corresponding DNA sequences is precisely described, for example, in WO 90/12084. Such DNA molecules preferably encode an RNA having a high degree of homology to transcripts of the target gene. It is, however, not absolutely necessary that the coding RNA is translatable into a protein. The principle of the co-suppression effect is known to the person skilled in the art and is, for example, described in Jorgensen, Trends Biotechnol. 8 (1990), 340-344; Niebel, Curr. Top. Microbiol. Immunol. 197 (1995), 91-103; Flavell, Curr. Top. Microbiol. Immunol. 197 (1995), 43-36; Palaqui and Vaucheret, Plant. Mol. Biol. 29 (1995), 149- 159; Vaucheret, Mol. Gen. Genet. 248 (1995), 311-317; de Borne, Mol. Gen. Genet. 243 (1994), 613-621 and in other sources.

Likewise, DNA molecules encoding an RNA molecule with ribozyme activity which specifically cleaves transcripts of a gene encoding the relevant glucosinolate sulfatase can be used. Ribozymes are catalytically active RNA molecules capable of cleaving RNA molecules and specific target sequences. By means of recombinant DNA techniques, it is possible to alter the specificity of ribozymes. There are various classes of ribozymes. For practical applications aiming at the specific cleavage of the transcript of a certain gene, use is preferably made of representatives of the group of ribozymes belonging to the group I intron ribozyme type or of those ribozymes exhibiting the so-called "hammerhead" motif as a characteristic feature. The specific recognition of the target RNA molecule may be modified by altering the sequences flanking this motif. By base pairing with sequences in the target molecule these sequences determine the position at which the catalytic reaction and therefore the cleavage of the target molecule takes place. Since the sequence requirements for an efficient cleavage are low, it is in principle possible to develop specific ribozymes for practically each desired RNA molecule. In order to produce DNA molecules encoding a ribozyme which specifically cleaves transcripts of a gene encoding the relevant glucosinolate sulfatase, for example a DNA sequence encoding a catalytic domain of a ribozyme is bilaterally linked with DNA sequences which are complementary to sequences encoding the target protein. Sequences encoding the catalytic domain may for example be the catalytic domain of the satellite DNA of the SCMo virus (Davies, Virology 177 (1990), 216-224 and Steinecke, EMBO J. 11 (1992), 1525-1530) or that of the satellite DNA of the TobR virus (Haseloff and Gerlach, Nature 334 (1988), 585-591). The expression of ribozymes in order to decrease the activity of certain proteins in cells is known to the person skilled in the art and is, for example, described in EP-B1 0 321 201.

The nucleic acid molecule used in order to prepare a transgenic animal having a reduced or abolished activity of the polypeptide of the invention can also be a nucleic acid molecule, e.g. a heterologous DNA, suitable for disrupting the endogenous genes encoding the polypeptide in the genome of the organisms, i.e. a molecule which is suitable for the in vivo mutagenesis or the production of knock-out animals. The term "in vivo mutagenesis", relates to methods where the sequence of the gene encoding the relevant glucosinolate sulfatase is modified at its natural chromosomal location such as for instance by techniques applying homologous recombination. This may be achieved by using a hybrid RNA-DNA oligonucleotide ("chimeroplast") which is introduced into cells by transformation (TIBTECH 15 (1997), 441-447; WO95/15972; Kren, Hepatology 25 (1997), 1462-1468; Cole-Strauss, Science 273 (1996), 1386- 1389). Part of the DNA component of the RNA-DNA oligonucleotide is homologous to the target glucosinolate sulfatase gene sequence, however, displays in comparison to this sequence a mutation or a heterologous region which is surrounded by the homologous regions. The term "heterologuous region" corresponds to any sequence that can be introduced and encompasses, for instance, also sequences from the same glucosinolate sulfatase gene but from a different site than that which is to be mutagenized. By means of base pairing of the homologous regions with the target sequence followed by a homologous recombination, the mutation or the heterologous region contained in the DNA component of the RNA-DNA oligonucleotide can be transferred to the corresponding gene of the cell of the target organism. By means of in vivo mutagenesis, any part of the gene encoding the glucosinolate sulfatase can be modified as long as it results in a decrease of the activity of said enzyme. Thus, in vivo mutagenesis can for instance concern, the promoter, e.g. the RNA polymerase binding site, as well as the coding region.

The term "heterologous DNA sequence" refers to any DNA sequences which can be inserted into the target gene via appropriate techniques other than those described above in connection with in vivo mutagenesis. The insertion of such a heterologous DNA sequence may be accompanied by other mutations in the target gene such as the deletion, inversion or rearrangement of the sequence located at the insertion site. This embodiment of the method of the invention includes that the introduction of a nucleic acid molecule in step (a) leads to the generation of a pool, i.e. a plurality, of transgenic animals in the genome of which the nucleic acid molecule, i.e. the heterologous DNA sequence, is randomly spread over various chromosomal locations, and that step (c) is followed by selecting those transgenic animals out of the pool which show the desired genotype, i.e. an inactivating insertion in the relevant glucosinolate sulfatase gene and/or the desired phenotype, i.e. a reduced glucosinolate sulfatase activity

Suitable heterologous DNA sequences that can be taken for such an approach are described in the literature and include, for instance vector sequences capable of self- integration into the host genome or mobile genetic elements.

Another example of insertional mutations that may result in gene silencing includes the duplication of promoter sequences which may lead to a methylation and thereby an inactivation of the promoter (Morel, Current Biology 10 (2000), 1591-1594). It is also evident from the disclosure of the present invention that any combination of the above-identified strategies can be used for the generation of transgenic animals, which due to the one or more of the above-described nucleic acid molecules in their cells display a reduced expression and/or activity of the relevant glucosinolate sulfatase compared to corresponding non-genetically modified animals. In the context of the present invention, the term "transgenic" means that the animal contains cells in which the genome structurally deviates from that of corresponding source animals in such a way that the activity of a glucosinolate sulfatase according to the invention is reduced or abolished as explained above. Such a structural difference preferentially refers to the gene encoding this sulfatase, which includes for instance the inactivation due to a deletion. The prior art provides means and methods for providing transgenic animals wherein the activity of a specific protein is reduced. Methods for producing transgenic animals, in particular transgenic insects, are, e.g., described in Peloquin et al. (Insect Molecular Biology 9 (2000), 323-333), Kennerdell and Carthew, Nature Biotechnology 17 (2000), 896-898) and Atkinson et al. Annual Review of Entomology 46 (2001), 317-346).

It is, for example, possible to use so-called "PiggyBac" vectors, which are viral vectors, and which can be obtained from the USDA (Gainsville, Florida, USA). The transformation with such vectors is preferably carried out by microinjection into the eggs of insects as described, e.g., in Peloquin et al. (loc. cit.), since these are easiest to manipulate.

The present invention also relates to the mutants and transgenic animals obtainable by the above-described methods in which the activity of a polypeptide of the present invention is reduced or abolished.

Another object of the present invention is a method for reducing infestation of plants by an organism which expresses the polypeptide of the present invention, in particular by larvae of such an organism, comprising the step of artificially increasing in a natural population of such an organism the portion of individuals in which the activity of the glucosinolate sulfatase is reduced or abolished. In this context the terms "reduced" and "abolished" have the meaning as defined above. The plant is a plant which synthesizes glucosinolates, preferably it is a plant of the order Capparales.

The increase of the portion of individuals can be achieved, e.g., by setting free mutants or transgenic animals of such an organism which have a reduced or no glucosinolate sulfatase activity. Such mutants and/or transgenic animals can be produced as described above.

These and other embodiments are disclosed and obvious to a skilled person and embraced by the description and the examples of the present invention. Additional literature regarding one of the above-mentioned methods, means and applications, which can be used within the meaning of the present invention, can be obtained from the state of the art, for instance from public libraries for instance by the use of electronic means. This purpose can be served inter alia by public databases, such as the "medline", which are accessible via internet, for instance under the address httpJ/www.ncbi.nlm.nih.gov/PubMed/medline.html. Other databases and addresses are known to a skilled person and can be obtained from the internet, for instance under the address http://www. lycos . com . An overview of sources and information regarding patents and patent applications in biotechnology is contained in Berks, TIBTECH 12 (1994), 352-364.

All of the above cited disclosures of patents, publications and database entries are specifically incorporated herein by reference in their entirety to the same extent as if each such individual patent, publication or entry were specifically and individually indicated to be incorporated by reference.

Figure 1 shows the reactions catalyzed by plant myrosinase and Diamondback moth glucosinolate sulfatase (GSS). (A) Myrosinase removes glucose from glucosinolates (top), leading to the formation of toxic hydolysis products (isothiocyanates, nitriles, thiocyanates; bottom left). (B) GSS forms desulfo-glucosinolates (bottom right).

Figure 2 shows GSS activity in DBM larval tissue. Lane 1 , Tris-HCl control; lane 2, body w/o gut; lane 3, gut tissue (w/o hindgut and malphigian tubules); lane 4, gut content. Enzyme assays were performed as described . From each sample, 1 μg total protein was used. Guts were rinsed with Tris-HCl to remove remaining content. Figure 3 shows immunoblots which localize DBM GSS to larval guts. (A) SDS- PAGE protein gel (6 % stacking/10 % resolving gel), stained with Coomassie Blue G250. Molecular mass is indicated on the left. Lane 1 , 5 μg body w/o gut; lane 2, 5 μg gut tissue; lane 3, 3 μg gut content; lane 4, 0.1 μg native PAGE-purified DBM GSS (8 % PAGE; imidazole/HEPES, pH 7.4); lane 5, 1 μg Ni-Agarose purified GSS, heterologously expressed by E. coli; lane 6, 5 μg E. coli total protein with control vector w/o GSS insert; lane 7, 5 μg Helix pomatia sulfatase, crude extract, type H-1 (Sigma). (B) Immunoblot detection with polyclonal GSS antibody including a second anti-rabbit antibody. For immunoblotting 1/10^th of the protein amounts from (A) was loaded, except for lane 4, where VT. was used.

Figure 4 shows an RT-PCR analysis of GSS-specific mRNA. Upper bands indicate GAPDH control (549 bp), lower bands GSS cDNA (324 bp). cDNA was synthesized from 1 μg total RNA each using the Omniscript- Kit (Qiagen, Germany). 0.2 μl first-strand cDNA each were used for PCR with primers specific for GSS (Dsulf-F1 : GTGGTGCTCCTCGGCGCGGC (SEQ ID NO: 10) /Dsulf-R2: AGCGTCCTGTAGGTACTGCGAGA) (SEQ ID NO: 11) and GAPDH (GAPDH-F: CAGTGCCGATGCACCTATGTTC (SEQ ID NO: 12) /GAPDH-R: AAGTTGTCGTTGAGGGAGATGCC(SEQ ID NO: 13)). (A) Developmental transcript analysis of GSS in DBM. Lane 1 , eggs; lanes 2 to 5, 1^st, 2^nd, 3^rd, 4^th larval instars; lane 6, prepupae; lane 7, pupae; lane 8, adults. (B) Spatial transcript analysis of GSS in 4^th instar larvae. Lane 1 , head; lane 2, body (w/o gut); lane 3, silk glands; lanes 4 to 9, gut parts, from anterior to posterior; lane 10, hindgut with malphigian tubules.

Figure 5 shows HPLC chromatograms of glucosinolate profiles. (A) A. thaliana Cvi-0 leaf halves. (B) Faeces of 4^th instar larvae feeding on the other halves. Extraction, separation and identification were performed as described . Shown are signals from 7.5 to 17.5 minutes. Secondary structures indicate major peaks caused, from left to right, by sinigrin (2- propenyl GS), gluconapin (3-butenyl GS), glucohirsutin (8- methylsulfinyloctyl GS), and glucobrassicin (3-indolylmethyI GS).

Figure 6 shows the postulated pathway for chain elongation of amino acids in glucosinolate biosynthesis.

For methionine, R = H₃C-S-CH₂-; for homomethionine, R = H3C-S- (CH2)2-; for dihomomethionine, R = H₃C-S-(CH2)3-; etc. For phenylalanine, R = 0-; for homophenylalanine, R = 0-CH₂-; for dihomophenylalanine, R = 0-(CH2) 2-; etc. For tyrosine, R = OH-0-; for homotyrosine, R = OH-0-CH₂-; for dihomotyrosine, R = OH-0-(CH₂) 2-; etc.. 0 = benzyl-.

Figure 7 shows the isoelectric focussing, activity and immunoblotting of purified GSS.

(A) Isoelectric focussing of 10 μg natively purified DBM-GSS at pH 10 to 3, double stained with Bio-Safe Coomassie G250 and silver (Bio-Rad).

(B) A further lane from the IEF gel, loaded with 10 μg natively purified GSS, was divided vertically into 28 parts a 2 mm length. Gel pieces were suspended in 100 μl Tris, pH 7.5, and 30 μl each were tested for GSS activity. Vertical axis shows relative amounts of sinigrin converted to its desulfo-form, as measured by HPLC. Maximum GSS activity was detected in fraction 15. (C) SDS-PAGE (12 %) loaded with 30 μl from IEF fractions 12 to 17. Gel was double stained with Coomassie G250 and silver. (D) Immunoblot of a SDS-PAGE gel performed unter conditions as in (C), but with half the amounts of protein loaded. The anti-GSS antibody reacts strongest with fraction 15.

Figure 8 Top: Exon-intron structure of the DBM GSS gene. Triangles mark binding sites for primers used in RT-PCR. Bottom: Amino acid sequence of DBM GSS. PROSITE sulfatase signatures are shaded grey. A predicted (Nakai and Horton, Trends Biochem. Sci. 24 (1999), 34-36) signal sequence for extracellular targeting is printed in italics and underlined. Conserved amino acids of the catalytic center are underlined and printed extra bold. A highly conserved cystein residue posttranslationally converted into C -formylglycin in eukaryotic sulfatases (Dierks et al., EMBO J. 18 (1999), 2084-2091) is shaded black.

The following Examples further illustrate the invention. In the Examples the following methods and materials were used.

Methods and Materials

1. Plant materials

Arabidopsis thaliana seeds from the Col-0 (accession N1092) and Cvi-0 (N1096) ecotypes were kindly provided by the Nottingham Arabidopsis Stock Center, Nasturtium officinale was obtained from Saatgut GmbH (Quedlinburg, Germany). Plants were grown under 11.5 h light/12.5 h dark cycles at 23 °C in 5 x 5 cm² pots on a 1 :3 vermiculite:standard soil (Einheitserdenwerk, Frόndenberg, Germany) mix; N. officinale was grown partly submerged in 15 x 15 x 25 cm³ plastic boxes filled with standard soil and water under otherwise identical conditions.

2. Insects

A Plutella xylostella stock culture (G88 colony) was kindly provided by A.M Shelton from Cornell University (Geneva, NY, USA). Larvae were reared on a wheat germ based artificial diet at 27 °C and 16 h light/8 h dark cycles.

3. GSS assay

4^th instar larval tissue was ground in 100 mM Tris, pH 7.5. Debris was removed by centrifugation. Protein concentration in the supernatant was determined using standard methods. 1 μg protein in 50 μl 100 mM Tris, pH 7.5, was incubated with 50 μl of an aequeous solution of 5 mM glucosinolate for 3 min at room temperature. Reaction was stopped with 500 μl methanol. After purification with 10 mg DEAE sephadex A-25 and centrifugation, supernatant was dried and dissolved in 400 μl deionized water. 40 μl were HPLC analyzed as described below.

4. Glucosinolate extraction and HPLC analysis

Leaf samples (50 - 70 mg) were frozen in liquid nitrogen, freeze-dried, and ground to a fine powder. Glucosinolates were extracted with 500 μl methanol for 6 hours, and twice with 500 μl 60 % methanol for 1 hour. After centrifugation to remove debris, the supernatant was dried and dissolved in 400 μl deionized water. After o/n incubation with 50 μg sulfatase from Helix pomatia, crude extract, type H-1 (Sigma), purification with 10 mg DEAE sephadex A-25 and centrifugation, 40 μl from the supernatant were separated on a water (Solvent A)-acetonitrile (Solvent B) gradient at a flow rate of 1 ml/min and at 20 °C by HPLC (Agilent HP1100 Series) fitted with a Lichrocart 250-4 RP18e 5 μm column. The 25 min run consisted of 1.5 % B (1 min), 1.5 - 5.0 % B (5 min), 5.0 - 7.0 % B (2 min), 7.0 - 25.0 % B (7 min), 25.0 - 92.0 % B (3 min), and a 6 min hold at 92.0 % B, followed by 92.0 - 1.5 % B (1 min). Eluent was monitored by diode array detection between 190 and 360 nm (2 nm interval). Desulfo-glucosinolates were identified by retention time and UV spectra as compared with those of purified standards. Response factors determined from pure desulfo-glucosinolates were used to calculate molar concentrations of individual glucosinolates. Analysis of faeces followed a similar protocol, but omitting the sulfatase step.

The gut content of a 4^th instar DBM larva reared on artificial diet contains approximately 5 - 7 μg protein, equal to a GSS activity of 20 - 28 nmol/min. Total glucosinolate amount of 100 mg fresh leaves is approximately 150 nmol for A. thaliana Col-0, and 700 nmol for Cvi-0 or N. officinale. Therefore, one larva could desulfate the glucosinolates present in 100 mg fresh leaves in 30 min. According to the observations of the inventors, one DBM larva devours approximately 100 mg fresh leaves in 50 hours. Thus, actual GSS activity exceeds the minimal activity necessary to convert total glucosinolates in the plant meal 100-fold.

Example 1 Identification of a glucosinolate sulfatase in Plutella xylostella

A common procedure to analyze glucosinolates involves sulfatase from Helix pomatia. Glucosinolates are bound to anion exchange columns and released as desulfo-glucosinolates by treating with sulfatase (Thies, Naturwissenschaften 66 (1979), 364). HPLC analysis of water extracted faeces from DBM larvae fed on a variety of Arabidopsis thaliana ecotypes revealed the presence of desulfo- glucosinolates. This suggested that DBM contains a sulfatase activity which may desulfate glucosinolates, a ,glucosinolate sulfatase' (GSS) (Fig. 1 B). To test this hypothesis, crude protein extracts from fourth instar larval guts were separated on native PAGE or IEF gels. In both cases, GSS activity was monitored with sinigrin, and correlated with a single band on Coomassie Blue or silver stained gels. This band was excised from the native gels and subjected to SDS PAGE, where it also migrated as a single band, with a molecular weight of approximately 62 kDa. These results were confirmed by isoelectric focusing (Figure 7). Moreover, this band was excised from native gels, purified, desalted and concentrated with Centricon spin columns (Millipore) and, after lyophilization, subjected to MALDI mass fingerprinting and ESI-MS/MS (WITA PROTEOMICS, Teltow, Germany). Only a single polypeptide could be identified. Further analyses located GSS activity in the gut content and, in lesser amounts, in gut tissue but not in the remaining parts of the larval body (Fig. 2), indicating that the enzyme is secreted into the gut lumen.

For the isolation of a corresponding cDNA sequence, total RNA was isolated from DBM larvae, was transcribed into cDNA and cloned into a PCRII Topo vector (Invitrogen). After preparation of plasmid DNA several thousand clones were partially sequenced in order to obtain "expressed sequence tags" (ESTs). In this EST collection several fragments were identified which showed similarity to sulfatase sequences available in data bases. Primers were designed for specifically amplifying corresponding sequences.

Full length cDNA sequence was obtained using 5^' and 3^' RACEs (Frohman et al., Proc. Natl. Acad. Sci. USA 85 (1988), 8998), with an open reading frame of 1641 bp, equivalent to a polypeptide of 547 amino acids or 62 kDa. PSORT II (Nakai and Horton, Trends Biochem. Sci. 24 (1999), 34) predicted a 19 amino acid signal peptide for extracellular targeting. Several consensus sequences were obtained resulting from allelic variation in our DBM laboratory strain of the inventors. DNA blots were compatible with the presence of a single GSS gene locus. The sequences of three full-length cDNA sequences and of three genomic sequences are shown in SEQ ID NOs: 1 , 3 and 5 and in SEQ ID NOs: 7, 8 and 9, respectively. The genomic clones were isolated from genomic DBM DNA by using the cDNA sequences. Figure 8 shows the exon-intron structure of the gene.

Example 2 Expression of the GSS of DBM in E. coli

GSS was heterologously expressed in E. coli strain BL21 Star (Invitrogen) using a pET-28a vector (Novagen). The expressed polypeptide did not show sulfatase activity, reflecting a lack of posttranslational modifications necessary to activate sulfatases (Dierks, EMBO J. 18 (1999), 2084). A polyclonal rabbit antibody was raised against Nickel-Agarose column purified protein extracts from E. coli heterologously expressing GSS (Eurogentec, Belgium). In immunoblots including a second, horse-radish peroxidase coupled anti-rabbit antibody (Amersham Pharmacia), the first antibody strongly reacted with a single band of appr. 62 kDa in the gut content sample (Fig. 3). Most importantly, the antibody detected the natively purified polypeptide that comigrated with the GSS activity, demonstrating that the GSS gene from DBM was successfully cloned. Further evidence was obtained by functional expression in transformed insect SF9 cells, which yielded low GSS activity, clearly distinct from experiments with control constructs which lacked GSS activity.

Example 3 Expression pattern of GSS in DBM GSS expression was analyzed with RT PCR (Fig. 4). Specific primer pairs were multiplexed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers to control for variation in cDNA amounts synthesized from different RNA samples. Transcripts were detected in gut tissue but not the remaining body parts, which corresponds well with the localization of GSS activity. Furthermore, transcripts were detected in all four larval instars but not in DBM eggs, pupae or adults, indicating that GSS expression is both under tissue specific and developmental control.

Example 4 Glucosinolates used as substrate by GSS from DBM

Previous experiments by the inventors showed no correlation between glucosinolate profiles of 38 Arabidopsis thaliana ecotypes and resistance to DBM feeding. These ecotypes represented a collection of samples with very different mixtures of glucosinolate types and amounts (Kliebenstein et al., Plant Phys. 126 (2001), 811- 825), suggesting that GSS is able to act on different glucosinolate classes. Indeed, commercially available sinigrin (2-propenyl glucosinolate, Aldrich) and glucotropaeolin (benzyl glucosinolate, Calbiochem) and purified intact (Kliebenstein et al., Plant Cell 13 (2001), 681-693) glucobrassicin (3-indoyImethyl glucosinolate), which have distinct side chain structures, were all rapidly converted to desulfo- glucosinolates by crude DBM gut extract as is evident from the following Table:

Table 2

GSS enzymatic reaction constants for aliphatic, aromatic and indolic glucosinolates. GSS amount was quantified photometrically. 0.2 μg natively purified protein were used in each assay. Reactions were carried out for 3 minutes in 75 mM Tris-HCl, pH 7.5, at 24 °C, then 400 μl methanol were added to stop the reaction, and desulfo- glucosinolates were quantified by HPLC. Sinigrin and glucotropaeolin were tested in concentrations from 0.1 mM to 25 mM; for glucobrassicin the maximum concentration was 10 mM, due to limitations in the amount of purified substrate. Given are mean values + standard error; n indicates the number of experimental replicates.

Moreover, GSS activity was tested in vivo on two A. thaliana ecotypes (Col-0 and Cvi-0) and Nasturtium officinale, containing high amounts of aliphatic, indolyl or aromatic glucosinolates. Leaves from these plants were divided along their major veins, and veins were discarded. Glucosinolates were extracted directly from one half, including a Helix sulfatase treatment. The other halves were fed to fourth instar DBM larvae reared on artificial diet, (Shelton et al., J. Entomol. Sci. 26 (1991), 17), and faeces were HPLC analyzed without prior Helix sulfatase treatment. In each case, HPLC profiles from corresponding leaf and faeces samples revealed identical desulfo-glucosinolate peaks (Fig. 5). Faeces samples contained approximately 25 - 40 % less desulfo-glucosinolates than the direct extracted samples, indicating some glucosinolate degradation. This is evident from the following Table.

Table 3

Quantification of glucosinolates in leaves and desulfo-glucosinolates in larval faeces. Listed are plant sources and predominant glucosinolates, number of experimental replicates (n), glucosinolate content in leaf halves and desulfo-glucosinolate content in faeces from 4^th instar larvae fed on corresponding leaf halves, and recovery ratios in faeces vs. leaves. Given are mean values + standard deviation.

As DBM larvae do not feed continually, i.e. periods of heavy feeding alternate with pauses of various length, this difference may be explained by myrosinase activity in damaged leaf areas degrading glucosinolates pre ingestion.

In the insect gut, GSS has to compete for glucosinolates with myrosinase released from leaves. As myrosinases are not able to use desulfo-glucosinolates as a substrate (Ettlinger et al., Proc. Natl. Acad. Sci. USA 47 (1961), 1875) and sulfate competitively inhibits myrosinase (Shikita et al., Biochem. J 341 (1999), 725), GSS could act in two ways, directly by removing the myrosinase^'s substrate, glucosinolates, and indirectly by reducing its activity via the released sulfate.

A simplified, but fundamental, concept of chemical ecology assumes that antagonistic chemical interactions between plants and herbivorous insects coevolve in a stepwise process: an advance in plant defenses exerts selective pressure on the insect^'s ability to overcome these defenses, and vice versa (Ehrlich and Raven, Evolution 18 (1964), 586). Diversification of defenses in a plant species is, thus, the outcome of a historical process, which may result from an ,arms race' between the host and its pests. However, DBM can evade host plant diversification of glucosinolate structures, which are rapidly degraded by GSS. Also, variation in total glucosinolate content among crucifers does not affect herbivory by DBM (Bodnaryk, Can. J. Plant Sci. 77 (1997), 283; Li et al., J. Chem. Ecol. 26 (2000), 2401), which can be explained by an excess of GSS activity in the larval gut. Consequently, in DBM herbivory, competition between myrosinase and GSS activity may determine the efficacy of glucosinolate defenses. Indeed, elevated myrosinase activity leads to reduced DBM damage in Brassica (Li et al., loc. cit.). Diamondback moth feeds on many species of Brassicaceae. The wide substrate range of GSS is a prerequisite for the survival of newly hatched larvae on different cruciferous host plants, as female DBM are equally attracted by different glucosinolates when searching for oviposition sites (Reed et al., Entomol. Exp. Appl. 52 (1989), 277). GSS may have been recruited from other metabolic pathways as DBM evolved specialization to crucifer host plants. Tight developmental and tissue specific control ensures that GSS expression is limited to the stage at which DBM is exposed to glucosinolates and to the organ where these compounds are released from the ingested plant meal, consistent with an advanced stage in the evolution of a novel metabolic pathway (Copley, TIBS 25 (2000), 261). Nonetheless, Helix pomatia, a generalist herbivore, also possesses a sulfatase that can desulfate glucosinolates. A crude fraction containing this activity is widely used for biochemical applications, e.g. for glucosinolate analysis. The gene encoding this activity has not yet been cloned (Wittstock et al., Life 49 (2000), 71-76). Therefore, it is difficult to determine whether the DBM and the Helix enzymes share a common origin. However, a polyclonal DBM GSS antibody did not cross-hybridize with the commercially available Helix sulfatase (Figure 3). Moreover, desulfo-glucosinolates were detectable in feces from H. pomatia only when animals were fed with filter paper soaked with 5 mM sinigrin solution, but not when snails were allowed to feed on crucifers. Thus, Helix sulfatase appears unable to compete with plant myrosinase for the glucosinolate substrates, suggesting that the Helix glucosinolate sulfatase activity does not play a major role under natural conditions.

As glucosinolate breakdown products are toxic to DBM (Li et al., loc. cit), impairing GSS activity would render the larvae susceptible to the host plant's defenses. Therefore, GSS could serve as a new target for DBM pest management. This may be achieved by developing GSS inhibitors. Alternately, DBM mutants lacking GSS activity could be raised and released to mate with wild-type DBM.

Example 5 Heterologous expression in Spodoptera frugiperda S/9 cells

GSS cDNA was amplified by PCR using the primers SulfFEcoRV: 5^'- ATATGATATCAACATGGCGATTCTGCATCAAGC-3^' (SEQ ID NO:14) and SuIfRNotl: 5^'-ATATGCGGCCGCTTACAACTTTCACGGCGAACTGC-3^' (SEQ ID NO: 15). The PCR product was digested with EcoRV and Notl, and ligated into plZTΛ/5-His insect cell expression vector (Invitrogen). The GSS vector construct was propagated in E. coli TOP 10 cells. Correct orientation and sequence was confirmed by sequencing. S 9 cells were maintained at 27 °C in TNM-FH medium (Invitrogen) supplemented with 10 % heat-inactivated fetal bovine serum and 10μg/ml of gentamycin. Transfection of S/9 cells with either the GSS construct or a control vector construct (plZTΛ 5-His/CAT) was performed using Insectin-Plus liposomes as recommended by the manufacturer (Invitrogen). Transfected cells were grown in TNM-FH medium for 2, 3 and 5 days, respectively. Expression was driven by the Qrygia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 (OplE2) promoter. Supernatant from transfected S 9 cells was collected at various times starting 24 h post transfection and pooled. 3 ml of pooled supernatant from either the GSS or the control constructs was concentrated and buffer was exchanged using Ultrafree-15 centrifugation units (Millipore). From 200 μl protein in 100 mM Tris- HCl, pH 7.5, 10 μl were used for enzyme assays as described above; the CAT construct was used as a negative control.

Claims

1. A nucleic acid molecule encoding a glucosinolate modifying enzyme selected from the group consisting of

(a) nucleic acid molecules encoding a protein which comprises the amino acid sequence indicated in SEQ ID NO: 2, 4 or 6;

(b) nucleic acid molecules comprising the nucleotide sequence indicated in SEQ ID NO: 1 , 3 or 5;

(c) nucleic acid molecules encoding a protein, the amino acid sequence of which has a homology of at least 40% to the amino acid sequence indicated in SEQ ID NO: 2, 4 or 6;

(d) nucleic acid molecules the complementary strand of which hybridizes to a nucleic acid molecule as defined in (a) or (b);

(e) nucleic acid molecules comprising a nucleotide sequence encoding an enzymatically active fragment of the protein which is encoded by any one of the nucleic acid molecules as defined in (a), (b), (c) or (d); and

(f) nucleic acid molecules, the nucleotide sequence of which deviates because of the degeneracy of the genetic code from the sequence of the nucleic acid molecules as defined in any one of (a), (b), (c), (d) or (e).

2. An oligonucleotide which specifically hybridizes with the nucleic acid molecule of claim 1.

3. A vector containing the nucleic acid molecule of claim 1.

4. The vector of claim 3, wherein the nucleic acid molecule is linked to regulatory elements ensuring transcription in eukaryotic and prokaryotic cells.

5. A host cell, which is genetically modified with a nucleic acid molecule of claim 1 or with a vector of claim 3 or 4.

6. The host cell of claim 5, which is a prokaryotic host cells.

7. The host cell of claim 5 or 6, which is a eukaryotic host cell.

8. A method for the production of a polypeptide encoded by a nucleic acid molecule of claim 1 in which the host cell of any one of claims 5 to 7 is cultivated under conditions allowing for the expression of the polypeptide and in which the polypeptide is isolated from the cells and/or the culture medium.

9. A polypeptide encoded by the nucleic acid molecule of claim 1 or obtainable by the method of claim 8.

10. An antibody specifically recognizing the polypeptide of claim 9.

11. A method for identifying a compound which can act as an antagonist/inhibitor of the polypeptide of claim 9 comprising the steps of:

(a) contacting the polypeptide of claim 9 with a compound to be tested in the presence of a glucosinolate under appropriate conditions; and

(b) determining whether the compound reduces or abolishes the activity of the polypeptide to desulfate the glucosinolate.

12. A method for preparing an insecticidal composition comprising steps (a) and

(b) of the method of claim 11 and further comprising the step of

(c) formulating the compound identified in step (b) as having the ability to reduce or abolish the activity of the polypeptide to desulfate the glucosinolate into an insecticidal composition.

13. Use of the polynucleotide of claim 1 , of the host cell of any one of claims 5 to 7 or of the polypeptide of claim 9 for the identification of a compound which reduces or abolishes the activity of the polypeptide of claim 9 to desulfate glucosinolate.

14. A method for preventing the infestation of plants by an organism which expresses a polypeptide of claim 9 comprising the step of applying to the plant or parts thereof a compound which reduces or abolishes the activity of the polypeptide of claim 9 to desulfate glucosinolate.

15. The method of claim 14, wherein the organism is Plutella xylostella.

16. A method for producing mutants of an organism which expresses the polypeptide of claim 9, wherein said mutant shows reduced or abolished activity of a polypeptide of claim 9 comprising the steps of

(a) subjecting the organism to a mutagenesis step;

(b) testing the mtuants obtained according to step (a) for the expression and/or activity of the polypeptide of claim 9; and

(c) selecting those mutants in which the expression and/or activity of the polypeptide of claim 9 is reduced or abolished.

17. A method for producing a transgenic animal of an organism which expresses the polypeptide of claim 9 and wherein said transgenic animal shows a reduced or abolished activity of the polypeptide of claim 9 comprising the step of genetically modifying the organism with a nucleic acid molecule the presence or expression of which in the cells of the organism reduces or abolishes the expression of the polypeptide of claim 9.

18. A mutant or a transgenic animal obtainable by the method of claim 16 or 17, in which the activity of the polypeptide of claim 9 is reduced or abolished.

19. A method for reducing infestation of plants by an organism which expresses the polypeptide of claim 9 comprising the step of artificially increasing in a natural population of such an organism the portion of individuals in which the activity of the polypeptide of claim 9 is reduced or abolished.

20. The method of claim 19, wherein the step of artificially increasing the portion of individuals in which the activity of the polypeptide of claim 9 is reduced or abolished is achieved by setting free mutants or transgenic animals of claim 18 in a natural population of the corresponding organism.