# Patent

## Title
Use of methionine synthase inhibitors for the treatment of fungal diseases of crops

## Abstract

The invention relates to the use of methionine synthase inhibitors for the treatment of fungal diseases of crops. The invention further relates to methods for treatment of crops against fungal diseases comprising the application of a methionine synthase inhibitor also methods for the identification of novel fungicidal compounds comprising a step for identification of methionine synthase inhibitors.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a 35 U.S.C. §371 national phase conversion of PCT/EP2005/014209 filed Dec. 20, 2005, which claims priority of French Application No. 04/13628 filed Dec. 21, 2004.
The present invention relates to the use of methionine synthase inhibitors for the treatment of fungal diseases, and more particularly the treatment of fungal diseases of crop plant species.
Fungi are responsible for devastating epidemics which can result in substantial losses of crops of various plant species. The principle of employing inhibitors of enzymes of pathogenic fungi, and of using these enzymes in tests in order to identify new molecules that are active against these fungi, are known per se. However, merely characterizing a fungal enzyme is not sufficient to achieve this objective, the enzyme chosen as a target for potential antifungal molecules must also be essential to the life of the fungus, its inhibition by the antifungal molecule resulting in death of the fungus, or essential to the pathogenesis of the fungus, in which case its inhibition is not lethal for the fungus but merely inhibits its pathogenic capacity. The identification of metabolic pathways and enzymes essential to the pathogenesis and to the survival of the fungus is therefore necessary for the development of novel antifungal products.
The sulfur assimilation pathway comprises incorporation of the sulfate ion (SO42−), activation thereof, and reduction thereof to reduced sulfur (S2−). These steps are catalyzed successively by an ATP sulfurylase (EC 2.7.7.4), an APS kinase (EC 2.7.1.25), a PAPS reductase (EC 1.8.4.8) (APS reductase in photosynthetic organisms, EC 1.8.4.9), and an (NADPH 2) sulfite reductase (EC 1.8.1.2) (a ferredoxin-dependent enzyme in photosynthetic organisms, EC 1.8.7.1). In all autotrophic organisms, the sulfate ion assimilation, activation and reduction pathway is conserved in terms of its general principle; the incorporation of the reduced sulfur into a carbon backbone exhibits considerable variations according to the organisms: bacteria1 (for example: Escherichia coli), plants2 (for example: Arabidopsis thaliana), yeasts (for example: Saccharomyces cerevisiae3) and filamentous fungi4. In fact, in plants and bacteria, the reduced sulfur is incorporated into a molecule at C3 which derives from serine, to form cysteine. The sulfur is then transferred to a molecule at C4 which derives from homoserine, to form homocysteine. This series of reactions forms the direct transsulfuration pathway. Conversely, in Saccharomyces cerevisiae (S. cerevisae), the sulfur is directly incorporated into a molecule at C4 which derives from homoserine, to form homocysteine (direct sulfhydrylation)3. Cysteine is then synthesized from the homocysteine by means of a series of reactions which make up the reverse transsulfuration pathway. In filamentous fungi, the synthesis of homocysteine is carried out both by the direct pathway in plants (direct transsulfuration) and by that of S. cerevisiae (direct sulfhydrylation). Furthermore, the synthesis of cysteine is carried out either by means of serine or from homocysteine via the reverse transsulfuration pathway. These various metabolic pathways were defined following the characterization of mutants auxotrophic for cysteine and for methionine in Neurospora crassa (N. crassa)5 and Aspergillus nidulans (A. nidulans)6. This model can be extrapolated to all filamentous fungi, including pathogenic fungi of plants (for example, Magnaporthe grisea, M. grisea) and of animals (for example, Aspergillus fumigatus (A. fumigatus)). M. grisea, an ascomycete-type pathogen responsible for considerable damage on rice crops, is a model of choice for such an approach. Methionine synthesis in filamentous fungi requires the action of a methionine synthase of vitamin B12-independent type as in plants. The approach described in the study of the methionine synthase gene of Cryptococcus neoformans28, a human pathogen, differs from the present invention. In fact, while animals (including humans) are capable of synthesizing methionine, this step is catalyzed by a vitamin B12-dependent type methionine synthase very different from that of the other eukaryotes such as plants and fungi. The plant methionine synthase exhibits strong homologies at the protein level with that of M. grisea, but also exhibits structural-type differences according to the modeling carried out9,12,27. Thus, identification of the fungal enzyme and characterization thereof are required in order to determine its specific characteristics, allowing the identification of solely fungal inhibitors. The choice and the application of such inhibitors in methods for treating plant crops will then be specific. Thus, the present invention describes the fact that the mutants of the MET6 gene, and more particular the deletion mutants of the MET6 gene encoding the methionine synthase of M. grisea are auxotrophic for methionine and are nonpathogenic. In these mutants, the infectious process is greatly effected at the level of the phase of penetration of the pathogen into the plant cell, but also in terms of its ability to progress in the infected tissues. The pathogenic capacity of the M. grisea methionine synthase mutants is partially restored when methionine is added during infection. These results show that the absence of methionine synthase activity is lethal to the fungus during infection. Similar results have been obtained in Utilago maydis (U. maydis) and Phytophthora infestans (P. infestans).



BRIEF DESCRIPTION OF THE DRAWING(S)
FIG. 1 is a comparative gene map of wild type Magnaporthe grisea methionine synthase gene and a MET6-deficient mutant thereof.



DESCRIPTION OF THE SEQUENCE LISTING


    SEQ ID No. 1: Magnaporthe grisea methionine synthase gene
    SEQ ID No. 2: Magnaporthe grisea methionine synthase cDNA
    SEQ ID No. 3: Magnaporthe grisea methionine synthase protein sequence
    SEQ ID No. 4: Met6-5 primer
    SEQ ID No. 5: Met6-6 primer
    SEQ ID No. 6: HphRP10 primer
    SEQ ID No. 7: Met6-7 primer
    SEQ ID No. 8: Met6-10 primer
    SEQ ID No. 9: dCGS-hph-end(−) primer
    SEQ ID No. 10: Met6-8 primer
    SEQ ID No. 11: Met6-9 primer
    SEQ ID No. 12: Met6-1 primer
    SEQ ID No. 13: Met6-2 primer
    SEQ ID No. 14: Met6-3 primer
    SEQ ID No. 15: Met6-4 primer
    SEQ ID No. 16: U. maydis methionine synthase gene
    SEQ ID No. 17: U. maydis methionine synthase cDNA
    SEQ ID No. 18: U. maydis methionine synthase protein sequence
    SEQ ID No. 19: P. infestans methionine synthase EST sequence
    SEQ ID No. 20: deduced P. infestans methionine synthase protein sequence.


DESCRIPTION OF THE INVENTION
A subject of the present invention is methods for treating crops against fungal diseases by application of an effective amount of a methionine synthase inhibitor.
In the context of the present invention, the fungal diseases are defined as diseases due to pathogenic plant fungi belonging to the ascomycete, basidiomycete and oomycete families.
A subject of the invention is a method for controlling, in a curative or preventive capacity, phytopathogenic fungi of crops, characterized in that an effective (agronomically effective) and nonphytotoxic amount of a methionine synthase inhibitor is applied to the soil where the plants grow or are liable to grow, to the leaves and/or the fruit of the plants or to the seeds of the plants. The term “effective and nonphytotoxic amount” is intended to mean an amount of inhibitor that is sufficient to allow the control of the developmental cycle or the destruction of the fungi which are present or which may appear on the crops, and that does not result in any notable symptom of phytotoxicity for said crops. Such an amount may vary within broad limits depending on the fungal family to be controlled, the type of crop, the climatic conditions and the compounds included in the antifungal composition according to the invention.
This amount can be determined by systematic field trials, which are within the scope of those skilled in the art.
The methods according to the invention are of use for treating the seeds of cereals (wheat, rye, triticale and barley, in particular), potato, cotton, pea, rapeseed, maize or flax, alternatively the seeds of forest trees or else genetically modified seeds of these plants. The present invention also relates to foliar application to plant crops i.e. to the foliage, the leaves, the fruit and/or the stems of the plants concerned, but also any other type of application. Among the plants targeted by the methods according to the invention, mention may be made of rice, maize, cotton, cereals, such as wheat, barley or triticale, fruit trees, in particular apple trees, pear trees, peach trees, vine, banana trees, orange trees, lemon trees, etc., oil-yielding crops, for example rapeseed or sunflower, market garden and vegetable crops, tomatoes, salads, protein-yielding crops, pea, Solanaceae, for example potato, beetroot, flax, and forest trees, and also genetically modified homologs of these crops.
Among the plants targeted by the method according to the invention, mention may be made of:

    
    
        wheat, as regards controlling the following seed diseases: fusaria (Microdochium nivale and Fusarium roseum), stinking smut (Tilletia caries, Tilletia controversa or Tilletia indica), septoria disease (Septoria nodorum); loose smut (Ustilago tritici);
        wheat, as regards controlling the following diseases of the parts of the plant above ground: cereal eyespot (Tapesia yallundae, Tapesia acuiformis), take-all (Gaeumannomyces graminis), foot blight (F. culmorum, F. graminearum), head blight (F. culmorum, F. graminearum, Microdochium nivale), black speck (Rhizoctonia cerealis, powdery mildew (Erysiphe graminis form a specie tritici), rusts (Puccinia striiformis and Puccinia recondita) and septoria diseases (Septoria tritici and Septoria nodorum), net blotch (Drechslera tritici-repentis);
        barley, as regards controlling the following seed diseases: net blotch (Pyrenophora graminea, Pyrenophora teres and Cochliobolus sativus), loose smut (Ustilago nuda) and fusaria (Microdochium nivale and Fusarium roseum);
        barley, as regards controlling the following diseases of the parts of the plant above ground: cereal eyespot (Tapesia yallundae), net blotch (Pyrenophora teres and Cochliobolus sativus), powdery mildew (Erysiphe graminis form a specie hordei), dwarf leaf rust (Puccinia hordei) and leaf blotch (Rhynchosporium secalis);
        potato, as regards controlling tuber diseases (in particular Helminthosporium solani, Phoma tuberosa, Rhizoctonia solani, Fusarium solani), and mildew (Phytophthora infestans);
        potato, as regards controlling the following foliage diseases: early blight (Alternaria solani), mildew (Phytophthora infestans);
        cotton, as regards controlling the following diseases of young plants grown from seeds: damping-off and collar rot (Rhizoctonia solani, Fusarium oxysporum), black root rot (Thielaviopsis basicola);
        protein-yielding crops, for example pea, as regards controlling the following seed diseases: anthracnose (Ascochyta pisi, Mycosphaerella pinodes), fusaria (Fusarium oxysporum), gray mold (Botrytis cinerea), mildew (Peronospora pisi);
        oil-yielding crops, for example rapeseed, as regards controlling the following seed diseases: Phoma lingam, Alternaria brassicae and Sclerotinia sclerotiorum; 
        maize, as regards controlling seed diseases: (Rhizopus sp., Penicillium sp., Trichoderma sp., Aspergillus sp. and Gibberella fujikuroi);
        flax, as regards controlling seed diseases: Alternaria linicola; 
        forest trees, as regards controlling damping-off (Fusarium oxysporum, Rhizoctonia solani);
        rice, as regards controlling the following diseases of the parts above ground: blast disease (Magnaporthe grisea), black speck (Rhizoctonia solani);
        vegetable crops, as regards controlling the following diseases of seeds or of young plants grown from seeds: damping-off and collar rot (Fusarium oxysporum, Fusarium roseum, Rhizoctonia solani, Pythium sp.);
        vegetable crops, as regards controlling the following diseases of the parts above ground: gray mold (Botrytis sp.), powdery mildews (in particular Erysiphe cichoracearum, Sphaerotheca fuliginea, Leveillula taurica), fusaria (Fusarium oxysporum, Fusarium roseum), leaf spot (Cladosporium sp.), alternaria leaf spot (Alternaria sp.), anthracnose (Colletotrichum sp.), septoria leaf spot (Septoria sp.), black speck (Rhizoctonia solani), mildews (for example, Bremia lactucae, Peronospora sp., Pseudoperonospora sp., Phytophthora sp.);
        fruit trees, as regards diseases of the parts above ground: monilia disease (Monilia fructigenae, M. laxa), scab (Venturia inaequalis), powdery mildew (Podosphaera leucotricha);
        grapevine, as regards foliage diseases: in particular, gray mold (Botrytis cinerea), powdery mildew (Uncinula necator), black rot (Guignardia biwelli), mildew (Plasmopara viticola);
        beetroot, as regards the following diseases of the parts above ground: cercosporia blight (Cercospora beticola), powdery mildew (Erysiphe beticola), leaf spot (Ramularia beticola).
    
    


Methionine synthase is a well characterized enzyme that is found in plants and microorganisms (bacteria, yeasts, fungi). The methods of the present invention use methionine synthase inhibitors. In a first embodiment, the invention relates to the use of inhibitors of fungal methionine synthase, more preferably of inhibitors of the methionine synthase of a phytopathogenic fungus, for the treatment of fungal diseases of crops.
Preferably, the methionine synthase is isolated, purified or partially purified from its natural environment. The methionine synthase can be prepared by means of various methods. These methods are in particular purification from natural sources such as cells that naturally express these polypeptides, production of recombinant polypeptides by appropriate host cells and subsequent purification thereof, production by chemical synthesis or, finally, a combination of these various approaches. These various methods of production are well known to those skilled in the art.
In one of the embodiments of the invention, the methionine synthase is purified from an organism that naturally produces this enzyme, for instance bacteria such as E. coli, yeasts such as S. cerevisiae, or fungi such as N. crassa or M. grisea. 
In a preferred embodiment of the invention, the methionine synthase is overexpressed in a recombinant host organism. The methods of engineering DNA fragments and the expression of polypeptides in host cells are well known to those skilled in the art and have, for example, been described in “Current Protocols in Molecular Biology” Volumes 1 and 2, Ausubel F. M. et al., published by Greene Publishing Associates and Wiley-Interscience (1989) or in Molecular Cloning, T. Maniatis, E. F. Fritsch, J. Sambrook (1982).
In a specific embodiment of the invention, the methionine synthase inhibitors inhibit the methionine synthase of M. grisea, of U. maydis, and more particularly represented by a sequence comprising the sequence identifier SEQ ID No. 18, or else of P. infestans, in particular represented by a sequence comprising the sequence identifier SEQ ID No. 20; said methionine synthase can be encoded by the gene of M. grisea represented by a sequence comprising the sequence identifier SEQ ID No. 1, or by the cDNA represented by a sequence comprising the sequence identifier SEQ ID No. 2, by the gene of U. maydis represented by a sequence comprising the sequence identifier SEQ ID No. 16, or by the cDNA represented by a sequence comprising the sequence identifier SEQ ID No. 17, or else by the gene of P. infectans represented by a sequence comprising the sequence identifier SEQ ID No. 19.
A subject of the present invention is also antifungal compositions comprising a methionine synthase inhibitor and another antifungal compound. Mixtures with other antifungal compounds are particularly advantageous, especially mixtures with acibenzolar-S-methyl, azoxystrobin, benalaxyl, benomyl, blasticidin-S, bromuconazole, captafol, captan, carbendazim, carboxin, carpropamide, chlorothalonil, antifungal compositions based on copper or on copper derivatives such as copper hydroxide or copper oxychloride, cyazofamide, cymoxanil, cyproconazole, cyprodinyl, dichloran, diclocymet, dicloran, diethofencarb, difenoconazole, diflumetorim, dimethomorph, diniconazole, discostrobin, dodemorph, dodine, edifenphos, epoxyconazole, ethaboxam, ethirimol, famoxadone, fenamidone, fenarimol, fenbuconazole, fenhexamid, fenpiclonil, fenpropidine, fenpropimorph, ferimzone, fluazinam, fludioxonil, flumetover, fluquinconazole, flusilazole, flusulfamide, flutolanil, flutriafol, folpet, furalaxyl, furametpyr, guazatine, hexaconazole, hymexazol, imazalil, iprobenphos, iprodione, isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb, maneb, mefenoxam, mepanipyrim, metalaxyl and its enantiomers such as metalaxyl-M, metconazole, metiram-zinc, metominostrobin, oxadixyl, pefurazoate, penconazole, pencycuron, phosphoric acid and its derivatives such as fosetyl-Al, phthalide, picoxystrobin, probenazole, prochloraz, procymidone, propamocarb, propiconazole, pyraclostrobin, pyrimethanil, pyroquilon, quinoxyfen, silthiofam, simeconazole, spiroxamine, tebuconazole, tetraconazole, thiabendazole, thifluzamide, thiophanate, e.g. thiophanate-methyl, thiram, triadimefon, triadimenol, tricyclazole, tridemorph, trifloxystrobin, triticonazole, valinamide derivatives, for instance iprovalicarb, vinclozolin, zineb and zoxamide. The mixtures thus obtained have a broader spectrum of activity. The compositions according to the invention can also comprise one or more insecticides, bactericides, acaricides or pheromones, or other compounds that have a biological activity.
A subject of the present invention is also methods for producing an antifungal composition using a methionine synthase inhibitor.
A subject of the present invention is also methods for preparing antifungal compounds, comprising the identification of compounds which inhibit the enzymatic activity of methionine synthase.
The enzymatic reaction is carried out in the presence of the test compound in order to measure the inhibition of the enzymatic activity of the methionine synthase. All biochemical tests for measuring the enzymatic activity of methionine synthase and therefore identifying compounds which inhibit this enzymatic activity can be used in the methods according to the invention.
A high-throughput biochemical assay is proposed in order to screen for specific inhibitors of this enzyme.
Preferably, the methods for identifying compounds which inhibit the activity of methionine synthase comprise bringing these compounds into contact with methionine synthase in the presence of its substrates: homocysteine, methyl tetrahydrofolate or polyglutamate derivatives of methyl tetrahydrofolate ((CH3—H4)PteGlun), and of various cofactors such as phosphate, magnesium and zinc; and measuring the enzymatic activity.
Measuring the enzymatic activity of methionine synthase can be associated with measuring the formation of methionine, of tetrahydrofolate or else of methenyl tetrahydrofolate or of any product thus obtained, or else measuring said activity by any other chemical or enzymatic reaction.
The measurement of the enzymatic activity of methionine synthase can also be carried out in the presence of a coupling enzyme. S-Adenosylmethionine synthase (AdoMetS) can be used as such; it catalyzes the formation of S-adenosylmethionine (S-AdoMet) in the presence of methionine, ATP and magnesium. The measurement of the enzymatic activity of methionine synthase can then be associated with the measurement of the formation of S-adenosylmethionine, of phosphate or of pyrophosphate.
According to another aspect of the invention, the methods for identifying compounds which inhibit the enzymatic activity of methionine synthase comprise expressing methionine synthase in a host organism, purifying the methionine synthase produced by the host organism, bringing these compounds into contact with the purified methionine synthase and its substrates, and measuring the enzymatic activity.
In a preferred embodiment, all these methods comprise an additional step in which it is determined whether said compounds which inhibit the enzymatic activity of methionine synthase inhibit fungal growth and/or pathogenesis.
The present invention therefore relates to methods for identifying compounds which inhibit fungal growth and/or pathogenesis by inhibiting the enzymatic activity of methionine synthase. These methods consist in subjecting a compound, or a mixture of compounds, to an appropriate assay for identifying the compounds which inhibit methionine synthase, and in selecting the compounds which react positively to said assay, where appropriate in isolating them, and then in identifying them.
Preferably, the appropriate assay is an assay of the enzymatic activity of methionine synthase as defined above.
Preferably, a compound identified according to these methods is subsequently tested for these antifungal properties according to methods known to those skilled in the art. Preferably, the compound is evaluated by means of phenotypic tests such as pathogenesis assays on detached leaves or on whole plants.
The term “compound” is intended to mean, according to the invention, any chemical compound or mixture of chemical compounds, including peptides and proteins.
The term “mixture of compounds” is understood to mean, according to the invention, at least two different compounds, such as, for example, the (dia)stereoisomers of a molecule, mixtures of natural origin derived from the extraction of biological material (plants, plant tissues, bacterial cultures, cultures of yeasts or of fungi, insects, animal tissues, etc.) or reaction mixtures that have not been purified or have been completely or partially purified, alternatively mixtures of products derived from combinatorial chemistry techniques.
Finally, the present invention relates to novel fungal pathogenesis-inhibiting compounds which inhibit the enzymatic activity of methionine synthase, in particular the compounds identified by means of the methods according to the invention and/or the compounds derived from the compounds identified by means of the methods according to the invention.
Preferably, the fungal pathogenesis-inhibiting compounds which inhibit the enzymatic activity of methionine synthase are not general inhibitors of enzymes. Also preferably, the compounds according to the invention are not compounds already known to have an antifungal activity and/or an activity on fungal pathogenesis.
EXAMPLE 1
Characterization of the Methionine Synthase Gene in Fungi
The methionine synthase gene was identified in the genome of M. grisea version V2 using the protein sequence of the methionine synthase of A. nidulans7 (NCBI, accession number: AAF82115) as model. The complete nucleotide sequence of the methionine synthase gene located on Contig 2.150 (MG_contig—2.150, position 6196-8629, complementary strand, SEQ ID No. 1) comprises 3 exons corresponding to a cDNA of 2301 bp (SEQ ID No. 2) which encodes a polypeptide of 766 amino acids (SEQ ID No. 3). The sequence of the g ene and the splicing resulting in the definitive messenger could be confirmed by virtue of the numerous ESTs identified in the various public and private bases. The M. grisea methionine synthase is encoded by a single gene as in A. nidulans7. Analysis of the primary protein sequence deduced from the putative cDNA shows from 48% to 79% homology with the vitamin B12-independent methionine synthases of S. cerevisae (P05694), of A. nidulans (AAF82115), of the bacterium E. coli (P13009) and of the plant A. thaliana (AAF00639).
The primary sequence of the M. grisea methionine synthase has two conserved domains corresponding to the methionine synthase domain (334 residues, E=4e−116, pfam01717) characteristic of this enzyme. This domain allows the production of methionine by transfer of a methyl group from methyl tetrahydrofolate triglutamate to homocysteine. This region is located in the C-terminal part of the protein. A second domain, COG0620 or methionine synthase II (methyltransferase) concerns the N-terminal part of the protein (330 amino acids)8. It has recently been possible to determine the specificity of each of these domains with respect to the substrates of the enzyme, homocysteine and methyl tetrahydrofolate, and to the reaction product, methionine, on the enzyme crystallized from A. thaliana9.
The primary sequence of the M. grisea methionine synthase was used to search for orthologs in the various fungal species whose genome is partially or completely sequenced. These various primary sequences were subsequently compared with the methionine synthases described in various organisms such as plants, bacteria and animals. The characterization of the structure of the genes (introns+exons) and of the primary amino acid sequences was carried out using the appropriate programs (tblastn; FGENSH; PSI-PHI-BLAST). According to this procedure, methionine synthase could be characterized in several fungi (ascomycetes and basidiomycetes) and a phytopathogenic oomycete (P. sojae and P. infestans). A phylogenetic tree for methionine synthase could be established and the representation obtained shows that the M. grisea methionine synthase belongs to the methionine synthases of ascomycetes and that it is distant from those of basidiomycetes. Overall, the tree obtained is in agreement with that which retraces the phylogenetic origin of these organisms10.
EXAMPLE 2
Deletion of the Magnaporthe grisea Methionine Synthase Gene
The study of the role of the methionine synthase gene in the development and the infectious process of M. grisea was carried out by studying the phenotype of deletion mutants of this gene. The strategy for obtaining deletion mutants is based on replacing the MET6 gene with a mutant allele in which the MET6 open reading frame has been replaced with a cassette for resistance to an antibiotic for selection of the transformants (hygromycin).
The construction of this vector for replacing the M. grisea MET6 gene is carried out in two steps: (i) PCR amplification of the regions which border this gene and which correspond respectively to genomic regions of approximately 1 kb located on either side of MET6, (ii) ligation of these genomic DNA fragments to a gene for resistance to an antibiotic that makes it possible to select the transformants. Thus, the PCR fragments used to replace the gene consist of the two regions which are referred to as left border and right border of the gene studied (FIG. 1). We selected the hygromycin resistance gene (HYG, comprising the PtrpC promoter, the coding portion of the hygromycin resistance gene hph) as selectable gene. Ligation of the HYG gene is first carried out via the SacII/BglII sites and the EcoRI/SacII sites of the left border (Met6-1/Met6-2 primers) of the MET6 gene. The right border of the MET6 gene (Met6-3/Met6-4 primers) is then introduced via the PmeI site downstream of the hph gene (FIG. 1). The replacement vector therefore comprises the left border (BG) of the MET6 gene (promoter region of 1475 bp), the HYG cassette (1400 bp) and the right border (BD) of the MET6 gene (terminator region of 1251 bp). The ligation product (BG-hph-BD) was subsequently cloned into a plasmid vector. The MET6 gene replacement cassette was subsequently amplified from the corresponding plasmid by PCR with primers specific for the ends of the borders of the MET6 gene (Met6-5/Met6-6 primers). Sequencing of the junctions between the borders and the HYG cassette of the replacement vector made it possible to verify the construction. The PCR product (1 μg), purified by agarose gel electrophoresis, is then used to transform protoplasts of the M. grisea P1.2 wild-type strain according to conventional techniques developed in the laboratory. The products derived from the transformation are selected on a medium containing the corresponding antibiotic (hygromycin).
EXAMPLE 3
Identification and Trophic Characterization of the met6Δ::hph Deletion Mutants Obtained by Gene Replacement
The primary transformants are selected for their ability to develop in the presence of hygromycin. The identification of the met6Δ::hph mutants is carried out by measuring the differential growth of the transformants on a minimum medium containing hygromycin, supplemented or not supplemented with 1 mM methionine. The met6Δ::hph mutants are incapable of developing on this minimum medium, but they have a normal growth on this minimum medium supplemented with methionine. The frequency of the mutants is of the order of 20% of the primary transformants analyzed under our experimental conditions. These mutants were subsequently genetically purified by isolation of monosphores.
The five met6Δ::hph mutants obtained (4.1, 15.1, 22.1 and 23.1) are incapable of developing on a minimum medium that allows growth of the P1.2 wild-type strain. The addition of methionine to the minimum medium restores growth of the mutants. The trophic complementation of the met6Δ::hph mutants by the addition of methionine indicates that the methionine synthase is affected by the MET6 gene deletion.
The addition of sulfur donors such as cysteine or glutathione (precursors of homocysteine, a substrate from methionine synthase) is not sufficient to restore the growth of the M. grisea met6 Δ::hph mutants. Consequently, there is not parallel pathway which would use methyl tetrahydrofolate or its polyglutamate derivatives for the synthesis of methionine in this fungus. Thus, the de novo synthesis of methionine is catalyzed solely by methionine synthase. The activity of this enzyme is therefore essential to the development of the fungus.
On the other hand, in the presence of S-adenosylmethionine (SAM or AdoMet), a compound which derives directly from methionine and which is essential to the cell cycle, or of S-methylmethionine (SMM), a compound which is synthesized in plants, the M. grisea met6Δ::hph mutants are capable of developing, although with a reduced growth compared to the P1.2 wild-type strain. SAM and SMM are capable of penetrating and of being metabolized to methionine by M. grisea. This mechanism is probably similar to that described in the yeast S. cerevisae. In fact, in this ascomycete fungus,
SAM and SMM are incorporated into the cell via the transporters SAM3 and MPP1, and are subsequently converted to methionine in the presence of homocysteine by homocysteine-5-methyltransferases (SAM4 and MHT1) which use SAM or SMM, respectively, as methyl group donor (S. cerevisae)11. According to our experimental conditions, the addition of SAM (1 mM) to the minimum medium is more effective than that of SMM in restoring the growth of the met6Δ::hph mutants. These results suggest that M. grisea has transporters and homocysteine methyltransferases similar to those described in S. cerevisae. An analysis of the M. grisea genome by sequence homology search (tBlastN) with respect to the yeast SAM4 and MHT1 proteins makes it possible to demonstrate a gene which is an ortholog of SAM4 in M. grisea. It would appear that filamentous fungi have only one gene (no ortholog of MHT1). According to our results (better growth on SAM than on SMM), this protein could have a greater affinity for SAM than for SMM.
Several “ectopic” transformants, corresponding to transformants which have integrated the replacement vector BG-hph-BD at a locus other than that of the MET6 gene, were also analyzed. These hygromycin-resistant ectopic transformants are capable of developing on a minimum medium. The methionine synthase gene is therefore functional in these ectopic transformants and the vector has been inserted at a locus of the genome which has no effect on the development of the pathogen under our experimental conditions (ability to develop on the minimum or complete medium, sporulation).
EXAMPLE 4
Molecular Characterization of the met6Δ::hph Mutants
The met6Δ::hph mutants are cultured in a medium containing methionine (1 mM) in order to extract the genomic DNA, which will be used to perform a molecular analysis of the MET6 locus by PCR and by Southern hybridization.
The molecular analysis of the transformants is carried out by amplification of the genomic regions of the MET6 locus using the various specific primers for replacing the wild-type allele of the MET6 gene of P1.2 with the mutant allele met6Δ::hph. These PCRs are carried out for each mutant with specific oligonucleotides. The reactions use oligonucleotides which hybridize: firstly, with the hygromycin resistance gene hph and, secondly, with a genomic sequence of the MET6 locus located outside the MET6 region used to construct the replacement vector (left junction and right junction); with the sequences homologous to the MET6 gene. Thus, the amplification of a fragment of 1969 bp (left junction) or of 2447 bp (right junction) occurs only in the case of replacement of the wild-type gene with the met6Δ::hph gene of the construct (primers HphRP01rev (SEQ ID No. 6)/Met6-7(−) (SEQ ID No. 7) and Met6-10 (SEQ ID No. 8)/dCGS-hph-end(−) (SEQ ID No. 9) (FIG. 1)). No amplification is obtained in the case of the P1.2 wild-type strain or of the ectopic transformants. Similarly, the absence of amplification of the MET6 gene in the met6Δ::hph mutants (primers Met6-8, SEQ ID No. 10/Met6-9, SEQ ID No. 11) (FIG. 1) is an indication that the MET6 gene is indeed absent in these transformants. On the other hand, with these primers, a fragment of 2424 bp is amplified in the P1.2 wild-type strain and in the ectopic transformants. Only the PCRs carried out with the mutants 22.2 and 23.1 give the expected results with these three types of PCR. No amplification could be obtained, for unexplained reasons, with the mutants 4.1 and 15.1 in the PCRs Met6-10 (SEQ ID No. 8)/dCGS-hph-end (SEQ ID No. 9) and hphRP01 (SEQ ID No. 6)/Met6-7(−), SEQ ID No. 7).
An analysis by Southern hybridization after digestion of the genomic DNA with the BamH1 restriction enzyme was carried out and the hybridization signals obtained for the 4 mutants were compared with those obtained with the P1.2 wild-type strain and the ectopic transformant (19.1). Using the (Met6-1 (SEQ ID No. 12)/Met6-2 (SEQ ID No. 13)) PCR fragment corresponding to the MET6 left border present in the replacement vector (MET6 promoter region) as probe, 2 bands are observed for the mutants, the sizes of which are different from those of the P1.2 wild-type strain and of the ectopic transformant (19.1). The signal corresponds to the MET6 promoter region in the P1.2 wild-type strain and the ectopic transformant 19.1. The latter also shows a hybridization signal corresponding to the replacement vector inserted into another genomic region. A similar result is obtained using a (Met6-3 (SEQ ID No. 14)/Met6-4 (SEQ ID No. 15)) PCR fragment corresponding to the MET6 right border present in the replacement vector (MET6 terminator region). With a probe specific for the inserted gene (hph), only the met6Δ::hph mutants and the ectopic transformant 19.1 show a hybridization signal corresponding either to the presence of hph at the MET6 locus (mutants) or to the replacement vector BG-met6Δ::hph-BD (ectopic). The latter results indicate that the various mutants analyzed are identical at the molecular level and contain just one copy of the hph gene inserted as the MET6 locus in place of the MET6 coding phase.
EXAMPLE 5
Analysis of the Pathogenic Capacity of the Magnaporthe grisea met6Δ::hph Mutants
The pathogenic capacity of the M. grisea met6 Δ::hph mutants was evaluated by means of an infection test on barley leaves under survival conditions and on whole barley and rice plants. This analysis was followed by measurement of the spore germination rate, of appressorial differentiation and of penetration into barley leaves. The spores of the P1.2 wild-type strain and of the met6Δ::hph mutants 4.1, 15.1, 22.1, 23.1 and 24.1 are harvested after growth for 14 days on a rice flour-based medium containing 1 mM. The plant material used is barley (cv. Express) and rice (cv. Sariceltik).
In the experiments carried out on barley leaves under survival conditions, the leaves are incubated on an agar medium (1% agar-H2O) containing kinetin (2 mg·ml−1) in a temperate climatic chamber (26° C.) at high humidity (100%) and under light of 100 microeinsteins. During infection, the spores are deposited onto the leaves either in the form of droplets (35 μl) or by coating the surface of the leaves with the suspension of spores using a cotton wool bud. These experiments are carried out in the absence or in the presence of 1 mM methionine throughout the incubation or for only 24 hours. The spore concentration is 3×104 spores/ml to 1×105 spores/ml in water. The appearance of the symptoms caused by M. grisea is then observed for at least 7 days of incubation. Inoculation of barley leaves under survival conditions, with spores of the M. grisea wild-type strain P1.2, causes necroses characteristic of the development of the fungus in the infected leaf (sporulating lesions). Conversely, the met6Δ::hph mutants 4.1, 15.1, 22.1, 23.1 and 24.1 do not cause any symptom on the barley leaves, regardless of the method of inoculation, and are therefore nonpathogenic. The addition of methionine to the met6Δ::hph mutant spores makes it possible to partially restore their pathogenic capacity (development of characteristic but nonsporulating lesions). Furthermore, under our experimental conditions, we did not observe any marked differences between the infections carried out with injured or noninjured leaves. These observations suggest that the met6Δ::hph mutants are incapable of penetrating barley leaves, even injured barley leaves. The yellowing of the leaves observed in the experiments carried out with spreading of the spores by coating is probably due to premature ageing of the leaves under our experimental conditions. The results are summarized in the following table:







TABLE I







Estimation of the pathogenic capacity of the Magnaporthe


grisea met6Δ::hph mutants with respect to barley










Barley leaves under survival conditions -




Magnaporthe grisea 3 × 104 spores/ml










Without
With 1 mM



methionine
methionine











Wild-type strains











P1.2
+++
+++







Ectopic mutant strains











Ectopic 19.1
+++
+++



Ectopic 20.1
+++
+++







met6Δ::hph mutants











 4.1
0
+



15.1
0
+



22.1
0
+



23.1
0
+







Legend: 0: no lesions; +: nonsporulating necroses (lesions); +++: sporulating lesions






The observation and the quantification of the various steps of the infection (germination, formation of the appressorium and penetration) are carried out based on an inoculation of barley leaves under survival conditions with drops of 35 μl of a suspension of spores (3×105 spores/ml). After 24 hours, the epidermis of the leaf is peeled in order to observe, under the microscope, the number of germinated spores that have differentiated an appressorium and the number of events of penetration into the epidermal cell. A solution of calcofluor at 0.01% makes it possible to cause an intense fluorescence of the walls of the fungal cells located at the surface of the plant (the hyphae located in the epidermal cell are not colored). These observations (Table II) demonstrate that the met6Δ::hph mutants have a slightly reduced germination (−10% to −40% compared to the wild-type strain). Their appressorial differentiation rate is also slightly reduced (0% to −30% compared to the wild-type strain). On the other hand, these mutant appressoria are incapable of penetrating into the foliar tissues.







TABLE II







Development of the Magnaporthe grisea


met6Δ::hph mutants on barley












%





appressorium/


M. grisea
%
germinated
%


strains
germination
spores
penetration













ΔMET6-4.1
45
52
0


ΔMET6-15.1
70
66
ND


ΔMET6-22.1
62
80
ND


ΔMET6-23.1
37
63
0


Wild-type P1.2
80
80
100


Ectopic 19.1
80
80
100


Ectopic 20.1
80
80
ND





ND: not determined






The formation of appressoria is also observed under an artificial condition where the spores are germinated on teflon membranes with or without the addition of methionine (1 mM concentration under our experimental conditions). These very hydrophobic membranes mimic the surface of the leaf, thereby making it possible to induce the formation of appressoria and to readily measure the appressorial differentiation rate.
In conclusion, the M. grisea met6Δ::hph mutants are therefore incapable of penetrating into the plant, although they differentiate appressoria. These results show that these mutants have nonfunctional appressoria.
Pots containing 3-week-old barley plants (corresponding to the emergence of the second leaf) are subjected to spraying with a suspension of M. grisea spores (10 ml of water containing spores at the concentration of 3×104 spores/ml and 0.3% gelatin for adhesion of the water droplets to the leaves). The observations of the symptoms are carried out for at least 8 days after spraying. The barley plants treated with the wild-type strain (P1.2) and the ectopic transformants (19.1 and 20.1) show necrotic lesions caused by the development of the fungus in the infected leaves. On the other hand, no symptom of disease was observed in the case of the inoculation with the M. grisea met6Δ::hph mutants. These mutants are therefore considered to be nonpathogenic. Furthermore, this study indicates, like the analysis carried out with the barley leaves under survival conditions, that the amount of methionine (or of other compounds that derive from methionine, such as SAM and SMM) in the tissues of the leaf is insufficient to complement the deficiency in methionine synthesis of the met6Δ::hph mutants. Thus, the pathway demonstrated in the in vitro experiments (complementation in the presence of SAM and of SMM) does not appear to be functional in the fungus while it is growing in the plant.
The met6Δ::hph mutants obtained correspond to the deletion of the coding phase of the MET6 gene which is replaced by the hph gene, conferring hygromycin resistance. These mutants are incapable of synthesizing methionine from homocysteine and are therefore incapable of multiplying on a minimum medium. The addition of methionine to this minimum medium is essential for the development of these mutants. The met6Δ::hph mutants do not cause any symptom of disease when they are used to inoculate barley leaves either under survival conditions, or whole plants, even at high spore concentrations. Thus, methionine synthesis by methionine synthase is essential to the development of the fungus, both in vitro and in planta.
The met6Δ::hph mutants can differentiate an appressorium in the absence of methionine. This aspect indicates that the spore can possess a not insignificant store of methionine allowing synthesis of proteins and metabolites necessary for the development of this cell. This store must come from the methionine that was supplied to the mutant in order to allow its growth and its sporulation on the rice flour-based medium used (containing 1 mM of methionine). On the other hand, the absence of penetration of the met6Δ::hph mutants into the leaves indicates that said mutants differentiate nonfunctional appressoria incapable of directing penetration of the fungus into the plant. It is probable that the mutant appressoria have rapidly exhausted their methionine stores. In fact, the addition of methionine to the mutant spores allows penetration into the plant and the beginning of development in the leaf. However, said development is not complete and the absence of formation of sporulating lesions suggests that, once the methionine store supplied to the spores is exhausted, the plant is incapable of covering the methionine needs of the mutants.
EXAMPLE 6
Methods for Assaying and Characterizing Molecules which Inhibit the Enzymatic Activity of Methionine Synthase
The method involves the characterization of all molecules whose action inhibits the consumption of substrates or the formation of products determined according to direct or indirect techniques (which include the use of a “coupling” enzyme for measuring the activity of methionine synthase). Methionine synthase catalyzes an irreversible reaction in the presence of homocysteine, of methyl tetrahydrofolate (n=1) or of its polyglutamate derivatives (n≧3) in the presence of various cofactors (phosphate, magnesium, zinc) according to known assays described in the literature12,13,14,15,27. The methodology includes determining methionine or the folate derivatives produced, by techniques for separating compounds by reverse phase HPLC chromatography16. The assaying of the tetrahydrofolate produced during the reaction after it has been converted to methenyl tetrahydrofolate can be carried out on a spectrophotometer at 350 nm, since the methyl tetrahydrofolate substrate is not detectable under the experimental conditions described in the procedure17. A proposed alternative is to assay the methionine synthase activity in the presence of S-adenosylmethionine synthetase. In this assay, the S-adenosylmethionine synthetase (AdoMetS) of M. grisea will preferably be used, but any of the AdoMetS can be used as a “coupling” enzyme. The AdoMetS enzyme catalyzes the irreversible reaction which, in the presence of methionine, ATP and magnesium, produces S-adenosylmethionine (SAM), phosphate and pyrophosphate.
The methionine synthase activity is assayed, in the end, through the amount of SAM, of phosphate or of pyrophosphate produced, by means of a colorimetric method and/or on a spectrophotometer after conversion of the products in the presence of a coupling enzyme or by means of any other chemical or enzymatic reaction for measuring methionine synthase activity. These various methodologies are the subject of many descriptions in the literature and can be adapted according to the experimenter18,19,20,21,22,23. For example, the sensitivity of the method for assaying methionine synthase activity in the presence of SAM synthetase can be improved through the addition of a pyrophosphatase which converts the pyrophosphate into 2 mol of phosphate. Thus, for each mole of methionine synthesized, the method produces 3 mol of phosphate.
Purification of Magnaporthe grisea Methionine Synthase
The production of a large amount of methionine synthase is carried out using techniques that use expression vectors for overproduction of the protein in bacteria or yeast. The technique preferably uses cloning of the cDNA into an expression vector which makes it possible to integrate a His-Tag extension at the N-terminal or C-terminal end of the protein. For example, when the pET-28b(+) vector (Novagen)24 is selected, the 2301 bp cDNA is cloned into NdEI and EcoRI according to conventional molecular biology techniques. The construct obtained, called pET-28-MgMET6, is introduced into the Escherichia coli BL21 type DE3 (pLysS) bacterial strain and the expression is produced after induction with IPTG (0.5 mM). The recombinant bacteria are cultured at 28° C. for 4 hours. The cells are then harvested by centrifugation and the pellet obtained is resuspended in lysis buffer suitable for the stability of the protein. After sonication of the preparation, the soluble fraction containing the recombinant protein, obtained after centrifugation, is loaded onto a column of Ni-NTA agarose type. The purification and the elution of the enzyme are then carried out after several successive washes of the matrix with an imidazole solution. The procedure follows the protocol define by Qiagen25.
After elution, the protein fraction containing the recombinant methionine synthase is concentrated by ultrafiltration and subjected to molecular filtration on PD10 (Pharmacia)27 in order to remove all traces of imidazole. The purification of the recombinant protein can be accompanied by a second step consisting of molecular filtration by chromatography on Superdex S200 (Pharmacia)26 or of ion exchange chromatography on MonoQ HR10/10 (Pharmacia)26. The activity of the methionine synthase is followed during the purification, using the appropriate direct measurement assay.
BIBLIOGRAPHY


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## Claims

1. A method for identifying a compound that is an inhibitor of methionine synthase comprising:
(A) bringing said compound into contact with methionine synthase in the presence of homocysteine and of methyl tetraglutamate or its polyglutamate derivatives, and of cofactors and measuring the reduction in the formation of methionine as compared to a control carried out in the absence of said compound; or
(B) bringing said compound into contact with methionine synthase in the presence of homocysteine and of methyl tetraglutamate or its polyglutamate derivatives, of S-adenosylmethionine synthetase, of ATP and of Mg, and of cofactors, and measuring the reduction in the formation of S-adenosylmethionine, phosphate or pyrophosphate as compared to a control carried out in the absence of said compound;
wherein the methionine synthase is selected from the group consisting of:
(1) the methionine synthase that is derived from Magnaporthe grisea and comprises SEQ ID No. 3, and is encoded by a sequence comprising SEQ ID No. 1 or SEQ ID No. 2;
(2) the methionine synthase that is derived from Ustilago maydis and comprises SEQ ID No. 18, and is encoded by a sequence comprising SEQ ID No. 16 or SEQ ID No. 17; and
(3) the methionine synthase that is derived from Phytophora infestans and comprises SEQ ID No. 20, and is encoded by a sequence comprising SEQ ID No. 19; and

(C) identifying the compound that reduces enzymatic activity and/or reduces the formation of methionine as an inhibitor of methionine.

2. The method of claim 1 wherein the methionine synthase is derived from Magnaporthe grisea.

3. The method of claim 1 wherein the methionine synthase is derived from Ustilago maydis.

4. The method of claim 1 wherein the methionine synthase is derived from Phytophthora infestans.

5. The method of claim 1 wherein the identification of compounds that inhibit the enzymatic activity of methionine synthase comprises the steps of:
bringing said compound into contact with methionine synthase in the presence of homocysteine and of methyltetraglutamate or its polyglutamate derivatives, and of cofactors; and
measuring the reduction in the formation of methionine as compared to a control carried out in the absence of said compound.

6. The method of claim 5 wherein the identification of compounds that inhibit the enzymatic activity of methionine synthase comprises the steps of:
bringing said compound into contact with methionine synthase in the presence of homocysteine, of methyl tetrahydrofolate and of phosphate, magnesium and zinc; and
measuring the reduction in the formation of methionine as compared to a control carried out in the absence of said compound.

7. The method of claim 1 wherein the identification of compounds that inhibit the enzymatic activity of methionine synthase comprises the steps of:
bringing said compound into contact with methionine synthase in the presence of homocysteine, of methyl tetrahydrofolate or its polyglutamate derivatives, of S-adenosylmethionine synthetase, of ATP and of Mg, and of cofactors; and
measuring the reduction in the formation of S-adenosylmethionine, phosphate or pyrophosphate as compared to a control carried out in the absence of said compound.

8. The method of claim 7 wherein the identification of compounds that inhibit the enzymatic activity of methionine synthase comprises the steps of:
bringing said compound into contact with methionine synthase in the presence of homocysteine, of methyl tetrahydrofolate or its polyglutamate derivatives, of S-adenosylmethionine synthetase, of ATP and of Mg, and of cofactors; and
measuring the reduction in the formation of phosphate as compared to a control carried out in the absence of said compound.

9. The method of claim 1 wherein the identification of compounds that inhibit the enzymatic activity of methionine synthase comprises the steps of:
expressing methionine synthase in a host organism;
purifying the methionine synthase produced by said host organism;
bringing said compound into contact with said purified methionine synthase in the presence of homocysteine, of methyl tetrahydrofolate, and of phosphate, magnesium and zinc; and
measuring the reduction in the formation of methionine as compared to a control carried out in the absence of said compound.

10. The method of claim 1 wherein the identification of compounds that inhibit the enzymatic activity of methionine synthase comprises the steps of:
expressing methionine synthase in a host organism;
purifying the methionine synthase produced by said host organism;
bringing said compound into contact with said purified methionine synthase in the presence of homocysteine, of methyl tetrahydrofolate or its polyglutamate derivatives, of S-adenosylmethionine synthetase, of ATP and of Mg, and of cofactors; and
measuring the reduction in the formation of S-adenosylmethionine, phosphate, or of pyrophosphate as compared to a control carried out in the absence of said compound.

11. A method for identifying a compound that is an inhibitor of methionine synthase comprising:
(A) bringing said compound into contact with methionine synthase in the presence of homocysteine and of methyl tetraglutamate or its polyglutamate derivatives, and of cofactors and measuring the reduction in the formation of methionine as compared to a control carried out in the absence of said compound; or
(B) bringing said compound into contact with methionine synthase in the presence of homocysteine and of methyl tetraglutamate or its polyglutamate derivatives, of S-adenosylmethionine synthetase, of ATP and of Mg, and of cofactors, and measuring the reduction in the formation of S-adenosylmethionine, phosphate or pyrophosphate as compared to a control carried out in the absence of said compound;
wherein the methionine synthase is derived from a phytopathogenic fungus; and
(C) identifying the compound that reduces enzymatic activity and/or reduces the formation of methionine as an inhibitor of methionine.