JP2005524390A - Methods for identifying inhibitors of asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate and threonine synthase as antibiotics - Google Patents

Abstract

The inventors of the present invention have discovered that asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate dehydratase and threonine synthase are each essential for fungal virulence. Specifically, inhibiting asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate dehydratase or threonine synthase gene expression in fungi does not result in any sign of successful infection or lesion. Thus, asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate dehydratase and threonine synthase can be used as targets for identifying antibiotics, preferably antifungal agents. Accordingly, the present invention provides methods for identifying compounds that inhibit asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate dehydratase or threonine synthase expression or activity. The method of the present invention is useful for identifying antibiotics, preferably antifungal agents.

Description

Field of Invention:
The present invention relates generally to methods for identifying antibiotics, preferably antifungal agents that affect L-asparagine, heme, L-histidine, L-leucine or L-threonine biosynthesis.

Background of the invention:
Filamentous fungi are the causative pathogens involved in many serious pathogenic infections of plants and animals. Because fungi are eukaryotes and, therefore, are more like host organisms than bacteria, for example, treatment of infections with fungi presents special risks and challenges that do not occur with other types of infections. One such fungus is Magnaporthe grisea , a fungus that causes rice blast. The fungus is an organism that poses a serious threat to the world food supply. Other examples of economically important plant pathogens, Agaricus, Alternaria, Anisogramma, Anthracoidea , Antrodia, Apiognomonia, Apiosporina, Armillaria, Ascochyta, Aspergillus, Bipolaris, Bjerkandera, Botryosphaeria, Botrytis, Ceratobasidium, Ceratocystis, Cercospora, Cercosporidium, Cerotelium, Cerrena, Chondrostereum, Chryphonectria, Chrysomyxa, Cladosporium, Claviceps, Co hliobolus, Coleosporium, Colletotrichium, Colletotrichum, Corticium, Corynespora, Cronartium, Cryphonectria, Cryptosphaeria, Cyathus, Cymadothea, Cytospora, Daedaleopsis, Diaporthe, Didymella, Diplocarpon, Diplodia, Discohainesia, Discula, Dothistroma, Drechslera, Echinodontium, Elsinoe, Endocronartium, Endothia, Entyloma, Epichloe, Erysip e, Exobasidium, Exserohilum, Fomes, Fomitopsis, Fusarium, Gaeumannomyces, Ganoderma, Gibberella, Gloeocercospora, Gloeophyllum, Gloeoporus, Glomerella, Gnomoniella, Guignardia, Gymnosporangium, Helminthosporium, Herpotrichia, Heterobasidion, Hirschioporus, Hypodermella, Inonotus, Irpex, Kabatiella, Kabatina, Laetiporus , Laetisaria , Lasiodip lodia, Laxitextum, Leptographium, Leptosphaeria, Leptosphaerulina, Leucytospora, Linospora, Lophodermella, Lophodermium, Macrophomina, Magnaporthe, Marssonina, Melampsora, Melampsorella, Meria, Microdochium, Microsphaera, Monilinia, Monochaetia, Morchella, Mycosphaerella, Myrothecium, Nectria, Nigrospora, Ophiosphaerella, Ophiostoma , Penici llium, Perenniporia, Peridermium, Pestalotia, Phaeocryptopus, Phaeolus, Phakopsora, Phellinus, Phialophora, Phoma, Phomopsis, Phragmidium, Phyllachora, Phyllactinia, Phyllosticta, Phymatotrichopsis, Pleospora, Podosphaera, Pseudopeziza, Pseudoseptoria, Puccinia, Pucciniastrum, Pyricularia, Rhabdocline, Rhizoctonia, Rhizopus, Rhizosphaera, R ynchosporium, Rhytisma, Schizophyllum, Schizopora, Scirrhia, Sclerotinia, Sclerotium, Scytinostroma, Septoria, Setosphaera, Sirococcus, Spaerotheca, Sphaeropsis, Sphaerotheca, Sporisorium, Stagonospora, Stemphylium, Stenocarpella, Stereum, Taphrina, Thielaviopsis, Tilletia, Trametes, Tranzschelia, Trichoderma, Tubakia , Typhula , Uncinula , Ur These include pathogens of Ocystis , Uromyces , Ustilago , Valsa , Venturia , Verticillium , Xylaria , and other genera. Albugo, Aphanomyces, Bremia, Peronospora, Phytophthora, Plasmodiophora, Plasmopara, Pseudoperonospora, Pythium, Sclerophthora, and other genera, also related organisms are classified as Oomycetes also an important plant pathogens, and when true Classified with fungi. Human diseases caused by filamentous fungi include life-threatening lung and disseminated diseases, which are often the result of Aspergillus fumigatus infection. Other fungal disease in animals is caused Fusarium, Blastomyces, Microsporum, Trichophyton, Epidermophyton, Candida, Histoplamsa, Pneumocystis, Cryptococcus, other Aspergilli, and other genera. Control of fungal diseases in plants and animals is usually mediated by chemicals that inhibit the growth, proliferation, and / or pathogenicity of fungal organisms. To date, less than 20 modes of action of plant defense fungicides and human antifungal compounds are known.

Pathogenic organisms have been defined as organisms that cause or are capable of causing disease. Pathogenic organisms can grow on or in tissues and obtain nutrients and other essential substances from the host. A significant amount of research involving filamentous fungal pathogens has been carried out using the human pathogen Aspergillus fumigatus . Shibuya et al. (Shibuya, K., M. Takaoka et al. (1999) Microb Pathog 27: 123-31 (PMID: 10455003)) code for alkaline protease or hydrophobin (rodlet), respectively. Thus, it has been shown that deletion of either of the two genes suspected to be pathogenic-related does not reduce the mortality of mice infected with these mutants. Smith et al. (Smith, JM, CM Tang et al. (1994) Infect Immun 62: 5247-54 (PMID: 7960101)) showed similar results with alkaline protease and ribotoxin, restrictocin. Shown; Aspergillus fumigatus strains mutated for any of these genes were completely pathogenic to mice. Reichard et al. (Reichard, U., M. Monod et al. (1997) J Med Vet Mycol 35: 189-96 (PMID: 9229335)) in Aspergillus fumigatus (aspergillopepsin) Deletion of genes suspected of having sex has been shown to have no effect on mortality in the guinea pig model system, and Aufauvre-Brown et al. (Aufauvre-Brown, A., E. Melado et al.). (1997) Fungal Genet Biol 21: 141-52 (PMID: 9073488)) indicates that chitin synthase mutations have no effect on virulence. showed that. However, not all experiments yielded negative results. Ergosterol is an important membrane component found in fungal organisms. Because neither plant nor animal hosts contain this particular sterol, pathogenic fungi lacking key enzymes in this biochemical pathway can be expected to be non-pathogenic. A number of antifungal compounds that affect this biochemical pathway have been described (Onishi, JC and AA Patchcht (1990a, b, c, d, and e) US Pat. No. 4,920, 109; 4,920,111; 4,920,112; 4,920,113; and 4,921,844, Merck & Co. Inc. (Rahway, NJ)) and (Hewitt, HG (1998). Fungicides in Crop Protection Cambridge, University Press). D'Enfert et al. (D'Enfert, C., M. Diaquin et al. (1996) Infect Immuno 64: 4401-5 (PMID: 8926121)) is an Aspergillus fumigatus mutated in the orotidine 5'-phosphate decarboxylase gene. The strain has been shown to be completely avirulent in mice, and Brown et al. (Brown, JS, A. Aufaubre-Brown et al. (2000) Mol Microbiol 36: 1371-80 (PMID: 1093287)) Observed that mutating genes involved in the synthesis of para-aminobenzoic acid resulted in non-pathogenicity. Several specific target genes have been described as having utility in screening for inhibitors of phytopathogenic fungi. Bacot et al. (Bacot, K.O., D.B. Jordan et al. (2000) US Pat. No. 6,074,830, E. I. du Pont de Nemours & Company (Wilmington, Del.)) Is 3,4- The use of dihydroxy-2-butanone 4-phosphate synthase is described, and Davis et al. (Davis, GE, GD Gustafson et al. (1999) US Pat. No. 5,976,848, Dow AgroSciences LLC (Indiana) Indianapolis)) describes the use of dihydroorotic acid dehydrogenase for potential screening purposes.

There are also several papers reporting less clear results where the mutants do not show complete pathogenicity or non-pathogenicity. Hensel et al (Hensel, M., H. N. Arst , Jr. et al. (1998) Mol Gen Genet 258: 553-7 (PMID: 9669338)) , even by deletion of areA transcriptional activator, Aspergillus fumigatus It showed that the pathogenicity of was only moderately effective. Tang et al. (Tang, CM, JM Smith et al. (1994) Infect Immun 62: 5255-60 (PMID: 7960102)) uses a related fungus, Aspergillus nidulans , in the synthesis of para-aminobenzoic acid. Although the mutation prevented death in mice, it was observed that mutations in lysine biosynthesis did not significantly affect mortality in infected mice.

Thus, it is currently impossible for a pathogen to determine which specific growth substances can be easily obtained from a host and which substances cannot be obtained. The inventors of the present invention have found that Magnaporthe grisea , which is unable to synthesize its own L-asparagine, is non-pathogenic to the host organism. Previous studies using the Saccharomyces cerevisiae asparagine synthase gene, ASN1 and ASN2, showed that disrupting only ASN1 or ASN2 had no effect on proliferation (Dang et al. (1996) Mol Microbiol 22: 681-92 (PMID: 8951815)), which is the opposite of our findings. To date, there have been no publications demonstrating the anti-pathogenic effects of knockout, overexpression, antisense expression, or inhibition of genes or gene products involved in L-asparagine biosynthesis in filamentous fungi. Thus, de novo biosynthesis of L-asparagine has not been shown to be essential for fungal virulence. Therefore, it would be desirable to determine the utility of enzymes involved in L-asparagine biosynthesis in order to evaluate antibiotic compounds, particularly fungicides. If a fungal biochemical pathway or a specific gene product of this pathway has been shown to be required for fungal virulence, various forms of in vitro and in vivo screening assays can be established to establish effective target genes, It is possible to discover chemical compound species that react with gene products, or biochemical pathways, and thus are candidates for antifungal, biocide, and biostatic agents.

The inventors of the present invention have found that Magnaporthe grisea , which is unable to synthesize its heme, is non-pathogenic to the host organism. In addition to being an important component of respiratory cytochromes and hemoglobin, heme is a prosthetic group of many enzymes involved in oxygen radical detoxification and fatty acid and sterol metabolism. In the yeast, Saccharomyces cerevisiae , mutants with deficient heme biosynthesis have been isolated and studied in detail genetically (Golrub et al. (1977) J Biol Chem 252: 2846-54 (PMID: 323256). ). Synthase gene 5-aminolevulinic acid, cloned from Aspergillus oryzae, and A. It has been shown to be used as a selectable marker for oryzae transformation (Elrod et al. (2000) Curr Genet 38: 291-8 (PMID: 11191214)). In humans, two 5-aminolevulinate synthase genes have been identified. One of these mutations encodes an erythrocyte isoform, resulting in X chromosome-linked ironblastic anemia (Cox et al. (1994) N Engl J Med 330: 675-9 (PMID: 8107717)). 5-Aminolevulinate synthase has been proposed as a novel antimalarial target (Padmanaban and Rangarajan (2000) Biochem Biophys Res Commun 268: 665-8 (PMID: 10679261)).

  To date, there have been no publications demonstrating the anti-pathogenic effects of knockout, overexpression, antisense expression, or inhibition of genes or gene products involved in heme biosynthesis in filamentous fungi. Therefore, de novo biosynthesis of heme has not been shown to be essential for fungal virulence. Thus, it would be desirable to determine the utility of enzymes involved in heme biosynthesis in order to evaluate antibiotic compounds, particularly fungicides. If a fungal biochemical pathway or a specific gene product of this pathway has been shown to be required for fungal virulence, various forms of in vitro and in vivo screening assays can be established to establish effective target genes, It is possible to discover chemical compound species that react with gene products or biochemical pathways and are therefore candidates for antifungal agents, biocides, and biostatics.

The inventors of the present invention have found that Magnaporthe grisea , which is unable to synthesize its own L-histidine, has reduced virulence to the host organism. M.M. The grisea HISP1 enzyme has the greatest similarity to Schizosaccharomyces pombe His9 and some similarity to Saccharomyces cerevisiae His2p and His9. These genes encode a distantly related family of histidinol phosphate phosphatases (HolPases) that catalyze the dephosphorylation of histidinol phosphate to histidinol. This family includes HolPase encoded by the HisJ (or ytvP) gene found in Bacillus subtilis . HisJ knockout has resulted in an auxotrophic mutant that cannot grow without histidine supplementation (le Coq et al. (1999) J Bacteriol 181: 3277-3280 (PMID: 10322033)). There were no references in which SpHis2, ScHis2p or ScHis9 were known targets for antifungal / fungicidal development. However, S. cerevisiae containing a knockout in the His1-His7 gene . cerevisiae mutants were found to be unable to grow in elevated levels of Cu, Co, or Ni at near neutral pH (Pearce and Sherman (1999) J Bacteriol 181: 4774-4777 ( PMID: 10438744)).

  To date, there have been no publications demonstrating the anti-pathogenic effects of knockout, overexpression, antisense expression, or inhibition of genes or gene products involved in L-histidine biosynthesis in filamentous fungi. Thus, de novo biosynthesis of L-histidine has not been shown to be essential for fungal virulence. And therefore, it would be desirable to determine the utility of enzymes involved in L-histidine biosynthesis to evaluate antibiotic compounds, particularly fungicides. If a fungal biochemical pathway or a specific gene product of this pathway has been shown to be required for fungal virulence, various forms of in vitro and in vivo screening assays can be established to establish effective target genes, It is possible to discover chemical compound species that react with gene products or biochemical pathways and are therefore candidates for antifungal agents, biocides, and biostatics.

The inventors of the present invention have found that Magnaporthe grisea , which is unable to synthesize its own L-leucine, is non-pathogenic to the host organism. To date, there have been no publications demonstrating the anti-pathogenic effects of knockout, overexpression, antisense expression, or inhibition of genes or gene products involved in L-leucine biosynthesis in filamentous fungi. Thus, de novo biosynthesis of L-leucine has not been shown to be essential for fungal pathogenesis. And therefore, it would be desirable to determine the utility of enzymes involved in L-leucine biosynthesis in order to evaluate antibiotic compounds, particularly fungicides. If a fungal biochemical pathway or a specific gene product of this pathway has been shown to be required for fungal virulence, various forms of in vitro and in vivo screening assays can be established to establish effective target genes, It is possible to discover chemical compound species that react with gene products or biochemical pathways and are therefore candidates for antifungal agents, biocides, and biostatics.

The inventors of the present invention have found that Magnaporthe grisea , which is unable to synthesize its own L-threonine, is non-pathogenic to the host organism. To date, there have been no publications demonstrating the anti-pathogenic effects of knockout, overexpression, antisense expression, or inhibition of genes or gene products involved in L-threonine biosynthesis in filamentous fungi. Thus, de novo biosynthesis of L-threonine has not been shown to be essential for fungal pathogenesis. Therefore, it would be desirable to determine the utility of enzymes involved in L-threonine biosynthesis in order to evaluate antibiotic compounds, particularly fungicides. If a fungal biochemical pathway or a specific gene product of this pathway has been shown to be required for fungal virulence, various forms of in vitro and in vivo screening assays can be established to establish effective target genes, It is possible to discover chemical compound species that react with gene products or biochemical pathways and are therefore candidates for antifungal agents, biocides, and biostatics.

Summary of the invention:
The inventors of the present invention, in Magnaporthe grisea , in vivo disruption of a gene encoding asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate dehydratase or threonine synthase is responsible for the pathogenicity of the fungus. Found to protect or inhibit. Thus, the inventors of the present invention found that asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate dehydratase and threonine synthase are essential for normal rice blast pathogenesis and antibiotics, preferably Discovered that it can be used as a target to identify fungicides. Accordingly, the present invention provides methods for identifying compounds that inhibit the expression or activity of asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate dehydratase or threonine synthase. The method of the invention is useful for the identification of antibiotics, preferably fungicides.

Detailed description of the invention:
Unless otherwise indicated, the following terms are intended to have the following meanings in interpreting the present invention.

  The term “active against” in the context of a compound, agent or composition having antibiotic activity refers to the organism in which the compound has one or more targets for a particular target or targets. It shows a deleterious effect on proliferation in vitro and / or in vivo. In particular, a compound that is active against a gene exerts an effect on a target that affects the expression product of that gene. This does not necessarily mean that the compound acts directly on the expression product of the gene, indicating that the compound affects the expression product in a detrimental manner. Thus, a direct target of a compound can be, for example, an upstream component that reduces transcription from a gene and produces a lower expression level. Similarly, a compound can affect the level of translation of a polypeptide expression product or act on a downstream component of a biochemical pathway in which the gene expression product plays a major biological role. Is possible. As a result, such compounds can be said to be active against a gene, against a gene product, or related components either upstream or downstream of the gene or expression product. The term “activity against” includes a wide range of potential activities, but also exhibits some target specificity. Thus, for example, common proteases are not “active” against a particular gene that produces a polypeptide product. In contrast, a compound that inhibits a particular enzyme is active against that enzyme and active against the gene encoding that enzyme.

As used herein, the term “allele” refers to any alternative form of a gene that can occur at a given locus.
As used herein, the term “α-aminoadipate reductase (EC 1.2.1.31), α-aminoadipate reductase polypeptide, α-aminoadipate semialdehyde dehydrogenase, or alpha-aminoadipate reductase” Is synonymous with “AAR1 gene product” or “ASD1 gene product” and L-2-aminoadipate, NADPH, and ATP and L-2-aminoadipate 6-semialdehyde, NADP +, AMP, pyrophosphate , Refers to an enzyme that catalyzes the interconversion of H ₂ O.

  The term “antibiotic” refers to any substance or compound that, when contacted with a living cell, organism, virus, or other replicable entity, causes a decrease in the growth, viability, or pathogenicity of that entity.

As used herein, the term “ALAS1” refers to a gene that encodes 5-aminolevulinate synthase activity and catalyzes the interconversion of succinyl-CoA and glycine with 5-aminolevulinate, CoA, and CO _2. It can be used to refer to an enzyme and also to refer to its gene product.

As used herein, the terms “5-aminolevulinate synthase” (EC 2.3.3.17) and “5-aminolevulinate synthase polypeptide” are synonymous with “ALAS1 gene product” and succinyl-CoA and glycine. refers If, 5-amino-levulinate, CoA, and an enzyme that catalyzes the interconversion of CO _2.

  As used herein, the term “ASN1” refers to a gene that encodes asparagine synthase activity, and is an interrelation of L-aspartate, L-glutamine, and ATP with L-asparagine, L-glutamate, AMP, and pyrophosphate. It refers to an enzyme that catalyzes conversion and can also be used to refer to its gene product.

  As used herein, the terms “asparagine synthase” (EC 6.3.5.4) and “asparagine synthase polypeptide” are synonymous with “ASN1 gene product” and L-aspartate, L-glutamine, and ATP. And an enzyme that catalyzes the interconversion of L-asparagine, L-glutamate, AMP, and pyrophosphate.

  The term “binding” refers to a non-covalent or covalent interaction, preferably a non-covalent interaction, that holds two molecules together. For example, two such molecules can be an enzyme and an inhibitor of that enzyme. Non-covalent interactions include hydrogen bonds, ionic interactions between charged groups, van der Waals interactions, and hydrophobic interactions between nonpolar groups. One or more of these interactions can mediate the binding of two molecules to each other.

  The term “biochemical pathway” or “pathway” refers to a series of linked biochemical reactions that normally occur in a cell, or in a broader sense, cellular events such as cell division or DNA replication. Typically, these biochemical pathway steps act in a coordinated manner to produce a specific product or products, or some other specific biochemical effect. In the absence of a gene expression product, the completion of one or more steps of the biochemical pathway is directly or indirectly prevented, thereby preventing or significant production of one or more of the normal products or effects of that pathway. If so, such biochemical pathways require the expression product of that gene. Thus, if the presence of an agent stops or substantially reduces the completion of a sequence of biochemical pathways that require the expression product of a particular gene, that agent specifically directs the biochemical pathway. Inhibit. Such agents may, but need not, act directly on the expression product of that particular gene.

As used herein, the term “cDNA” means complementary deoxyribonucleic acid.
As used herein, the term “CoA” means coenzyme A.
As used herein, the term “conditional lethal” refers to a mutation that allows growth and / or survival only under certain growth or environmental conditions.

  As used herein, the term “cosmid” is used for gene cloning and refers to a hybrid vector containing a cos site (derived from lambda bacteriophage). The vector also contains a drug resistance marker gene and other plasmid genes. Cosmids are particularly suitable for cloning large genes or multigene fragments.

  As used herein, the term “dominant allele” refers to a mutant expression that can be distinguished when the mutation is present in an organism that also contains a wild type (non-mutant), recessive allele, or other dominant allele. Refers to a dominant mutant allele whose type can be detected.

As used herein, the term “DNA” means deoxyribonucleic acid.
As used herein, the term “ELISA” means an enzyme linked immunosorbent assay.
“Fungi” refers to whole fungi, fungal organs and tissues (eg, ascending, hyphae, pseudohyphae, pseudoroots, mycorrhiza, infarct, spore, conidia, spore sac, conidia Bundles, conidia, ascosac, closed sac, mycelium, ascossa, basidiomycetes, etc.), spores, fungal cells and their progeny. Fungi are a group of organisms (including about 50,000 species) including, but not limited to, the fungus kingdom, including mushrooms, mildew, mold, yeast and the like. These may exist as single cells or constitute multicellular bodies called mycelium consisting of fibers known as mycelia. Most fungal cells are multinucleated and have a cell wall composed primarily of chitin. Fungi are mainly parasites of other organisms because they exist mainly in the wet conditions on the ground, and chlorophyll is not present, and therefore it is impossible to produce their own food by photosynthesis, Or it is either a humus that feeds on dead biological material. The basic criteria used for classification are the nature of the spores produced and the presence or absence of transverse walls within the mycelium. Fungi are distributed throughout the world in land, freshwater, and marine environments. Some live in the ground. Many pathogenic fungi cause disease in animals and humans or plants, while others are destructive to timber, textiles and other materials. Some fungi form associations with other organisms, especially lichens with algae.

  As used herein, the term “fungicide”, “antifungal”, or “antimycotic” kills at least one of a fungus, fungal cell, fungal tissue or spore. Or an antibiotic or compound that inhibits its growth, viability, or pathogenicity.

  In the context of this disclosure, “gene” should be understood to refer to a unit of heredity. Each gene is composed of a linear chain of deoxyribonucleotides that can be referred to by the sequence of nucleotides that form the chain. Thus, “sequence” is used to indicate both the ordered listing of nucleotides that form a chain, and the chain itself that has the sequence of nucleotides (“sequence” is a linear chain made of ribonucleotides, The same method is used when referring to the RNA strand). Genes can include regulatory and regulatory sequences, sequences that can be transcribed into RNA molecules, and can contain sequences with unknown functions. The majority of RNA transcripts are messenger RNA (mRNA), which includes sequences that are translated into polypeptides and can also include sequences that are not translated. It should be recognized that there may be minor differences in the nucleotide sequence of the same gene that do not alter the identity of the gene between different fungal strains or even within a particular fungal strain.

  In this disclosure, the term “growth” or “cell proliferation” of an organism refers to an increase in the mass, density, or number of cells of the organism. Some common methods of measuring proliferation include measuring the optical density of a cell suspension, counting the number of cells in a fixed volume, counting the number of cells by measuring cell division, measuring the cell mass or cell volume Measurement etc. are included.

  In the present disclosure, the term “growth condition phenotype” means that a fungal strain having such a phenotype is more responsive to changes in one or more culture parameters than a strain that is similar except that it does not have a growth condition phenotype, It shows that the growth rate shows a significantly greater difference. Typically, the growth condition phenotype is described in terms of a single growth culture parameter, such as temperature. Thus, temperature (or heat-sensitive) mutants (ie fungal strains having a heat-sensitive phenotype) show significantly different growth under non-permissive temperature conditions when compared to growth under permissive conditions, and Preferably it does not show any growth. Furthermore, such mutants also preferably exhibit a moderate growth rate at moderate or semi-permissive temperatures. Similar reactions also occur from appropriate growth changes of other types of growth condition phenotypes.

As used herein, the term “H ₂ O” means water.
As used herein, the term “heterologous ALAS1 gene” is not derived from Magnaporthe grisea , and: at least 50% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5, preferably 60%, 70%, 80%, 90 %, 95%, 99% sequence identity, and in ascending order 50-100% of each integer unit of sequence identity; or at least 10%, preferably 25% of Magnaporthe grisea 5-aminolevulinate synthase activity , 50%, 75%, 90%, 95%, 99%, and in ascending order, a gene having 10 to 100% of each integer unit of activity.

As used herein, the term “heterologous ASN1 gene” is not derived from Magnaporthe grisea , and: at least 50% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, preferably 60%, 70%, 80%, 90 %, 95%, 99% sequence identity, and in ascending order, 50-100% of each integer unit of sequence identity; or at least 10%, preferably 25%, 50% of Magnaporthe grisea asparagine synthase activity , 75%, 90%, 95%, 99%, and in ascending order, 10-100% of each gene having an integer unit of activity.

As used herein, the term “heterologous HISP1 gene” is not derived from Magnaporthe grisea , and: at least 50% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8, preferably 60%, 70%, 80%, 90 %, 95%, 99% sequence identity and, in ascending order, 50-100% of each integer unit of sequence identity; or at least 10%, preferably 25%, 50% of Magnaporthe grisea histidinol-phosphatase activity %, 75%, 90%, 95%, 99% and in ascending order 10-100% of the genes with each integer unit activity.

As used herein, the terms “histidinol-phosphatase” (EC 3.1.3.15) and “histidinol-phosphatase polypeptide” are synonymous with “HISP1 gene product” and L-histidinol phosphate and H _2. Refers to an enzyme that catalyzes the interconversion of O with L-histidinol and orthophosphate.

As used herein, the term “heterologous IPMD1 gene” is not derived from Magnaporthe grisea , and: at least 50% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11, preferably 60%, 70%, 80%, 90 %, 95%, 99% sequence identity, and in ascending order, 50-100% of each integer unit sequence identity; or at least 10% of Magnaporthe grisea 3-isopropylmalate dehydratase activity, preferably 25 %, 50%, 75%, 90%, 95%, 99%, and in ascending order, 10-100% of the gene having an integer unit of activity.

As used herein, the term “heterologous THR4 gene” is not derived from Magnaporthe grisea and is: SEQ ID NO: 13 or SEQ ID NO: 14 with at least 50% sequence identity, preferably 60%, 70%, 80%, 90 %, 95%, 99% sequence identity, and in ascending order, 50-100% of each integer unit sequence identity; or at least 10%, preferably 25%, 50% of Magnaporthe grisea threonine synthase activity , 75%, 90%, 95%, 99%, and in ascending order, 10-100% of each gene having an integer unit of activity.

As used herein, the term “HISP1” refers to a gene encoding histidinol-phosphatase activity, and refers to an enzyme that catalyzes the interconversion of L-histidinol phosphate and H ₂ O with L-histidinol and orthophosphate. , And can also be used to refer to the gene product.

As used herein, the term “His tag” refers to an encoded polypeptide consisting of a number of consecutive histidine amino acids.
As used herein, the term “HPLC” refers to high pressure liquid chromatography.

  As used herein, the terms “hph”, “hygromycin B phosphotransferase”, and “hygromycin resistance gene” refer to the E. coli hygromycin phosphotransferase gene or gene product.

As used herein, the term “hygromycin B” refers to an aminoglycoside antibiotic used for the selection and maintenance of eukaryotic cells containing the E. coli hygromycin resistance gene.
“Hypersensitivity” refers to a phenotype in which cells are more sensitive to antibiotic compounds than wild-type cells with a similar or identical genetic background.

“Desensitized” refers to a phenotype in which cells are less sensitive to antibiotic compounds than wild-type cells with similar or identical genetic backgrounds.
As used herein, the term “incomplete state” refers to a class of fungal organisms that do not have a sexual life stage that can be verified.

  The term “inhibitor” as used herein inactivates the enzyme activity of any one of asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate dehydratase or threonine synthase, or the enzyme A chemical substance that substantially reduces the activity level, “substantially” is a reduction that is at least as great as the standard deviation of the measurement, preferably a 50% reduction, more preferably at least an order of magnitude reduction, ie 10 Means a decrease to%. Inhibitors can function by interacting directly with either an enzyme, an enzyme cofactor, an enzyme substrate, or a combination thereof.

  A polynucleotide can be “introduced” into a fungal cell by any means known to those of skill in the art including transfection, transformation or transduction, transposable elements, electroporation, particle bombardment, infection, and the like. is there. The introduced polynucleotide can be stably maintained in the cell when incorporated into a non-chromosomal autonomous replicon or integrated into a fungal chromosome. Alternatively, the introduced polynucleotide can be present on an extrachromosomal non-replicating vector and transiently expressed or transiently active.

In the present specification, the term “IPMD1” means a gene encoding 3-isopropylmalate dehydratase activity, and an enzyme that catalyzes the interconversion of 2-isopropylmalate and H ₂ O with 3-isopropylmalate. And can also be used to refer to the gene product.

As used herein, the terms “3-isopropylmalate dehydratase” (EC 4.2.3.33), “α-isopropylmalate dehydratase polypeptide” and “3-isopropylmalate dehydratase polypeptide” are referred to as “IPMD1 gene product”. And refers to an enzyme that catalyzes the interconversion of 2-isopropylmalate and H ₂ O with 3-isopropylmalate.

  As used herein, the term “knockout” or “gene disruption” refers to null mutations (mutations without an active gene product), single or multiple partial null mutations, or insertion of a foreign piece of DNA. Refers to the production of an organism carrying one or more alterations in gene regulation by disruption of the DNA sequence. Usually, the foreign DNA encodes a selectable marker.

As used herein, the term “LB agar” means Luria's broth agar.
The term “screening method” means that the method is suitable and typically used for testing for a particular property or effect in a number of compounds. Typically, more than one compound is tested simultaneously (as in a 96 well microtiter plate) and preferably a significant portion of the method is automatable. “Screening method” also refers to the simultaneous measurement of different properties or sets of effects of a compound.

As used herein, the term “mRNA” means messenger ribonucleic acid.
As used herein, the term “mutant” of a gene refers to a gene that has been altered naturally or artificially and whose nucleotide sequence has changed. The changes in the base sequence can be of several different types and include changes such as one or more base changes to different bases, deletions, and / or transposons. In contrast, the normal type (wild type) of a gene is the type commonly found in the natural population of an organism. In general, a single type of gene will dominate the natural population. In general, such genes are suitable as normal forms of the gene, but other forms that provide similar functional properties can also be used as normal genes. In particular, a normal gene does not confer a growth condition phenotype on a strain carrying that gene, but a mutant gene suitable for use in these methods provides such a growth condition phenotype.

As used herein, the term “Ni” refers to nickel.
As used herein, the term “Ni-NTA” refers to nickel sepharose.
As used herein, a “normal” form (wild type) of a gene is a form commonly found in the natural population of an organism. In general, a single type of gene will dominate the natural population. In general, such genes are suitable as normal forms of the gene, but other forms that provide similar functional properties can also be used as normal genes. In particular, a normal gene does not confer a growth condition phenotype on a strain carrying that gene, but a mutant gene suitable for use in these methods provides such a growth condition phenotype.

  As used herein, the term “one type” of a gene is synonymous with the term “gene” and a “different type” of a gene is greater than 49% sequence identity with the first type. , And refers to genes with sequence identity less than 100% sequence identity.

As used herein, the term “pathogenic” refers to the ability to cause disease. The term applies to a parasitic microorganism in relation to its host.
As used herein, the term “PCR” means polymerase chain reaction.

The “percent sequence identity (%)” between two polynucleotide or two polypeptide sequences was determined by the National Center for Biotechnology BLAST program (Basic Local Alignment Search Tool; (Altschul, SF, W. Gish et al. 1990) Smith Waterman Parallel (Smith, TF and MS Waterman (1981), according to J Mol Biol 215: 403-10 (PMID: 2231712)) or incorporated in GeneMatcher Plus ^™. J Mol Biol 147: 195-7 (PMID: 7265238)) DN for the purpose of determining sequence identity. When comparing the sequences to the RNA sequence, a thymine nucleotide is understood to be equivalent to a uracil nucleotide.

  By “polypeptide” is meant a chain of at least two amino acids linked by peptide bonds. The chain can be linear, branched, cyclic, or combinations thereof. Preferably, the polypeptide is about 10 to about 1000 amino acids in length, more preferably 10 to 50 amino acids in length. Polypeptides can contain amino acid analogs and other modifications, including but not limited to glycosylated or phosphorylated residues.

As used herein, the term “proliferation” is synonymous with the term “growth”.
As used herein, the term “reverse transcriptase PCR” means reverse transcription-polymerase chain reaction.

As used herein, the term “RNA” means ribonucleic acid.
As used herein, a “semi-permissive condition” is a condition where the corresponding culture parameter for a particular growth condition phenotype is intermediate between permissive and non-permissive conditions. As a result, in semi-permissive conditions, organisms with a growth condition phenotype will exhibit growth rates that are intermediate to those exhibited in permissive and non-permissive conditions. In general, these intermediate growth rates are partially functional under semi-permissive conditions, essentially fully functional under permissive conditions, and do not function under non-permissive conditions, or have very low function. It may be due to the mutant cell component not having, in which case the functional level of this component is related to the growth rate of the organism. The intermediate growth rate may also be the result of one or more nutrients present in an amount that is insufficient to achieve an optimal growth rate.

“Sensitivity phenotype” refers to a phenotype exhibiting hypersensitivity or reduced sensitivity.
The term “specific binding” refers to an interaction between any one of asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate dehydratase or threonine synthase and a molecule or compound comprising asparagine synthase, Refers to interactions depending on the primary amino acid sequence and / or conformation of 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate dehydratase or threonine synthase.

  As used herein, the term “THR4” refers to a gene that encodes threonine synthase activity, refers to an enzyme that catalyzes the interconversion of O-phospho-L-homoserine and water with L-threonine and orthophosphate, and It can also be used to refer to the gene product.

  As used herein, the terms “threonine synthase” (EC 4.2.99.2) and “threonine synthase polypeptide” are synonymous with “THR4 gene product” and O-phospho-L-homoserine and water; It refers to an enzyme that catalyzes the interconversion of L-threonine and orthophosphate.

As used herein, the term “TLC” means thin layer chromatography.
“Transformation” as used herein refers to the introduction of a polynucleotide (single-stranded or double-stranded DNA, RNA, or a combination thereof) into a living cell by any means. Transformation can be accomplished by a variety of methods including, but not limited to, electroporation, polyethylene glycol mediated uptake, particle gun, agrotransformation, and the like. This process can result in transient or stable expression of the transformed polynucleotide. By “stable transformation” is meant that a sequence of interest is integrated into a replicon, eg, a chromosome or episome, in a cell. A transformed cell contains not only the final product of the transformation process, but also its progeny that retain the polynucleotide of interest.

  For the purposes of the present invention, “transgenic” refers to any cell, spore, tissue or part that contains all or part of at least one recombinant polynucleotide. In many cases, the recombinant polynucleotide is stably or completely integrated into the chromosome or into a stable extrachromosomal element, such as to be passed on to successive generations.

  As used herein, the term “transposase” refers to an enzyme that catalyzes rearrangement. Preferred transposons are described in WO 00/55346, PCT / US00 / 07317, and US 09/6588859.

  As used herein, the term “translocation” often moves a polynucleotide (transposon) from one position within or between a single genome or multiple genomes, or DNA constructs such as plasmids, bacmids, and cosmids. Or refers to a complex gene rearrangement process that involves being copied and inserted at another location.

  As used herein, the term “transposon” (or also known as “translocation element”, “translocation gene element”, “mobile element”, or “jump gene”) is, for example, WO 00/55346, PCT / US00 / 07317. And refers to mobile DNA elements, such as those described in US 09/6588859. Transposons can disrupt gene expression or cause deletions and inversions and can thus affect both the genotype and phenotype of the organism involved. The mobility of transposable elements has long been used for genetic manipulation and has introduced genes or other information into the genome of specific model systems.

As used herein, the term “Tween 20” means sorbitan mono-9-octadecanoate poly (oxy-1,1-ethanediyl).
In this disclosure, the term “viability” of an organism refers to the ability of the organism to show growth or to exhibit active cell function under conditions suitable for the organism. Some examples of active cell function include respiration as measured by gas evolution, secretion of proteins and / or other compounds, dye exclusion, mobility, dye oxidation, dye reduction, pigment production, medium acidity Changes are included.

The inventors of the present invention have discovered that disruption of the ASN1 gene and / or gene product inhibits the virulence of Magnaporthe grisea . Thus, the inventors of the present invention have demonstrated for the first time that asparagine synthase is the target of antibiotics, preferably antifungal agents.

  Accordingly, the present invention provides methods for identifying compounds that inhibit ASN1 gene expression or the biological activity of its gene product (s). Such methods include ligand binding assays, enzyme activity assays, cell-based assays, and assays for ASN1 gene expression. Any compound that is a ligand for asparagine synthase can have antibiotic activity. For the purposes of the present invention, “ligand” refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the present invention are useful as antibiotics.

Accordingly, in one embodiment, the present invention is a method of identifying a test compound as an antibiotic candidate:
a) contacting an asparagine synthase polypeptide with a test compound; and b) detecting the presence or absence of a bond between the test compound and the asparagine synthase polypeptide, wherein the test compound is an antibiotic. The method is provided to indicate that it is a candidate.

An asparagine synthase protein can have the amino acid sequence of a naturally occurring asparagine synthase found in fungi, animals, plants, or microorganisms, and can have an amino acid sequence derived from a naturally occurring sequence. Preferably, the asparagine synthase is a fungal asparagine synthase. CDNA encoding asparagine synthase protein (SEQ ID NO: 1), M. Genomic DNA (SEQ ID NO: 2) encoding the grisea protein and polypeptide (SEQ ID NO: 3) can be found herein.

In one aspect, the present invention also provides a polypeptide consisting essentially of SEQ ID NO: 3. For purposes of the present invention, a polypeptide consisting essentially of SEQ ID NO: 3 has at least 80% sequence identity with SEQ ID NO: 3, and at least 10% of the activity of SEQ ID NO: 3 with L-aspartate , L-glutamine, and ATP catalyze the interconversion of L-asparagine, L-glutamate, AMP, and pyrophosphate. Preferably, the polypeptide consisting essentially of SEQ ID NO: 3 has at least 85% sequence identity with SEQ ID NO: 3, more preferably the sequence identity is at least 90%, most preferably the sequence identity Sex is at least 95% or 97% or 99%, or in ascending order with any integer sequence identity of 80-100%. And preferably, a polypeptide consisting essentially of SEQ ID NO: 3, M. It has an activity of at least 25%, at least 50%, at least 75%, or at least 90% of the activity of grisea asparagine synthase, or any integer from 60 to 100% in ascending order.

  A “fungal asparagine synthase” can be found in at least one fungus and interconverts L-aspartate, L-glutamine, and ATP with L-asparagine, L-glutamate, AMP, and pyrophosphate. It means an enzyme that catalyzes. Asparagine synthase can be derived from any fungus, including ascomycetes, zygomycetes, basidiomycetes, fungi, and lichens.

In one embodiment, the asparagine synthase is Magnaporthe asparagine synthase. The Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae, as well as Magnaporthe incomplete state of Pyricularia sp. Preferably, the Magnaporte asparagine synthase is derived from Magnaporthe grisea .

In various embodiments, asparagine synthase, powdery scab fungus (Spongospora subterranea), gray mold (Botrytis cinerea), white rot fungus (Armillaria mellea), core rot fungus (Ganoderma adspersum), brown rot fungi (Piptoporus betulinus) , corn smut (Ustilago maydis), heartwood rot fungus (Polyporus squamosus), black spot fungus (Cercospora zeae-maydis), honey fungus (Armillaria gallica), root rot fungus (Armillaria luteobubalina), shoelaces rot fungus (Armillaria ostoyae) , Bana Anthracnose fungus (Colletotrichum musae), apples rot fungus (Monilinia fructigena), apples rot fungi (Penicillium expansum), root kelp fungus (Plasmodiophora brassicae), potato blight fungus (Phytophthora infestans), root pathogens (Heterobasidion annosum), wilt fungus (Gaeumannomyces graminis), Orandanire fungus (Ophiostoma ulm i), bean rust (Uromyces appendiculatus), northern spot fungus (Cochliobolus carbonum), Miro fungus (Periconia circinata) Southern corn blight fungus (Cochliobolus heterostrophus), leaf spot fungus (Cochliobolus lunata),褐条fungus (Cochliobolus stenospilus), Panama fungus (Fusarium oxysporum), wheat scab fungus (Fusarium graminearum), cereal root rot fungus (Fusarium culmorum ), Potato black rust fungus ( Rhizotonia solani ), wheat black rust ( Puccinia graminis ), white mold ( Sclerotinia sclerotiorum ) and the like.

  A fragment of an asparagine synthase polypeptide preferably comprises an intact or nearly intact epitope that occurs in a wild-type asparagine synthase where the fragment is biologically active. Can be used in the method. The fragment comprises at least 10 consecutive amino acids of asparagine synthase. Preferably, the fragment is at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, asparagine synthase, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, or at least 580 consecutive amino acid residues. In one embodiment, the fragment is derived from Magnaporte asparagine synthase. Preferably, the fragment contains an amino acid sequence that is conserved among fungal asparagine synthases.

  Polypeptides having at least 50% sequence identity with fungal asparagine synthase are also useful in the methods of the invention. Preferably the sequence identity is at least 60%, more preferably the sequence identity is at least 70%, most preferably the sequence identity is at least 80% or 90% or 95% or 99%, or in ascending order. , An integer of 60 to 100%.

Furthermore, it is preferred that the polypeptide has at least 10% of fungal asparagine synthase activity. More preferably, the polypeptide has at least 25%, at least 50%, at least 75% or at least 90% of the fungal asparagine synthase activity. Most preferably, the polypeptide is M.P. have at least 10%, at least 25%, at least 50%, at least 75% or at least 90% of the grisea asparagine synthase protein activity.

Thus, in another aspect, the invention provides a method for identifying a test compound as a candidate for a fungicide:
a) with a test compound: a polypeptide having at least 10 consecutive amino acids of fungal asparagine synthase; a polypeptide having at least 50% sequence identity with fungal asparagine synthase; and a polypeptide having at least 10% of the activity of fungal asparagine synthase Contacting with at least one polypeptide selected from the group consisting of; and b) detecting the presence and / or absence of binding between said test compound and said polypeptide, wherein binding comprises said test compound Wherein said method is shown to be an antibiotic candidate.

  Any technique that detects binding of a ligand to a target can be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting binding of a ligand to a target are known in the art and include, but are not limited to, detection of a fixed ligand-target complex, or target characteristics when a target binds to a ligand. Change detection is included. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligand is contacted with the asparagine synthase protein, or a fragment or variant thereof, the unbound protein is removed, and the bound asparagine synthase is detected. In a preferred embodiment, a bound asparagine synthase is detected using a labeled binding partner such as a labeled antibody. In a variation of this assay, asparagine synthase is labeled prior to contacting the immobilized candidate ligand. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetry.

  Once a compound has been identified as an antibiotic candidate, it can be tested for its ability to inhibit asparagine synthase enzyme activity. Compounds can be tested using either in vitro assays or cell based assays. Alternatively, the compound can be applied directly to the fungus or fungal cell or expressed in the fungus or fungal cell and monitored for growth, development, viability, pathogenic change or reduction, or gene expression modification Can be tested. Accordingly, in one aspect, the present invention is a method for determining whether a compound identified as an antibiotic candidate by the method described above has antifungal activity comprising: a fungus or fungal cell and said antifungal agent candidate And detecting a decrease in growth, viability, or pathogenicity of the fungus or fungal cell.

  By reducing the growth is meant that the antifungal candidate causes at least a 10% reduction in fungal or fungal cell growth as compared to the fungal or fungal cell growth in the absence of the antifungal candidate. . By reduced viability is meant that at least 20% of the fungal cells, or fungal parts, contacted with the antifungal agent candidate are not viable. Preferably, proliferation or viability will be reduced by at least 40%. More preferably, proliferation or viability will be reduced by at least 50%, 75% or at least 90% or more. Methods for measuring fungal growth and cell viability are known to those skilled in the art. Reduced virulence causes an antifungal candidate to cause at least a 10% reduction in disease caused by contact of the fungal pathogen with its host when compared to disease caused in the absence of the antifungal candidate Means. Preferably, the disease will be reduced by at least 40%. More preferably, the disease will be reduced by at least 50%, 75% or at least 90% or more. Methods for measuring fungal diseases are well known to those skilled in the art and include metrics such as lesion formation, lesion size, sporulation, respiratory failure, and / or death.

  The ability of a compound to inhibit asparagine synthase activity can be detected using an in vitro enzyme assay that directly or indirectly detects the disappearance of a substrate or the appearance of a product. Asparagine synthase catalyzes irreversible or reversible reactions, L-aspartate, L-glutamine, and ATP = L-asparagine, L-glutamate, AMP, and pyrophosphate (see FIG. 1). Methods for detecting L-aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, and / or pyrophosphate include spectrophotometry, mass spectrometry, thin layer chromatography (TLC) and reverse phase HPLC is included.

Accordingly, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) contacting asparagine synthase with L-aspartate, L-glutamine, and ATP;
b) contacting L-aspartate, L-glutamine, and ATP with asparagine synthase and test compound; and c) the following: L-aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, And / or measuring at least one concentration change of pyrophosphate, wherein the concentration change of any of the above substances indicates that the test compound is a candidate for an antibiotic.

The present invention is a further method for identifying test compounds as antibiotic candidates:
a) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with asparagine synthase;
b) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with asparagine synthase and a test compound; and c) the following: L-aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, The method is provided comprising measuring a change in the concentration of at least one of AMP and / or pyrophosphate, wherein a change in the concentration of any of the substances indicates that the test compound is a candidate for an antibiotic.

  Enzymatically active fragments of fungal asparagine synthase are also useful in the methods of the invention. For example, a polypeptide that contains at least 100 contiguous amino acid residues of fungal asparagine synthase and is enzymatically active can be used in the methods of the invention. Further, a polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity and fungal asparagine synthase is enzymatically active. Can be used. Most preferably, the polypeptide has at least 50% sequence identity with the fungal asparagine synthase and has at least 10%, 25%, 75%, or at least 90% of its activity.

Accordingly, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) with L-aspartate, L-glutamine, and ATP: a polypeptide having at least 50% sequence identity with asparagine synthase, having at least 50% sequence identity with asparagine synthase, and at least 10 of its activity Contacting a polypeptide selected from the group consisting of a polypeptide having a% and a polypeptide comprising at least 100 contiguous amino acids of asparagine synthase;
b) contacting L-aspartate, L-glutamine, and ATP with said polypeptide and test compound; and c) the following: L-aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP And / or measuring at least one change in concentration of pyrophosphate, wherein the change in concentration of any of the substances indicates that the test compound is a candidate for an antibiotic.

The present invention is a further method of identifying a test compound as an antibiotic candidate:
a) with L-asparagine, L-glutamate, AMP and pyrophosphate: a polypeptide having at least 50% sequence identity with asparagine synthase, having at least 50% sequence identity with asparagine synthase and of its activity Contacting a polypeptide selected from the group consisting of a polypeptide having at least 10% and a polypeptide comprising at least 100 contiguous amino acids of asparagine synthase;
b) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with the polypeptide and the test compound; and c) the following: L-aspartate, L-glutamine, L-asparagine, L-glutamate, ATP Measuring the concentration change of at least one of AMP, AMP, and / or pyrophosphate, wherein the change in concentration of any of the agents indicates that the test compound is a candidate for an antibiotic .

  For in vitro enzyme assays, asparagine synthase proteins and derivatives thereof can be purified from fungi or produced recombinantly in archaea, bacteria, fungi, or other eukaryotic cell cultures and It is possible to purify from this culture. Preferably, these proteins are produced using E. coli, yeast, or filamentous fungal expression systems. Methods for purifying asparagine synthase can be those described in Van Heeke and Schuster (1989) J Biol Chem 264: 5503-9 (PMID: 2564390). Other methods of purifying asparagine synthase proteins and polypeptides are known to those skilled in the art.

As an alternative to in vitro assays, the present invention also provides cell-based assays. In one embodiment, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) measuring the expression of asparagine synthase in the absence of the test compound in a single cell, a plurality of cells, tissues or organisms;
b) contacting the test compound with the single cell, cells, tissue or organism and measuring the expression of the asparagine synthase in the single cell, cells, tissue or organism. And c) comparing the expression of asparagine synthase in steps (a) and (b), wherein the expression is lower in the presence of the test compound, the compound is a candidate for an antibiotic; Wherein said method is provided.

  Asparagine synthase expression can be measured by detecting ASN1 primary transcript or mRNA, asparagine synthase polypeptide, or asparagine synthase enzyme activity. Methods of detecting RNA and protein expression are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1995. The detection method is not critical to the present invention. Methods for detecting ASN1 RNA include, but are not limited to, amplification assays such as quantitative reverse transcriptase PCR and / or hybridization assays such as Northern analysis, dot blot, slot blot, in-situ hybridization. , Transcription fusion using the ASN1 promoter fused to a reporter gene, DNA assay, and microarray assay.

  Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Western blots, ELISA assays, polyacrylamide gel electrophoresis, mass spectrometry, and enzyme assays. It is also possible to detect ASN1 protein expression using any reporter gene system. For detection using a gene reporter system, a polynucleotide encoding a reporter protein is fused in frame to ASN1 so as to produce a chimeric polypeptide. Methods using reporter systems are known to those skilled in the art.

  Thereafter, fungal growth can be controlled using the chemicals, compounds or compositions identified as modulators, preferably inhibitors, of ASN1 expression or activity by the methods described above. Diseases such as rust, powdery mildew, and blight can quickly spread once established. Therefore, fungicides are applied to growing and stored crops as a protective measure, routinely, generally as a foliar application or seed dressing. For example, to protect against fungal growth, compounds that inhibit fungal growth can be applied to or expressed in the fungus. Accordingly, the present invention provides a method for inhibiting fungal growth comprising contacting a fungus with a compound identified by the method of the present invention as having antifungal activity.

  Using the antifungal and antifungal inhibitor candidates identified by the methods of the present invention to control the growth of unwanted fungi, including ascomycetes, zygotes, basidiomycetes, astragalus, and lichens Is possible.

Examples of undesired fungi include, but are not limited to, Spongospora subterranea , Gray mold ( Botrytis cinerea ), White rot fungus ( Armillaria mellea ), Heartwood rot fungus ( umanoder ) Brown rot fungus (Piptoporus betulinus), corn smut (Ustilago maydis), heartwood rot fungus (Polyporus squamosus), black spot fungus (Cercospora zeae-maydis), honey fungus (Armillaria gallica), root rot fungus (Armillaria luteobubalina), shoes string rot fungus (Armillaria ostoyae) Banana anthracnose fungi (Colletotrichum musae), apples rot fungus (Monilinia fructigena), apples rot fungi (Penicillium expansum), root kelp fungus (Plasmodiophora brassicae), potato blight fungus (Phytophthora infestans), root pathogens (Heterobasidion annosum) , Withering fungi ( Gaeumanomyces graminis ), Dutch elm fungus ( Ophiostoma ulmi ), bean rust fungus ( Uromyces appendiculatus ), northern spot fungus ( Cochliobolus carbinorum bacterium), a), Southern corn blight fungus (Cochliobolus heterostrophus), leaf spot fungus (Cochliobolus lunata),褐条fungus (Cochliobolus stenospilus), Panama fungus (Fusarium oxysporum), wheat scab fungus (Fusarium graminearum), cereal root rot fungus ( Fusarium culmorum ), potato black husk fungus ( Rhizotonia solani ), wheat black rust ( Puccinia graminis ), white mold ( Sclerotinia sclerotiorum ), lung, blood, brain, skin, scalp, etc. animal disease (Aspergillus fumigatus, A pergillus species, Fusraium species, Trichophyton species, Epidermophyton species, and Microsporum species, etc.).

  Also provided is a test compound for an identified gene (SEQ ID NO: 1 or SEQ ID NO: 2), its gene product (SEQ ID NO: 3), or one or more biochemical pathways in which the gene product functions. Antibiotic screening method by determining whether it is active.

  In one particular embodiment, the first type of gene corresponding to either SEQ ID NO: 1 or SEQ ID NO: 2, either a normal, mutant, homolog, or heterologous ASN1 gene that performs a function similar to ASN1 The method is performed by providing an organism having The first type of ASN1 may or may not provide a growth condition phenotype, ie, an L-asparagine requirement phenotype, and / or a hypersensitivity or hyposensitivity phenotype of an organism having a modified type. In one particular embodiment, the mutant form contains a transposon insertion. A comparative organism having a second type of ASN1 different from the first type of gene is also provided, and the two organisms are contacted with the test compound separately. The growth of the two organisms is then compared in the presence of the test compound.

Accordingly, in one embodiment, the present invention is a method of identifying a test compound as an antibiotic candidate:
a) providing a cell having one type of asparagine synthase gene and providing a comparative cell having a different type of asparagine synthase gene; and b) contacting said cell and said comparative cell with a test compound and testing Measuring the proliferation of the cell and the comparative cell in the presence of a compound, wherein the test compound is an antibiotic that there is a difference in proliferation between the cell and the comparative cell in the presence of the test compound. The method is provided to indicate that a substance is a candidate.

In some cases, in the absence of all test compounds, the growth of the first organism and the second comparative organism is measured to determine that the genetic difference results in a unique difference in growth. It is recognized that both can be managed. It will also be appreciated that any combination of two different types of ASN1 gene can be used in the present methods, including normal genes, mutant genes, homologues, and functional homologues. The growth and / or proliferation of the organism is measured by methods well known in the art such as optical density measurement. In a preferred embodiment, the organism is Magnaporthe grisea .

Conditional lethal mutants can identify specific biochemical and / or genetic pathways if at least one identified target gene is present in the pathway. Knowledge of these pathways makes it possible to screen test compounds as antibiotic candidates as inhibitors of pathway substrates, products and enzymes. A route known in the art can be found in Kyoto Encyclopedia of Genes and Genomes and a standard biochemistry textbook (Lehninger, A., D. Nelson et al. (1993) Principles of Biochemistry, New York, New York, New York, United States). is there.

Accordingly, in one aspect, the present invention is a method of screening for test compounds that act on one or more biochemical and / or genetic pathways in which ASN1 functions:
a) providing a cell having one type of gene in the L-asparagine biochemical pathway and / or gene pathway and providing a comparative cell having a different type of said gene;
b) contacting said cell and said comparative cell with a test compound; and c) measuring the proliferation of said cell and said comparative cell in the presence of said test compound, wherein in the presence of said test compound, The method is provided wherein a difference in proliferation between the cell and the comparative cell indicates that the test compound is an antibiotic candidate.

  The use of multiwell plates for screening is a format that readily accommodates a number of different assays that characterize diverse compounds, compound concentrations, and fungal strains in a variety of combinations and formats. Certain test parameters of the screening method can significantly affect the identification of growth inhibitors, and therefore these parameters can be manipulated to optimize screening efficiency and / or reliability. is there. Notable among these factors are the various sensitivities of different mutants, hypersensitivity that increases as conditions become less permissive, hypersensitivity that apparently increases as compound concentrations increase, and Other factors known to those skilled in the art.

Conditional lethal mutants can identify specific biochemical and / or genetic pathways if at least one identified target gene is present in the pathway. Knowledge of these pathways makes it possible to screen test compounds as antibiotic candidates. Pathways known in the art can be found in Kyoto Encyclopedia of Genes and Genomes and standard biochemistry textbooks (Lehninger et al. (1993) Principles of Biochemistry ).

Accordingly, in one aspect, the present invention is a method of screening for test compounds that act on one or more biochemical and / or genetic pathways in which ASN1 functions:
(A) providing a paired growth medium comprising a first medium and a second medium, wherein the second medium contains a higher level of L-asparagine than the first medium;
(B) contacting the organism with the test compound;
(C) inoculating said first medium and said second medium with said organism; and (d) measuring the growth of said organism, said first medium and second medium Providing a method wherein the difference in organism growth indicates that the test compound is an antibiotic candidate.

In the art, measurement of the growth of the organism in a pair of media in the absence of all test compounds is performed to control any differences in media resulting in unique differences in growth. Is recognized as possible. The growth and / or proliferation of the organism is measured by methods well known in the art such as optical density measurement. In a preferred embodiment, the organism is Magnaporthe grisea .

The inventors of the present invention have discovered that disruption of the ALAS1 gene and / or gene product inhibits the virulence of Magnaporthe grisea . Thus, the inventors of the present invention have demonstrated for the first time that 5-aminolevulinate synthase is the target of antibiotics, preferably antifungal agents.

  Accordingly, the present invention provides methods for identifying compounds that inhibit ALAS1 gene expression or biological activity of its gene product (s). Such methods include ligand binding assays, enzyme activity assays, cell-based assays, and assays for ALAS1 gene expression. Any compound that is a ligand for 5-aminolevulinate synthase can have antibiotic activity. For the purposes of the present invention, “ligand” refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the present invention are useful as antibiotics.

Accordingly, in one embodiment, the present invention is a method of identifying a test compound as an antibiotic candidate:
a) contacting a test compound with a 5-aminolevulinate synthase polypeptide; and b) detecting the presence or absence of binding between the test compound and the 5-aminolevulinate synthase polypeptide;
The method is provided wherein binding indicates that the test compound is an antibiotic candidate.

The 5-aminolevulinate synthase protein can have the amino acid sequence of a naturally occurring 5-aminolevulinate synthase found in fungi, animals, plants, or microorganisms, and has an amino acid sequence derived from the naturally occurring sequence. It is possible. Preferably, the 5-aminolevulinate synthase is a fungal 5-aminolevulinate synthase. M.M. A cDNA encoding the grisea 5-aminolevulinate synthase protein (SEQ ID NO: 4), genomic DNA encoding the protein (SEQ ID NO: 5), and a polypeptide (SEQ ID NO: 6) may be found herein.

In one aspect, the present invention also provides a polypeptide consisting essentially of SEQ ID NO: 6. For purposes of the present invention, a polypeptide consisting essentially of SEQ ID NO: 6 has at least 80% sequence identity with SEQ ID NO: 6 and has at least 10% of the activity of SEQ ID NO: 6 with succinyl-CoA and glycine, 5-amino-levulinate, CoA, and the interconversion of CO ₂ to the catalyst. Preferably, the polypeptide consisting essentially of SEQ ID NO: 6 has at least 85% sequence identity with SEQ ID NO: 6, more preferably the sequence identity is at least 90%, most preferably the sequence identity Sex is at least 95% or 97% or 99%, or in ascending order with any integer sequence identity of 80-100%. And preferably, a polypeptide consisting essentially of SEQ ID NO: 6, M. It has at least 25%, at least 50%, at least 75%, or at least 90% of the activity of grisea 5-aminolevulinate synthase, or any integer between 60 and 100% in ascending order.

A “fungal 5-aminolevulinate synthase” is an enzyme that can be found in at least one fungus and catalyzes the interconversion of succinyl-CoA and glycine with 5-aminolevulinate, CoA, and CO _2. means. 5-Aminolevulinate synthase can be derived from any fungus, including ascomycetes, zygomycetes, basidiomycetes, fungi, and lichens.

In one embodiment, the 5-aminolevulinate synthase is Magnaporte 5-aminolevulinate synthase. The Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae, as well as Magnaporthe incomplete state of Pyricularia sp. Preferably, the Magnaporte 5-aminolevulinate synthase is derived from Magnaporthe grisea .

In various embodiments, 5-aminolevulinic acid synthase is a powdery scab ( Spongospora subterranea ), gray mold ( Botrytis cinerea ), white rot fungus ( Armillaria mellea ), heartwood rot fungus ( umanoderma fungus), Piptoporus betulinus), corn smut (Ustilago maydis), heartwood rot fungus (Polyporus squamosus), black spot fungus (Cercospora zeae-maydis), honey fungus (Armillaria gallica), root rot fungus (Armillaria luteobubalina), shoelaces rot fungus ( Armillaria ostoyae , Banana anthracnose fungi (Colletotrichum musae), apples rot fungus (Monilinia fructigena), apples rot fungi (Penicillium expansum), root kelp fungus (Plasmodiophora brassicae), potato blight fungus (Phytophthora infestans), root pathogens (Heterobasidion annosum ), Withering fungi ( Gaeumnomyces graminis ), Dutch elm fungus ( Ophiostoma ulmi ), legume rust fungus ( Uromyces appendiculatus ), northern spot fungus ( Cochliobolus carbinorum disease) ta), Southern corn blight fungus (Cochliobolus heterostrophus), leaf spot fungus (Cochliobolus lunata),褐条fungus (Cochliobolus stenospilus), Panama fungus (Fusarium oxysporum), wheat scab fungus (Fusarium graminearum), cereal root rot fungus ( Fusarium culmorum ), potato black rust fungus ( Rhizotonia solani ), wheat black rust ( Puccinia graminis ), white mold ( Sclerotinia sclerotiorum ) and the like.

  A fragment of a 5-aminolevulinate synthase polypeptide preferably contains an intact or nearly intact epitope that occurs in wild-type 5-aminolevulinate synthase where the fragment is biologically active. It can be used in the method of the present invention. The fragment comprises at least 10 consecutive amino acids of 5-aminolevulinate synthase. Preferably, the fragment is at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 of 5-aminolevulinate synthase. , 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, or at least 610 consecutive amino acid residues. In one embodiment, the fragment is derived from Magnaporte 5-aminolevulinate synthase. Preferably, the fragment contains an amino acid sequence that is conserved among fungal 5-aminolevulinate synthases.

  Polypeptides having at least 50% sequence identity with fungal 5-aminolevulinate synthase are also useful in the methods of the invention. Preferably the sequence identity is at least 60%, more preferably the sequence identity is at least 70%, most preferably the sequence identity is at least 80% or 90% or 95% or 99%, or in ascending order. , An integer of 60 to 100%.

Furthermore, it is preferred that the polypeptide has at least 10% of fungal 5-aminolevulinate synthase activity. More preferably, the polypeptide has at least 25%, at least 50%, at least 75% or at least 90% of fungal 5-aminolevulinate synthase activity. Most preferably, the polypeptide is M.P. has at least 10%, at least 25%, at least 50%, at least 75% or at least 90% of the grisea 5-aminolevulinate synthase protein activity.

Thus, in another aspect, the invention provides a method for identifying a test compound as a candidate for a fungicide:
a) with a test compound: a polypeptide having at least 10 consecutive amino acids of fungal 5-aminolevulinate synthase; a polypeptide having at least 50% sequence identity with fungal 5-aminolevulinate synthase; and of fungal 5-aminolevulinate synthase Contacting at least one polypeptide selected from the group consisting of polypeptides having at least 10% of activity; and b) detecting the presence and / or absence of binding between said test compound and said polypeptide Including:
The method is provided wherein binding indicates that the test compound is an antibiotic candidate.

  Any technique that detects binding of a ligand to a target can be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting binding of a ligand to a target are known in the art and include, but are not limited to, detection of a fixed ligand-target complex, or target characteristics when a target binds to a ligand. Change detection is included. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligand is contacted with the 5-aminolevulinate synthase protein, or a fragment or variant thereof, the unbound protein is removed, and the bound 5-aminolevulinate synthase is detected. In a preferred embodiment, a labeled binding partner such as a labeled antibody is used to detect bound 5-aminolevulinate synthase. In a variation of this assay, 5-aminolevulinate synthase is labeled prior to contacting the immobilized candidate ligand. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetry.

  Once a compound has been identified as an antibiotic candidate, it can be tested for its ability to inhibit 5-aminolevulinate synthase enzyme activity. Compounds can be tested using either in vitro assays or cell based assays. Alternatively, the compound can be applied directly to the fungus or fungal cell or expressed in the fungus or fungal cell and monitored for growth, development, viability, pathogenic change or reduction, or gene expression modification Can be tested. Accordingly, in one aspect, the present invention is a method for determining whether a compound identified as an antibiotic candidate by the method described above has antifungal activity comprising: a fungus or fungal cell and said antifungal agent candidate And detecting a decrease in growth, viability, or pathogenicity of the fungus or fungal cell.

  By reducing the growth is meant that the antifungal candidate causes at least a 10% reduction in fungal or fungal cell growth as compared to the fungal or fungal cell growth in the absence of the antifungal candidate. . By reduced viability is meant that at least 20% of the fungal cells, or fungal parts, contacted with the antifungal agent candidate are not viable. Preferably, proliferation or viability will be reduced by at least 40%. More preferably, proliferation or viability will be reduced by at least 50%, 75% or at least 90% or more. Methods for measuring fungal growth and cell viability are known to those skilled in the art. Reduced virulence causes an antifungal candidate to cause at least a 10% reduction in disease caused by contact of the fungal pathogen with its host when compared to disease caused in the absence of the antifungal candidate Means. Preferably, the disease will be reduced by at least 40%. More preferably, the disease will be reduced by at least 50%, 75% or at least 90% or more. Methods for measuring fungal diseases are well known to those skilled in the art and include metrics such as lesion formation, lesion size, sporulation, respiratory failure, and / or death.

The ability of a compound to inhibit 5-aminolevulinate synthase activity can be detected using an in vitro enzyme assay that directly or indirectly detects the disappearance of a substrate or the appearance of a product. 5-aminolevulinate synthase catalyzes an irreversible or reversible reaction, succinyl-CoA and glycine = 5-aminolevulinate, CoA, and CO ₂ (see FIG. 1). Succinyl-CoA, glycine, 5-amino-levulinate, the method of detecting the CoA, and / or CO _2, spectrophotometry, mass spectrometry, include thin layer chromatography (TLC) and Reversed Phase HPLC.

Accordingly, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) contacting succinyl-CoA and glycine with 5-aminolevulinate synthase;
b) contacting succinyl-CoA and glycine with 5-aminolevulinate synthase and a test compound; and c) the following: at least one of succinyl-CoA, glycine, 5-aminolevulinate, CoA, and / or CO ₂ Including measuring concentration changes,
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

The present invention is a further method for identifying test compounds as antibiotic candidates:
a) contacting 5-aminolevulinate, CoA, and CO ₂ with 5-aminolevulinate synthase;
b) contacting 5-aminolevulinate, CoA, and CO ₂ with 5-aminolevulinate synthase and a test compound; and c) the following: succinyl-CoA, glycine, 5-aminolevulinate, CoA, and / or Or measuring at least one concentration change of CO ₂ ;
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

  Enzymatically active fragments of fungal 5-aminolevulinate synthase are also useful in the methods of the invention. For example, a polypeptide comprising at least 100 contiguous amino acid residues of fungal 5-aminolevulinate synthase and being enzymatically active can be used in the methods of the invention. In addition, a polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity and fungal 5-aminolevulinate synthase is enzymatically active. It can be used in the inventive method. Most preferably, the polypeptide has at least 50% sequence identity with fungal 5-aminolevulinate synthase and has at least 10%, 25%, 75%, or at least 90% of its activity.

Accordingly, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) with succinyl-CoA and glycine: a polypeptide having at least 50% sequence identity with 5-aminolevulinate synthase; having at least 50% sequence identity with 5-aminolevulinate synthase and at least 10 of its activity Contacting a polypeptide selected from the group consisting of a polypeptide comprising at least 100 contiguous amino acids of 5-aminolevulinate synthase;
b) contacting said polypeptide and test compound with succinyl-CoA and glycine; and c) following: at least one concentration change of succinyl-CoA, glycine, 5-aminolevulinate, CoA, and / or CO ₂ Including measuring;
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

The present invention is a further method of identifying a test compound as an antibiotic candidate:
a) 5-Amino levulinate, CoA, and CO ₂ and 5-aminolevulinic acid synthase polypeptide having at least 50% sequence identity; with 5-aminolevulinic acid synthase having at least 50% sequence identity And a polypeptide having at least 10% of its activity; and a polypeptide selected from the group consisting of a polypeptide comprising at least 100 contiguous amino acids of 5-aminolevulinate synthase;
b) 5-amino-levulinate, CoA, and the CO _2, wherein contacting the polypeptide and a test compound; and c) below: succinyl-CoA, glycine, 5-amino-levulinate, CoA, and / or CO ₂ comprises measuring at least one change in density;
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

  For in vitro enzyme assays, the 5-aminolevulinate synthase protein and its derivatives can be purified from fungi or produced recombinantly in archaea, bacteria, fungi, or other eukaryotic cell cultures. And can be purified from these cultures. Preferably, these proteins are produced using E. coli, yeast, or filamentous fungal expression systems. A method for purifying 5-aminolevulinate synthase can be that described in Volland and Felix (1984) Eur J Biochem 142: 551-7 (PMID: 6381051). Other methods of purifying 5-aminolevulinate synthase proteins and polypeptides are known to those skilled in the art.

As an alternative to in vitro assays, the present invention also provides cell-based assays. In one embodiment, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) measuring the expression of 5-aminolevulinate synthase in the absence of the test compound in a single cell, a plurality of cells, tissues or organisms;
b) contacting the test compound with the single cell, cells, tissues or organism and expressing the 5-aminolevulinate synthase in the single cell, cells, tissues or organism And c) comparing the expression of 5-aminolevulinate synthase in steps (a) and (b);
The method is provided wherein lower expression in the presence of the test compound indicates that the compound is an antibiotic candidate.

  The expression of 5-aminolevulinate synthase can be measured by detecting ALAS1 primary transcript or mRNA, 5-aminolevulinate synthase polypeptide, or 5-aminolevulinate synthase enzyme activity. Methods of detecting RNA and protein expression are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1995. The detection method is not critical to the present invention. Methods for detecting ALAS1 RNA include, but are not limited to, amplification assays such as quantitative reverse transcriptase PCR and / or hybridization assays such as Northern analysis, dot blot, slot blot, in-situ hybridization. , Transcription fusion using the ALAS1 promoter fused to a reporter gene, DNA assays, and microarray assays.

  Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Western blots, ELISA assays, polyacrylamide gel electrophoresis, mass spectrometry, and enzyme assays. It is also possible to detect ALAS1 protein expression using any reporter gene system. For detection using a gene reporter system, a polynucleotide encoding a reporter protein is fused in frame to ALAS1 to produce a chimeric polypeptide. Methods using reporter systems are known to those skilled in the art.

  Thereafter, fungal growth can be controlled using the chemicals, compounds or compositions identified as modulators, preferably inhibitors, of ALAS1 expression or activity by the methods described above. Diseases such as rust, powdery mildew, and blight can quickly spread once established. Therefore, fungicides are applied to growing and stored crops as a protective measure, routinely, generally as a foliar application or seed dressing. For example, to protect against fungal growth, compounds that inhibit fungal growth can be applied to or expressed in the fungus. Accordingly, the present invention provides a method for inhibiting fungal growth comprising contacting a fungus with a compound identified by the method of the present invention as having antifungal activity.

  Using the antifungal and antifungal inhibitor candidates identified by the methods of the present invention to control the growth of unwanted fungi, including ascomycetes, zygotes, basidiomycetes, astragalus, and lichens Is possible.

Examples of undesired fungi include, but are not limited to, Spongospora subterranea , Gray mold ( Botrytis cinerea ), White rot fungus ( Armillaria mellea ), Heartwood rot fungus ( umanoder ) Brown rot fungus (Piptoporus betulinus), corn smut (Ustilago maydis), heartwood rot fungus (Polyporus squamosus), black spot fungus (Cercospora zeae-maydis), honey fungus (Armillaria gallica), root rot fungus (Armillaria luteobubalina), shoes string rot fungus (Armillaria ostoyae) Banana anthracnose fungi (Colletotrichum musae), apples rot fungus (Monilinia fructigena), apples rot fungi (Penicillium expansum), root kelp fungus (Plasmodiophora brassicae), potato blight fungus (Phytophthora infestans), root pathogens (Heterobasidion annosum) , Withering fungi ( Gaeumanomyces graminis ), Dutch elm fungus ( Ophiostoma ulmi ), bean rust fungus ( Uromyces appendiculatus ), northern spot fungus ( Cochliobolus carbinorum bacterium), a), Southern corn blight fungus (Cochliobolus heterostrophus), leaf spot fungus (Cochliobolus lunata),褐条fungus (Cochliobolus stenospilus), Panama fungus (Fusarium oxysporum), wheat scab fungus (Fusarium graminearum), cereal root rot fungus ( Fusarium culmorum ), potato black husk fungus ( Rhizotonia solani ), wheat black rust ( Puccinia graminis ), white mold ( Sclerotinia sclerotiorum ), lung, blood, brain, skin, scalp, etc. animal disease (Aspergillus fumigatus, A pergillus species, Fusraium species, Trichophyton species, Epidermophyton species, and Microsporum species, etc.).

  Also provided is a test compound for an identified gene (SEQ ID NO: 4 or SEQ ID NO: 5), its gene product (SEQ ID NO: 6), or one or more biochemical pathways in which the gene product functions. Antibiotic screening method by determining whether it is active.

  In one particular embodiment, the first type of the gene corresponding to either SEQ ID NO: 4 or SEQ ID NO: 5, either normal, mutant, homolog, or a heterologous ALAS1 gene that performs a function similar to ALAS1 The method is performed by providing an organism having The first type of ALAS1 may or may not provide a growth condition phenotype, ie, a 5-aminolevulinate requirement phenotype, and / or a hypersensitivity or reduced sensitivity phenotype of an organism having a modified type. Good. In one particular embodiment, the mutant form contains a transposon insertion. A comparative organism having a second type of ALAS1 that is different from the first type of gene is also provided, and the two organisms are contacted separately with the test compound. The growth of the two organisms is then compared in the presence of the test compound.

Accordingly, in one embodiment, the present invention is a method of identifying a test compound as an antibiotic candidate:
a) providing a cell having one type of 5-aminolevulinate synthase gene and providing a comparative cell having a different type of 5-aminolevulinate synthase gene; and b) said cell and said comparative cell and a test compound And measuring the proliferation of said cells and said comparative cells in the presence of a test compound,
The method is provided wherein a difference in proliferation between the cell and the comparative cell in the presence of the test compound indicates that the test compound is an antibiotic candidate.

In some cases, in the absence of all test compounds, the growth of the first organism and the second comparative organism is measured to determine that the genetic difference results in a unique difference in growth. It is recognized that both can be managed. It will also be appreciated that any combination of two different types of ALAS1 gene can be used in the present method, including normal genes, mutant genes, homologues, and functional homologues. The growth and / or proliferation of the organism is measured by methods well known in the art such as optical density measurement. In a preferred embodiment, the organism is Magnaporthe grisea .

Conditional lethal mutants can identify specific biochemical and / or genetic pathways if at least one identified target gene is present in the pathway. Knowledge of these pathways makes it possible to screen test compounds as antibiotic candidates as inhibitors of pathway substrates, products and enzymes. A route known in the art can be found in Kyoto Encyclopedia of Genes and Genomes and a standard biochemistry textbook (Lehninger, A., D. Nelson et al. (1993) Principles of Biochemistry, New York, New York, New York, United States). is there.

Accordingly, in one aspect, the invention is a method of screening for a test compound that acts on one or more biochemical and / or genetic pathways in which ALAS1 functions:
d) providing a cell having one type of gene in the heme biochemical pathway and / or gene pathway, and providing a comparative cell having a different type of said gene;
e) contacting the cell and the comparative cell with a test compound; and f) measuring the proliferation of the cell and the comparative cell in the presence of the test compound;
The method is provided wherein a difference in proliferation between the cell and the comparative cell in the presence of the test compound indicates that the test compound is an antibiotic candidate.

  The use of multiwell plates for screening is a format that readily accommodates a number of different assays that characterize diverse compounds, compound concentrations, and fungal strains in a variety of combinations and formats. Certain test parameters of the screening method can significantly affect the identification of growth inhibitors, and therefore these parameters can be manipulated to optimize screening efficiency and / or reliability. is there. Notable among these factors are the various sensitivities of different mutants, hypersensitivity that increases as conditions become less permissive, hypersensitivity that apparently increases as compound concentrations increase, and Other factors known to those skilled in the art.

Conditional lethal mutants can identify specific biochemical and / or genetic pathways if at least one identified target gene is present in the pathway. Knowledge of these pathways makes it possible to screen test compounds as antibiotic candidates. A route known in the art can be found in Kyoto Encyclopedia of Genes and Genomes and a standard biochemistry textbook (Lehninger, A., D. Nelson et al. (1993) Principles of Biochemistry, New York, New York, New York, United States). is there.

Accordingly, in one aspect, the invention is a method of screening for a test compound that acts on one or more biochemical and / or genetic pathways in which ALAS1 functions:
(A) providing a paired growth medium comprising a first medium and a second medium, wherein the second medium contains a higher level of 5-aminolevulinate than the first medium; And
(B) contacting the organism with the test compound;
(C) inoculating said first medium and said second medium with said organism; and (d) measuring the growth of said organism;
The method is provided wherein a difference in organism growth between the first medium and the second medium indicates that the test compound is a candidate antibiotic.

In the art, measurement of the growth of the organism in a pair of media in the absence of all test compounds is performed to control any differences in media resulting in unique differences in growth. Is recognized as possible. The growth and / or proliferation of the organism is measured by methods well known in the art such as optical density measurement. In a preferred embodiment, the organism is Magnaporthe grisea .

The inventors of the present invention have discovered that disruption of the HISP1 gene and / or gene product inhibits the virulence of Magnaporthe grisea . Thus, the inventors of the present invention have demonstrated for the first time that histidinol-phosphatase is a target for antibiotics, preferably antifungal agents.

  Accordingly, the present invention provides methods for identifying compounds that inhibit HISP1 gene expression or the biological activity of its gene product (s). Such methods include ligand binding assays, enzyme activity assays, cell based assays, and assays for HISP1 gene expression. Any compound that is a ligand for histidinol-phosphatase can have antibiotic activity. For the purposes of the present invention, “ligand” refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the present invention are useful as antibiotics.

Accordingly, in one embodiment, the present invention is a method of identifying a test compound as an antibiotic candidate:
a) contacting the test compound with a histidinol-phosphatase polypeptide; and b) detecting the presence or absence of binding between the test compound and the histidinol-phosphatase polypeptide;
The method is provided wherein binding indicates that the test compound is an antibiotic candidate.

The histidinol-phosphatase protein can have the amino acid sequence of naturally occurring histidinol-phosphatase found in fungi, animals, plants, or microorganisms, and can have an amino acid sequence derived from the naturally occurring sequence. is there. Preferably the histidinol-phosphatase is a fungal histidinol-phosphatase. A cDNA encoding the histidinol-phosphatase protein (SEQ ID NO: 7), M.P. Genomic DNA encoding the grisea protein (SEQ ID NO: 8), and polypeptide (SEQ ID NO: 9) can be found herein.

In one aspect, the present invention also provides a polypeptide consisting essentially of SEQ ID NO: 9. For the purposes of the present invention, a polypeptide consisting essentially of SEQ ID NO: 9 has at least 80% sequence identity with SEQ ID NO: 9 and has at least 10% of the activity of SEQ ID NO: 9 with L-histidine. and Nord phosphate and H _{2 O,} the L- histidinol and orthophosphate mutual conversion catalyst. Preferably, the polypeptide consisting essentially of SEQ ID NO: 9 has at least 85% sequence identity with SEQ ID NO: 9, more preferably the sequence identity is at least 90%, most preferably the sequence identity Sex is at least 95% or 97% or 99%, or in ascending order with any integer sequence identity of 80-100%. And preferably, a polypeptide consisting essentially of SEQ ID NO: 9, M. It has an activity of at least 25%, at least 50%, at least 75%, or at least 90% of the activity of grisea histidinol-phosphatase, or any integer from 60-100% in ascending order.

“Fungus histidinol-phosphatase” means an enzyme that can be found in at least one fungus and catalyzes the interconversion of L-histidinol phosphate and H ₂ O with L-histidinol and orthophosphate. Histidinol-phosphatase can be derived from any fungus, including ascomycetes, zygomycetes, basidiomycetes, fungi, and lichens.

In one embodiment, the histidinol-phosphatase is Magnaporte histidinol-phosphatase. The Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae, as well as Magnaporthe incomplete state of Pyricularia sp. Preferably, the Magnaporte histidinol-phosphatase is derived from Magnaporthe grisea .

In various embodiments, histidinol - phosphatase, powdery scab fungus (Spongospora subterranea), gray mold (Botrytis cinerea), white rot fungus (Armillaria mellea), core rot fungus (Ganoderma adspersum), brown rot fungi (Piptoporus betulinus ), corn smut (Ustilago maydis), heartwood rot fungus (Polyporus squamosus), black spot fungus (Cercospora zeae-maydis), honey fungus (Armillaria gallica), root rot fungus (Armillaria luteobubalina), shoelaces rot fungus (Armillaria ostoyae , Banana anthracnose fungi (Colletotrichum musae), apples rot fungus (Monilinia fructigena), apples rot fungi (Penicillium expansum), root kelp fungus (Plasmodiophora brassicae), potato blight fungus (Phytophthora infestans), root pathogens (Heterobasidion annosum ), Withering fungi ( Gaeumnomyces graminis ), Dutch elm fungus ( Ophiostoma ulmi ), legume rust fungus ( Uromyces appendiculatus ), northern spot fungus ( Cochliobolus carbinorum disease) ta), Southern corn blight fungus (Cochliobolus heterostrophus), leaf spot fungus (Cochliobolus lunata),褐条fungus (Cochliobolus stenospilus), Panama fungus (Fusarium oxysporum), wheat scab fungus (Fusarium graminearum), cereal root rot fungus ( Fusarium culmorum ), potato black rust fungus ( Rhizotonia solani ), wheat black rust ( Puccinia graminis ), white mold ( Sclerotinia sclerotiorum ) and the like.

  A fragment of a histidinol-phosphatase polypeptide preferably comprises the intact or near intact epitope that occurs in a wild-type histidinol-phosphatase where the fragment is biologically active. Can be used. The fragment comprises at least 10 contiguous amino acids of histidinol-phosphatase. Preferably, the fragment is at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 of histidinol-phosphatase. , 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, or at least 330 consecutive amino acid residues. In one embodiment, the fragment is derived from Magnaporte histidinol-phosphatase. Preferably, the fragment contains an amino acid sequence that is conserved between fungal histidinol-phosphatases.

  Polypeptides having at least 50% sequence identity with the fungal histidinol-phosphatase are also useful in the methods of the invention. Preferably the sequence identity is at least 60%, more preferably the sequence identity is at least 70%, most preferably the sequence identity is at least 80% or 90% or 95% or 99%, or in ascending order. , An integer of 60 to 100%.

Furthermore, it is preferred that the polypeptide has at least 10% of fungal histidinol-phosphatase activity. More preferably, the polypeptide has at least 25%, at least 50%, at least 75% or at least 90% of the fungal histidinol-phosphatase activity. Most preferably, the polypeptide is M.P. have at least 10%, at least 25%, at least 50%, at least 75% or at least 90% of the grisea histidinol-phosphatase protein activity.

Thus, in another aspect, the invention provides a method for identifying a test compound as a candidate for a fungicide:
a) with a test compound: a polypeptide having at least 10 consecutive amino acids of fungal histidinol-phosphatase; a polypeptide having at least 50% sequence identity with fungal histidinol-phosphatase; and at least 10% of the activity of fungal histidinol-phosphatase Contacting at least one polypeptide selected from the group consisting of polypeptides having; and b) detecting the presence and / or absence of binding between said test compound and said polypeptide;
The method is provided wherein binding indicates that the test compound is an antibiotic candidate.

  Any technique that detects binding of a ligand to a target can be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting binding of a ligand to a target are known in the art and include, but are not limited to, detection of a fixed ligand-target complex, or target characteristics when a target binds to a ligand. Change detection is included. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligand is contacted with a histidinol-phosphatase protein, or a fragment or variant thereof, the unbound protein is removed, and the bound histidinol-phosphatase is detected. In a preferred embodiment, a labeled binding partner such as a labeled antibody is used to detect bound histidinol-phosphatase. In a variation of this assay, the histidinol-phosphatase is labeled prior to contacting the immobilized candidate ligand. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetry.

  Once a compound has been identified as an antibiotic candidate, it can be tested for its ability to inhibit histidinol-phosphatase enzyme activity. Compounds can be tested using either in vitro assays or cell based assays. Alternatively, the compound can be applied directly to the fungus or fungal cell or expressed in the fungus or fungal cell and monitored for growth, development, viability, pathogenic change or reduction, or gene expression modification Can be tested. Accordingly, in one aspect, the present invention is a method for determining whether a compound identified as an antibiotic candidate by the method described above has antifungal activity comprising: a fungus or fungal cell and said antifungal agent candidate And detecting a decrease in growth, viability, or pathogenicity of the fungus or fungal cell.

  By reducing the growth is meant that the antifungal candidate causes at least a 10% reduction in fungal or fungal cell growth as compared to the fungal or fungal cell growth in the absence of the antifungal candidate. . By reduced viability is meant that at least 20% of the fungal cells, or fungal parts, contacted with the antifungal agent candidate are not viable. Preferably, proliferation or viability will be reduced by at least 40%. More preferably, proliferation or viability will be reduced by at least 50%, 75% or at least 90% or more. Methods for measuring fungal growth and cell viability are known to those skilled in the art. Reduced virulence causes an antifungal candidate to cause at least a 10% reduction in disease caused by contact of the fungal pathogen with its host when compared to disease caused in the absence of the antifungal candidate Means. Preferably, the disease will be reduced by at least 40%. More preferably, the disease will be reduced by at least 50%, 75% or at least 90% or more. Methods for measuring fungal diseases are well known to those skilled in the art and include metrics such as lesion formation, lesion size, sporulation, respiratory failure, and / or death.

The ability of a compound to inhibit histidinol-phosphatase activity can be detected using an in vitro enzyme assay that directly or indirectly detects the disappearance of a substrate or the appearance of a product. Histidinol-phosphatase catalyzes irreversible or reversible reactions, L-histidinol phosphate and H ₂ O = L-histidinol and orthophosphate (see FIG. 1). Methods for detecting L-histidinol phosphate, H ₂ O, L-histidinol, and / or orthophosphate include spectrophotometry, mass spectrometry, thin layer chromatography (TLC) and reverse phase HPLC.

Accordingly, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) the L- histidinol phosphate and H _{2 O,} histidinol - contacting the phosphatase;
b) contacting L-histidinol phosphate and H ₂ O with histidinol-phosphatase and said test compound; and c) the following: L-histidinol phosphate, H ₂ O, L-histidinol, and / or orthophosphate Measuring at least one concentration change of
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

The present invention is a further method for identifying test compounds as antibiotic candidates:
a) contacting L-histidinol and orthophosphate with histidinol-phosphatase;
b) contacting L-histidinol and orthophosphate with histidinol-phosphatase and test compound; and c) at least one concentration of L-histidinol phosphate, H ₂ O, L-histidinol, and / or orthophosphate Including measuring changes,
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

  Enzymatically active fragments of the fungal histidinol-phosphatase are also useful in the methods of the invention. For example, a polypeptide comprising at least 100 contiguous amino acid residues of fungal histidinol-phosphatase and being enzymatically active can be used in the methods of the invention. Furthermore, a polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity and fungal histidinol-phosphatase is enzymatically active according to the invention. Can be used in the way. Most preferably, the polypeptide has at least 50% sequence identity with the fungal histidinol-phosphatase and has at least 10%, 25%, 75%, or at least 90% of its activity.

Accordingly, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) with L-histidinol phosphate and H ₂ O: a polypeptide having at least 50% sequence identity with histidinol-phosphatase; having at least 50% sequence identity with histidinol-phosphatase and at least of its activity Contacting a polypeptide selected from the group consisting of: a polypeptide having 10%; and a polypeptide comprising at least 100 contiguous amino acids of histidinol-phosphatase;
b) contacting L-histidinol phosphate and H ₂ O with said polypeptide and test compound; and c) the following: L-histidinol phosphate, H ₂ O, L-histidinol, and / or orthophosphate Measuring at least one concentration change,
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

The present invention is a further method of identifying a test compound as an antibiotic candidate:
a) with L-histidinol and orthophosphate: a polypeptide having at least 50% sequence identity with histidinol-phosphatase; having at least 50% sequence identity with histidinol-phosphatase and having at least 10% of its activity Contacting a polypeptide selected from the group consisting of a polypeptide; and a polypeptide comprising at least 100 contiguous amino acids of histidinol-phosphatase;
b) contacting L-histidinol and orthophosphate with said polypeptide and test compound; and c) the following: at least one concentration of L-histidinol phosphate, H ₂ O, L-histidinol, and / or orthophosphate Including measuring changes;
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

  For in vitro enzyme assays, the histidinol-phosphatase protein and its derivatives can be purified from fungi or produced recombinantly in archaea, bacteria, fungi, or other eukaryotic cell cultures, and It is possible to purify from these cultures. Preferably, these proteins are produced using E. coli, yeast, or filamentous fungal expression systems. Methods for purifying histidinol-phosphatase can be those described in Millay and Houston (1973) Biochemistry 12: 2591-2596 (PMID: 4351203). Other methods of purifying histidinol-phosphatase proteins and polypeptides are known to those skilled in the art.

As an alternative to in vitro assays, the present invention also provides cell-based assays. In one embodiment, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) measuring the expression of histidinol-phosphatase in a single cell, a plurality of cells, tissues or organisms in the absence of the test compound;
b) contacting the test compound with the cell, cells, tissues or organism and measuring the expression of the histidinol-phosphatase in the cell, cells, tissues or organism And c) comparing the expression of histidinol-phosphatase in steps (a) and (b);
The method is provided wherein lower expression in the presence of the test compound indicates that the compound is an antibiotic candidate.

  The expression of histidinol-phosphatase can be measured by detecting HISP1 primary transcript or mRNA, histidinol-phosphatase polypeptide, or histidinol-phosphatase enzyme activity. Methods of detecting RNA and protein expression are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1995. The detection method is not critical to the present invention. Methods for detecting HISP1 RNA include but are not limited to amplification assays such as quantitative reverse transcriptase PCR and / or hybridization assays such as Northern analysis, dot blot, slot blot, in-situ hybridization. , Transcription fusion using the HISP1 promoter fused to a reporter gene, DNA assays, and microarray assays.

  Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Western blots, ELISA assays, polyacrylamide gel electrophoresis, mass spectrometry, and enzyme assays. It is also possible to detect HISP1 protein expression using any reporter gene system. For detection using a gene reporter system, a polynucleotide encoding a reporter protein is fused in-frame to HISP1 to produce a chimeric polypeptide. Methods using reporter systems are known to those skilled in the art.

  Thereafter, fungal growth can be controlled by the methods described above using chemicals, compounds or compositions identified as modulators, preferably inhibitors, of HISP1 expression or activity. Diseases such as rust, powdery mildew, and blight can quickly spread once established. Therefore, fungicides are applied to growing and stored crops as a protective measure, routinely, generally as a foliar application or seed dressing. For example, to protect against fungal growth, compounds that inhibit fungal growth can be applied to or expressed in the fungus. Accordingly, the present invention provides a method for inhibiting fungal growth comprising contacting a fungus with a compound identified by the method of the present invention as having antifungal activity.

  Using the antifungal and antifungal inhibitor candidates identified by the methods of the present invention to control the growth of unwanted fungi, including ascomycetes, zygotes, basidiomycetes, astragalus, and lichens Is possible.

Examples of undesired fungi include, but are not limited to, Spongospora subterranea , Gray mold ( Botrytis cinerea ), White rot fungus ( Armillaria mellea ), Heartwood rot fungus ( umanoder ) Brown rot fungus (Piptoporus betulinus), corn smut (Ustilago maydis), heartwood rot fungus (Polyporus squamosus), black spot fungus (Cercospora zeae-maydis), honey fungus (Armillaria gallica), root rot fungus (Armillaria luteobubalina), shoes string rot fungus (Armillaria ostoyae) Banana anthracnose fungi (Colletotrichum musae), apples rot fungus (Monilinia fructigena), apples rot fungi (Penicillium expansum), root kelp fungus (Plasmodiophora brassicae), potato blight fungus (Phytophthora infestans), root pathogens (Heterobasidion annosum) , Withering fungi ( Gaeumanomyces graminis ), Dutch elm fungus ( Ophiostoma ulmi ), bean rust fungus ( Uromyces appendiculatus ), northern spot fungus ( Cochliobolus carbinorum bacterium), a), Southern corn blight fungus (Cochliobolus heterostrophus), leaf spot fungus (Cochliobolus lunata),褐条fungus (Cochliobolus stenospilus), Panama fungus (Fusarium oxysporum), wheat scab fungus (Fusarium graminearum), cereal root rot fungus ( Fusarium culmorum ), potato black husk fungus ( Rhizotonia solani ), wheat black rust ( Puccinia graminis ), white mold ( Sclerotinia sclerotiorum ), lung, blood, brain, skin, scalp, etc. animal disease (Aspergillus fumigatus, A pergillus species, Fusraium species, Trichophyton species, Epidermophyton species, and Microsporum species, etc.).

  Also provided is a test compound for the identified gene (SEQ ID NO: 7 or SEQ ID NO: 8), its gene product (SEQ ID NO: 9), or the biochemical pathway or functions for which the gene product functions. Antibiotic screening method by determining whether it is active.

  In one particular embodiment, the first type of gene corresponding to either SEQ ID NO: 7 or SEQ ID NO: 8, either normal, mutant, homologous, or a heterologous HISP1 gene that performs a function similar to HISP1 The method is performed by providing an organism having The first type of HISP1 may or may not give a growth condition phenotype, ie an L-histidine requirement phenotype, and / or a hypersensitivity or reduced sensitivity phenotype of an organism having a modified type. In one particular embodiment, the mutant form contains a transposon insertion. A comparative organism having a second type of HISP1 that differs from the first type of gene is also provided, and the two organisms are contacted with the test compound separately. The growth of the two organisms is then compared in the presence of the test compound.

Accordingly, in one embodiment, the present invention is a method of identifying a test compound as an antibiotic candidate:
a) providing a cell having one type of histidinol-phosphatase gene and providing a comparative cell having a different type of histidinol-phosphatase gene; and b) contacting said cell and said comparative cell with a test compound; And measuring the proliferation of the cell and the comparative cell in the presence of a test compound,
The method is provided wherein a difference in proliferation between the cell and the comparative cell in the presence of the test compound indicates that the test compound is an antibiotic candidate.

In some cases, in the absence of all test compounds, the growth of the first organism and the second comparative organism is measured to determine that the genetic difference results in a unique difference in growth. It is recognized that both can be managed. It will also be appreciated that any combination of two different types of HISP1 genes can be used in the present methods, including normal genes, mutant genes, homologues, and functional homologues. The growth and / or proliferation of the organism is measured by methods well known in the art such as optical density measurement. In a preferred embodiment, the organism is Magnaporthe grisea .

Conditional lethal mutants can identify specific biochemical and / or genetic pathways if at least one identified target gene is present in the pathway. Knowledge of these pathways makes it possible to screen test compounds as antibiotic candidates as inhibitors of pathway substrates, products and enzymes. A route known in the art can be found in Kyoto Encyclopedia of Genes and Genomes and a standard biochemistry textbook (Lehninger, A., D. Nelson et al. (1993) Principles of Biochemistry, New York, New York, New York, United States). is there.

Accordingly, in one aspect, the invention is a method of screening for test compounds that act on one or more biochemical and / or genetic pathways in which HISP1 functions:
g) providing a cell having one type of gene in the L-histidine biochemical pathway and / or gene pathway, and providing a comparative cell having a different type of said gene;
h) contacting said cell and said comparative cell with a test compound; and i) measuring the proliferation of said cell and said comparative cell in the presence of said test compound;
The method is provided wherein a difference in proliferation between the cell and the comparative cell in the presence of the test compound indicates that the test compound is an antibiotic candidate.

  The use of multiwell plates for screening is a format that readily accommodates a number of different assays that characterize diverse compounds, compound concentrations, and fungal strains in a variety of combinations and formats. Certain test parameters of the screening method can significantly affect the identification of growth inhibitors, and therefore these parameters can be manipulated to optimize screening efficiency and / or reliability. is there. Notable among these factors are the various sensitivities of different mutants, hypersensitivity that increases as conditions become less permissive, hypersensitivity that apparently increases as compound concentrations increase, and Other factors known to those skilled in the art.

Conditional lethal mutants can identify specific biochemical and / or genetic pathways if at least one identified target gene is present in the pathway. Knowledge of these pathways makes it possible to screen test compounds as antibiotic candidates. A route known in the art can be found in Kyoto Encyclopedia of Genes and Genomes and a standard biochemistry textbook (Lehninger, A., D. Nelson et al. (1993) Principles of Biochemistry, New York, New York, New York, United States). is there.

Accordingly, in one aspect, the invention is a method of screening for test compounds that act on one or more biochemical and / or genetic pathways in which HISP1 functions:
(A) providing a paired growth medium comprising a first medium and a second medium, wherein the second medium contains a higher level of L-histidine than the first medium;
(B) contacting the organism with the test compound;
(C) inoculating said first medium and said second medium with said organism; and (d) measuring the growth of said organism;
The method is provided wherein a difference in organism growth between the first medium and the second medium indicates that the test compound is a candidate antibiotic.

In the art, measurement of the growth of the organism in a pair of media in the absence of all test compounds is performed to control any differences in media resulting in unique differences in growth. Is recognized as possible. The growth and / or proliferation of the organism is measured by methods well known in the art such as optical density measurement. In a preferred embodiment, the organism is Magnaporthe grisea .

The inventors of the present invention have discovered that disruption of the IPMD1 gene and / or gene product inhibits the virulence of Magnaporthe grisea . Thus, the inventors of the present invention have demonstrated for the first time that 3-isopropylmalate dehydratase is a target for antibiotics, preferably antifungal agents.

  Accordingly, the present invention provides methods for identifying compounds that inhibit IPMD1 gene expression or biological activity of its gene product (s). Such methods include ligand binding assays, enzyme activity assays, cell based assays, and assays for IPMD1 gene expression. Any compound that is a ligand for 3-isopropylmalate dehydratase can have antibiotic activity. For the purposes of the present invention, “ligand” refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the present invention are useful as antibiotics.

Accordingly, in one embodiment, the present invention is a method of identifying a test compound as an antibiotic candidate:
a) contacting a test compound with a 3-isopropylmalate dehydratase polypeptide; and b) detecting the presence or absence of a bond between the test compound and the 3-isopropylmalate dehydratase polypeptide;
The method is provided wherein binding indicates that the test compound is an antibiotic candidate.

The 3-isopropylmalate dehydratase protein can have the amino acid sequence of naturally occurring 3-isopropylmalate dehydratase found in fungi, animals, plants, or microorganisms, and can also be derived from a naturally occurring sequence. It is possible to have Preferably, the 3-isopropylmalate dehydratase is a fungal 3-isopropylmalate dehydratase. CDNA encoding 3-isopropyl malate dehydratase protein (SEQ ID NO: 10), M. Genomic DNA (SEQ ID NO: 11) encoding the grisea protein, and polypeptide (SEQ ID NO: 12) can be found herein.

In one aspect, the present invention also provides a polypeptide consisting essentially of SEQ ID NO: 12. For purposes of the present invention, a polypeptide consisting essentially of SEQ ID NO: 12 has at least 80% sequence identity with SEQ ID NO: 12, and at least 10% of the activity of SEQ ID NO: 12, It catalyzes the interconversion of rate and H ₂ O with 3-isopropylmalate. Preferably, the polypeptide consisting essentially of SEQ ID NO: 12 has at least 85% sequence identity with SEQ ID NO: 12, more preferably the sequence identity is at least 90%, most preferably the sequence identity Sex is at least 95% or 97% or 99%, or in ascending order with any integer sequence identity of 80-100%. And preferably, a polypeptide consisting essentially of SEQ ID NO: 12, M. It has an activity of at least 25%, at least 50%, at least 75%, or at least 90% of the activity of grisea 3-isopropylmalate dehydratase, or any integer from 60-100% in ascending order.

“Fungus 3-isopropylmalate dehydratase” means an enzyme that can be found in at least one fungus and catalyzes the interconversion of 2-isopropylmalate and H ₂ O with 3-isopropylmalate. . 3-Isopropylmalate dehydratase can be derived from any fungus including ascomycetes, zygomycetes, basidiomycetes, amber fungi, and lichens.

In one embodiment, the 3-isopropylmalate dehydratase is Magnaporte 3-isopropylmalate dehydratase. The Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae, as well as Magnaporthe incomplete state of Pyricularia sp. Preferably, the Magnaporte 3-isopropylmalate dehydratase is derived from Magnaporthe grisea .

In various embodiments, 3-isopropylmalate dehydratase is a powdery scab fungus ( Spongospora subterranea ), gray mold ( Botrytis cinerea ), white rot fungus ( Armillaria mellea ), heartwood rot fungus (a umspor fungus) (Piptoporus betulinus), corn smut (Ustilago maydis), heartwood rot fungus (Polyporus squamosus), black spot fungus (Cercospora zeae-maydis), honey fungus (Armillaria gallica), root rot fungus (Armillaria luteobubalina), shoelaces rot fungus (Armillaria ost yae), banana anthracnose fungi (Colletotrichum musae), apples rot fungus (Monilinia fructigena), apples rot fungi (Penicillium expansum), root kelp fungus (Plasmodiophora brassicae), potato blight fungus (Phytophthora infestans), root pathogens ( Heterobasidion annosum), wilt fungi (Gaeumannomyces graminis), Orandanire fungus (Ophiostoma ulm i), bean rust (Uromyces appendiculatus), northern spot fungus (Cochliobolus carbonum), Miro fungus (Periconia cir cinata), Southern corn blight fungus (Cochliobolus heterostrophus), leaf spot fungus (Cochliobolus lunata),褐条fungus (Cochliobolus stenospilus), Panama fungus (Fusarium oxysporum), wheat scab fungus (Fusarium graminearum), cereal root rot fungus ( Fusarium culmorum ), potato black rust fungus ( Rhizotonia solani ), wheat black rust ( Puccinia graminis ), white mold ( Sclerotinia sclerotiorum ) and the like.

  A fragment of a 3-isopropylmalate dehydratase polypeptide preferably comprises an intact or nearly intact epitope that occurs in wild-type 3-isopropylmalate dehydratase where the fragment is biologically active. Can be used in the method of the present invention. The fragment comprises at least 10 consecutive amino acids of 3-isopropylmalate dehydratase. Preferably, the fragment is at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 3-isopropylmalate dehydratase, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 7 0, or at least 770 contiguous amino acid residues. In one embodiment, the fragment is derived from Magnaporte 3-isopropylmalate dehydratase. Preferably, the fragment contains an amino acid sequence that is conserved among the fungi 3-isopropylmalate dehydratase.

  Polypeptides having at least 50% sequence identity with the fungal 3-isopropylmalate dehydratase are also useful in the methods of the invention. Preferably the sequence identity is at least 60%, more preferably the sequence identity is at least 70%, most preferably the sequence identity is at least 80% or 90% or 95% or 99%, or in ascending order. , An integer of 60 to 100%.

Furthermore, it is preferred that the polypeptide has at least 10% of fungal 3-isopropylmalate dehydratase activity. More preferably, the polypeptide has at least 25%, at least 50%, at least 75% or at least 90% of the fungal 3-isopropylmalate dehydratase activity. Most preferably, the polypeptide is M.P. has at least 10%, at least 25%, at least 50%, at least 75% or at least 90% of the grisea 3-isopropylmalate dehydratase protein activity.

Thus, in another aspect, the invention provides a method for identifying a test compound as a candidate for a fungicide:
a) with a test compound: a polypeptide having at least 10 consecutive amino acids of fungal 3-isopropylmalate dehydratase; a polypeptide having at least 50% sequence identity with fungal 3-isopropylmalate dehydratase; and fungal 3-isopropylapple Contacting at least one polypeptide selected from the group consisting of polypeptides having at least 10% of the activity of acid dehydratase; and b) the presence and / or absence of binding between said test compound and said polypeptide Including detecting;
The method is provided wherein binding indicates that the test compound is an antibiotic candidate.

  Any technique that detects binding of a ligand to a target can be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting binding of a ligand to a target are known in the art and include, but are not limited to, detection of a fixed ligand-target complex, or target characteristics when a target binds to a ligand. Change detection is included. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligand is contacted with 3-isopropylmalate dehydratase protein, or a fragment or variant thereof to remove unbound protein and detect bound 3-isopropylmalate dehydratase. In preferred embodiments, a labeled binding partner such as a labeled antibody is used to detect bound 3-isopropylmalate dehydratase. In a variation of this assay, 3-isopropylmalate dehydratase is labeled prior to contacting the immobilized candidate ligand. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetry.

  Once a compound has been identified as an antibiotic candidate, it can be tested for its ability to inhibit 3-isopropylmalate dehydratase enzyme activity. Compounds can be tested using either in vitro assays or cell based assays. Alternatively, the compound can be applied directly to the fungus or fungal cell or expressed in the fungus or fungal cell and monitored for growth, development, viability, pathogenic change or reduction, or gene expression modification Can be tested. Accordingly, in one aspect, the present invention is a method for determining whether a compound identified as an antibiotic candidate by the method described above has antifungal activity comprising: a fungus or fungal cell and said antifungal agent candidate And detecting a decrease in growth, viability, or pathogenicity of the fungus or fungal cell.

  By reducing the growth is meant that the antifungal candidate causes at least a 10% reduction in fungal or fungal cell growth as compared to the fungal or fungal cell growth in the absence of the antifungal candidate. . By reduced viability is meant that at least 20% of the fungal cells, or fungal parts, contacted with the antifungal agent candidate are not viable. Preferably, proliferation or viability will be reduced by at least 40%. More preferably, proliferation or viability will be reduced by at least 50%, 75% or at least 90% or more. Methods for measuring fungal growth and cell viability are known to those skilled in the art. Reduced virulence causes an antifungal candidate to cause at least a 10% reduction in disease caused by contact of the fungal pathogen with its host when compared to disease caused in the absence of the antifungal candidate Means. Preferably, the disease will be reduced by at least 40%. More preferably, the disease will be reduced by at least 50%, 75% or at least 90% or more. Methods for measuring fungal diseases are well known to those skilled in the art and include metrics such as lesion formation, lesion size, sporulation, respiratory failure, and / or death.

The ability of a compound to inhibit 3-isopropylmalate dehydratase activity can be detected using an in vitro enzyme assay that directly or indirectly detects the disappearance of a substrate or the appearance of a product. 3-Isopropylmalate dehydratase catalyzes an irreversible or reversible reaction, 2-isopropylmalate and H ₂ O = 3-isopropylmalate (see FIG. 1). 2-isopropyl _malate, the method of detecting _H 2 O, and / or 3-isopropyl malate, spectrophotometry, mass spectrometry, include thin layer chromatography (TLC) and Reversed Phase HPLC.

Accordingly, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) contacting 2-isopropylmalate and H ₂ O with 3-isopropylmalate dehydratase;
b) 2- isopropyl and malate and _{H 2} O, by contacting the 3-isopropyl malate dehydratase and the test compound; and c) the following: 2-isopropyl _{malate, H} 2 O, and / or 3-isopropyl malate Measuring at least one concentration change of
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

The present invention is a further method for identifying test compounds as antibiotic candidates:
a) contacting 3-isopropylmalate with 3-isopropylmalate dehydratase;
b) contacting 3-isopropylmalate with 3-isopropylmalate dehydratase and a test compound; and c) the following: at least one concentration of 2-isopropylmalate, H ₂ O, and / or 3-isopropylmalate Including measuring changes,
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

  Enzymatically active fragments of the fungal 3-isopropylmalate dehydratase are also useful in the methods of the invention. For example, a polypeptide comprising at least 100 consecutive amino acid residues of fungal 3-isopropylmalate dehydratase and being enzymatically active can be used in the methods of the invention. Furthermore, an enzymatically active polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity with fungal 3-isopropylmalate dehydratase, It can be used in the method of the present invention. Most preferably, the polypeptide has at least 50% sequence identity with the fungal 3-isopropylmalate dehydratase and has at least 10%, 25%, 75%, or at least 90% of its activity.

Accordingly, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) with 2-isopropylmalate and H ₂ O: a polypeptide having at least 50% sequence identity with 3-isopropylmalate dehydratase; having at least 50% sequence identity with 3-isopropylmalate dehydratase; And contacting with a polypeptide having at least 10% of its activity; and a polypeptide selected from the group consisting of polypeptides comprising at least 100 contiguous amino acids of 3-isopropylmalate dehydratase;
b) contacting the polypeptide and the test compound with 2-isopropylmalate and H ₂ O; and c) the following: at least one of 2-isopropylmalate, H ₂ O, and / or 3-isopropylmalate Including measuring concentration changes;
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

The present invention is a further method of identifying a test compound as an antibiotic candidate:
a) with 3-isopropylmalate: a polypeptide having at least 50% sequence identity with 3-isopropylmalate dehydratase; having at least 50% sequence identity with 3-isopropylmalate dehydratase and of its activity Contacting with a polypeptide selected from the group consisting of a polypeptide comprising at least 10%; and a polypeptide comprising at least 100 consecutive amino acids of 3-isopropylmalate dehydratase;
b) contacting 3-isopropylmalate with said polypeptide and test compound; and c) measuring the following: at least one concentration change of 2-isopropylmalate, H ₂ O, and / or 3-isopropylmalate Including doing;
The method is provided wherein a change in concentration of any of the above substances indicates that the test compound is a candidate antibiotic.

  For in vitro enzyme assays, 3-isopropylmalate dehydratase protein and its derivatives can be purified from fungi or produced recombinantly in archaea, bacteria, fungi, or other eukaryotic cell cultures And can be purified from these cultures. Preferably, these proteins are produced using E. coli, yeast, or filamentous fungal expression systems. Methods for purifying 3-isopropylmalate dehydratase are described in Bigelis and Umberger (1975) J Biol Chem 250: 4315-21 (PMID: 1126953); Kohlhow (1988) Meth Enzymol 166: 423-9 (PMID: 3071717). It is possible that Other methods of purifying 3-isopropylmalate dehydratase proteins and polypeptides are known to those skilled in the art.

As an alternative to in vitro assays, the present invention also provides cell-based assays. In one embodiment, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) measuring the expression of 3-isopropylmalate dehydratase in the absence of the test compound in a single cell, a plurality of cells, tissues or organisms;
b) contacting the test compound with the single cell, cells, tissues, or organism, and in the single cell, cells, tissue, or organism, the 3-isopropylmalate dehydratase Measuring expression; and c) comparing the expression of 3-isopropylmalate dehydratase in steps (a) and (b);
The method is provided wherein lower expression in the presence of the test compound indicates that the compound is an antibiotic candidate.

  The expression of 3-isopropylmalate dehydratase can be measured by detecting IPMD1 primary transcript or mRNA, 3-isopropylmalate dehydratase polypeptide, or 3-isopropylmalate dehydratase enzyme activity. Methods of detecting RNA and protein expression are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1995. The detection method is not critical to the present invention. Methods for detecting IPMD1 RNA include but are not limited to amplification assays such as quantitative reverse transcriptase PCR and / or hybridization assays such as Northern analysis, dot blot, slot blot, in-situ hybridization. , Transcription fusion using the IPMD1 promoter fused to a reporter gene, DNA assays, and microarray assays.

  Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Western blots, ELISA assays, polyacrylamide gel electrophoresis, mass spectrometry, and enzyme assays. It is also possible to detect IPMD1 protein expression using any reporter gene system. For detection using a gene reporter system, a polynucleotide encoding a reporter protein is fused in frame to IPMD1 to produce a chimeric polypeptide. Methods using reporter systems are known to those skilled in the art.

  Thereafter, fungal growth can be controlled using the chemicals, compounds or compositions identified as modulators, preferably inhibitors, of IPMD1 expression or activity by the methods described above. Diseases such as rust, powdery mildew, and blight can quickly spread once established. Therefore, fungicides are applied to growing and stored crops as a protective measure, routinely, generally as a foliar application or seed dressing. For example, to protect against fungal growth, compounds that inhibit fungal growth can be applied to or expressed in the fungus. Accordingly, the present invention provides a method for inhibiting fungal growth comprising contacting a fungus with a compound identified by the method of the present invention as having antifungal activity.

  Using the antifungal and antifungal inhibitor candidates identified by the methods of the present invention to control the growth of unwanted fungi, including ascomycetes, zygotes, basidiomycetes, astragalus, and lichens Is possible.

Examples of undesired fungi include, but are not limited to, Spongospora subterranea , Gray mold ( Botrytis cinerea ), White rot fungus ( Armillaria mellea ), Heartwood rot fungus ( umanoder ) Brown rot fungus (Piptoporus betulinus), corn smut (Ustilago maydis), heartwood rot fungus (Polyporus squamosus), black spot fungus (Cercospora zeae-maydis), honey fungus (Armillaria gallica), root rot fungus (Armillaria luteobubalina), shoes string rot fungus (Armillaria ostoyae) Banana anthracnose fungi (Colletotrichum musae), apples rot fungus (Monilinia fructigena), apples rot fungi (Penicillium expansum), root kelp fungus (Plasmodiophora brassicae), potato blight fungus (Phytophthora infestans), root pathogens (Heterobasidion annosum) , Withering fungi ( Gaeumanomyces graminis ), Dutch elm fungus ( Ophiostoma ulmi ), bean rust fungus ( Uromyces appendiculatus ), northern spot fungus ( Cochliobolus carbinorum bacterium), a), Southern corn blight fungus (Cochliobolus heterostrophus), leaf spot fungus (Cochliobolus lunata),褐条fungus (Cochliobolus stenospilus), Panama fungus (Fusarium oxysporum), wheat scab fungus (Fusarium graminearum), cereal root rot fungus ( Fusarium culmorum ), potato black husk fungus ( Rhizotonia solani ), wheat black rust ( Puccinia graminis ), white mold ( Sclerotinia sclerotiorum ), lung, blood, brain, skin, scalp, etc. animal disease (Aspergillus fumigatus, A pergillus species, Fusraium species, Trichophyton species, Epidermophyton species, and Microsporum species, etc.).

  Also provided is a test compound for the identified gene (SEQ ID NO: 10 or SEQ ID NO: 11), its gene product (SEQ ID NO: 12), or one or more biochemical pathways in which the gene product functions. Antibiotic screening method by determining whether it is active.

  In one particular embodiment, the first type of the gene corresponding to either SEQ ID NO: 10 or SEQ ID NO: 11, either normal, mutant, homolog, or a heterologous IPMD1 gene that performs a function similar to IPMD1 The method is performed by providing an organism having The first type of IPMD1 may or may not provide a growth condition phenotype, ie, an L-leucine requirement phenotype, and / or a hypersensitivity or reduced sensitivity phenotype of an organism having a modified type. In one particular embodiment, the mutant form contains a transposon insertion. A comparative organism having a second type of IPMD1 that differs from the first type of gene is also provided, and the two organisms are contacted separately with the test compound. The growth of the two organisms is then compared in the presence of the test compound.

Accordingly, in one embodiment, the present invention is a method of identifying a test compound as an antibiotic candidate:
a) providing a cell having one type of 3-isopropylmalate dehydratase gene and providing a comparative cell having a different type of 3-isopropylmalate dehydratase gene; and b) said cell and said comparative cell; Contacting the test compound and measuring the proliferation of the cell and the comparative cell in the presence of the test compound,
The method is provided wherein a difference in proliferation between the cell and the comparative cell in the presence of the test compound indicates that the test compound is an antibiotic candidate.

In some cases, in the absence of all test compounds, the growth of the first organism and the second comparative organism is measured to determine that the genetic difference results in a unique difference in growth. It is recognized that both can be managed. It will also be appreciated that any combination of two different types of IPMD1 gene can be used in the present methods, including normal genes, mutant genes, homologues, and functional homologues. The growth and / or proliferation of the organism is measured by methods well known in the art such as optical density measurement. In a preferred embodiment, the organism is Magnaporthe grisea .

Conditional lethal mutants can identify specific biochemical and / or genetic pathways if at least one identified target gene is present in the pathway. Knowledge of these pathways makes it possible to screen test compounds as antibiotic candidates as inhibitors of pathway substrates, products and enzymes. A route known in the art can be found in Kyoto Encyclopedia of Genes and Genomes and a standard biochemistry textbook (Lehninger, A., D. Nelson et al. (1993) Principles of Biochemistry, New York, New York, New York, United States). is there.

Accordingly, in one aspect, the invention is a method of screening for test compounds that act on one or more biochemical and / or genetic pathways in which IPMD1 functions:
j) providing a cell having one type of gene in the L-leucine biochemical pathway and / or gene pathway, and providing a comparative cell having a different type of said gene;
k) contacting said cell and said comparative cell with a test compound; and 1) measuring the proliferation of said cell and said comparative cell in the presence of said test compound;
The method is provided wherein a difference in proliferation between the cell and the comparative cell in the presence of the test compound indicates that the test compound is an antibiotic candidate.

  The use of multiwell plates for screening is a format that readily accommodates a number of different assays that characterize diverse compounds, compound concentrations, and fungal strains in a variety of combinations and formats. Certain test parameters of the screening method can significantly affect the identification of growth inhibitors, and therefore these parameters can be manipulated to optimize screening efficiency and / or reliability. is there. Notable among these factors are the various sensitivities of different mutants, hypersensitivity that increases as conditions become less permissive, hypersensitivity that apparently increases as compound concentrations increase, and Other factors known to those skilled in the art.

Conditional lethal mutants can identify specific biochemical and / or genetic pathways if at least one identified target gene is present in the pathway. Knowledge of these pathways makes it possible to screen test compounds as antibiotic candidates. A route known in the art can be found in Kyoto Encyclopedia of Genes and Genomes and a standard biochemistry textbook (Lehninger, A., D. Nelson et al. (1993) Principles of Biochemistry, New York, New York, New York, United States). is there.

Accordingly, in one aspect, the invention is a method of screening for test compounds that act on one or more biochemical and / or genetic pathways in which IPMD1 functions:
(A) providing a paired growth medium comprising a first medium and a second medium, wherein the second medium contains a higher level of L-leucine than the first medium;
(B) contacting the organism with the test compound;
(C) inoculating said first medium and said second medium with said organism; and (d) measuring the growth of said organism;
The method is provided wherein a difference in organism growth between the first medium and the second medium indicates that the test compound is a candidate antibiotic.

In the art, measurement of the growth of the organism in a pair of media in the absence of all test compounds is performed to control any differences in media resulting in unique differences in growth. Is recognized as possible. The growth and / or proliferation of the organism is measured by methods well known in the art such as optical density measurement. In a preferred embodiment, the organism is Magnaporthe grisea .

The inventors of the present invention have discovered that disruption of the THR4 gene and / or gene product inhibits the virulence of Magnaporthe grisea . Thus, the inventors of the present invention have demonstrated for the first time that threonine synthase is the target of antibiotics, preferably antifungal agents.

  Accordingly, the present invention provides methods for identifying compounds that inhibit THR4 gene expression or the biological activity of its gene product (s). Such methods include ligand binding assays, enzyme activity assays, cell based assays, and assays for THR4 gene expression. Any compound that is a ligand for threonine synthase can have antibiotic activity. For the purposes of the present invention, “ligand” refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the present invention are useful as antibiotics.

Accordingly, in one embodiment, the present invention is a method of identifying a test compound as an antibiotic candidate:
a) contacting the test compound with a threonine synthase polypeptide; and b) detecting the presence or absence of a bond between the test compound and the threonine synthase polypeptide, wherein the test compound is an antibiotic. The method is provided to indicate that it is a candidate.

A threonine synthase protein can have the amino acid sequence of a naturally occurring threonine synthase found in fungi, animals, plants, or microorganisms, and can have an amino acid sequence derived from a naturally occurring sequence. Preferably, the threonine synthase is a fungal threonine synthase. CDNA which encodes threonine synthase protein (SEQ ID NO: 13), M. Genomic DNA (SEQ ID NO: 14) encoding grisea protein and polypeptide (SEQ ID NO: 15) can be found herein.

In one aspect, the invention also provides a polypeptide consisting essentially of SEQ ID NO: 15. For purposes of the present invention, a polypeptide consisting essentially of SEQ ID NO: 15 has at least 80% sequence identity with SEQ ID NO: 15, and at least 10% of the activity of SEQ ID NO: 15 with O-phospho- It catalyzes the interconversion of L-homoserine and water with L-threonine and orthophosphate. Preferably, the polypeptide consisting essentially of SEQ ID NO: 15 has at least 85% sequence identity with SEQ ID NO: 15, more preferably the sequence identity is at least 90%, most preferably the sequence identity Sex is at least 95% or 97% or 99%, or in ascending order with any integer sequence identity of 80-100%. And preferably, a polypeptide consisting essentially of SEQ ID NO: 15, M. It has an activity of at least 25%, at least 50%, at least 75%, or at least 90% of the activity of grisea threonine synthase, or any integer from 60 to 100% in ascending order.

  By “fungal threonine synthase” is meant an enzyme that can be found in at least one fungus and catalyzes the interconversion of O-phospho-L-homoserine and water with L-threonine and orthophosphate. Threonine synthase can be derived from any fungus, including ascomycetes, zygomycetes, basidiomycetes, fungi, and lichens.

In one embodiment, the threonine synthase is Magnaporte threonine synthase. The Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae, as well as Magnaporthe incomplete state of Pyricularia sp. Preferably, the Magnaporte threonine synthase is derived from Magnaporthe grisea .

In various embodiments, threonine synthase, powdery scab fungus (Spongospora subterranea), gray mold (Botrytis cinerea), white rot fungus (Armillaria mellea), core rot fungus (Ganoderma adspersum), brown rot fungi (Piptoporus betulinus) , corn smut (Ustilago maydis), heartwood rot fungus (Polyporus squamosus), black spot fungus (Cercospora zeae-maydis), honey fungus (Armillaria gallica), root rot fungus (Armillaria luteobubalina), shoelaces rot fungus (Armillaria ostoyae) ,banana疽病fungi (Colletotrichum musae), apples rot fungus (Monilinia fructigena), apples rot fungi (Penicillium expansum), root kelp fungus (Plasmodiophora brassicae), potato blight fungus (Phytophthora infestans), root pathogens (Heterobasidion annosum), wilt fungus (Gaeumannomyces graminis), Orandanire fungus (Ophiostoma ulm i), bean rust (Uromyces appendiculatus), northern spot fungus (Cochliobolus carbonum), Miro fungus (Periconia circinata), Square corn blight fungus (Cochliobolus heterostrophus), leaf spot fungus (Cochliobolus lunata),褐条fungus (Cochliobolus stenospilus), Panama fungus (Fusarium oxysporum), wheat scab fungus (Fusarium graminearum), cereal root rot fungus (Fusarium culmorum ), Potato black rust fungus ( Rhizotonia solani ), wheat rust ( Puccinia graminis ), white mold ( Sclerotinia sclerotiorum ), and the like.

  A fragment of a threonine synthase polypeptide is preferably used in the methods of the invention provided that the fragment contains an intact or nearly intact epitope that occurs in a biologically active wild-type threonine synthase. Is possible. The fragment comprises at least 10 consecutive amino acids of threonine synthase. Preferably, the fragment is at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, threonine synthase, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, or at least 540 consecutive amino acid residues. In one embodiment, the fragment is derived from Magnaporte threonine synthase. Preferably, the fragment contains an amino acid sequence that is conserved among fungal threonine synthases.

  Polypeptides having at least 50% sequence identity with fungal threonine synthase are also useful in the methods of the invention. Preferably the sequence identity is at least 60%, more preferably the sequence identity is at least 70%, most preferably the sequence identity is at least 80% or 90% or 95% or 99%, or in ascending order. , An integer of 60 to 100%.

Furthermore, it is preferred that the polypeptide has at least 10% of fungal threonine synthase activity. More preferably, the polypeptide has at least 25%, at least 50%, at least 75% or at least 90% of fungal threonine synthase activity. Most preferably, the polypeptide is M.P. have at least 10%, at least 25%, at least 50%, at least 75% or at least 90% of the grisea threonine synthase protein activity.

Thus, in another aspect, the invention provides a method for identifying a test compound as a candidate for a fungicide:
a) with a test compound: a polypeptide having at least 10 consecutive amino acids of fungal threonine synthase; a polypeptide having at least 50% sequence identity with fungal threonine synthase; and a polypeptide having at least 10% of the activity of fungal threonine synthase Contacting with at least one polypeptide selected from the group consisting of; and b) detecting the presence and / or absence of binding between said test compound and said polypeptide, wherein binding comprises said test compound Wherein said method is shown to be an antibiotic candidate.

  Any technique that detects binding of a ligand to a target can be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting binding of a ligand to a target are known in the art and include, but are not limited to, detection of a fixed ligand-target complex, or target characteristics when a target binds to a ligand. Change detection is included. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligand is contacted with the threonine synthase protein, or a fragment or variant thereof, the unbound protein is removed, and the bound threonine synthase is detected. In a preferred embodiment, a labeled binding partner such as a labeled antibody is used to detect bound threonine synthase. In a variation of this assay, threonine synthase is labeled prior to contacting the immobilized candidate ligand. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetry.

  Once a compound has been identified as an antibiotic candidate, it can be tested for its ability to inhibit threonine synthase enzyme activity. Compounds can be tested using either in vitro assays or cell based assays. Alternatively, the compound can be applied directly to the fungus or fungal cell or expressed in the fungus or fungal cell and monitored for growth, development, viability, pathogenic change or reduction, or gene expression modification Can be tested. Accordingly, in one aspect, the present invention is a method for determining whether a compound identified as an antibiotic candidate by the method described above has antifungal activity comprising: a fungus or fungal cell and said antifungal agent candidate And detecting a decrease in growth, viability, or pathogenicity of the fungus or fungal cell.

  By reducing the growth is meant that the antifungal candidate causes at least a 10% reduction in fungal or fungal cell growth as compared to the fungal or fungal cell growth in the absence of the antifungal candidate. . By reduced viability is meant that at least 20% of the fungal cells, or fungal parts, contacted with the antifungal agent candidate are not viable. Preferably, proliferation or viability will be reduced by at least 40%. More preferably, proliferation or viability will be reduced by at least 50%, 75% or at least 90% or more. Methods for measuring fungal growth and cell viability are known to those skilled in the art. Reduced virulence causes an antifungal candidate to cause at least a 10% reduction in disease caused by contact of the fungal pathogen with its host when compared to disease caused in the absence of the antifungal candidate Means. Preferably, the disease will be reduced by at least 40%. More preferably, the disease will be reduced by at least 50%, 75% or at least 90% or more. Methods for measuring fungal diseases are well known to those skilled in the art and include metrics such as lesion formation, lesion size, sporulation, respiratory failure, and / or death.

  The ability of a compound to inhibit threonine synthase activity can be detected using an in vitro enzyme assay that directly or indirectly detects the disappearance of a substrate or the appearance of a product. Threonine synthase catalyzes irreversible or reversible reactions, O-phospho-L-homoserine and water = L-threonine and orthophosphate (see FIG. 1). Methods for detecting O-phospho-L-homoserine, L-threonine, orthophosphate, and water include spectrophotometry, mass spectrometry, thin layer chromatography (TLC) and reverse phase HPLC.

Accordingly, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) contacting O-phospho-L-homoserine and water with threonine synthase;
b) contacting O-phospho-L-homoserine and water with threonine synthase and said test compound; and c) the following: at least one concentration of O-phospho-L-homoserine, L-threonine, orthophosphate, and water The method is provided comprising measuring a change, wherein a change in concentration of any of the above substances indicates that the test compound is a candidate for an antibiotic.

The present invention is a further method for identifying test compounds as antibiotic candidates:
a) contacting L-threonine and orthophosphate with threonine synthase;
b) contacting L-threonine and orthophosphate with threonine synthase and test compound; and c) measuring the concentration change of at least one of: O-phospho-L-homoserine, L-threonine, orthophosphate, and water Wherein the change in concentration of any of the above substances indicates that the test compound is an antibiotic candidate.

  Enzymatically active fragments of fungal threonine synthase are also useful in the methods of the invention. For example, an enzymatically active polypeptide comprising at least 100 consecutive amino acid residues of fungal threonine synthase can be used in the methods of the invention. Further, a polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity and fungal threonine synthase is enzymatically active. Can be used. Most preferably, the polypeptide has at least 50% sequence identity with fungal threonine synthase and has at least 10%, 25%, 75%, or at least 90% of its activity.

Accordingly, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) O-phospho-L-homoserine and water: a polypeptide having at least 50% sequence identity with threonine synthase, and at least 50% sequence identity with threonine synthase and at least 10% of its activity And a polypeptide selected from the group consisting of a polypeptide comprising at least 100 contiguous amino acids of threonine synthase;
b) contacting the polypeptide and test compound with O-phospho-L-homoserine and water; and c) the following: at least one concentration of O-phospho-L-homoserine, L-threonine, orthophosphate, and water. The method is provided comprising measuring a change, wherein a change in concentration of any of the above substances indicates that the test compound is a candidate for an antibiotic.

The present invention is a further method of identifying a test compound as an antibiotic candidate:
a) L-threonine and orthophosphate: a polypeptide having at least 50% sequence identity with threonine synthase, and a poly having at least 50% sequence identity with threonine synthase and having at least 10% of its activity Contacting a peptide selected from the group consisting of a peptide and a polypeptide comprising at least 100 contiguous amino acids of threonine synthase;
b) contacting L-threonine and orthophosphate with the polypeptide and test compound; and c) measuring the concentration change of at least one of the following: O-phospho-L-homoserine, L-threonine, orthophosphate, and water Providing a method wherein a change in concentration of any of the substances indicates that the test compound is a candidate for an antibiotic.

  For in vitro enzyme assays, threonine synthase proteins and derivatives thereof can be purified from fungi or produced recombinantly in archaea, bacteria, fungi, or other eukaryotic cell cultures, and It is possible to purify from this culture. Preferably, these proteins are produced using E. coli, yeast, or filamentous fungal expression systems. The method of purifying threonine synthase can be as described in Malumbres et al. (1994) Appl Environ Microbiol 60: 2209-19 (PMID: 8074505). Other methods of purifying threonine synthase proteins and polypeptides are known to those skilled in the art.

As an alternative to in vitro assays, the present invention also provides cell-based assays. In one embodiment, the present invention is a method of identifying a test compound as a candidate antibiotic:
a) measuring the expression of threonine synthase in the absence of the test compound in a single cell, a plurality of cells, tissues or organisms;
b) contacting the test compound with the singular cell, cells, tissue or organism and measuring the expression of the threonine synthase in the singular cell, cells, tissue or organism. And c) comparing the expression of threonine synthase in steps (a) and (b), wherein the expression is lower in the presence of the test compound, the compound is a candidate for an antibiotic; Wherein said method is provided.

  Threonine synthase expression can be measured by detecting THR4 primary transcript or mRNA, threonine synthase polypeptide, or threonine synthase enzyme activity. Methods of detecting RNA and protein expression are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1995. The detection method is not critical to the present invention. Methods for detecting THR4 RNA include, but are not limited to, amplification assays such as quantitative reverse transcriptase PCR, and / or hybridization assays such as Northern analysis, dot blot, slot blot, in-situ hybridization. , Transcription fusion using the THR4 promoter fused to a reporter gene, DNA assays, and microarray assays.

  Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Western blots, ELISA assays, polyacrylamide gel electrophoresis, mass spectrometry, and enzyme assays. It is also possible to detect THR4 protein expression using any reporter gene system. For detection using a gene reporter system, a polynucleotide encoding a reporter protein is fused in-frame to THR4 to produce a chimeric polypeptide. Methods using reporter systems are known to those skilled in the art.

  Thereafter, fungal growth can be controlled using chemicals, compounds or compositions identified as modulators, preferably inhibitors, of THR4 expression or activity by the methods described above. Diseases such as rust, powdery mildew, and blight can quickly spread once established. Therefore, fungicides are applied to growing and stored crops as a protective measure, routinely, generally as a foliar application or seed dressing. For example, to protect against fungal growth, compounds that inhibit fungal growth can be applied to or expressed in the fungus. Accordingly, the present invention provides a method for inhibiting fungal growth comprising contacting a fungus with a compound identified by the method of the present invention as having antifungal activity.

  Using the antifungal and antifungal inhibitor candidates identified by the methods of the present invention to control the growth of unwanted fungi, including ascomycetes, zygotes, basidiomycetes, astragalus, and lichens Is possible.

Examples of undesired fungi include, but are not limited to, Spongospora subterranea , Gray mold ( Botrytis cinerea ), White rot fungus ( Armillaria mellea ), Heartwood rot fungus ( umanoder ) Brown rot fungus (Piptoporus betulinus), corn smut (Ustilago maydis), heartwood rot fungus (Polyporus squamosus), black spot fungus (Cercospora zeae-maydis), honey fungus (Armillaria gallica), root rot fungus (Armillaria luteobubalina), shoes string rot fungus (Armillaria ostoyae) Banana anthracnose fungi (Colletotrichum musae), apples rot fungus (Monilinia fructigena), apples rot fungi (Penicillium expansum), root kelp fungus (Plasmodiophora brassicae), potato blight fungus (Phytophthora infestans), root pathogens (Heterobasidion annosum) , Withering fungi ( Gaeumanomyces graminis ), Dutch elm fungus ( Ophiostoma ulmi ), bean rust fungus ( Uromyces appendiculatus ), northern spot fungus ( Cochliobolus carbinorum bacterium), a), Southern corn blight fungus (Cochliobolus heterostrophus), leaf spot fungus (Cochliobolus lunata),褐条fungus (Cochliobolus stenospilus), Panama fungus (Fusarium oxysporum), wheat scab fungus (Fusarium graminearum), cereal root rot fungus ( Fusarium culmorum ), potato black husk fungus ( Rhizotonia solani ), wheat black rust ( Puccinia graminis ), white mold ( Sclerotinia sclerotiorum ), lung, blood, brain, skin, scalp, etc. animal disease (Aspergillus fumigatus, A pergillus species, Fusraium species, Trichophyton species, Epidermophyton species, and Microsporum species, etc.).

  Also provided is a test compound for the identified gene (SEQ ID NO: 13 or SEQ ID NO: 14), its gene product (SEQ ID NO: 15), or the biochemical pathway (s) in which the gene product functions. Antibiotic screening method by determining whether it is active.

  In one particular embodiment, the first form of the gene corresponding to either SEQ ID NO: 13 or SEQ ID NO: 14, either normal, mutant, homolog, or a heterologous THR4 gene that performs a function similar to THR4 The method is performed by providing an organism having The first type of THR4 may or may not provide a growth condition phenotype, ie, an L-threonine requirement phenotype, and / or a hypersensitivity or reduced sensitivity phenotype of an organism. In one particular embodiment, the mutant form contains a transposon insertion. A comparative organism having a second type of THR4 that is different from the first type of gene is also provided, and the two organisms are contacted with the test compound separately. The growth of the two organisms is then compared in the presence of the test compound.

Accordingly, in one embodiment, the present invention is a method of identifying a test compound as an antibiotic candidate:
a) providing a cell having one type of threonine synthase gene and providing a comparative cell having a different type of threonine synthase gene; and b) contacting said cell and said comparative cell with a test compound and testing Measuring the proliferation of the cell and the comparative cell in the presence of a compound, wherein the test compound is an antibiotic that there is a difference in proliferation between the cell and the comparative cell in the presence of the test compound. The method is provided to indicate that a substance is a candidate.

In some cases, in the absence of all test compounds, the growth of the first organism and the second comparative organism is measured to determine that the genetic difference results in a unique difference in growth. It is recognized that both can be managed. It will also be appreciated that any combination of two different types of THR4 genes can be used in the present methods, including normal genes, mutant genes, homologues, and functional homologues. The growth and / or proliferation of the organism is measured by methods well known in the art such as optical density measurement. In a preferred embodiment, the organism is Magnaporthe grisea .

Conditional lethal mutants can identify specific biochemical and / or genetic pathways if at least one identified target gene is present in the pathway. Knowledge of these pathways makes it possible to screen test compounds as antibiotic candidates as inhibitors of pathway substrates, products and enzymes. Routes known in the art can be found in Kyoto Encyclopedia of Genes and Genomes and standard biochemistry textbooks (Lehninger, A., D. Nelson et al. (1993) Principles of Biochemistry , New York, New York, United States). is there.

Accordingly, in one aspect, the invention is a method of screening for test compounds that act on one or more biochemical and / or genetic pathways in which THR4 is functional:
m) providing a cell having one type of gene in the L-threonine biochemical pathway and / or gene pathway, and providing a comparative cell having a different type of said gene;
n) contacting the cell and the comparative cell with a test compound; and o) measuring the proliferation of the cell and the comparative cell in the presence of the test compound, and in the presence of the test compound, The method is provided wherein a difference in proliferation between the cell and the comparative cell indicates that the test compound is an antibiotic candidate.

  The use of multiwell plates for screening is a format that readily accommodates a number of different assays that characterize diverse compounds, compound concentrations, and fungal strains in a variety of combinations and formats. Certain test parameters of the screening method can significantly affect the identification of growth inhibitors, and therefore these parameters can be manipulated to optimize screening efficiency and / or reliability. is there. Notable among these factors are the various sensitivities of different mutants, hypersensitivity that increases as conditions become less permissive, hypersensitivity that apparently increases as compound concentrations increase, and Other factors known to those skilled in the art.

Conditional lethal mutants can identify specific biochemical and / or genetic pathways if at least one identified target gene is present in the pathway. Knowledge of these pathways makes it possible to screen test compounds as antibiotic candidates. Routes known in the art can be found in Kyoto Encyclopedia of Genes and Genomes and standard biochemistry textbooks (Lehninger, A., D. Nelson et al. (1993) Principles of Biochemistry .).

Accordingly, in one aspect, the invention is a method of screening for test compounds that act on one or more biochemical and / or genetic pathways in which THR4 is functional:
(A) providing a paired growth medium comprising a first medium and a second medium, wherein the second medium contains a higher level of L-threonine than the first medium;
(B) contacting the organism with the test compound;
(C) inoculating said first medium and said second medium with said organism; and (d) measuring the growth of said organism, said first medium and said second medium There is provided the method, wherein the difference in growth of the organisms between the different media indicates that the test compound is a candidate antibiotic.

In the art, measurement of the growth of the organism in a pair of media in the absence of all test compounds is performed to control any differences in media resulting in unique differences in growth. Is recognized as possible. The growth and / or proliferation of the organism is measured by methods well known in the art such as optical density measurement. In a preferred embodiment, the organism is Magnaporthe grisea .

Experiment:

Construction of a plasmid with a transposon containing a selectable marker:
Construction of Sif transposon: As the main chain, New England Biolabs, Inc. Sif was constructed using the GPS3 vector of the GPS-M mutagenesis system (Beverly, Massachusetts). This system is based on the bacterial transposon Tn7. According to Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press, the following operations were performed on GPS3. EcoRV digestion removed the kanamycin resistance gene (npt) contained between the Tn7 arms. Bacterial hygromycin under the control of the Aspergillus nidulans trpC promoter and terminator (Mullaney et al. (1985) Mol Gen Genet 199: 37-45 (PMID: 3158996)) by blunt ligation of the HpaI / EcoRV to the Tn7 arm of the GPS3 vector. The transferase (hph) gene (Gritz and Davies (1983) Gene 25: 179-88 (PMID: 6319235)) was cloned to yield pSif1. Ampicillin resistance from pSif1 by cutting pSif1 with XmnI and BglI, followed by treatment with T4 DNA polymerase to remove the 3 ′ overhang left by BglI digestion and religation to the plasmid to obtain pSif Gene (bla) excision was achieved. Top10F ′ electrocompetent E. coli cells (Invitrogen) were transformed with the ligation mixture according to the manufacturer's recommendations. Transformants containing Sif transposons were selected on LB agar containing 50 μg / ml hygromycin B (Sigma Chem. Co., St. Louis, MO) (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual ) .

Construction of fungal cosmid library:
Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual as described in pcosKA5 vector (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 112296265)). . The quality of the cosmid library was checked by pulse field gel electrophoresis, restriction digest analysis, and single gene PCR identification.

Construction of cosmid using transposon inserted in fungal gene:
Sif rearrangement to cosmid: Translocation of Sif to cosmid framework was performed as described in the GPS-M mutagenesis system (New England Biolabs, Inc.). Briefly, 2 μl of 10 × GPS buffer, 70 ng supercoiled pSIF, 8-12 μg of target cosmid DNA were mixed and brought to a final volume of 20 μl with water. 1 μl of transposase (TnsABC) was added to the assembly reaction and incubated at 37 ° C. for 10 minutes. Following the assembly reaction, 1 μl of the starting solution was added to the tube, mixed well, and incubated for 1 hour at 37 ° C., after which the protein was heat inactivated at 75 ° C. for 10 minutes. The remaining non-translocated pSif was destroyed by inactivating the protein by digesting with PISceI for 2 hours at 37 ° C. and then incubating at 75 ° C. for 10 minutes. Top10F ′ electrocompetent cells (Invitrogen) were transformed according to the manufacturer's recommendations. Sif-containing cosmid transformants were selected by growth on LB agar plates containing 50 μg / ml hygromycin B (Sigma Chem. Co.) and 100 μg / ml ampicillin (Sigma Chem. Co.).

M.M. High-throughput preparation and validation of transposon insertions into the grisea ASN1 gene:
Escherichia coli strains containing cosmids containing transposon insertions were prepared from 1.5 ml TB (Terrific Broth, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratories) supplemented with 50 μg / ml ampicillin. To a 96 well growth block (Beckman Co.). Incubate overnight at 37 ° C. while shaking the block. E. coli cells were pelleted by centrifugation and cosmids were isolated by a modified alkaline lysis method (Marra et al. (1997) Genome Res 7: 1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis on an agarose gel. Cosmids were sequenced using primers from the end of each transposon and a commercial dideoxy sequencing kit (Big Dye Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNA sequencer (Perkin Elmer Co.).

DNA sequences flanking the insertion site were collected and used to search DNA and protein databases using the BLAST algorithm (Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402 (PMID: 9254694)). . A single insertion of SIF into the Magnaporthe grisea ASN1 gene was chosen for further analysis. This construct is referred to as cpgmra0011008a10 and contains a SIF transposon between approximately amino acids 345 and 346, compared to the Saccharomyces cerevisiae homolog (full length 572 amino acids, GENBANK: 6325403).

Preparation of ASN1 cosmid DNA and transformation of Magnaporthe grisea:
Cosmid DNA was prepared from cosmid clones tagged with ASN1 transposon using the QIAGEN Plasmid Maxi kit (QIAGEN) and digested with PI-PspI (New England Biolabs, Inc.). Fungal electrotransformation was performed as essentially described (Wu et al. (1997) MPMI 10: 700-708). For brevity, M.M. grisea strain Guy11 was grown in complete liquid medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) for 3 days at 25 ° C. in the dark with shaking at 120 rpm. Mycelium was harvested and washed with sterile H ₂ O and digested with 4 mg / ml beta-glucanase (InterSpex) for 4-6 hours to produce protoplasts. Protoplasts were collected by centrifugation and resuspended in 20% sucrose at a concentration of 2 × 10 ⁸ protoplasts / ml. 50 μl protoplast suspension was mixed with 10-20 μg cosmid DNA and pulsed with Gene Pulser II (BioRad) set to the following parameters: resistance 200 ohm, capacitance 25 μF, voltage 0.6 kV. Transformed protoplasts were regenerated one day in complete agar medium (CM, Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) supplemented with 20% sucrose, followed by hygromycin B (250 μg / Transformants were selected by overlaying CM agar medium containing (ml). Transformants were screened by PCR for homologous recombination events in the target gene (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)). Two independent strains have been identified and are referred to herein as KO1-2 and KO1-8, respectively.

Effect of transposon insertion on Magnaporte pathogenicity:
The target fungal strains obtained in Example 5, KO1-2 and KO1-8, and the wild type strain Guy11 were subjected to virulence assays and observed for infection over a period of one week. Rice infection assays were performed using the Indian rice variety CO39, essentially as described in Valent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). All three strains were grown for spore production on complete agar medium. Spores were collected and the spore concentration was adjusted for whole plant inoculation. Two-week-old seedlings of variety CO39 were sprayed with 12 ml of conidia suspension (5 × 10 ⁴ conidia per ml of 0.01% Tween-20 (polyoxyethylene sorbitan monolaurate) solution). Inoculated plants were incubated in a humid chamber (dew chamber) at 27 ° C. in the dark for 36 hours and transferred to the growth chamber (27 ° C. 12 hours / 21 ° C. 12 hours, 70% humidity) for an additional 5.5 days. At 3, 5, and 7 days after inoculation, leaf samples were taken and examined for signs of successful infection (ie lesions). FIG. 2 shows the effect of ASN1 gene disruption on Magnaporte infection 5 days after inoculation.

Verification of ASN1 gene function by analysis of nutritional requirements:
Biolog, Inc. The fungal strains KO1-2 and KO1-8 containing the ASN1 disruption gene obtained in Example 5 were analyzed for nutritional requirements for L-asparagine using a PM5 phenotype microarray from Hayward, Calif. PM5 plates are tested for nutritional requirements for 94 different metabolites. The inoculum is 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO ₃ , 6.7 mM KCl, 3.5 mM Na ₂ SO ₄ , 11 mM KH ₂ PO ₄ , 0.01% p- iodonitrotetrazolium violet, 0.1 mM MgCl _2, consists 1.0 mM CaCl ₂ and trace elements, to adjust the pH to 6.0 with NaOH. The final concentrations of the trace elements are: 7.6 μM ZnCl ₂ , 2.5 μM MnCl ₂ ^. 4H ₂ O, 1.8 μM FeCl ₂ ^. 4H ₂ O, 0.71 μM CoCl ₂ ^. 6H ₂ O, 0.64 μM CuCl ₂ ^. 2H ₂ O, 0.62 μM Na ₂ MoO ₄ , 18 μM H ₃ BO ₃ . Spores of each strain were collected in the inoculum. The spore concentration was adjusted to 2 × 10 ⁵ spores / ml. 100 μl of spore suspension was placed in each well of the microtiter plate. Plates were incubated for 7 days at 25 ° C. Optical density (OD) measurements were taken daily at 490 nm and 750 nm. OD ₄₉₀ measures the degree of tetrazolium dye reduction and proliferation level, and OD ₇₅₀ measures proliferation only. Turbidity = OD ₄₉₀ + OD ₇₅₀ . Data confirming the annotated gene function as a graph of turbidity versus time showing both mutant fungus and wild-type control in the absence (Figure 3A) and presence (Figure 3B) of L-asparagine. Show.

Cloning and expression strategy, extraction and purification of asparagine synthase protein:
The following protocol can be used to obtain purified asparagine synthase protein.

Cloning and expression strategies:
The ASN1 cDNA gene can be cloned into Escherichia coli (pET vector, Novagen), baculovirus (Pharmingen) and yeast (Invitrogen) expression vectors containing His / fusion protein tags and assembled by SDS-PAGE and Western blot analysis. The expression of the replacement protein can be evaluated.

Extraction:
Recombinant protein is extracted from 250 ml cell pellet into 3 ml extraction buffer by sonicating 6 times with 4 ° C., 6 sec pulses. The extract is centrifuged at 15000 xg for 10 minutes and the supernatant is collected. The biological activity of the recombinant protein is assessed by an activity assay.

Purification:
Recombinant protein is purified by Ni-NTA affinity chromatography (Qiagen).
Purification protocol: All steps are performed at 4 ° C .:
Use 3 ml Ni beads (Qiagen) Equilibrate column with buffer Load protein extract Wash with equilibration buffer Elute bound protein with 0.5 M imidazole

Assay to test the binding of a test compound to asparagine synthase:
The following protocol can be used to identify test compounds that bind to asparagine synthase protein.

• Purified full-length asparagine synthase polypeptide (Example 8) containing a His / fusion protein tag is bound to HisGrab ^™ nickel-coated plates (Pierce, Rockford, IL) according to the manufacturer's instructions.

Optimize buffer conditions (eg, ionic strength or pH) for binding of radiolabeled L- [4- ¹⁴ C] aspartate (Dearing and Walker (1960) Nature 185: 690-691) to bound asparagine synthase Luhr and Schuster (1980) J Biochem Biophys Methods 3: 151-61 (PMID: 6108975)).

Test compounds by adding test compounds and L- [4- ¹⁴ C] aspartate (Dearing and Walker (1960) Nature 185: 690-691) to the wells of HisGrab ^™ plates containing bound asparagine synthase Screening.

  Wash wells to remove excess labeled ligand and add scintillation fluid (Scintiver®, Fisher Scientific) to each well.

• Read the plate in a microplate scintillation counter.
Identify candidate compounds as wells with lower radioactivity compared to control wells to which no test compound has been added.

In addition, M.M. Purified polypeptides containing 10-50 amino acids from grisea asparagine synthase are screened in the same manner. A polypeptide containing 10-50 amino acids is generated by subcloning a portion of the ASN1 gene into a protein expression vector that adds a His tag when expressed (see Example 8). Oligonucleotide primers are designed to amplify a portion of the ASN1 gene using the polymerase chain reaction amplification method. As described in Example 8 above, a DNA fragment encoding a 10-50 amino acid polypeptide is cloned into an expression vector, expressed in a host organism, and purified.

Test compounds that bind to ASN1 are further tested for antibiotic activity. As described (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 831740)) for spore production on oatmeal agar medium . Grisea is grown. Spores are harvested and the culture is split to a concentration of 2 × 10 ⁵ spores / ml in minimal medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Test compounds are added to one culture to a final concentration of 20-100 μg / ml. Only the solvent is added to the second culture. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Compare the growth curves of the solvent control sample and the test compound sample. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is inferior to that of the control culture.

Assays to test inhibitors or candidate inhibitors of asparagine synthase activity:
Asparagine synthase enzyme activity in the presence and absence of candidate compounds in an appropriate reaction mixture as described in Luehr and Schuster (1980) J Biochem Biophys Methods 3: 151-61 (PMID: 6108975). taking measurement. Candidate compounds are identified when a reduction in product or lack of substrate is detected in a reaction that proceeds in either direction.

Furthermore, Luehr and Schuster (1980) J Biochem Biophys Methods 3: 151-61: as described in (PMID 6,108,975), in a suitable reaction mixture, in the presence and absence of a candidate compound, M. The enzymatic activity of a polypeptide comprising 10 to 50 amino acids derived from grisea asparagine synthase is measured. A polypeptide containing 10-50 amino acids is generated by subcloning a portion of the ASN1 gene into a protein expression vector that adds a His tag when expressed (see Example 8). Oligonucleotide primers are designed to amplify a portion of the ASN1 gene using the polymerase chain reaction amplification method. As described in Example 8 above, a DNA fragment encoding a 10-50 amino acid polypeptide is cloned into an expression vector, expressed and purified.

Test compounds identified as inhibitors of ASN1 activity are further tested for antibiotic activity. Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. As described (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 831740)) for spore production on oatmeal agar medium . Grisea is grown. Spores are harvested and the culture is split to a concentration of 2 × 10 ⁵ spores / ml in minimal medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Test compounds are added to one culture to a final concentration of 20-100 μg / ml. Only the solvent is added to the second culture. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Compare the growth curves of the solvent control sample and the test compound sample. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is inferior to that of the control culture.

Assays to test compounds that alter the expression of the asparagine synthase gene:
Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. After growing on complete agar or oatmeal agar medium for 10-13 days in the light at 25 ° C., wild type M. pneumoniae was grown from the grown culture using a wet cotton swab . Collect grisea spores. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. A 25 ml culture is prepared, to which test compounds are added at various concentrations. Cultures in the absence of test compound are included as controls. After incubation of the culture for 3 days at 25 ° C., only the solvent is added to the test compound or control. The culture is further incubated for 18 hours. The fungal mycelium is collected by filtration through Miracloth (CalBiochem®, La Jolla, Calif.), Washed with water and frozen in liquid nitrogen. Total RNA is extracted with TRIZOL® reagent using methods provided by the manufacturer (Life Technologies, Rockville, Md.). Expression is analyzed by Northern analysis of RNA samples as described (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press) using a radiolabeled fragment of the ASN1 gene as a probe. Test compounds that cause a decrease in ASN1 mRNA levels compared to untreated control samples are identified as candidate antibiotic compounds.

In vivo cell-based assay screening protocol using a fungal strain containing a mutant form of inactive asparagine synthase:
Magnaporthe grisea fungal cells containing mutant forms of the ASN1 gene that abolish enzymatic activity, such as genes containing transposon insertions (see Examples 4 and 5), are well known in the art, and Grow under standard fungal growth conditions as described. After growing on complete agar medium containing 4 mM L-asparagine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium containing 100 μM L-asparagine at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores are added to each well of a 96-well plate, to which test compounds (various concentrations) are added. The total volume of each well is 200 μl. Wells without test compound (growth control) and wells without cells are included as controls (negative control). Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutated form of asparagine synthase with reduced activity:
Magnaporthe grisea fungal cells containing mutant forms of the ASN1 gene, such as promoter truncations that reduce expression, are grown under standard fungal growth conditions well known and described in the art. Let One of the promoters upstream of the transcription initiation site using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press). A part of the promoter is excised by deleting the part. After growing on complete agar medium containing 4 mM L-asparagine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores are added to each well of a 96-well plate, to which test compounds (various concentrations) are added. The total volume of each well is 200 μl. Wells without test compound (growth control) and wells without cells are included as controls (negative control). Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. Similar protocols are found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221).

In vivo cell-based assay screening protocol using a fungal strain containing a mutant form of the inactive L-asparagine biosynthetic gene:
Magnaporthe grisea fungal cells containing mutant forms of L-asparagine biosynthetic pathway genes (eg, formiminoaspartate deiminase (EC 3.5.3.5.)) Are well known in the art. And grown under standard fungal growth conditions as described. After growing on complete agar medium containing 4 mM L-asparagine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium containing 100 μM L-asparagine at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. Similar protocols are found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221).

In vivo cell-based assay screening protocol using a fungal strain containing a mutated form of the L-asparagine biosynthetic gene with reduced activity:
Magnaporthe grisea fungus containing a mutant form of a gene of the L-asparagine biosynthetic pathway (eg, formiminoaspartate deiminase (EC 3.5.3.5.)), Such as partial excision of the promoter that reduces expression Cells are grown under standard fungal growth conditions, which are well known and described in the art. One of the promoters upstream of the transcription initiation site using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press). A part of the promoter is excised by deleting the part. Magnaporthe grisea fungal cells containing the mutant form are grown under standard fungal growth conditions well known and described in the art. After growing on complete agar medium containing 4 mM L-asparagine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. Similar protocols are found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221).

In vivo cell-based assay screening protocol using a fungal strain containing the fungal ASN1 gene and a second fungal strain containing the heterologous ASN1 gene:
M. lacks wild-type Magnaporthe grisea fungal cells and a functioning ASN1 gene and contains an asparagine synthase B gene (Genbank accession number 112726666, 50% sequence identity) from Vibrio cholerae . Grisea fungal cells are grown under standard fungal growth conditions, which are well known and described in the art. M. carrying a heterologous ASN1 gene The grisea strain is created as follows:
-M. having a non-functional ASN1 gene, such as those containing a transposon insertion in the natural gene . A grisea strain is created (see Examples 4 and 5).

• Using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual ), fungal expression vectors containing trpC promoter and terminator (eg, pCB1003 Carroll et al. (1994) Fungal Gen News Lett 41:22) create a construct containing the heterologous ASN1 gene by cloning the asparagine synthase B gene from Vibrio cholerae .

• Using the construct, M. lacking a functioning ASN1 gene . The grisea strain is transformed (see Example 5). Transformants are selected on minimal agar medium lacking L-asparagine. Only transformants carrying a functional ASN1 gene will grow.

Magnaporthe grisea wild-type strains and strains containing a heterologous form of ASN1 are grown under standard fungal growth conditions well known and described in the art. Magnaporthe grisea spores are harvested from the grown cultures using wet cotton swabs after growth on complete agar medium for 10-13 days in the light at 25 ° C. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. The effect of each compound on wild-type and heterologous fungal strain, measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition. Percent growth inhibition is compared as a result of test compounds against wild type and heterologous fungal strains. Compounds that show differential growth inhibition between wild type and heterologous strains are identified as potential antifungal compounds with specificity for natural or heterologous ASN1 gene products. Similar protocols are found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221).

Pathway-specific in vivo assay screening protocol:
Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. After growing on oatmeal agar medium for 10-13 days in the light at 25 ° C., wild type M. pneumoniae was grown from the grown culture using a wet cotton swab . Collect grisea spores. Spore concentration was measured using a hemocytometer and spore suspension in minimal growth medium containing minimal growth medium and 4 mM L-asparagine (Sigma-Aldrich Co.) at a concentration of 2 × 10 ⁵ spores / ml. To prepare. Minimal growth medium contains carbon, nitrogen, phosphate, and sulfate sources, as well as magnesium, calcium, and trace elements (see, eg, the inoculum of Example 7). Add a spore suspension to each well of a 96 well microtiter plate (approximately ⁴ × 10 ⁴ spores / well). For each well containing spore suspension in minimal medium, there are additional wells containing spore suspension in minimal medium containing 4 mM L-asparagine. Test compounds are added to wells containing spores in minimal medium and wells containing spores in minimal medium containing L-asparagine. The total volume of each well is 200 μl. Both minimal medium wells and medium wells containing L-asparagine without test compound are provided as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. If the growth observed in a well containing minimal medium is inferior to that observed in a well containing L-asparagine as a result of the addition of the test compound, the compound acts on the L-asparagine biosynthetic pathway. To identify antibiotic candidates. Similar protocols are found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221).

M.M. High-throughput preparation and validation of transposon insertions into the grisea ALAS1 gene:
Escherichia coli strains containing cosmids containing transposon insertions were prepared from 1.5 ml TB (Terrific Broth, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratories) supplemented with 50 μg / ml ampicillin. To a 96 well growth block (Beckman Co.). Incubate overnight at 37 ° C. while shaking the block. E. coli cells were pelleted by centrifugation and cosmids were isolated by a modified alkaline lysis method (Marra et al. (1997) Genome Res 7: 1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis on an agarose gel. Cosmids were sequenced using primers from the end of each transposon and a commercial dideoxy sequencing kit (Big Dye Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNA sequencer (Perkin Elmer Co.).

DNA sequences flanking the insertion site were collected and used to search DNA and protein databases using the BLAST algorithm (Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402 (PMID: 9254694)). . A single insertion of SIF into the Magnaporthe grisea ALAS1 gene was chosen for further analysis. This construct is called cpgmra0011005e01 and contains a SIF transposon at approximately amino acid 100 compared to the Aspergillus nidulans homolog HEMA (full length 648 amino acids, GENBANK: 585244 (SWISS-PROT: P38092)).

Preparation of ALAS1 cosmid DNA and transformation of Magnaporthe grisea:
Cosmid DNA was prepared from cosmid clones tagged with the ALAS1 transposon using the QIAGEN Plasmid Maxi kit (QIAGEN) and digested with PI-PspI (New England Biolabs, Inc.). Fungal electrotransformation was performed as essentially described (Wu et al. (1997) MPMI 10: 700-708). For brevity, M.M. grisea strain Guy11 was grown in complete liquid medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) for 3 days at 25 ° C. in the dark with shaking at 120 rpm. Mycelium was harvested and washed with sterile H ₂ O and digested with 4 mg / ml beta-glucanase (InterSpex) for 4-6 hours to produce protoplasts. Protoplasts were collected by centrifugation and resuspended in 20% sucrose at a concentration of 2 × 10 ⁸ protoplasts / ml. 50 μl protoplast suspension was mixed with 10-20 μg cosmid DNA and pulsed with Gene Pulser II (BioRad) set to the following parameters: resistance 200 ohm, capacitance 25 μF, voltage 0.6 kV. Transformation in complete agar medium (CM, Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) supplemented with 20% sucrose and 200 μM 5-aminolevulinic acid (Sigma-Aldrich Co.) Protoplasts were regenerated and then transformants were selected by overlaying CM agar medium containing hygromycin B (250 μg / ml). Transformants were screened by PCR for homologous recombination events in the target gene (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)). Two independent strains have been identified and are referred to herein as KO1-1 and KO1-106, respectively.

Effect of transposon insertion on Magnaporte pathogenicity:
The target fungal strains obtained in Example 19, KO1-1 and KO1-106, and the wild type strain Guy11 were subjected to virulence assays and observed for infection over a period of one week. Rice infection assays were performed using Maccomo variety CO39, essentially as described by Valent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). All three strains were grown for spore production on complete agar medium. Spores were collected and the spore concentration was adjusted for whole plant inoculation. Two-week-old seedlings of variety CO39 were sprayed with 12 ml of conidia suspension (5 × 10 ⁴ conidia per ml of 0.01% Tween-20 (polyoxyethylene sorbitan monolaurate) solution). Inoculated plants were incubated in a humidified chamber at 27 ° C. in the dark for 36 hours and transferred to the growth chamber (27 ° C. 12 hours / 21 ° C. 12 hours, 70% humidity) for an additional 5.5 days. At 3, 5, and 7 days after inoculation, leaf samples were taken and examined for signs of successful infection (ie lesions). FIG. 2 shows the effect of ALAS1 gene disruption on Magnaporte infection 5 days after inoculation.

Verification of ALAS1 gene function by analysis of nutritional requirements:
Biolog, Inc. The fungal strains KO1-1 and KO1-106 containing the ALAS1 disruption gene obtained in Example 19 were analyzed for nutritional requirements for 5-aminolevulinic acid using a PM5 phenotype microarray from Hayward, Calif. PM5 plates are tested for nutritional requirements for 94 different metabolites. The inoculum is 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO ₃ , 6.7 mM KCl, 3.5 mM Na ₂ SO ₄ , 11 mM KH ₂ PO ₄ , 0.01% p- iodonitrotetrazolium violet, 0.1 mM MgCl _2, consisting of 1.0 mM CaCl ₂ and trace elements. The final concentrations of the trace elements are: 7.6 μM ZnCl ₂ , 2.5 μM MnCl ₂ ^. 4H ₂ O, 1.8 μM FeCl ₂ ^. 4H ₂ O, 0.71 μM CoCl ₂ ^. 6H ₂ O, 0.64 μM CuCl ₂ ^. 2H ₂ O, 0.62 μM Na ₂ MoO ₄ , 18 μM H ₃ BO ₃ . The pH was adjusted to 6.0 with NaOH. Spores of each strain were collected in the inoculum. The spore concentration was adjusted to 2 × 10 ⁵ spores / ml. 100 μl of spore suspension was placed in each well of the microtiter plate. Plates were incubated for 7 days at 25 ° C. Optical density (OD) measurements were taken daily at 490 nm and 750 nm. OD ₄₉₀ measures the degree of tetrazolium dye reduction and proliferation level, and OD ₇₅₀ measures proliferation only. Turbidity = OD ₄₉₀ + OD ₇₅₀ . Confirmed annotated gene function as a graph of turbidity versus time showing both mutant fungus and wild type control in the absence (Figure 3A) and presence (Figure 3B) of 5-aminolevulinate Data to be displayed.

Cloning and expression strategy, extraction and purification of 5-aminolevulinate synthase protein:
The following protocol can be used to obtain purified 5-aminolevulinate synthase protein.

Cloning and expression strategies:
The ALAS1 cDNA gene can be cloned into Escherichia coli (pET vector, Novagen), baculovirus (Pharmingen) and yeast (Invitrogen) expression vectors containing His / fusion protein tags and assembled by SDS-PAGE and Western blot analysis. The expression of the replacement protein can be evaluated.

Extraction:
Recombinant protein is extracted from 250 ml cell pellet into 3 ml extraction buffer by sonicating 6 times with 4 ° C., 6 sec pulses. The extract is centrifuged at 15000 xg for 10 minutes and the supernatant is collected. The biological activity of the recombinant protein is assessed by an activity assay.

Purification:
Recombinant protein is purified by Ni-NTA affinity chromatography (Qiagen).
Purification protocol: All steps are performed at 4 ° C .:
Use 3 ml Ni beads (Qiagen) Equilibrate column with buffer Load protein extract Wash with equilibration buffer Elute bound protein with 0.5 M imidazole

Assay to test binding of test compound to 5-aminolevulinate synthase:
The following protocol can be used to identify test compounds that bind to 5-aminolevulinate synthase protein.

• Purified full-length 5-aminolevulinate synthase polypeptide (Example 22) containing His / fusion protein tag is bound to HisGrab ^™ nickel coated plates (Pierce, Rockford, IL) according to the manufacturer's instructions.

  • Optimize buffer conditions (eg, ionic strength or pH, Coolingin- Jordan et al. (1997) Methods Enzymol 281: 309-16 (PMID: 9250995)).

Add the test compound and radiolabeled succinyl-CoA (custom made, PerkinElmer Life Sciences, Inc., Boston, Mass.) To the well of a HisGrab ^™ plate containing bound 5-aminolevulinate synthase Perform screening.

  Wash wells to remove excess labeled ligand and add scintillation fluid (Scintiver®, Fisher Scientific) to each well.

• Read the plate in a microplate scintillation counter.
Identify candidate compounds as wells with lower radioactivity compared to control wells to which no test compound has been added.

In addition, M.M. Purified polypeptides containing 10-50 amino acids from grisea 5-aminolevulinate synthase are screened in the same manner. A polypeptide containing 10-50 amino acids is generated by subcloning a portion of the ALAS1 gene into a protein expression vector that adds a His tag when expressed (see Example 22). Oligonucleotide primers are designed to amplify a portion of the ALAS1 gene using the polymerase chain reaction amplification method. As described in Example 22 above, a DNA fragment encoding a 10-50 amino acid polypeptide is cloned into an expression vector, expressed in a host organism, and purified.

Test compounds that bind to ALAS1 are further tested for antibiotic activity. As described (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 831740)) for spore production on oatmeal agar medium . Grisea is grown. Spores are harvested and the culture is split to a concentration of 2 × 10 ⁵ spores / ml in minimal medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Test compounds are added to one culture to a final concentration of 20-100 μg / ml. Only the solvent is added to the second culture. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Compare the growth curves of the solvent control sample and the test compound sample. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is inferior to that of the control culture.

Assays to test inhibitors or candidate inhibitors of 5-aminolevulinate synthase activity:
Enzymes of 5-aminolevulinate synthase in the presence and absence of candidate compounds in a suitable reaction mixture as described in Shoelingin-Jordan et al. (1997) Methods Enzymol 281: 309-16 (PMID: 9250995) Measure activity. Candidate compounds are identified when a reduction in product or lack of substrate is detected in a reaction that proceeds in either direction.

Furthermore, Shoolingin-Jordan et al. (1997) Methods Enzymol 281: 309-16 (PMID: 9250995) as described, in a suitable reaction mixture, in the presence and absence of a candidate compound, M. The enzyme activity of a polypeptide comprising 10 to 50 amino acids derived from grisea 5-aminolevulinate synthase is measured. A polypeptide containing 10-50 amino acids is generated by subcloning a portion of the ALAS1 gene into a protein expression vector that adds a His tag when expressed (see Example 22). Oligonucleotide primers are designed to amplify a portion of the ALAS1 gene using the polymerase chain reaction amplification method. As described in Example 22 above, a DNA fragment encoding a 10-50 amino acid polypeptide is cloned into an expression vector, expressed, and purified.

Test compounds identified as inhibitors of ALAS1 activity are further tested for antibiotic activity. Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. As described (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 831740)) for spore production on oatmeal agar medium . Grisea is grown. Spores are harvested and the culture is split to a concentration of 2 × 10 ⁵ spores / ml in minimal medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Test compounds are added to one culture to a final concentration of 20-100 μg / ml. Only the solvent is added to the second culture. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Compare the growth curves of the solvent control sample and the test compound sample. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is inferior to that of the control culture.

Assays to test compounds or compound candidates that alter the expression of the 5-aminolevulinate synthase gene:
Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. After growing on complete agar or oatmeal agar medium for 10-13 days in the light at 25 ° C., wild type M. pneumoniae was grown from the grown culture using a wet cotton swab . Collect grisea spores. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. A 25 ml culture is prepared, to which test compounds are added at various concentrations. Cultures in the absence of test compound are included as controls. After incubation of the culture for 3 days at 25 ° C., only the solvent is added to the test compound or control. The culture is further incubated for 18 hours. The fungal mycelium is collected by filtration through Miracloth (CalBiochem®, La Jolla, Calif.), Washed with water and frozen in liquid nitrogen. Total RNA is extracted with TRIZOL® reagent using methods provided by the manufacturer (Life Technologies, Rockville, Md.). Expression is analyzed by Northern analysis of RNA samples as described (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press) using radiolabeled fragments of the ALAS1 gene as probes. Test compounds that cause a decrease in ALAS1 mRNA levels compared to untreated control samples are identified as candidate antibiotic compounds.

In vivo cell-based assay screening protocol using a fungal strain containing a mutant form of 5-aminolevulinate synthase with no activity:
Magnaporthe grisea fungal cells containing mutant forms of the ALAS1 gene that abolish enzymatic activity, such as genes containing transposon insertions (see Examples 18 and 19), are well known in the art, and Grow under standard fungal growth conditions as described. After growing on complete agar medium containing 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., wet cotton swabs were grown from the grown cultures. Use to collect Magnaporthe grisea spores. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium containing 20 μM 5-aminolevulinate at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores are added to each well of a 96-well plate, to which test compounds (various concentrations) are added. The total volume of each well is 200 μl. Wells without test compound (growth control) and wells without cells are included as controls (negative control). Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutated form of 5-aminolevulinate synthase with reduced activity:
Magnaporthe grisea fungal cells containing a mutated form of the ALAS1 gene, such as promoter partial excision that reduces expression, are grown under standard fungal growth conditions well known and described in the art. One of the promoters upstream of the transcription initiation site using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press). A part of the promoter is excised by deleting the part. After growing on complete agar medium containing 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., wet cotton swabs were grown from the grown cultures. Use to collect Magnaporthe grisea spores. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores are added to each well of a 96-well plate, to which test compounds (various concentrations) are added. The total volume of each well is 200 μl. Wells without test compound (growth control) and wells without cells are included as controls (negative control). Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutant form of the inactive heme biosynthetic gene:
Magnaporthe grisea fungal cells containing mutant forms of the genes of the heme biosynthetic pathway (eg aminolevulinate dehydratase (EC 4.2.1.24)) are well known in the art and described Grow under standard fungal growth conditions. After growing on complete agar medium containing 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., wet cotton swabs were grown from the grown cultures. Use to collect Magnaporthe grisea spores. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium containing 20 μM 5-aminolevulinate at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutant form of the heme biosynthetic gene with reduced activity:
Magnaporthe grisea fungal cells containing a mutant form of a gene of the heme biosynthetic pathway (eg, aminolevulinate dehydratase (EC 4.2.2.14)), such as partial excision of the promoter that reduces expression, are obtained in the art. Grow under standard fungal growth conditions well known and described in the art. One of the promoters upstream of the transcription initiation site using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press). A part of the promoter is excised by deleting the part. Magnaporthe grisea fungal cells containing the mutant form are grown under standard fungal growth conditions well known and described in the art. After growing on complete agar medium containing 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., wet cotton swabs were grown from the grown cultures. Use to collect Magnaporthe grisea spores. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

M.M. In vivo cell-based assay screening protocol using a fungal strain containing the grisea ALAS1 gene and a second fungal strain containing the heterologous ALAS1 gene:
M. lacks wild-type Magnaporthe grisea fungal cells and a functional ALAS1 gene and contains a 5-aminolevulinate synthase gene from Candida albicans (Genbank: 107200014, SWISS-PROT: O9406, 54% sequence identity) . Grisea fungal cells are grown under standard fungal growth conditions, which are well known and described in the art. M. carrying the heterologous ALAS1 gene The grisea strain is created as follows:
-M. having a non-functional ALAS1 gene, such as those containing a transposon insertion in the natural gene . A grisea strain is created (see Examples 18 and 19).

Using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press), including the trpC promoter and terminator A construct containing a heterologous ALAS1 gene is created by cloning a 5-aminolevulinate synthase gene from Candida albicans into a fungal expression vector (eg, pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41:22).

• Using the construct, M. lacking a functional ALAS1 gene . The grisea strain is transformed (see Example 19). Transformants are selected on minimal agar medium lacking 5-aminolevulinate. Only transformants carrying a functional ALAS1 gene will grow.

Magnaporthe grisea wild-type strains and strains containing a heterologous form of ALAS1 are grown under standard fungal growth conditions well known and described in the art. Magnaporthe grisea spores are harvested from the grown cultures using wet cotton swabs after growth on complete agar medium for 10-13 days in the light at 25 ° C. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. The effect of each compound on wild-type and heterologous fungal strain, measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition. Percent growth inhibition is compared as a result of test compounds against wild type and heterologous fungal strains. Compounds that show differential growth inhibition between wild-type and heterologous strains are identified as potential antifungal compounds with specificity for natural or heterologous ALAS1 gene products. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

Pathway-specific in vivo assay screening protocol:
Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. After growing on oatmeal agar medium for 10-13 days in the light at 25 ° C., wild type M. pneumoniae was grown from the grown culture using a wet cotton swab . Collect grisea spores. Spore concentration was measured using a hemocytometer and in a minimal growth medium containing minimal growth medium and 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) at a concentration of 2 × 10 ⁵ spores / ml. A suspension is prepared. Minimal growth medium contains carbon, nitrogen, phosphate and sulfate sources, as well as magnesium, calcium and trace elements (see, eg, the inoculum of Example 21). Add a spore suspension to each well of a 96 well microtiter plate (approximately ⁴ × 10 ⁴ spores / well). For each well containing spore suspension in minimal medium, there are additional wells containing spore suspension in minimal medium containing 200 μM 5-aminolevulinate. Test compounds are added to wells containing spores in minimal media and wells containing spores in minimal media containing 5-aminolevulinate. The total volume of each well is 200 μl. Both minimal media wells and 5-aminolevulinate containing media wells containing no test compound are provided as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. If the addition of the test compound results in the growth observed in wells containing minimal media being inferior to that observed in wells containing 5-aminolevulinate, the compound is directed against the heme biosynthetic pathway. Identify candidate antibiotics to act. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

M.M. High-throughput preparation and validation of transposon insertions into the grisea HISP1 gene:
Escherichia coli strains containing cosmids containing transposon insertions were prepared from 1.5 ml TB (Terrific Broth, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratories) supplemented with 50 μg / ml ampicillin. To a 96 well growth block (Beckman Co.). Incubate overnight at 37 ° C. while shaking the block. E. coli cells were pelleted by centrifugation and cosmids were isolated by a modified alkaline lysis method (Marra et al. (1997) Genome Res 7: 1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis on an agarose gel. Cosmids were sequenced using primers from the end of each transposon and a commercial dideoxy sequencing kit (Big Dye Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNA sequencer (Perkin Elmer Co.).

DNA sequences flanking the insertion site were collected and used to search DNA and protein databases using the BLAST algorithm (Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402 (PMID: 9254694)). . A single insertion of SIF into the Magnaporthe grisea HISP1 gene was chosen for further analysis. This construct is designated cpgmra0012021b05 and contains a SIF transposon approximately 100 nucleotides before the start codon compared to the Schizosaccharomyces pombe homolog (full length 315 amino acids, GENBANK: 3183028, SWISS-PROT: O14059), and Is expected to eliminate or reduce gene function.

Preparation of HISP1 cosmid DNA and transformation of Magnaporthe grisea:
Cosmid DNA was prepared from cosmid clones tagged with the HISP1 transposon using the QIAGEN Plasmid Maxi kit (QIAGEN) and digested with PI-PspI (New England Biolabs, Inc.). Fungal electrotransformation was performed as essentially described (Wu et al. (1997) MPMI 10: 700-708). For brevity, M.M. grisea strain Guy11 was grown in complete liquid medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) for 3 days at 25 ° C. in the dark with shaking at 120 rpm. Mycelium was harvested and washed with sterile H ₂ O and digested with 4 mg / ml beta-glucanase (InterSpex) for 4-6 hours to produce protoplasts. Protoplasts were collected by centrifugation and resuspended in 20% sucrose at a concentration of 2 × 10 ⁸ protoplasts / ml. 50 μl protoplast suspension was mixed with 10-20 μg cosmid DNA and pulsed with Gene Pulser II (BioRad) set to the following parameters: resistance 200 ohm, capacitance 25 μF, voltage 0.6 kV. Transformed protoplasts were regenerated one day in complete agar medium (CM, Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) supplemented with 20% sucrose, followed by hygromycin B (250 μg / Transformants were selected by overlaying CM agar medium containing (ml). Transformants were screened by PCR for homologous recombination events in the target gene (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)). Two independent strains have been identified and are referred to herein as KO1-1 and KO1-3, respectively.

Effect of transposon insertion on Magnaporte pathogenicity:
The target fungal strains obtained in Example 33, KO1-1 and KO1-3, and the wild type strain Guy11 were subjected to virulence assays and observed for infection over a period of one week. Rice infection assays were performed using Maccomo variety CO39, essentially as described by Valent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). All three strains were grown for spore production on complete agar medium. Spores were collected and the spore concentration was adjusted for whole plant inoculation. Two-week-old seedlings of variety CO39 were sprayed with 12 ml of conidia suspension (5 × 10 ⁴ conidia per ml of 0.01% Tween-20 (polyoxyethylene sorbitan monolaurate) solution). Inoculated plants were incubated in a humidified chamber at 27 ° C. in the dark for 36 hours and transferred to the growth chamber (27 ° C. 12 hours / 21 ° C. 12 hours, 70% humidity) for an additional 5.5 days. At 3, 5, and 7 days after inoculation, leaf samples were taken and examined for signs of successful infection (ie lesions). FIG. 2 shows the effect of HISP1 gene disruption on Magnaporte infection 5 days after inoculation.

Verification of HISP1 gene function by analysis of nutritional requirements:
Biolog, Inc. The fungal strains KO1-1 and KO1-3 containing the HISP1 disruption gene obtained in Example 33 were analyzed for nutritional requirements for histidine using PM5 phenotype microarrays (Hayward, Calif.). PM5 plates are tested for nutritional requirements for 94 different metabolites. The inoculum is 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO ₃ , 6.7 mM KCl, 3.5 mM Na ₂ SO ₄ , 11 mM KH ₂ PO ₄ , 0.01% p- iodonitrotetrazolium violet, 0.1 mM MgCl _2, consisting of 1.0 mM CaCl ₂ and trace elements. The final concentrations of the trace elements are: 7.6 μM ZnCl ₂ , 2.5 μM MnCl ₂ ^. 4H ₂ O, 1.8 μM FeCl ₂ ^. 4H ₂ O, 0.71 μM CoCl ₂ ^. 6H ₂ O, 0.64 μM CuCl ₂ ^. 2H ₂ O, 0.62 μM Na ₂ MoO ₄ , 18 μM H ₃ BO ₃ . The pH was adjusted to 6.0 with NaOH. Spores of each strain were collected in the inoculum. The spore concentration was adjusted to 2 × 10 ⁵ spores / ml. 100 μl of spore suspension was placed in each well of the microtiter plate. Plates were incubated for 7 days at 25 ° C. Optical density (OD) measurements were taken daily at 490 nm and 750 nm. OD ₄₉₀ measures the degree of tetrazolium dye reduction and proliferation level, and OD ₇₅₀ measures proliferation only. Turbidity = OD ₄₉₀ + OD ₇₅₀ . Data confirming the annotated gene function as a graph of turbidity versus time showing both mutant fungus and wild-type control in the absence (Figure 3A) and presence (Figure 3B) of L-histidine. Show.

Cloning and expression strategy, extraction and purification of histidinol-phosphatase protein:
The following protocol can be used to obtain purified histidinol-phosphatase protein.

Cloning and expression strategies:
The HISP1 cDNA gene can be cloned into Escherichia coli (pET vector, Novagen), baculovirus (Pharmingen) and yeast (Invitrogen) expression vectors containing His / fusion protein tags and assembled by SDS-PAGE and Western blot analysis. The expression of the replacement protein can be evaluated.

Extraction:
Recombinant protein is extracted from 250 ml cell pellet into 3 ml extraction buffer by sonicating 6 times with 4 ° C., 6 sec pulses. The extract is centrifuged at 15000 xg for 10 minutes and the supernatant is collected. The biological activity of the recombinant protein is assessed by an activity assay.

Purification:
Recombinant protein is purified by Ni-NTA affinity chromatography (Qiagen).
Purification protocol: All steps are performed at 4 ° C .:
Use 3 ml Ni beads (Qiagen) Equilibrate column with buffer Load protein extract Wash with equilibration buffer Elute bound protein with 0.5 M imidazole

Assay to test binding of test compound to histidinol-phosphatase:
The following protocol can be used to identify test compounds that bind to the histidinol-phosphatase protein.

• Purified full-length histidinol-phosphatase polypeptide (Example 36) containing a His / fusion protein tag is bound to HisGrab ^™ nickel-coated plates (Pierce, Rockford, IL) according to the manufacturer's instructions.

  Optimize buffer conditions (eg, ionic strength or pH, Milly) for binding of radiolabeled L-histidinol phosphate (Custom-made, PerkinElmer Life Sciences, Inc., Boston, Mass.) To bound histidinol-phosphatase And Houston (1973) Biochemistry 12: 2591-2596 (PMID: 4351203)).

Screening of test compounds by adding test compounds and L-histidinol phosphatase (custom made, PerkinElmer Life Sciences, Inc., Boston, Mass.) To the wells of HisGrab ^™ plates containing bound histidinol-phosphatase I do.

  Wash wells to remove excess labeled ligand and add scintillation fluid (Scintiver®, Fisher Scientific) to each well.

• Read the plate in a microplate scintillation counter.
Identify candidate compounds as wells with lower radioactivity compared to control wells to which no test compound has been added.

In addition, M.M. Purified polypeptides containing 10-50 amino acids from grisea histidinol-phosphatase are screened in the same manner. A polypeptide containing 10-50 amino acids is generated by subcloning a portion of the HISP1 gene into a protein expression vector that adds a His tag when expressed (see Example 36). Oligonucleotide primers are designed to amplify a portion of the HISP1 gene using the polymerase chain reaction amplification method. As described in Example 36 above, a DNA fragment encoding a 10-50 amino acid polypeptide is cloned into an expression vector, expressed in a host organism, and purified.

Test compounds that bind to HISP1 are further tested for antibiotic activity. As described (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 831740)) for spore production on oatmeal agar medium . Grisea is grown. Spores are harvested and the culture is split to a concentration of 2 × 10 ⁵ spores / ml in minimal medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Test compounds are added to one culture to a final concentration of 20-100 μg / ml. Only the solvent is added to the second culture. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Compare the growth curves of the solvent control sample and the test compound sample. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is inferior to that of the control culture.

Assays to test inhibitors or candidate inhibitors of histidinol-phosphatase activity:
Measure the enzymatic activity of histidinol-phosphatase in the presence and absence of candidate compounds in an appropriate reaction mixture as described in Millay and Houston (1973) Biochemistry 12: 2591-2596 (PMID: 4351203). . Candidate compounds are identified when a reduction in product or lack of substrate is detected in a reaction that proceeds in either direction.

Further, as described in Millay and Houston (1973) Biochemistry 12: 2591-2596 (PMID: 4351203), in the presence and absence of candidate compounds, M. The enzymatic activity of a polypeptide comprising 10-50 amino acids derived from grisea histidinol-phosphatase is measured. A polypeptide containing 10-50 amino acids is generated by subcloning a portion of the HISP1 gene into a protein expression vector that adds a His tag when expressed (see Example 36). Oligonucleotide primers are designed to amplify a portion of the HISP1 gene using the polymerase chain reaction amplification method. As described in Example 36 above, a DNA fragment encoding a 10-50 amino acid polypeptide is cloned into an expression vector, expressed and purified.

Test compounds identified as inhibitors of HISP1 activity are further tested for antibiotic activity. Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. As described (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 831740)) for spore production on oatmeal agar medium . Grisea is grown. Spores are harvested and the culture is split to a concentration of 2 × 10 ⁵ spores / ml in minimal medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Test compounds are added to one culture to a final concentration of 20-100 μg / ml. Only the solvent is added to the second culture. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Compare the growth curves of the solvent control sample and the test compound sample. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is inferior to that of the control culture.

Assay to test compounds that alter the expression of the histidinol-phosphatase gene:
Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. After growing on complete agar or oatmeal agar medium for 10-13 days in the light at 25 ° C., wild type M. pneumoniae was grown from the grown culture using a wet cotton swab . Collect grisea spores. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. A 25 ml culture is prepared, to which test compounds are added at various concentrations. Cultures in the absence of test compound are included as controls. After incubation of the culture for 3 days at 25 ° C., only the solvent is added to the test compound or control. The culture is further incubated for 18 hours. The fungal mycelium is collected by filtration through Miracloth (CalBiochem®, La Jolla, Calif.), Washed with water and frozen in liquid nitrogen. Total RNA is extracted with TRIZOL® reagent using methods provided by the manufacturer (Life Technologies, Rockville, Md.). Expression is analyzed by Northern analysis of RNA samples as described (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press) using radiolabeled fragments of the HISP1 gene as probes. Test compounds that cause a decrease in HISP1 mRNA levels compared to untreated control samples are identified as candidate antibiotic compounds.

In vivo cell-based assay screening protocol using a fungal strain containing a mutant form of inactive histidinol-phosphatase:
Magnaporthe grisea fungal cells containing mutant forms of the HISP1 gene that abolish enzymatic activity, such as genes containing transposon insertions (see Examples 32 and 33), are well known in the art, and Grow under standard fungal growth conditions as described. After growing on complete agar medium containing 4 mM L-histidine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium containing 100 μM L-histidine at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores are added to each well of a 96-well plate, to which test compounds (various concentrations) are added. The total volume of each well is 200 μl. Wells without test compound (growth control) and wells without cells are included as controls (negative control). Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutated form of histidinol-phosphatase with reduced activity:
Magnaporthe grisea fungal cells containing mutant forms of the HISP1 gene, such as promoter partial excisions that reduce expression, are grown under standard fungal growth conditions well known and described in the art. One of the promoters upstream of the transcription initiation site using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press). A part of the promoter is excised by deleting the part. After growing on complete agar medium containing 4 mM L-histidine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores are added to each well of a 96-well plate, to which test compounds (various concentrations) are added. The total volume of each well is 200 μl. Wells without test compound (growth control) and wells without cells are included as controls (negative control). Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutant form of the inactive L-histidine biosynthetic gene:
Magnaporthe grisea fungal cells containing mutant forms of L-histidine biosynthetic pathway genes such as histidinol dehydrogenase (EC 1.1.1.23) are well known in the art and Grow under standard fungal growth conditions as described. After growing on complete agar medium containing 4 mM L-histidine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium containing 100 μM L-histidine at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutated form of the L-histidine biosynthetic gene with reduced activity:
Magnaporthe grisea fungal cells containing a mutant form of a gene of the L-histidine biosynthetic pathway (eg, histidinol dehydrogenase (EC 1.1.1.23)), such as partial excision of the promoter that reduces expression Grow under standard fungal growth conditions well known and described in the art. One of the promoters upstream of the transcription initiation site using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press). A part of the promoter is excised by deleting the part. Magnaporthe grisea fungal cells containing the mutant form are grown under standard fungal growth conditions well known and described in the art. After growing on complete agar medium containing 4 mM L-histidine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing the fungal HISP1 gene and a second fungal strain containing the heterologous HISP1 gene:
Wild-type Magnaporthe grisea fungal cells and M. lacking a functioning HISP1 gene and contain a heterologous HISP1 gene . Grisea fungal cells are grown under standard fungal growth conditions, which are well known and described in the art. M. possessing a heterologous HISP1 gene . The grisea strain is created as follows:
M. having a non-functional HISP1 gene, such as those containing a transposon insertion in the natural gene . A grisea strain is created (see Examples 32 and 33).

Using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press), including the trpC promoter and terminator A construct containing the heterologous HISP1 gene is created by cloning the heterologous HISP1 gene into a fungal expression vector (eg, pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41:22).

• Using the construct, M. lacking a functioning HISP1 gene . The grisea strain is transformed (see Example 33). Transformants are selected on minimal agar medium lacking L-histidine. Only transformants carrying a functional HISP1 gene will grow.

Magnaporthe grisea wild-type strains and strains containing heterologous forms of HISP1 are grown under standard fungal growth conditions well known and described in the art. Magnaporthe grisea spores are harvested from the grown cultures using wet cotton swabs after growth on complete agar medium for 10-13 days in the light at 25 ° C. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. The effect of each compound on wild-type and heterologous fungal strain, measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition. Percent growth inhibition is compared as a result of test compounds against wild type and heterologous fungal strains. Compounds that show differential growth inhibition between wild-type and heterologous strains are identified as potential antifungal compounds with specificity for natural or heterologous HISP1 gene products. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

Pathway-specific in vivo assay screening protocol:
Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. After growing on oatmeal agar medium for 10-13 days in the light at 25 ° C., wild type M. pneumoniae was grown from the grown culture using a wet cotton swab . Collect grisea spores. Spore concentration was measured using a hemocytometer and spore suspension in minimal growth medium containing minimal growth medium and 4 mM L-histidine (Sigma-Aldrich Co.) at a concentration of 2 × 10 ⁵ spores / ml. To prepare. Minimal growth medium contains carbon, nitrogen, phosphate, and sulfate sources, and magnesium, calcium, and trace elements (see, eg, the inoculum of Example 35). Add a spore suspension to each well of a 96 well microtiter plate (approximately ⁴ × 10 ⁴ spores / well). For each well containing spore suspension in minimal medium, there are additional wells containing spore suspension in minimal medium containing 4 mM L-histidine. Test compounds are added to wells containing spores in minimal medium and wells containing spores in minimal medium containing L-histidine. The total volume of each well is 200 μl. Both minimal medium wells and medium wells containing L-histidine without test compound are provided as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. If the growth observed in the wells containing minimal medium is inferior to that observed in the wells containing L-histidine as a result of adding the test compound, the compound acts on the L-histidine biosynthetic pathway. To identify antibiotic candidates. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

M.M. High-throughput preparation and validation of transposon insertions into the grisea IPMD1 gene:
Escherichia coli strains containing cosmids containing transposon insertions were prepared from 1.5 ml TB (Terrific Broth, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratories) supplemented with 50 μg / ml ampicillin. To a 96 well growth block (Beckman Co.). Incubate overnight at 37 ° C. while shaking the block. E. coli cells were pelleted by centrifugation and cosmids were isolated by a modified alkaline lysis method (Marra et al. (1997) Genome Res 7: 1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis on an agarose gel. Cosmids were sequenced using primers from the end of each transposon and a commercial dideoxy sequencing kit (Big Dye Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNA sequencer (Perkin Elmer Co.).

DNA sequences flanking the insertion site were collected and used to search DNA and protein databases using the BLAST algorithm (Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402 (PMID: 9254694)). . A single insertion of SIF into the Magnaporthe grisea IPMD1 gene was chosen for further analysis. This construct is designated cpgmra0014081f03 and contains a SIF transposon between approximately amino acids 560 and 561 compared to the Rhizomucor pusillus homologue LeuA (full length 755 amino acids, SWISS-PROT: P55251).

Preparation of IPMD1 cosmid DNA and transformation of Magnaporthe grisea:
Cosmid DNA was prepared from cosmid clones tagged with IPMD1 transposon using the QIAGEN Plasmid Maxi kit (QIAGEN) and digested with PI-PspI (New England Biolabs, Inc.). Fungal electrotransformation was performed as essentially described (Wu et al. (1997) MPMI 10: 700-708). For brevity, M.M. grisea strain Guy11 was grown in complete liquid medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) for 3 days at 25 ° C. in the dark with shaking at 120 rpm. Mycelium was harvested and washed with sterile H ₂ O and digested with 4 mg / ml beta-glucanase (InterSpex) for 4-6 hours to produce protoplasts. Protoplasts were collected by centrifugation and resuspended in 20% sucrose at a concentration of 2 × 10 ⁸ protoplasts / ml. 50 μl protoplast suspension was mixed with 10-20 μg cosmid DNA and pulsed with Gene Pulser II (BioRad) set to the following parameters: resistance 200 ohm, capacitance 25 μF, voltage 0.6 kV. Transformed protoplasts were regenerated one day in complete agar medium (CM, Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) supplemented with 20% sucrose, followed by hygromycin B (250 μg / Transformants were selected by overlaying CM agar medium containing (ml). Transformants were screened by PCR for homologous recombination events in the target gene (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)). Two independent strains have been identified and are referred to herein as KO1-3 and KO1-7, respectively.

Effect of transposon insertion on Magnaporte pathogenicity:
The target fungal strains obtained in Example 47, KO1-3 and KO1-7, and the wild type strain Guy11 were subjected to virulence assays and observed for infection over a period of one week. Rice infection assays were performed using Maccomo variety CO39, essentially as described by Valent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). All three strains were grown for spore production on complete agar medium. Spores were collected and the spore concentration was adjusted for whole plant inoculation. Two-week-old seedlings of variety CO39 were sprayed with 12 ml of conidia suspension (5 × 10 ⁴ conidia per ml of 0.01% Tween-20 (polyoxyethylene sorbitan monolaurate) solution). Inoculated plants were incubated in a humidified chamber at 27 ° C. in the dark for 36 hours and transferred to the growth chamber (27 ° C. 12 hours / 21 ° C. 12 hours, 70% humidity) for an additional 5.5 days. At 3, 5, and 7 days after inoculation, leaf samples were taken and examined for signs of successful infection (ie lesions). FIG. 2 shows the effect of IPMD1 gene disruption on Magnaporte infection 5 days after inoculation.

Verification of IPMD1 gene function by analysis of nutritional requirements:
Biolog, Inc. The fungal strains KO1-3 and KO1-7 containing the IPMD1 disruption gene obtained in Example 47 were analyzed for nutritional requirements for L-leucine using PM5 phenotype microarray (Hayward, Calif.). PM5 plates are tested for nutritional requirements for 94 different metabolites. The inoculum is 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO ₃ , 6.7 mM KCl, 3.5 mM Na ₂ SO ₄ , 11 mM KH ₂ PO ₄ , 0.01% p- iodonitrotetrazolium violet, 0.1 mM MgCl _2, consisting of 1.0 mM CaCl ₂ and trace elements. The final concentrations of the trace elements are: 7.6 μM ZnCl ₂ , 2.5 μM MnCl ₂ ^. 4H ₂ O, 1.8 μM FeCl ₂ ^. 4H ₂ O, 0.71 μM CoCl ₂ ^. 6H ₂ O, 0.64 μM CuCl ₂ ^. 2H ₂ O, 0.62 μM Na ₂ MoO ₄ , 18 μM H ₃ BO ₃ . The pH was adjusted to 6.0 with NaOH. Spores of each strain were collected in the inoculum. The spore concentration was adjusted to 2 × 10 ⁵ spores / ml. 100 μl of spore suspension was placed in each well of the microtiter plate. Plates were incubated for 7 days at 25 ° C. Optical density (OD) measurements were taken daily at 490 nm and 750 nm. OD ₄₉₀ measures the degree of tetrazolium dye reduction and proliferation level, and OD ₇₅₀ measures proliferation only. Turbidity = OD ₄₉₀ + OD ₇₅₀ . Data confirming the annotated gene function as a graph of turbidity versus time showing both mutant fungus and wild type control in the absence (Figure 3A) and presence (Figure 3B) of L-leucine. Show.

Cloning and expression strategy, extraction and purification of 3-isopropylmalate dehydratase protein:
The following protocol can be used to obtain purified 3-isopropylmalate dehydratase protein.

Cloning and expression strategies:
The IPMD1 cDNA gene can be cloned into Escherichia coli (pET vector, Novagen), baculovirus (Pharmingen) and yeast (Invitrogen) expression vectors containing His / fusion protein tags and assembled by SDS-PAGE and Western blot analysis. The expression of the replacement protein can be evaluated.

Extraction:
Recombinant protein is extracted from 250 ml cell pellet into 3 ml extraction buffer by sonicating 6 times with 4 ° C., 6 sec pulses. The extract is centrifuged at 15000 xg for 10 minutes and the supernatant is collected. The biological activity of the recombinant protein is assessed by an activity assay.

Purification:
Recombinant protein is purified by Ni-NTA affinity chromatography (Qiagen).
Purification protocol: All steps are performed at 4 ° C .:
Use 3 ml Ni beads (Qiagen) Equilibrate column with buffer Load protein extract Wash with equilibration buffer Elute bound protein with 0.5 M imidazole

Assay to test binding of test compound to 3-isopropylmalate dehydratase:
The following protocol can be used to identify test compounds that bind to 3-isopropylmalate dehydratase protein.

• Purified full-length 3-isopropylmalate dehydratase polypeptide (Example 50) containing a His / fusion protein tag is bound to a HisGrab ^™ nickel coated plate (Pierce, Rockford, IL) according to the manufacturer's instructions. .

  Optimize buffer conditions (eg ionic strength or pH) for binding of radiolabeled 2-isopropylmalate (custom made, PerkinElmer Life Sciences, Inc., Boston, Mass.) To bound 3-isopropylmalate dehydratase Satyanarayana et al. ((1968B) J Bacteriol 96: 2018-24 (PMID: 5724970)) and / or Kohlhow ((1988) Methods Enzymol 166: 423-9 (PMID: 30771717)).

Test compounds by adding test compounds and 2-isopropyl malate (custom made, PerkinElmer Life Sciences, Inc., Boston, Mass.) To wells of HisGrab ^™ plates containing bound 3-isopropylmalate dehydratase Screening.

  Wash wells to remove excess labeled ligand and add scintillation fluid (Scintiver®, Fisher Scientific) to each well.

• Read the plate in a microplate scintillation counter.
Identify candidate compounds as wells with lower radioactivity compared to control wells to which no test compound has been added.

In addition, M.M. Purified polypeptides containing 10-50 amino acids from grisea 3-isopropylmalate dehydratase are screened in the same manner. A polypeptide containing 10-50 amino acids is generated by subcloning a portion of the IPMD1 gene into a protein expression vector that adds a His tag when expressed (see Example 50). Oligonucleotide primers are designed to amplify a portion of the IPMD1 gene using the polymerase chain reaction amplification method. As described in Example 50 above, a DNA fragment encoding a 10-50 amino acid polypeptide is cloned into an expression vector, expressed in a host organism, and purified.

Test compounds that bind to IPMD1 are further tested for antibiotic activity. As described (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 831740)) for spore production on oatmeal agar medium . Grisea is grown. Spores are harvested and the culture is split to a concentration of 2 × 10 ⁵ spores / ml in minimal medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Test compounds are added to one culture to a final concentration of 20-100 μg / ml. Only the solvent is added to the second culture. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Compare the growth curves of the solvent control sample and the test compound sample. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is inferior to that of the control culture.

Assays to test inhibitors or candidate inhibitors of 3-isopropylmalate dehydratase activity:
Appropriate reactions, as described in Satyanarayana et al. ((1968B) J Bacteriol 96: 2018-24 (PMID: 5724970)) and / or Kohlhow ((1988) Methods Enzymol 166: 423-9 (PMID: 30771717)). The enzyme activity of 3-isopropylmalate dehydratase is measured in the mixture in the presence and absence of the candidate compound. Candidate compounds are identified when a reduction in product or lack of substrate is detected in a reaction that proceeds in either direction.

Further, as described in Satyanarayana et al. ((1968B) J Bacteriol 96: 2018-24 (PMID: 5724970)) and / or Kohlhow ((1988) Methods Enzymol 166: 423-9 (PMID: 30771717)) M. reaction mixture in the presence and absence of candidate compounds . The enzyme activity of a polypeptide containing 10 to 50 amino acids derived from grisea 3-isopropylmalate dehydratase is measured. A polypeptide containing 10-50 amino acids is generated by subcloning a portion of the IPMD1 gene into a protein expression vector that adds a His tag when expressed (see Example 50). Oligonucleotide primers are designed to amplify a portion of the IPMD1 gene using the polymerase chain reaction amplification method. As described in Example 50 above, a DNA fragment encoding a 10-50 amino acid polypeptide is cloned into an expression vector, expressed, and purified.

Test compounds identified as inhibitors of IPMD1 activity are further tested for antibiotic activity. Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. As described (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 831740)) for spore production on oatmeal agar medium . Grisea is grown. Spores are harvested and the culture is split to a concentration of 2 × 10 ⁵ spores / ml in minimal medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Test compounds are added to one culture to a final concentration of 20-100 μg / ml. Only the solvent is added to the second culture. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Compare the growth curves of the solvent control sample and the test compound sample. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is inferior to that of the control culture.

Assay to test compounds that modify the expression of the 3-isopropylmalate dehydratase gene:
Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. After growing on complete agar or oatmeal agar medium for 10-13 days in the light at 25 ° C., wild type M. pneumoniae was grown from the grown culture using a wet cotton swab . Collect grisea spores. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. A 25 ml culture is prepared, to which test compounds are added at various concentrations. Cultures in the absence of test compound are included as controls. After incubation of the culture for 3 days at 25 ° C., only the solvent is added to the test compound or control. The culture is further incubated for 18 hours. The fungal mycelium is collected by filtration through Miracloth (CalBiochem®, La Jolla, Calif.), Washed with water and frozen in liquid nitrogen. Total RNA is extracted with TRIZOL® reagent using methods provided by the manufacturer (Life Technologies, Rockville, Md.). Expression is analyzed by Northern analysis of RNA samples as described (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press) using a radiolabeled fragment of the IPMD1 gene as a probe. Test compounds that produce a decrease in IPMD1 mRNA levels relative to untreated control samples are identified as candidate antibiotic compounds.

In vivo cell-based assay screening protocol using a fungal strain containing a non-active mutant of 3-isopropylmalate dehydratase:
Magnaporthe grisea fungal cells containing mutant forms of the IPMD1 gene that abolish enzymatic activity, such as genes containing transposon insertions (see Examples 46 and 47), are well known in the art, and Grow under standard fungal growth conditions as described. After growing on complete agar medium containing 4 mM L-leucine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium containing 100 μM L-leucine at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores are added to each well of a 96-well plate, to which test compounds (various concentrations) are added. The total volume of each well is 200 μl. Wells without test compound (growth control) and wells without cells are included as controls (negative control). Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutated form of 3-isopropylmalate dehydratase with reduced activity:
Magnaporthe grisea fungal cells containing mutant forms of the IPMD1 gene, such as promoter partial excisions that reduce expression, are grown under standard fungal growth conditions well known and described in the art. One of the promoters upstream of the transcription initiation site using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press). A part of the promoter is excised by deleting the part. After growing on complete agar medium containing 4 mM L-leucine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores are added to each well of a 96-well plate, to which test compounds (various concentrations) are added. The total volume of each well is 200 μl. Wells without test compound (growth control) and wells without cells are included as controls (negative control). Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutant form of the inactive L-leucine biosynthesis gene:
Magnaporthe grisea fungal cells containing mutant forms of L-leucine biosynthetic pathway genes such as 3-isopropylmalate dehydrogenase (EC 1.1.1.85) are well known in the art. And grown under standard fungal growth conditions as described. After growing on complete agar medium containing 4 mM L-leucine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium containing 100 μM L-leucine at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutated form of the L-leucine biosynthetic gene with reduced activity:
Magnaporthe grisea fungus containing a mutant form of a gene of the L-leucine biosynthetic pathway (eg, 3-isopropylmalate dehydrogenase (EC 1.1.1.85)), such as partial excision of the promoter that reduces expression Cells are grown under standard fungal growth conditions, which are well known and described in the art. One of the promoters upstream of the transcription initiation site using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press). A part of the promoter is excised by deleting the part. Magnaporthe grisea fungal cells containing the mutant form are grown under standard fungal growth conditions well known and described in the art. After growing on complete agar medium containing 4 mM L-leucine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing the fungal IPMD1 gene and a second fungal strain containing the heterologous IPMD1 gene:
M. lacks wild-type Magnaporthe grisea fungal cells and a functioning IPMD1 gene and contains the 3-isopropylmalate dehydratase large subunit gene (Genbank accession number H82564, 63% sequence identity) from Xylella fastidiosa . Grisea fungal cells are grown under standard fungal growth conditions, which are well known and described in the art. M. carrying the heterologous IPMD1 gene The grisea strain is created as follows:
-M. having a non-functional IPMD1 gene, such as those containing a transposon insertion in the natural gene . A grisea strain is created (see Examples 46 and 47).

Using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press), including the trpC promoter and terminator Cloning the 3-isopropylmalate dehydratase large subunit gene from Xylella fastidiosa into a fungal expression vector (eg pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41:22) create.

• Using the construct, M. lacking a functioning IPMD1 gene . The grisea strain is transformed (see Example 47). Transformants are selected on minimal agar medium lacking L-leucine. Only transformants carrying a functioning IPMD1 gene will grow.

Magnaporthe grisea wild-type strains and strains containing a heterologous type of IPMD1 are grown under standard fungal growth conditions well known and described in the art. Magnaporthe grisea spores are harvested from the grown cultures using wet cotton swabs after growth on complete agar medium for 10-13 days in the light at 25 ° C. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. The effect of each compound on wild-type and heterologous fungal strain, measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition. Percent growth inhibition is compared as a result of test compounds against wild type and heterologous fungal strains. Compounds that show differential growth inhibition between wild-type and heterologous strains are identified as potential antifungal compounds with specificity for natural or heterologous IPMD1 gene products. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

Pathway-specific in vivo assay screening protocol:
Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. After growing on oatmeal agar medium for 10-13 days in the light at 25 ° C., wild type M. pneumoniae was grown from the grown culture using a wet cotton swab . Collect grisea spores. Spore concentration was measured using a haemocytometer and spore suspension in minimal growth medium and minimal growth medium containing 4 mM L-leucine (Sigma-Aldrich Co.) at a concentration of 2 × 10 ⁵ spores / ml. To prepare. Minimal growth medium contains carbon, nitrogen, phosphate, and sulfate sources, and magnesium, calcium, and trace elements (see, eg, the inoculum of Example 49). Add a spore suspension to each well of a 96 well microtiter plate (approximately ⁴ × 10 ⁴ spores / well). For each well containing spore suspension in minimal medium, there are additional wells containing spore suspension in minimal medium containing 4 mM L-leucine. Test compounds are added to wells containing spores in minimal medium and wells containing spores in minimal medium containing L-leucine. The total volume of each well is 200 μl. Both minimal medium wells and medium wells containing L-leucine without test compound are provided as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. If the growth observed in the well containing the minimal medium is inferior to that observed in the well containing L-leucine as a result of adding the test compound, the compound acts on the L-leucine biosynthetic pathway. To identify antibiotic candidates. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

M.M. High-throughput preparation and validation of transposon insertions into the grisea THR4 gene:
Escherichia coli strains containing cosmids containing transposon insertions were prepared from 1.5 ml TB (Terrific Broth, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratories) supplemented with 50 μg / ml ampicillin. To a 96 well growth block (Beckman Co.). Incubate overnight at 37 ° C. while shaking the block. E. coli cells were pelleted by centrifugation and cosmids were isolated by a modified alkaline lysis method (Marra et al. (1997) Genome Res 7: 1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis on an agarose gel. Cosmids were sequenced using primers from the end of each transposon and a commercial dideoxy sequencing kit (Big Dye Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNA sequencer (Perkin Elmer Co.).

DNA sequences flanking the insertion site were collected and used to search DNA and protein databases using the BLAST algorithm (Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402 (PMID: 9254694)). . A single insertion of SIF into the Magnaporthe grisea THR4 gene was chosen for further analysis. This construct is referred to as cpgmra0012020a04 and contains a SIF transposon between approximately amino acids 314 and 315 compared to the Schizosaccharomyces pombe homologue ThrC (full length 514 amino acids, GENBANK: 2501152).

Preparation of THR4 cosmid DNA and transformation of Magnaporthe grisea:
Cosmid DNA was prepared from cosmid clones tagged with a THR4 transposon using the QIAGEN Plasmid Maxi kit (QIAGEN) and digested with PI-PspI (New England Biolabs, Inc.). Fungal electrotransformation was performed as essentially described (Wu et al. (1997) MPMI 10: 700-708). For brevity, M.M. grisea strain Guy11 was grown in complete liquid medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) for 3 days at 25 ° C. in the dark with shaking at 120 rpm. Mycelium was harvested and washed with sterile H ₂ O and digested with 4 mg / ml beta-glucanase (InterSpex) for 4-6 hours to produce protoplasts. Protoplasts were collected by centrifugation and resuspended in 20% sucrose at a concentration of 2 × 10 ⁸ protoplasts / ml. 50 μl protoplast suspension was mixed with 10-20 μg cosmid DNA and pulsed with Gene Pulser II (BioRad) set to the following parameters: resistance 200 ohm, capacitance 25 μF, voltage 0.6 kV. Transformed protoplasts were regenerated one day in complete agar medium (CM, Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) supplemented with 20% sucrose, followed by hygromycin B (250 μg / Transformants were selected by overlaying CM agar medium containing (ml). Transformants were screened by PCR for homologous recombination events in the target gene (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)). Two independent strains have been identified and are referred to herein as KO1-3 and KO1-22, respectively.

Effect of transposon insertion on Magnaporte pathogenicity:
The target fungal strains obtained in Example 61, KO1-3 and KO1-22, and the wild type strain Guy11 were subjected to virulence assays and observed for infection over a period of 1 week. Rice infection assays were performed using Maccomo variety CO39, essentially as described by Valent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). All three strains were grown for spore production on complete agar medium. Spores were collected and the spore concentration was adjusted for whole plant inoculation. Two-week-old seedlings of variety CO39 were sprayed with 12 ml of conidia suspension (5 × 10 ⁴ conidia per ml of 0.01% Tween-20 (polyoxyethylene sorbitan monolaurate) solution). Inoculated plants were incubated in a humidified chamber at 27 ° C. in the dark for 36 hours and transferred to the growth chamber (27 ° C. 12 hours / 21 ° C. 12 hours, 70% humidity) for an additional 5.5 days. At 3, 5, and 7 days after inoculation, leaf samples were taken and examined for signs of successful infection (ie lesions). FIG. 2 shows the effect of THR4 gene disruption on Magnaporte infection 5 days after inoculation.

Verification of THR4 gene function by analysis of nutritional requirements:
Biolog, Inc. The fungal strains KO1-3 and KO1-22 containing the THR4 disruption gene obtained in Example 61 were analyzed for nutritional requirements for L-threonine using PM5 phenotype microarray (Hayward, Calif.). PM5 plates are tested for nutritional requirements for 94 different metabolites. The inoculum is 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO ₃ , 6.7 mM KCl, 3.5 mM Na ₂ SO ₄ , 11 mM KH ₂ PO ₄ , 0.01% p- iodonitrotetrazolium violet, 0.1 mM MgCl _2, consists 1.0 mM CaCl ₂ and trace elements, with NaOH, adjusting the pH to 6.0. The final concentrations of the trace elements are: 7.6 μM ZnCl ₂ , 2.5 μM MnCl ₂ ^. 4H ₂ O, 1.8 μM FeCl ₂ ^. 4H ₂ O, 0.71 μM CoCl ₂ ^. 6H ₂ O, 0.64 μM CuCl ₂ ^. 2H ₂ O, 0.62 μM Na ₂ MoO ₄ , 18 μM H ₃ BO ₃ . Spores of each strain were collected in the inoculum. The spore concentration was adjusted to 2 × 10 ⁵ spores / ml. 100 μl of spore suspension was placed in each well of the microtiter plate. Plates were incubated for 7 days at 25 ° C. Optical density (OD) measurements were taken daily at 490 nm and 750 nm. OD ₄₉₀ measures the degree of tetrazolium dye reduction and proliferation level, and OD ₇₅₀ measures proliferation only. Turbidity = OD ₄₉₀ + OD ₇₅₀ . Data confirming the annotated gene function as a graph of turbidity versus time showing both mutant fungus and wild type control in the absence (Figure 3A) and presence (Figure 3B) of L-threonine. Show.

Cloning and expression strategy, extraction and purification of threonine synthase protein:
The following protocol can be used to obtain purified threonine synthase protein.

Cloning and expression strategies:
The THR4 cDNA gene can be cloned into Escherichia coli (pET vector, Novagen), baculovirus (Pharmingen) and yeast (Invitrogen) expression vectors containing His / fusion protein tags and assembled by SDS-PAGE and Western blot analysis. The expression of the replacement protein can be evaluated.

Extraction:
Recombinant protein is extracted from 250 ml cell pellet into 3 ml extraction buffer by sonicating 6 times with 4 ° C., 6 sec pulses. The extract is centrifuged at 15000 xg for 10 minutes and the supernatant is collected. The biological activity of the recombinant protein is assessed by an activity assay.

Purification:
Recombinant protein is purified by Ni-NTA affinity chromatography (Qiagen).
Purification protocol: All steps are performed at 4 ° C .:
Use 3 ml Ni beads (Qiagen) Equilibrate column with buffer Load protein extract Wash with equilibration buffer Elute bound protein with 0.5 M imidazole

Assays that test the binding of a test compound to threonine synthase:
The following protocol can be used to identify test compounds that bind to threonine synthase protein.

• Purified full-length threonine synthase polypeptide (Example 64) containing the His / fusion protein tag is bound to HisGrab ^™ nickel-coated plates (Pierce, Rockford, IL) according to the manufacturer's instructions.

  Optimize buffer conditions (eg, ionic strength or pH, Ramos and Calderon (1994) FEBS Lett 351: 357-9 (PMID: 8082795)).

By adding test compounds and radiolabeled O-phospho-L-homoserine (Gening et al. (1994) Biokhemia 59: 1238-44 (PMID: 7819407)) to wells of HisGrab ^™ plates containing bound threonine synthase Screen for test compounds.

  Wash wells to remove excess labeled ligand and add scintillation fluid (Scintiver®, Fisher Scientific) to each well.

• Read the plate in a microplate scintillation counter.
Identify candidate compounds as wells with lower radioactivity compared to control wells to which no test compound has been added.

In addition, M.M. Purified polypeptides containing 10-50 amino acids from grisea threonine synthase are screened in the same manner. A polypeptide containing 10-50 amino acids is generated by subcloning a portion of the THR4 gene into a protein expression vector that adds a His tag when expressed (see Example 64). Oligonucleotide primers are designed to amplify a portion of the THR4 gene using the polymerase chain reaction amplification method. As described in Example 64 above, a DNA fragment encoding a 10-50 amino acid polypeptide is cloned into an expression vector, expressed in a host organism, and purified.

Test compounds that bind to THR4 are further tested for antibiotic activity. As described (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 831740)) for spore production on oatmeal agar medium . Grisea is grown. Spores are harvested and the culture is split to a concentration of 2 × 10 ⁵ spores / ml in minimal medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Test compounds are added to one culture to a final concentration of 20-100 μg / ml. Only the solvent is added to the second culture. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Compare the growth curves of the solvent control sample and the test compound sample. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is inferior to that of the control culture.

Assays to test inhibitors or candidate inhibitors of threonine synthase activity:
Measure the enzymatic activity of threonine synthase in the presence and absence of a candidate compound in an appropriate reaction mixture as described in Ramos and Calderon (1994) FEBS Lett 351: 357-9 (PMID: 8082795) . Candidate compounds are identified when a reduction in product or lack of substrate is detected in a reaction that proceeds in either direction.

In addition, as described in Ramos and Calderon (1994) FEBS Lett 351: 357-9 (PMID: 8082795), in the presence and absence of candidate compounds, M. The enzymatic activity of a polypeptide comprising 10 to 50 amino acids derived from grisea threonine synthase is measured. A polypeptide containing 10-50 amino acids is generated by subcloning a portion of the THR4 gene into a protein expression vector that adds a His tag when expressed (see Example 64). Oligonucleotide primers are designed to amplify a portion of the THR4 gene using the polymerase chain reaction amplification method. As described in Example 64 above, a DNA fragment encoding a 10-50 amino acid polypeptide is cloned into an expression vector, expressed and purified.

Test compounds identified as inhibitors of THR4 activity are further tested for antibiotic activity. Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. As described (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 831740)) for spore production on oatmeal agar medium . Grisea is grown. Spores are harvested and the culture is split to a concentration of 2 × 10 ⁵ spores / ml in minimal medium (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Test compounds are added to one culture to a final concentration of 20-100 μg / ml. Only the solvent is added to the second culture. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Compare the growth curves of the solvent control sample and the test compound sample. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is inferior to that of the control culture.

Assays to test compounds that alter threonine synthase gene expression:
Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. After growing on complete agar or oatmeal agar medium for 10-13 days in the light at 25 ° C., wild type M. pneumoniae was grown from the grown culture using a wet cotton swab . Collect grisea spores. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. A 25 ml culture is prepared, to which test compounds are added at various concentrations. Cultures in the absence of test compound are included as controls. After incubation of the culture for 3 days at 25 ° C., only the solvent is added to the test compound or control. The culture is further incubated for 18 hours. The fungal mycelium is collected by filtration through Miracloth (CalBiochem®, La Jolla, Calif.), Washed with water and frozen in liquid nitrogen. Total RNA is extracted with TRIZOL® reagent using methods provided by the manufacturer (Life Technologies, Rockville, Md.). Expression is analyzed by Northern analysis of RNA samples as described (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press) using a radiolabeled fragment of the THR4 gene as a probe. Test compounds that produce a decrease in THR4 mRNA levels compared to untreated control samples are identified as candidate antibiotic compounds.

In vivo cell-based assay screening protocol using a fungal strain containing a mutant form of inactive threonine synthase:
Magnaporthe grisea fungal cells containing mutant forms of the THR4 gene that abolish enzymatic activity, such as genes containing transposon insertions (see Examples 60 and 61), are well known in the art, and Grow under standard fungal growth conditions as described. After growing on complete agar medium containing 4 mM L-threonine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium containing 100 μM L-threonine at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores are added to each well of a 96-well plate, to which test compounds (various concentrations) are added. The total volume of each well is 200 μl. Wells without test compound (growth control) and wells without cells are included as controls (negative control). Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. A similar protocol can be seen in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

In vivo cell-based assay screening protocol using a fungal strain containing a mutated form of threonine synthase with reduced activity:
Magnaporthe grisea fungal cells containing mutant forms of the THR4 gene, such as promoter partial excisions that reduce expression, are grown under standard fungal growth conditions well known and described in the art. One of the promoters upstream of the transcription initiation site using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press). A part of the promoter is excised by deleting the part. After growing on complete agar medium containing 4 mM L-threonine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores are added to each well of a 96-well plate, to which test compounds (various concentrations) are added. The total volume of each well is 200 μl. Wells without test compound (growth control) and wells without cells are included as controls (negative control). Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. Similar protocols are found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221).

In vivo cell-based assay screening protocol using a fungal strain containing a mutant form of the inactive L-threonine biosynthetic gene:
Magnaporthe grisea fungal cells containing mutant forms of L-threonine biosynthetic pathway genes such as homoserine kinase (EC 2.7.1.39) are well known and described in the art. Grow under standard fungal growth conditions. After growing on complete agar medium containing 4 mM L-threonine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium containing 100 μM L-threonine at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. Similar protocols are found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221).

In vivo cell-based assay screening protocol using a fungal strain containing a mutated form of the L-threonine biosynthetic gene with reduced activity:
A Magnaporthe grisea fungal cell containing a mutant form of a gene of the L-threonine biosynthetic pathway (eg homoserine kinase (EC 2.7.1.39)), such as partial excision of a promoter that reduces expression, Grow under standard fungal growth conditions well known and described in the art. One of the promoters upstream of the transcription initiation site using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual , Cold Spring Harbor Laboratory Press). A part of the promoter is excised by deleting the part. Magnaporthe grisea fungal cells containing the mutant form are grown under standard fungal growth conditions well known and described in the art. After growing on complete agar medium containing 4 mM L-threonine (Sigma-Aldrich Co.) for 10-13 days in the light at 25 ° C., from the grown culture, using a wet cotton swab, Magnaporthe grisea spores are collected. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. Wild type cells are screened under the same conditions. The effect of each compound on mutant and wild-type fungal strains, as measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition . Percent growth inhibition is compared as a result of test compounds against fungal strains and wild type cells. Compounds that show differential growth inhibition between mutant and wild type are identified as potential antifungal compounds. Similar protocols are found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221).

In vivo cell-based assay screening protocol using a fungal strain containing a fungal THR4 gene and a second fungal strain containing a heterologous THR4 gene:
M. lacks wild-type Magnaporthe grisea fungal cells and a functional THR4 gene and contains the Thr4 gene from Saccharomyces cerevisiae (Genbank accession number 6319901, 50% sequence identity) . Grisea fungal cells are grown under standard fungal growth conditions, which are well known and described in the art. M. carrying a heterologous THR4 gene . The grisea strain is created as follows:
- the native gene in such as one containing transposon insertion, M. having a non-functional THR4 gene A grisea strain is created (see Examples 60 and 61).

• Using standard molecular biology techniques well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual ), fungal expression vectors containing trpC promoter and terminator (eg, pCB1003 Carroll et al. (1994) Fungal Gen News Lett 41:22) create a construct containing the heterologous THR4 gene by cloning the Thr4 gene from Saccharomyces cerevisiae .

• Using the construct, M. lacking a functioning THR4 gene . The grisea strain is transformed (see Example 61). Transformants are selected on minimal agar medium lacking L-threonine. Only transformants carrying a functional THR4 gene will grow.

Magnaporthe grisea wild-type strains and strains containing a heterologous form of THR4 are grown under standard fungal growth conditions well known and described in the art. Magnaporthe grisea spores are harvested from the grown cultures using wet cotton swabs after growth on complete agar medium for 10-13 days in the light at 25 ° C. Spore concentration is measured using a hemocytometer and a spore suspension is prepared in minimal growth medium at a concentration of 2 × 10 ⁵ spores / ml. Approximately ⁴ × 10 ⁴ spores or cells are harvested and added to each well of a 96-well plate, to which is added the amount of test compound (various concentrations) plus growth medium. The total volume of each well is 200 μl. Wells without test compound and wells without cells are included as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. The effect of each compound on wild-type and heterologous fungal strain, measured against the growth control, and the _{OD 590} (true strain plus test compound) _{/ OD 590} (growth control) x100, to calculate the percent inhibition. Percent growth inhibition is compared as a result of test compounds against wild type and heterologous fungal strains. Compounds that show differential growth inhibition between wild type and heterologous strains are identified as potential antifungal compounds with specificity for natural or heterologous THR4 gene products. Similar protocols are found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221).

Pathway-specific in vivo assay screening protocol:
Magnaporthe grisea fungal cells are grown under standard fungal growth conditions well known and described in the art. After growing on oatmeal agar medium for 10-13 days in the light at 25 ° C., wild type M. pneumoniae was grown from the grown culture using a wet cotton swab . Collect grisea spores. Spore concentration was measured using a hemocytometer and spore suspension in minimal growth medium and minimal growth medium containing 4 mM L-threonine (Sigma-Aldrich Co.) at a concentration of 2 × 10 ⁵ spores / ml. To prepare. Minimal growth medium contains carbon, nitrogen, phosphate, and sulfate sources, and magnesium, calcium, and trace elements (see, eg, inoculum in Example 63). Add a spore suspension to each well of a 96 well microtiter plate (approximately ⁴ × 10 ⁴ spores / well). For each well containing spore suspension in minimal medium, there are additional wells containing spore suspension in minimal medium containing 4 mM L-threonine. Test compounds are added to wells containing spores in minimal medium and wells containing spores in minimal medium containing L-threonine. The total volume of each well is 200 μl. Both minimal medium wells and medium wells containing L-threonine without test compound are provided as controls. Plates are incubated for 7 days at 25 ° C. and optical density measurements at 590 nm are performed daily. If the growth observed in a well containing minimal medium is inferior to that observed in a well containing L-threonine as a result of adding a test compound, the compound is acting on the L-threonine biosynthetic pathway. To identify antibiotic candidates. Similar protocols are found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221).

  While the above describes particular embodiments of the present invention, those skilled in the art will appreciate that variations and modifications can be made and still fall within the scope of the present invention. The foregoing examples are intended to illustrate various specific aspects of the invention and do not limit its scope in any way.

FIG. 1 shows the reaction performed by the asparagine synthase (ASN1) reaction. The substrate / product is L-aspartate, L-glutamine, and ATP, and the product / substrate is L-asparagine, L-glutamate, AMP, and pyrophosphate. The function of the asparagine synthase enzyme is the interconversion of L-aspartate, L-glutamine, and ATP to L-asparagine, L-glutamate, AMP, and pyrophosphate. This reaction is part of the L-asparagine biosynthetic pathway. FIG. 2 shows a digital image showing the effect of ASN1 gene disruption on Magnaporthe grisea virulence using a whole plant infection assay. Rice cultivar CO39 was inoculated with wild type and transposon insertion strains, KO1-2 and KO1-8. Five days after inoculation, the leaves were imaged. 3A and B. Verification of gene function by analysis of nutritional requirements. Wild type and transposon inserted strains, KO1-2 and KO1-8 were grown in (A) minimal medium and (B) minimal medium supplemented with L-asparagine, respectively. The x-axis shows time in days and the y-axis shows turbidity measured at 490 and 750 nanometers. Symbols indicate wild type (-♦-), transposon strain KO1-2 (-■-), and transposon strain KO1-8 (--black triangle--). FIG. 4 shows the reaction performed by the 5-aminolevulinate synthase (ALAS1) reaction. Substrate / product is succinyl -CoA and glycine, and product / substrate 5-amino levulinate, a CoA and CO _2. Function of 5-aminolevulinic acid synthase enzymes from succinyl -CoA and glycine, 5-amino-levulinate, the mutual conversion into CoA and CO _2. This reaction is part of the heme biosynthetic pathway. FIG. 5 shows a digital image showing the effect of ALAS1 gene disruption on Magnaporthe grisea virulence using a whole plant infection assay. Rice cultivar CO39 was inoculated with wild type and transposon insertion strains, KO1-1 and KO1-106. Five days after inoculation, the leaves were imaged. 6A and B. Verification of gene function by analysis of nutritional requirements. Wild type and transposon inserted strains, KO1-1 and KO1-106, were grown in minimal media supplemented with (A) minimal medium and (B) 5-aminolevulinate, respectively. The x-axis shows time in days and the y-axis shows turbidity measured at 490 and 750 nanometers. Symbols indicate wild type (-♦-), transposon strain KO1-1 (-■-), and transposon strain KO1-106 (--black triangle--). FIG. 7 shows the reaction performed by the histidinol-phosphatase (HISP1) reaction. The substrate / product is L-histidinol phosphate and H ₂ O, and the product / substrate is L-histidinol and orthophosphate. Histidinol - function of phosphatase enzymes, from L- histidinol phosphate and H _{2 O,} the mutual conversion into L- histidinol and orthophosphate. This reaction is part of the L-histidine biosynthetic pathway. FIG. 8 shows a digital image showing the effect of HISP1 gene disruption on Magnaporthe grisea virulence using a whole plant infection assay. Rice cultivar CO39 was inoculated with wild type and transposon insertion strains, KO1-1 and KO1-3. Five days after inoculation, the leaves were imaged. 9A and B. Verification of gene function by analysis of nutritional requirements. Wild-type and transposon inserted strains, KO1-1 and KO1-3, were grown in (A) minimal medium and (B) minimal medium supplemented with L-histidine, respectively. The x-axis shows time in days and the y-axis shows turbidity measured at 490 and 750 nanometers. Symbols indicate wild type (-♦-), transposon strain KO1-1 (-■-), and transposon strain KO1-3 (--black triangle--). FIG. 10 shows the reaction performed by the 3-isopropylmalate dehydratase (IPMD1) reaction. The substrate / product is 2-isopropylmalate and H ₂ O, and the product / substrate is 3-isopropylmalate. The function of the 3-isopropylmalate dehydratase enzyme is the interconversion of 2-isopropylmalate and H ₂ O to 3-isopropylmalate. This reaction is part of the L-leucine biosynthesis pathway. FIG. 11 shows a digital image showing the effect of IPMD1 gene disruption on Magnaporthe grisea virulence using a whole plant infection assay. Rice cultivar CO39 was inoculated with wild type and transposon insertion strains, KO1-3 and KO1-7. Five days after inoculation, the leaves were imaged. 12A and B. FIG. Verification of gene function by analysis of nutritional requirements. Wild type and transposon inserted strains, KO1-3 and KO1-7 were grown in (A) minimal medium and (B) minimal medium supplemented with L-leucine, respectively. The x-axis shows time in days and the y-axis shows turbidity measured at 490 and 750 nanometers. Symbols indicate wild type (-♦-), transposon strain KO1-3 (T1) (-■-), and transposon strain KO1-7 (T2) (--black triangle--). FIG. 13 shows the reaction performed by the threonine synthase (THR4) reaction. The substrate / product is O-phospho-L-homoserine and water, and the product / substrate is L-threonine and orthophosphate. The function of the threonine synthase enzyme is the interconversion of O-phospho-L-homoserine and water to L-threonine and orthophosphate. This reaction is part of the L-threonine biosynthetic pathway. FIG. 14 shows a digital image showing the effect of THR4 gene disruption on Magnaporthe grisea virulence using a whole plant infection assay. Rice cultivar CO39 was inoculated with wild type and transposon insertion strains, KO1-3 and KO1-22. Five days after inoculation, the leaves were imaged. 15A and B. Verification of gene function by analysis of nutritional requirements. Wild-type and transposon inserted strains, KO1-3 and KO1-22, were grown in (A) minimal medium and (B) minimal medium supplemented with L-threonine, respectively. The x-axis shows time in days and the y-axis shows turbidity measured at 490 and 750 nanometers. Symbols indicate wild type (-♦-), transposon strain KO1-3 (-■-), and transposon strain KO1-22 (--black triangle--).

Claims (223)

A method of identifying a test compound as a candidate antibiotic:
a) contacting the asparagine synthase polypeptide with a test compound; and b) detecting the presence or absence of a bond between the test compound and the asparagine synthase polypeptide, wherein the test compound is an antibiotic. Said method, indicating that it is a candidate.   2. The method of claim 1, wherein the asparagine synthase polypeptide is a fungal asparagine synthase polypeptide.   2. The method of claim 1, wherein the asparagine synthase polypeptide is a Magnaporthe asparagine synthase polypeptide.   The method of claim 1, wherein the asparagine synthase polypeptide is SEQ ID NO: 3. A method of identifying a test compound as a candidate antibiotic:
a) with a test compound: a polypeptide having at least 10 consecutive amino acids of fungal asparagine synthase, a polypeptide having at least 50% sequence identity with fungal asparagine synthase, and a polypeptide having at least 10% of the activity of fungal asparagine synthase Contacting with at least one polypeptide selected from the group consisting of; and b) detecting the presence and / or absence of binding between said test compound and said polypeptide, wherein binding comprises said test compound Wherein said method is an antibiotic candidate. A method of identifying a test compound as a candidate antibiotic:
a) contacting asparagine synthase with L-aspartate, L-glutamine, and ATP;
b) contacting L-aspartate, L-glutamine, and ATP with asparagine synthase and test compound; and c) the following: L-aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, And / or measuring at least one concentration change of pyrophosphate, wherein the concentration change of any of the above substances during steps (a) and (b) is such that the test compound is a candidate for an antibiotic Wherein said method.   7. The method of claim 6, wherein the asparagine synthase is a fungal asparagine synthase.   7. The method of claim 6, wherein the asparagine synthase is Magnaporte asparagine synthase.   7. The method of claim 6, wherein the asparagine synthase is SEQ ID NO: 3. A method of identifying a test compound as a candidate antibiotic:
a) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with asparagine synthase;
b) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with asparagine synthase and a test compound; and c) the following: L-aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, Measuring a change in at least one concentration of AMP and / or pyrophosphate, wherein a change in the concentration of any of the above substances during steps (a) and (b) is such that the test compound is a candidate for an antibiotic. Said method indicating that there is.   11. The method of claim 10, wherein the asparagine synthase is a fungal asparagine synthase.   11. The method of claim 10, wherein the asparagine synthase is Magnaporte asparagine synthase.   11. The method of claim 10, wherein the asparagine synthase is SEQ ID NO: 3. A method of identifying a test compound as a candidate antibiotic:
a) with L-aspartate, L-glutamine, and ATP: a polypeptide having at least 50% sequence identity with asparagine synthase, having at least 50% sequence identity with asparagine synthase, and at least 10 of its activity Contacting a polypeptide selected from the group consisting of a polypeptide having a% and a polypeptide comprising at least 100 contiguous amino acids of asparagine synthase;
b) contacting L-aspartate, L-glutamine, and ATP with said polypeptide and test compound; and c) the following: L-aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP And / or measuring at least one concentration change of pyrophosphate, wherein the concentration change of any of the above substances during steps (a) and (b) is such that the test compound is a candidate for an antibiotic Said method. A method of identifying a test compound as a candidate antibiotic:
a) with L-asparagine, L-glutamate, AMP and pyrophosphate: a polypeptide having at least 50% sequence identity with asparagine synthase, having at least 50% sequence identity with asparagine synthase and of its activity Contacting a polypeptide selected from the group consisting of a polypeptide having at least 10% and a polypeptide comprising at least 100 contiguous amino acids of asparagine synthase;
b) contacting the polypeptide and test compound with L-asparagine, L-glutamate, AMP, and pyrophosphate; and c) the following: L-aspartate, L-glutamine, L-asparagine, L-glutamate, ATP Measuring a change in at least one concentration of AMP, AMP, and / or pyrophosphate, wherein a change in the concentration of any of the above substances during steps (a) and (b) indicates that the test compound is an antibiotic candidate Said method indicating that A method of identifying a test compound as a candidate antibiotic:
a) measuring the expression of asparagine synthase in the absence of the test compound in a single cell, a plurality of cells, tissues or organisms;
b) contacting the test compound with the single cell, cells, tissue or organism and measuring the expression of the asparagine synthase in the single cell, cells, tissue or organism. And c) comparing the expression of asparagine synthase in steps (a) and (b), wherein the expression is lower in the presence of the test compound, the compound is a candidate for an antibiotic; Wherein said method.   17. The method of claim 16, wherein the singular cell, cells, tissue, or organism is fungal or derived from a fungus.   17. The method of claim 16, wherein the singular cell, cells, tissue, or organism are of, or derived from, a Magnaporte fungus or the fungal cell.   17. The method of claim 16, wherein the asparagine synthase is SEQ ID NO: 3.   17. The method of claim 16, wherein asparagine synthase expression is measured by detecting ASN1 mRNA.   17. The method of claim 16, wherein asparagine synthase expression is measured by detecting an asparagine synthase polypeptide. A method of identifying a test compound as a candidate antibiotic:
a) providing a cell having one type of asparagine synthase gene and providing a comparative cell having a different type of asparagine synthase gene; and b) contacting said cell and said comparative cell with a test compound and testing Measuring the proliferation of the cell and the comparative cell in the presence of a compound, wherein there is a difference in proliferation between the cell and the comparative cell in the presence of the compound, wherein the compound is an antibiotic. Said method, indicating that it is a candidate.   23. The method of claim 22, wherein the cell and comparative cell are fungal cells.   23. The method of claim 22, wherein the cell and the comparative cell are Magnaporte cells.   23. The method of claim 22, wherein the type of asparagine synthase and the different type are fungal asparagine synthases.   23. The method of claim 22, wherein at least one type is Magnaporte asparagine synthase.   23. The method of claim 22, wherein the type of asparagine synthase and the different type are non-fungal asparagine synthases.   23. The method of claim 22, wherein one type of asparagine synthase is a fungal asparagine synthase and the different type is a non-fungal asparagine synthase. A method of identifying a test compound as a candidate antibiotic:
a) providing a cell having one type of gene in the L-asparagine biochemical pathway and / or gene pathway and providing a comparative cell having a different type of said gene;
b) contacting said cell and said comparative cell with said test compound; and c) measuring the proliferation of said cell and said comparative cell in the presence of said test compound, in the presence of said test compound. The method, wherein a difference in proliferation between the cell and the comparative cell indicates that the test compound is an antibiotic candidate.   30. The method of claim 29, wherein the cells and comparative cells are fungal cells.   30. The method of claim 29, wherein the cell and the comparative cell are Magnaporte cells.   30. The method of claim 29, wherein the type of L-asparagine biosynthetic gene and the different type are fungal L-asparagine biosynthetic genes.   30. The method of claim 29, wherein at least one form is a Magnaporte L-asparagine biosynthetic gene.   30. The method of claim 29, wherein the type of L-asparagine biosynthetic gene and the different type are non-fungal L-asparagine biosynthetic genes.   30. The method of claim 29, wherein one type of L-asparagine biosynthesis gene is a fungal L-asparagine biosynthesis gene and a different type is a non-fungal L-asparagine biosynthesis gene.   30. A method for determining whether an antibiotic candidate of claim 29 has antifungal activity: contacting said antibiotic candidate with a fungal or fungal cell, and growth, viability, or pathogenesis of said fungal or fungal cell Said method further comprising detecting a decrease in sex, wherein said decrease in proliferation, viability, or pathogenicity of said fungus or fungal cell indicates that said antibiotic candidate has antifungal activity. A method of identifying a test compound as a candidate antibiotic:
(A) providing a paired growth medium comprising a first medium and a second medium, wherein the second medium contains a higher level of L-asparagine than the first medium;
(B) contacting the organism with the test compound;
(C) inoculating said first medium and said second medium with said organism; and (d) measuring the growth of said organism, said first medium and said second medium The method, wherein a difference in the growth of the organism among the culture media indicates that the test compound is a candidate for an antibiotic.   38. The method of claim 37, wherein the organism is a fungus.   38. The method of claim 37, wherein the organism is a genus Magnaporte.   An isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 3.   41. The nucleic acid of claim 40, comprising the nucleotide sequence of SEQ ID NO: 1.   41. An expression cassette comprising the nucleic acid of claim 40.   41. The isolated nucleic acid of claim 40, comprising a nucleotide sequence having at least 50% to at least 95% sequence identity to SEQ ID NO: 1.   A polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 3.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 3. A method of identifying a test compound as a candidate antibiotic:
contacting a test compound with a 5-aminolevulinate synthase polypeptide; and b) detecting the presence or absence of binding between the test compound and the 5-aminolevulinate synthase polypeptide,
The method wherein the binding indicates that the test compound is an antibiotic candidate.   48. The method of claim 46, wherein the 5-aminolevulinate synthase polypeptide is a fungal 5-aminolevulinate synthase polypeptide.   48. The method of claim 46, wherein the 5-aminolevulinate synthase polypeptide is a Magnaporte 5-aminolevulinate synthase polypeptide.   48. The method of claim 46, wherein the 5-aminolevulinate synthase polypeptide is SEQ ID NO: 6. A method of identifying a test compound as a candidate antibiotic:
a) with a test compound: a polypeptide having at least 10 consecutive amino acids of fungal 5-aminolevulinate synthase, a polypeptide having at least 50% sequence identity with fungal 5-aminolevulinate synthase, and fungal 5-aminolevulinate synthase Contacting at least one polypeptide selected from the group consisting of polypeptides having at least 10% of activity; and b) detecting the presence and / or absence of binding between said test compound and said polypeptide Including
The method wherein the binding indicates that the test compound is an antibiotic candidate. A method of identifying a test compound as a candidate antibiotic:
a) contacting succinyl-CoA and glycine with 5-aminolevulinate synthase;
b) contacting succinyl-CoA and glycine with 5-aminolevulinate synthase and a test compound; and c) the following: at least one of succinyl-CoA, glycine, 5-aminolevulinate, CoA, and / or CO ₂ Including measuring concentration changes,
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic.   52. The method of claim 51, wherein the 5-aminolevulinate synthase is a fungal 5-aminolevulinate synthase.   52. The method of claim 51, wherein the 5-aminolevulinate synthase is Magnaporte 5-aminolevulinate synthase.   52. The method of claim 51, wherein the 5-aminolevulinate synthase is SEQ ID NO: 6. A method of identifying a test compound as a candidate antibiotic:
a) contacting 5-aminolevulinate, CoA, and CO ₂ with 5-aminolevulinate synthase;
b) contacting 5-aminolevulinate, CoA, and CO ₂ with 5-aminolevulinate synthase and a test compound; and c) the following: succinyl-CoA, glycine, 5-aminolevulinate, CoA, and / or Or measuring at least one concentration change of CO ₂ ;
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic.   55. The method of claim 54, wherein the 5-aminolevulinate synthase is a fungal 5-aminolevulinate synthase.   55. The method of claim 54, wherein the 5-aminolevulinate synthase is Magnaporte 5-aminolevulinate synthase.   55. The method of claim 54, wherein the 5-aminolevulinate synthase is SEQ ID NO: 6. A method of identifying a test compound as a candidate antibiotic:
a) with succinyl-CoA and glycine: a polypeptide having at least 50% sequence identity with 5-aminolevulinate synthase; having at least 50% sequence identity with 5-aminolevulinate synthase and at least 10 of its activity Contacting a polypeptide selected from the group consisting of a polypeptide comprising at least 100 contiguous amino acids of 5-aminolevulinate synthase;
b) contacting said polypeptide and test compound with succinyl-CoA and glycine; and c) following: at least one concentration change of succinyl-CoA, glycine, 5-aminolevulinate, CoA, and / or CO ₂ Including measuring
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic. A method of identifying a test compound as a candidate antibiotic:
a) 5-Amino levulinate, CoA, and CO ₂ and 5-aminolevulinic acid synthase polypeptide having at least 50% sequence identity; with 5-aminolevulinic acid synthase having at least 50% sequence identity And a polypeptide having at least 10% of its activity; and a polypeptide selected from the group consisting of a polypeptide comprising at least 100 contiguous amino acids of 5-aminolevulinate synthase;
b) 5-amino-levulinate, CoA, and the CO _2, wherein contacting the polypeptide and a test compound; and c) below: succinyl-CoA, glycine, 5-amino-levulinate, CoA, and / or CO ₂ comprises measuring at least one change in density,
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic. A method of identifying a test compound as a candidate antibiotic:
a) measuring the expression of 5-aminolevulinate synthase in the absence of the test compound in a single cell, a plurality of cells, tissues or organisms;
b) contacting the test compound with the single cell, cells, tissues or organism and expressing the 5-aminolevulinate synthase in the single cell, cells, tissues or organism And c) comparing the expression of 5-aminolevulinate synthase in steps (a) and (b),
The method, wherein lower expression in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.   61. The method of claim 60, wherein said single cell, cells, tissue, or organism is fungal or derived from a fungus.   61. The method of claim 60, wherein the singular cell, cells, tissue, or organism are of, or derived from, a Magnaporte fungus or the fungal cell.   61. The method of claim 60, wherein the 5-aminolevulinate synthase is SEQ ID NO: 6.   61. The method of claim 60, wherein 5-aminolevulinate synthase expression is measured by detecting ALAS1 mRNA.   61. The method of claim 60, wherein 5-aminolevulinate synthase expression is measured by detecting a 5-aminolevulinate synthase polypeptide. A method of identifying a test compound as a candidate antibiotic:
a) providing a cell having one type of 5-aminolevulinate synthase gene and providing a comparative cell having a different type of 5-aminolevulinate synthase gene; and b) said cell and said comparative cell and a test compound And measuring the proliferation of said cells and said comparative cells in the presence of a test compound,
The method, wherein a difference in proliferation between the cell and the comparative cell in the presence of the compound indicates that the compound is a candidate for an antibiotic.   68. The method of claim 66, wherein the cell and the comparative cell are fungal cells.   68. The method of claim 66, wherein the cell and the comparative cell are Magnaporte cells.   68. The method of claim 66, wherein the form of 5-aminolevulinate synthase and the comparative form are fungal 5-aminolevulinate synthase.   67. The method of claim 66, wherein at least one type is Magnaporte 5-aminolevulinate synthase.   68. The method of claim 66, wherein the form of 5-aminolevulinate synthase and the comparative form are non-fungal 5-aminolevulinate synthase.   68. The method of claim 66, wherein one type of 5-aminolevulinate synthase is a fungal 5-aminolevulinate synthase and the other type is a non-fungal 5-aminolevulinate synthase. A method of identifying a test compound as a candidate antibiotic:
a) providing a cell having one type of gene in the heme biochemical pathway and / or gene pathway, and providing a comparative cell having a different type of said gene;
b) contacting the cell and the comparative cell with a test compound;
c) measuring the proliferation of the cells and the comparative cells in the presence of the test compound,
The method wherein a difference in proliferation between the cells and the comparative cells in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.   74. The method of claim 73, wherein the cells and comparative cells are fungal cells.   74. The method of claim 73, wherein the cell and the comparative cell are Magnaporte cells.   74. The method of claim 73, wherein said type of heme biosynthetic gene and said different type are fungal heme biosynthetic genes.   75. The method of claim 73, wherein at least one type is a Magnaporte heme biosynthetic gene.   74. The method of claim 73, wherein said type of heme biosynthetic gene and said different type are non-fungal heme biosynthetic genes.   74. The method of claim 73, wherein one type of heme biosynthetic gene is a fungal heme biosynthetic gene and the different type is a non-fungal heme biosynthetic gene. A method of identifying a test compound as a candidate antibiotic:
(A) providing a pair of growth media; the growth media comprising a first media and a second media, wherein the second media has a higher level of 5-aminole than the first media; Contains a blinate;
(B) contacting the organism with the test compound;
(C) inoculating said first medium and said second medium with said organism; and (d) measuring the growth of said organism;
The method, wherein a difference in organism growth between the first medium and the second medium indicates that the test compound is a candidate antibiotic.   81. The method of claim 80, wherein the organism is a fungus.   81. The method of claim 80, wherein the organism is Magnaporte.   An isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 6.   84. The nucleic acid of claim 83, comprising the nucleotide sequence of SEQ ID NO: 4.   84. An expression cassette comprising the nucleic acid of claim 83.   84. The isolated nucleic acid of claim 83, comprising a nucleotide sequence having at least 50% to at least 95% sequence identity to SEQ ID NO: 4.   A polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 6.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 6. A method of identifying a test compound as a candidate antibiotic:
contacting a test compound with a histidinol-phosphatase polypeptide; and b) detecting the presence or absence of binding between the test compound and the histidinol-phosphatase polypeptide,
The method wherein the binding indicates that the test compound is an antibiotic candidate.   90. The method of claim 89, wherein the histidinol-phosphatase polypeptide is a fungal histidinol-phosphatase polypeptide.   90. The method of claim 89, wherein the histidinol-phosphatase polypeptide is a Magnaporte histidinol-phosphatase polypeptide.   90. The method of claim 89, wherein the histidinol-phosphatase polypeptide is SEQ ID NO: 9. A method of identifying a test compound as a candidate antibiotic:
a) with a test compound: a polypeptide having at least 10 consecutive amino acids of fungal histidinol-phosphatase; a polypeptide having at least 50% sequence identity with fungal histidinol-phosphatase; and at least 10% of the activity of fungal histidinol-phosphatase Contacting at least one polypeptide selected from the group consisting of polypeptides having; and b) detecting the presence and / or absence of binding between said test compound and said polypeptide,
The method wherein the binding indicates that the test compound is an antibiotic candidate. A method of identifying a test compound as a candidate antibiotic:
a) the L- histidinol phosphate and H _{2 O,} histidinol - contacting the phosphatase;
b) the L- histidinol phosphate and H _{2 O,} histidinol - contacting a phosphatase and a test compound; and c) the following: L- histidinol phosphate, H 2 _O, L- histidinol, and / or orthophosphate Measuring at least one concentration change,
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic.   95. The method of claim 94, wherein the histidinol-phosphatase is a fungal histidinol-phosphatase.   95. The method of claim 94, wherein the histidinol-phosphatase is Magnaporte histidinol-phosphatase.   95. The method of claim 94, wherein the histidinol-phosphatase is SEQ ID NO: 9. A method of identifying a test compound as a candidate antibiotic:
a) contacting L-histidinol and orthophosphate with histidinol-phosphatase;
b) contacting L-histidinol and orthophosphate with histidinol-phosphatase and test compound; and c) at least one concentration of L-histidinol phosphate, H ₂ O, L-histidinol, and / or orthophosphate Including measuring changes,
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic.   99. The method of claim 98, wherein the histidinol-phosphatase is a fungal histidinol-phosphatase.   99. The method of claim 98, wherein said histidinol-phosphatase is Magnaporte histidinol-phosphatase.   99. The method of claim 98, wherein the histidinol-phosphatase is SEQ ID NO: 9. A method of identifying a test compound as a candidate antibiotic:
a) with L-histidinol phosphate and H ₂ O: a polypeptide having at least 50% sequence identity with histidinol-phosphatase; having at least 50% sequence identity with histidinol-phosphatase and at least of its activity Contacting a polypeptide selected from the group consisting of: a polypeptide having 10%; and a polypeptide comprising at least 100 contiguous amino acids of histidinol-phosphatase;
b) contacting L-histidinol phosphate and H ₂ O with said polypeptide and test compound; and c) the following: L-histidinol phosphate, H ₂ O, L-histidinol, and / or orthophosphate Measuring at least one concentration change,
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic. A method of identifying a test compound as a candidate antibiotic:
a) with L-histidinol and orthophosphate: a polypeptide having at least 50% sequence identity with histidinol-phosphatase; having at least 50% sequence identity with histidinol-phosphatase and having at least 10% of its activity Contacting a polypeptide selected from the group consisting of a polypeptide; and a polypeptide comprising at least 100 contiguous amino acids of histidinol-phosphatase;
b) contacting L-histidinol and orthophosphate with said polypeptide and test compound; and c) the following: at least one concentration of L-histidinol phosphate, H ₂ O, L-histidinol, and / or orthophosphate Including measuring changes,
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic. A method of identifying a test compound as a candidate antibiotic:
a) measuring the expression of histidinol-phosphatase in a single cell, a plurality of cells, tissues or organisms in the absence of the test compound;
b) contacting the test compound with the cell, cells, tissues or organism and measuring the expression of the histidinol-phosphatase in the cell, cells, tissues or organism And c) comparing the expression of histidinol-phosphatase in steps (a) and (b),
The method, wherein lower expression in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.   105. The method of claim 104, wherein the single cell, cells, tissue, or organism is fungal or derived from a fungus.   105. The method of claim 104, wherein the singular cell, cells, tissue, or organism is of, or derived from, a Magnaporte fungus or the fungal cell.   105. The method of claim 104, wherein the histidinol-phosphatase is SEQ ID NO: 9.   105. The method of claim 104, wherein histidinol-phosphatase expression is measured by detecting HISP1 mRNA.   105. The method of claim 104, wherein the histidinol-phosphatase expression is measured by detecting a histidinol-phosphatase polypeptide. A method of identifying a test compound as a candidate antibiotic:
a) providing a cell having one type of histidinol-phosphatase gene and providing a comparative cell having a different type of histidinol-phosphatase gene; and b) contacting said cell and said comparative cell with a test compound; And measuring the proliferation of the cell and the comparative cell in the presence of a test compound,
The method, wherein a difference in proliferation between the cell and the comparative cell in the presence of the compound indicates that the compound is a candidate for an antibiotic.   111. The method of claim 110, wherein the cells and comparative cells are fungal cells.   111. The method of claim 110, wherein the cell and the comparative cell are Magnaporte cells.   111. The method of claim 110, wherein the type of histidinol-phosphatase and the comparative type are fungal histidinol-phosphatases.   111. The method of claim 110, wherein at least one type is Magnaporte histidinol-phosphatase.   111. The method of claim 110, wherein the type of histidinol-phosphatase and the comparative type are non-fungal histidinol-phosphatases.   111. The method of claim 110, wherein one type of histidinol-phosphatase is a fungal histidinol-phosphatase and the other type is a non-fungal histidinol-phosphatase. A method of identifying a test compound as a candidate antibiotic:
a) providing a cell having one type of gene in the L-histidine biochemical pathway and / or gene pathway, and providing a comparative cell having a different type of said gene;
b) contacting the cell and the comparative cell with a test compound;
c) measuring the proliferation of the cells and the comparative cells in the presence of the test compound,
The method wherein a difference in proliferation between the cells and the comparative cells in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.   118. The method of claim 117, wherein the cells and comparative cells are fungal cells.   118. The method of claim 117, wherein the cell and the comparative cell are Magnaporte cells.   118. The method of claim 117, wherein said type of L-histidine biosynthetic gene and said different type are fungal L-histidine biosynthetic genes.   118. The method of claim 117, wherein at least one type is a Magnaporte L-histidine biosynthetic gene.   118. The method of claim 117, wherein said type of L-histidine biosynthetic gene and said different type are non-fungal L-histidine biosynthetic genes.   118. The method of claim 117, wherein one type of L-histidine biosynthetic gene is a fungal L-histidine biosynthetic gene and a different type is a non-fungal L-histidine biosynthetic gene. A method of identifying a test compound as a candidate antibiotic:
(A) providing a pair of growth media; the growth media comprises a first media and a second media, wherein the second media has a higher level of L-histidine than the first media; Containing;
(B) contacting the organism with the test compound;
(C) inoculating said first medium and said second medium with said organism; and (d) measuring the growth of said organism;
The method, wherein a difference in organism growth between the first medium and the second medium indicates that the test compound is a candidate antibiotic.   129. The method of claim 124, wherein the organism is a fungus.   129. The method of claim 124, wherein the organism is Magnaporte.   An isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 9.   128. The nucleic acid of claim 127, comprising the nucleotide sequence of SEQ ID NO :.   129. An expression cassette comprising the nucleic acid of claim 128.   128. The isolated nucleic acid of claim 127, comprising a nucleotide sequence having at least 50% to at least 95% sequence identity to SEQ ID NO: 7.   A polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 9.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 9. A method of identifying a test compound as a candidate antibiotic:
contacting a test compound with 3-isopropylmalate dehydratase polypeptide; and b) detecting the presence or absence of a bond between the test compound and the 3-isopropylmalate dehydratase polypeptide,
The method wherein the binding indicates that the test compound is an antibiotic candidate.   134. The method of claim 133, wherein the 3-isopropylmalate dehydratase polypeptide is a fungal 3-isopropylmalate dehydratase polypeptide.   134. The method of claim 133, wherein the 3-isopropylmalate dehydratase polypeptide is a Magnaporte 3-isopropylmalate dehydratase polypeptide.   134. The method of claim 133, wherein said 3-isopropylmalate dehydratase polypeptide is SEQ ID NO: 12. A method of identifying a test compound as a candidate antibiotic:
a) with a test compound: a polypeptide having at least 10 consecutive amino acids of fungal 3-isopropylmalate dehydratase; a polypeptide having at least 50% sequence identity with fungal 3-isopropylmalate dehydratase; and fungal 3-isopropylapple Contacting at least one polypeptide selected from the group consisting of polypeptides having at least 10% of the activity of acid dehydratase; and b) the presence and / or absence of binding between said test compound and said polypeptide Including detecting,
The method wherein the binding indicates that the test compound is an antibiotic candidate. A method of identifying a test compound as a candidate antibiotic:
a) contacting 2-isopropylmalate and H ₂ O with 3-isopropylmalate dehydratase;
b) 2-isopropyl malate and _{H 2} O, by contacting the 3-isopropyl malate dehydratase and a test compound; and c) the following: 2-isopropyl _malate, of _H 2 O, and / or 3-isopropyl malate Measuring at least one concentration change,
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic.   140. The method of claim 138, wherein said 3-isopropylmalate dehydratase is a fungal 3-isopropylmalate dehydratase.   138. The method of claim 138, wherein the 3-isopropylmalate dehydratase is Magnaporte 3-isopropylmalate dehydratase.   138. The method of claim 138, wherein the 3-isopropylmalate dehydratase is SEQ ID NO: 12. A method of identifying a test compound as a candidate antibiotic:
a) contacting 3-isopropylmalate with 3-isopropylmalate dehydratase;
b) contacting 3-isopropylmalate with 3-isopropylmalate dehydratase and a test compound; and c) the following: at least one concentration of 2-isopropylmalate, H ₂ O, and / or 3-isopropylmalate Including measuring changes,
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic.   143. The method of claim 142, wherein the 3-isopropylmalate dehydratase is a fungal 3-isopropylmalate dehydratase.   143. The method of claim 142, wherein the 3-isopropylmalate dehydratase is Magnaporte 3-isopropylmalate dehydratase.   143. The method of claim 142, wherein the 3-isopropylmalate dehydratase is SEQ ID NO: 12. 144. A method for determining whether an antibiotic candidate of claim 142 has antifungal activity comprising:
The method further comprising contacting the antibiotic candidate with the fungal or fungal cell and detecting a decrease in proliferation, viability, or pathogenicity of the fungal or fungal cell. A method of identifying a test compound as a candidate antibiotic:
a) with 2-isopropylmalate and H ₂ O: a polypeptide having at least 50% sequence identity with 3-isopropylmalate dehydratase; having at least 50% sequence identity with 3-isopropylmalate dehydratase; And contacting with a polypeptide having at least 10% of its activity; and a polypeptide selected from the group consisting of polypeptides comprising at least 100 contiguous amino acids of 3-isopropylmalate dehydratase;
b) contacting the polypeptide and the test compound with 2-isopropylmalate and H ₂ O; and c) the following: at least one of 2-isopropylmalate, H ₂ O, and / or 3-isopropylmalate Including measuring concentration changes,
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic. A method of identifying a test compound as a candidate antibiotic:
a) with 3-isopropylmalate: a polypeptide having at least 50% sequence identity with 3-isopropylmalate dehydratase; having at least 50% sequence identity with 3-isopropylmalate dehydratase and of its activity Contacting with a polypeptide selected from the group consisting of a polypeptide comprising at least 10%; and a polypeptide comprising at least 100 consecutive amino acids of 3-isopropylmalate dehydratase;
b) contacting 3-isopropylmalate with said polypeptide and test compound; and c) measuring the following: at least one concentration change of 2-isopropylmalate, H ₂ O, and / or 3-isopropylmalate Including
The method, wherein a change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic. A method of identifying a test compound as a candidate antibiotic:
a) measuring the expression of 3-isopropylmalate dehydratase in the absence of the test compound in a single cell, a plurality of cells, tissues or organisms;
b) contacting the test compound with the single cell, cells, tissues, or organism, and in the single cell, cells, tissue, or organism, the 3-isopropylmalate dehydratase Measuring expression; and c) comparing the expression of 3-isopropylmalate dehydratase in steps (a) and (b),
The method, wherein lower expression in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.   149. The method of claim 149, wherein said single cell, cells, tissue, or organism is fungal or derived from a fungus.   149. The method of claim 149, wherein said singular cell, cells, tissue, or organism is of, or derived from, Magnaporte fungus or the fungal cell.   149. The method of claim 149, wherein the 3-isopropylmalate dehydratase is SEQ ID NO: 12.   149. The method of claim 149, wherein 3-isopropylmalate dehydratase expression is measured by detecting IPMD1 mRNA.   149. The method of claim 149, wherein 3-isopropylmalate dehydratase expression is measured by detecting a 3-isopropylmalate dehydratase polypeptide. A method of identifying a test compound as a candidate antibiotic:
a) providing a cell having one type of 3-isopropylmalate dehydratase gene and providing a comparative cell having a different type of 3-isopropylmalate dehydratase gene; and b) said cell and said comparative cell; Contacting the test compound and measuring the proliferation of the cell and the comparative cell in the presence of the test compound,
The method, wherein a difference in proliferation between the cell and the comparative cell in the presence of the compound indicates that the compound is a candidate for an antibiotic.   165. The method of claim 155, wherein the cell and the comparative cell are fungal cells.   165. The method of claim 155, wherein the cell and the comparative cell are Magnaporte cells.   165. The method of claim 155, wherein said form of 3-isopropylmalate dehydratase and said comparative form are fungal 3-isopropylmalate dehydratase.   165. The method of claim 155, wherein at least one type is Magnaporte 3-isopropylmalate dehydratase.   165. The method of claim 155, wherein said form of 3-isopropylmalate dehydratase and said comparative form are non-fungal 3-isopropylmalate dehydratase.   165. The method of claim 155, wherein one form of 3-isopropylmalate dehydratase is a fungal 3-isopropylmalate dehydratase and the other form is a non-fungal 3-isopropylmalate dehydratase. A method of identifying a test compound as a candidate antibiotic:
a) providing a cell having one type of gene in the L-leucine biochemical pathway and / or gene pathway, and providing a comparative cell having a different type of said gene;
b) contacting the cell and the comparative cell with the test compound;
c) measuring the proliferation of the cells and the comparative cells in the presence of the test compound,
The method wherein a difference in proliferation between the cells and the comparative cells in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.   163. The method of claim 162, wherein the cell and the comparative cell are fungal cells.   163. The method of claim 162, wherein the cell and the comparative cell are Magnaporte cells.   163. The method of claim 162, wherein said type of L-leucine biosynthesis gene and said different type are fungal L-leucine biosynthesis genes.   163. The method of claim 162, wherein at least one type is a Magnaporte L-leucine biosynthesis gene.   163. The method of claim 162, wherein said type of L-leucine biosynthesis gene and said different type are non-fungal L-leucine biosynthesis genes.   163. The method of claim 162, wherein one type of L-leucine biosynthesis gene is a fungal L-leucine biosynthesis gene and the different type is a non-fungal L-leucine biosynthesis gene. A method of identifying a test compound as a candidate antibiotic:
(A) providing a pair of growth media; the growth media comprises a first media and a second media, wherein the second media has a higher level of L-leucine than the first media; Containing;
(B) contacting the organism with the test compound;
(C) inoculating said first medium and said second medium with said organism; and (d) measuring the growth of said organism;
The method, wherein a difference in organism growth between the first medium and the second medium indicates that the test compound is a candidate antibiotic.   169. The method of claim 169, wherein the organism is a fungus.   169. The method of claim 169, wherein the organism is a genus Magnaporte.   An isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 12.   173. The nucleic acid of 172, comprising the nucleotide sequence of SEQ ID NO: 10.   178. An expression cassette comprising the nucleic acid of 173.   175. The isolated nucleic acid of claim 172, comprising a nucleotide sequence having at least 50% to at least 95% sequence identity to SEQ ID NO: 10.   A polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 12.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 12. A method of identifying a test compound as a candidate antibiotic:
a) contacting the threonine synthase polypeptide with the test compound; and b) detecting the presence or absence of a bond between the test compound and the threonine synthase polypeptide, wherein the test compound is an antibiotic. Said method, indicating that it is a candidate.   178. The method of claim 178, wherein said threonine synthase polypeptide is a fungal threonine synthase polypeptide.   178. The method of claim 178, wherein said threonine synthase polypeptide is a Magnaporte threonine synthase polypeptide.   178. The method of claim 178, wherein said threonine synthase polypeptide is SEQ ID NO: 15. A method of identifying a test compound as a candidate antibiotic:
a) with a test compound: a polypeptide having at least 10 consecutive amino acids of fungal threonine synthase, and a polypeptide having at least 50% sequence identity with fungal threonine synthase, and a poly having at least 10% of the activity of fungal threonine synthase Contacting at least one polypeptide selected from the group consisting of peptides; and b) detecting the presence and / or absence of binding between said test compound and said polypeptide, wherein binding comprises said test The method wherein the compound is a candidate for antibiotic. A method of identifying a test compound as a candidate antibiotic:
a) contacting O-phospho-L-homoserine and water with threonine synthase;
b) contacting O-phospho-L-homoserine and water with threonine synthase and test compound; and c) the following: at least one concentration change of O-phospho-L-homoserine, L-threonine, orthophosphate, and water Wherein the change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic.   184. The method of claim 183, wherein said threonine synthase is a fungal threonine synthase.   184. The method of claim 183, wherein said threonine synthase is Magnaporte threonine synthase.   184. The method of claim 183, wherein said threonine synthase is SEQ ID NO: 15.   9. A method for determining whether an antibiotic candidate of claim 8 has antifungal activity comprising contacting said antibiotic candidate with said fungal or fungal cell, and said fungal or fungal cell growth, viability, or pathogenicity Further comprising detecting a decrease in sex. A method of identifying a test compound as a candidate antibiotic:
a) contacting L-threonine and orthophosphate with threonine synthase;
b) contacting L-threonine and orthophosphate with threonine synthase and test compound; and c) measuring the concentration change of at least one of: O-phospho-L-homoserine, L-threonine, orthophosphate, and water Wherein said change in concentration of any of the above substances during steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.   189. The method of claim 188, wherein said threonine synthase is a fungal threonine synthase.   189. The method of claim 188, wherein said threonine synthase is Magnaporte threonine synthase.   189. The method of claim 188, wherein said threonine synthase is SEQ ID NO: 15. A method of identifying a test compound as a candidate antibiotic:
a) O-phospho-L-homoserine and water: a polypeptide having at least 50% sequence identity with threonine synthase, and at least 50% sequence identity with threonine synthase and at least 10% of its activity And a polypeptide selected from the group consisting of a polypeptide comprising at least 100 contiguous amino acids of threonine synthase;
b) contacting the polypeptide and test compound with O-phospho-L-homoserine and water; and c) the following: at least one concentration of O-phospho-L-homoserine, L-threonine, orthophosphate, and water. Measuring the change, wherein the change in concentration of any of the above substances during steps (a) and (b) indicates that the test compound is a candidate antibiotic. A method of identifying a test compound as a candidate antibiotic:
a) L-threonine and orthophosphate: a polypeptide having at least 50% sequence identity with threonine synthase, and a poly having at least 50% sequence identity with threonine synthase and having at least 10% of its activity Contacting a peptide selected from the group consisting of a peptide and a polypeptide comprising at least 100 contiguous amino acids of threonine synthase;
b) contacting L-threonine and orthophosphate with said polypeptide and test compound; and c) measuring the concentration change of at least one of: O-phospho-L-homoserine, L-threonine, orthophosphate, and water Wherein said change in concentration of any of the above substances during steps (a) and (b) indicates that said test compound is a candidate antibiotic. A method of identifying a test compound as a candidate antibiotic:
a) measuring the expression of threonine synthase in the absence of the test compound in a single cell, a plurality of cells, tissues or organisms;
b) contacting the test compound with the singular cell, cells, tissue or organism and measuring the expression of the threonine synthase in the singular cell, cells, tissue or organism. And c) comparing the expression of threonine synthase in steps (a) and (b), wherein the test compound is a candidate for antibiotic if the expression is lower in the presence of the test compound; Said method.   195. The method of claim 194, wherein said single cell, cells, tissue, or organism is fungal or derived from a fungus.   195. The method of claim 194, wherein said singular cell, cells, tissue, or organism are of, or derived from, a Magnaporte fungus or the fungal cell.   195. The method of claim 194, wherein said threonine synthase is SEQ ID NO: 15.   195. The method of claim 194, wherein threonine synthase expression is measured by detecting THR4 mRNA.   195. The method of claim 194, wherein threonine synthase expression is measured by detecting a threonine synthase polypeptide. A method of identifying a test compound as a candidate antibiotic:
a) providing a cell having one type of threonine synthase gene and providing a comparative cell having a different type of threonine synthase gene; and b) contacting said cell and said comparative cell with a test compound and testing Measuring the proliferation of the cell and the comparative cell in the presence of a compound, wherein there is a difference in proliferation between the cell and the comparative cell in the presence of the compound, wherein the compound is an antibiotic. Said method, indicating that it is a candidate.   200. The method of claim 200, wherein the cells and comparative cells are fungal cells.   200. The method of claim 200, wherein the cells and comparative cells are Magnaporte cells.   200. The method of claim 200, wherein the type of threonine synthase and the different type are fungal threonine synthases.   202. The method of claim 200, wherein at least one type is Magnaporte threonine synthase.   200. The method of claim 200, wherein the type of threonine synthase and the different type are non-fungal threonine synthases.   200. The method of claim 200, wherein one type of threonine synthase is a fungal threonine synthase and the different type is a non-fungal threonine synthase. A method of identifying a test compound as a candidate antibiotic:
a) providing a cell having one type of gene in the L-threonine biochemical pathway and / or gene pathway, and providing a comparative cell having a different type of said gene;
b) contacting said cell and said comparative cell with said test compound; and c) measuring the proliferation of said cell and said comparative cell in the presence of said test compound, in the presence of said test compound. The method, wherein a difference in proliferation between the cell and the comparative cell indicates that the test compound is an antibiotic candidate.   207. The method of claim 207, wherein the cell and the comparative cell are fungal cells.   207. The method of claim 207, wherein the cell and the comparative cell are Magnaporte cells.   207. The method of claim 207, wherein said type of L-threonine biosynthetic gene and said different type are fungal L-threonine biosynthetic genes.   207. The method of claim 207, wherein at least one type is a Magnaporte L-threonine biosynthetic gene.   207. The method of claim 207, wherein said type of L-threonine biosynthetic gene and said different type are non-fungal L-threonine biosynthetic genes.   207. The method of claim 207, wherein one type of L-threonine biosynthesis gene is a fungal L-threonine biosynthesis gene and a different type is a non-fungal L-threonine biosynthesis gene. A method of identifying a test compound as a candidate antibiotic:
(A) providing a paired growth medium comprising a first medium and a second medium, wherein the second medium contains a higher level of L-threonine than the first medium;
(B) contacting the organism with the test compound;
(C) inoculating said first medium and said second medium with said organism; and (d) measuring the growth of said organism, said first medium and said second medium The method, wherein a difference in the growth of the organism among the culture media indicates that the test compound is a candidate for an antibiotic.   215. The method of claim 214, wherein the organism is a fungus.   224. The method of claim 214, wherein the organism is a genus Magnaporte.   An isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 15.   218. The nucleic acid of claim 217, comprising the nucleotide sequence of SEQ ID NO: 13.   218. An expression cassette comprising the nucleic acid of 218.   218. The isolated nucleic acid of claim 217, comprising a nucleotide sequence having at least 50% to at least 95% sequence identity to SEQ ID NO: 13.   A polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 15.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 15.

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