EP1461460A2 - Methods for the identification of inhibitors of asparagine synthase, 5-aminolevulinate synthase, histidinol-phosphatase, 3-isopropylmalate and threonine synthase as antibiotics - Google Patents

Abstract

The present inventors have discovered that Asparagine Synthase, 5-Aminolevulinate synthase, histidinol-phosphatase, 3-Isopropylmalate dehydratase and Threonine synthase are essential for fungal pathogenicity. Specifically, the inhibition of Asparagine Synthase, 5-Aminolevulinate synthase, histidinol-phosphatase, 3-Isopropylmalate dehydratase or Threonine synthase gene expression in fungi results in no signs 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 the identification of antibiotics, preferably antifungals. Accordingly, the present inventio provides methods for the identification of compounds that inhibit Asparagine Synthase, 5-Aminolevulinate synthase, histidinol-hosphatase, 3-Isopropylmalate dehydratase or Threonine synthase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably antifungals.

Description

METHODS FOR THE IDENTIFICATION OF INHIBITORS OF

ASPARAGINE SYNTHASE, 5-AMrNOLEVULINATE SYNTHASE, HISTTDINOL-

PHOSPHATASE, 3-ISOPROPYLMALATE AND THREONINE SYNTHASE AS

ANTIBIOTICS

FIELD OF THE INVENTION

The invention relates generally to methods for the identification of antibiotics, preferably antifungals that affect the biosynthesis of L-asparagine, heme, L-histidine, L- leucine or L-threonine.

BACKGROUND OF THE INVENTION

Filamentous fungi are the causal agents responsible for many serious pathogenic infections of plants and animals. Since fungi are eukaryotes, and thus more similar to their host organisms than, for example bacteria, the treatment of infections by fungi poses special risks and challenges not encountered with other types of infections. One such fungus is Magtiaporthe grisea, the fungus that causes rice blast disease. It is an organism that poses a significant threat to food supplies worldwide. Other examples of plant pathogens of economic importance include the pathogens in the genera 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, Cochliobolus, 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, Erysiphe, 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, Lasiodiplodia, Laxitextum, Leptographium, Leptosphaeria, Leptosphaerulina, Leucytospora, Linospora, Lophodermella, Lophodermium, Macrophomina, Magnaporthe, Marssonina, Melampsora, Melampsorella, Meria, Microdochium, Microsphaera, Monilinia, Monochaetia, Morchella, Mycosphaerella, Myrothecium, Nectria, Nigrospora, Ophiosphaerella, Ophiostbma, Penicillium, 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, Rhynchosporium, 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, Urocystis, Uromyces, Ustilago, Valsa, Venturia, Verticillium, Xylaria, and others. Related organisms in the classification, oomycetes, that include the genera Albugo, Aphanomyces, Bremia, Peronospora, Phytophthora, Plasmodiophora, Plasmopara, Pseudoperonospora, Pythium, Sclerophthora, and others are also significant plant pathogens and are sometimes classified along with the true fungi. Human diseases that are caused by filamentous fungi include life-threatening lung and disseminated diseases, often a result of infections by Aspergillus fumigatus. Other fungal diseases in animals are caused by fungi in the genera, Fusarium, Blastomyces, Microsporum, Trichophyton, Epidermophyton, Candida, Histoplamsa, Pneumocystis, Cryptococcus, other Aspergϊϊli, and others. The control of fungal diseases in plants and animals is usually mediated by chemicals that inhibit the growth, proliferation, and/or pathogenicity of the fungal organisms. To date, there are less than twenty known modes-of-action for plant protection fungicides and human antifungal compounds.

A pathogenic organism has been defined as an organism that causes, or is capable of causing disease. Pathogenic organisms propagate on or in tissues and may obtain nutrients and other essential materials from their hosts. A substantial amount of work concerning filamentous fungal pathogens has been performed with the human pathogen, Aspergillus fumigatus. Shibuya et al. (Shibuya, K., M. Takaoka, et al. (1999) Microb Pafhog 27: 123 - 31 (PMID: 10455003)) have shown that the deletion of either of two suspected pathogenicity related genes encoding an alkaline protease or a hydrophobin (rodlet) respectively, did not reduce mortality of mice infected with these mutant strains. Smith et al. (Smith, J. M., C. M. Tang, et al. (1994) Infect Immun 62: 5247 - 54 (PMID: 7960101)) showed similar results with alkaline protease and the ribotoxin restrictocin; Aspergillus fumigatus strains mutated for either of these genes were fully pathogenic to mice. Reichard et al. (Reichard, U., M. Monod, et al. (1997) J Med Net Mycol 35: 189 - 96 (PMID: 9229335)) showed that deletion of the suspected pathogenicity gene encoding aspergillopepsin (PEP) in Aspergillus fumigatus had no effect on mortality in a guinea pig model system, and Aufauvre-Brown et al (Aufauvre-Brown, A., E. Mellado, et al. (1997) Fungal Genet Biol 21: 141 - 52 (PMID: 9073488)) showed no effects of a chitin synthase mutation on pathogenicity. However, not all experiments produced negative results. Ergosterol is an important membrane component found in fungal organisms. Pathogenic fungi that lack key enzymes in this biochemical pathway might be expected to be non-pathogenic since neither the plant nor animal hosts contain this particular sterol. Many antifungal compounds that affect this biochemical pathway have been described (Onishi, J. C. and A. A. Patchett (1990a, b, c, d, and e) United States Patents 4,920,109; 4,920,111; 4,920,112; 4,920,113; and 4,921,844, Merck & Co. Inc. (Rahway NJ)) and (Hewitt, H. G. (1998) Fungicides in Crop Protection Cambridge, University Press). DΕnfert et al. ( D'Enfert, C, M. Diaquin, et al. (1996) Infect Immun 64: 4401 - 5 (PMID: 8926121)) showed that so. Aspergillus fumigatus strain mutated in an orotidine 5'-phosphate decarboxylase gene was entirely non-pathogenic in mice, and Brown et al. (Brown, J. S., A. Aufauvre-Brown, et al. (2000) Mol Microbiol 36: 1371-80 (PMID: 10931287)) observed a non-pathogenic result when genes involved in the synthesis of para-aminobenzoic acid were mutated. Some specific target genes have been described as having utility for the screening of inhibitors of plant pathogenic fungi. Bacot et al. (Bacot, K. O., D. B. Jordan, et al. (2000) United States Patent 6,074,830, E. I. du Pont de Nemours & Company (Wilmington DE)) describe the use of 3,4-dihydroxy-2-butanone 4-phosphate synthase, and Davis et al. (Davis, G. E., G. D. Gustafson, et al. (1999) United States Patent 5,976,848, Dow AgroSciences LLC (Indianapolis IN)) describe the use of dihydroorotate dehydrogenase for potential screening purposes.

There are also a number of papers that report less clear results, showing neither full pathogenicity nor non-pathogenicity of mutants. Hensel et al. (Hensel, M., H. N. Arst, Jr., et al. (1998) Mol Gen Genet 258: 553 - 7 (PMID: 9669338)) showed only moderate effects of the deletion of the areA transcriptional activator on the pathogenicity of Aspergillus fumigatus.

Therefore, it is not currently possible to determine which specific growth materials may be readily obtained by a pathogen from its host, and which materials may not. The present inventors have found that Magnaporthe grisea that cannot synthesize their own L-asparagine are non-pathogenic on their host organism. Previous studies of the Saccharomyces cerevisiae Asparagine Synthase genes, ASN1 and ASN2, indicated that disruption of ASN1 or ASN2 alone has no effect on growth (Dang et al. (1996) Mol Microbiol 22: 681 - 92 (PMID: 8951815)), teaching against our finding. To date there do not appear to be any publications demonstrating an anti-pathogenic effect of the knockout, over-expression, antisense expression, or inhibition of a gene or gene products involved in L-asparagine biosynthesis in filamentous fungi. Thus, it has not been shown that the de novo biosynthesis of L-asparagine is essential for fungal pathogenicity. Thus, it would be desirable to determine the utility of the enzymes involved in L-asparagine biosynthesis for evaluating antibiotic compounds, especially fungicides. If a fungal biochemical pathway or specific gene product in that pathway is shown to be required for fungal pathogenicity, various formats of in vitro and in vivo screening assays may be put in place to discover classes of chemical compounds that react with the validated target gene, gene product, or biochemical pathway, and are thus candidates for antifungal, biocide, and biostatic materials.

The present inventors have found that Magnaporthe grisea that cannot synthesize their own heme are non-pathogenic on their host organism. In addition to being a key component of respiratory cytochromes and hemoglobin, heme is the prosthetic group for many enzymes involved in the detoxification of oxygen radicals and in the metabolism of fatty acids and sterols. In yeast, Saccharomyces cerevisiae, mutants deficient in heme biosynthesis have been isolated and genetically studied in detail (Gollub et al. (1977) J Biol Chem 252: 2846 - 54 (PMID: 323256)). The 5-aminolevulinate synthase gene has been cloned from Aspergillus oryzae and shown to be used as a selectable marker for the transformation of A. oryzae (Elrod et al. (2000) Curr Genet 38: 291 - 8 (PMID: 11191214)). In humans, two 5-aminolevulinate synthase genes have been identified. Mutations in one of them, encoding an erythroid isoform, result in X-linked sideroblastic anemia (Cox et al. (1994) N Engl J Med 330: 675 - 9 (PMID: 8107717)). 5- aminolevulinate synthase has been proposed as a new antimalarial target (Padmanaban and Rangarajan (2000) Biochem Biophys Res Commun 268: 665 - 8 (PMID: 10679261)).

To date there do not appear to be any publications demonstrating an anti- pathogenic effect of the knock-out, over-expression, antisense expression, or inhibition of the genes or gene products involved in heme biosynthesis in filamentous fungi. Thus, it has not been shown that the de novo biosynthesis of heme is essential for fungal pathogenicity. And, thus, it would be desirable to determine the utility of the enzymes involved in heme biosynthesis for evaluating antibiotic compounds, especially fungicides. If a fungal biochemical pathway or specific gene product in that pathway is shown to be required for fungal pathogenicity, various formats of in vitro and in vivo screening assays may be put in place to discover classes of chemical compounds that react with the validated target gene, gene product, or biochemical pathway, and are thus candidates for antifungal, biocide, and biostatic materials.

The present inventors have found that Magnaporthe grisea that cannot synthesize their own L-histidine have reduced pathogenicity on their host organism. The M. grisea HISP1 enzyme has greatest similarity to Schizosaccharomyces pombe His9, as well as some similarity to Saccharomyces cerevisiae His2p and His9. These genes encode a distantly related family of Histidinol Phosphate Phosphatases (HolPase), which catalyzes the dephosphorylation of Histidinol Phosphate to Histidinol. This family includes the HolPase encoded by the HisJ (or ytvP) gene found in Bacillus subtilis. Knock-out of His J has yielded an auxotrophic mutant, unable to grow without Histidine supplementation (le Coq et al. (1999) J Bacteriol 181: 3277 - 3280 (PMID: 10322033)). No references were found where SpHis2, ScHis2p or ScHis9 are known targets for anti- fungal/ fungicide development. However, S. cerevisiae mutants containing knock-outs in the Hisl-His7 genes have been shown 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 - 4779 (PMID: 10438744)).

To date there do not appear to be any publications demonstrating an anti- pathogenic effect of the knock-out, over-expression, antisense expression, or inhibition of the genes or gene products involved in L-histidine biosynthesis in filamentous fungi. Thus, it has not been shown that the de novo biosynthesis of L-histidine is essential for fungal pathogenicity. And, thus, it would be desirable to determine the utility of the enzymes involved in L-histidine biosynthesis for evaluating antibiotic compounds, especially fungicides. If a fungal biochemical pathway or specific gene product in that pathway is shown to be required for fungal pathogenicity, various formats of in vitro and in vivo screening assays may be put in place to discover classes of chemical compounds that react with the validated target gene, gene product, or biochemical pathway, and are thus candidates for antifungal, biocide, and biostatic materials.

The present inventors have found that Magnaporthe grisea that cannot synthesize their own L-leucine are non-pathogenic on their host organism. To date there do not appear to be any publications demonstrating an anti-pathogenic effect of the knock-out, over-expression, antisense expression, or inhibition of the genes or gene products involved in L-leucine biosynthesis in filamentous fungi. Thus, it has not been shown that the de novo biosynthesis of L-leucine is essential for fungal pathogenicity. And, thus, it would be desirable to determine the utility of the enzymes involved in L-leucine biosynthesis for evaluating antibiotic compounds, especially fungicides. If a fungal biochemical pathway or specific gene product in that pathway is shown to be required for fungal pathogenicity, various formats of in vitro and in vivo screening assays may be put in place to discover classes of chemical compounds that react with the validated target gene, gene product, or biochemical pathway, and are thus candidates for antifungal, biocide, and biostatic materials.

The present inventors have found that Magnaporthe grisea that cannot synthesize their own L-threonine are non-pathogenic on their host organism. To date there do not appear to be any publications demonstrating an anti-pathogenic effect of the knock-out, over-expression, antisense expression, or inhibition of the genes or gene products involved in L-threonine biosynthesis in filamentous fungi. Thus, it has not been shown that the de novo biosynthesis of L-threonine is essential for fungal pathogenicity. Thus, it would be desirable to determine the utility of the enzymes involved in L-threonine biosynthesis for evaluating antibiotic compounds, especially fungicides. If a fungal biochemical pathway or specific gene product in that pathway is shown to be required for fungal pathogenicity, various formats of in vitro and in vivo screening assays may be put in place to discover classes of chemical compounds that react with the validated target gene, gene product, or biochemical pathway, and are thus candidates for antifungal, biocide, and biostatic materials.

SUMMARY OF THE INVENTION

The present inventors have discovered that in vivo disruption of the genes encoding Asparagine Synthase, 5-Aminolevulinate synthase, histidinol-phosphatase, 3- Isopropylmalate dehydratase or Threonine synthase in Magnaporthe grisea prevents or inhibits the pathogenicity of the fungus. Thus, the present inventors have discovered that Asparagine Synthase, 5-Aminolevulinate synthase, histidinol-phosphatase, 3- Isopropylmalate dehydratase and Threonine synthase are essential for normal rice blast pathogenicity, and can be used as targets for the identification of antibiotics, preferably fungicides. Accordingly, the present invention provides methods for the identification of compounds that inhibit Asparagine Synthase, 5-Aminolevulinate synthase, histidinol- phosphatase, 3-Isopropylmalate dehydratase or Threonine synthase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably fungicides.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the reaction performed by Asparagine Synthase (ASN1) reaction. The Substrates/Products are L-aspartate, L-glutamine, and ATP and the Products/Substrates are 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 biosynthesis pathway.

Figure 2 shows a digital image showing the effect of ASN1 gene disruption on Magnaporthe grisea pathogenicity using whole plant infection assays. Rice variety CO39 was inoculated with wild-type and the transposon insertion strains, KO1-2 and KOI -8. Leaf segments were imaged at five days post-inoculation.

Figure 3A&B. Verification of Gene Function by Analysis of Nutritional Requirements. Wild-type and transposon insertion strains, KO1-2 and KO1-8, were grown in (A) minimal media and (B) minimal media with the addition of L-asparagine, respectively. The x-axis shows time in days and the y-axis shows turbidity measured at 490 nanometers and 750 nanometers. The symbols represent wildtype (--♦--), transposon strain KO1-2 (--■--), and transposon strain KO1-8 (-- A--).

Figure 4 shows the reaction performed by 5-Aminolevulinate synthase (ALAS1) reaction. The Substrates/Products are succinyl-CoA and glycine and the Products/Substrates are 5-aminolevulinate, Co A, and CO₂. The function of the 5- Aminolevulinate synthase enzyme is the interconversion of succinyl-CoA and glycine to 5-aminolevulinate, CoA, and CO₂. This reaction is part of the heme biosynthesis pathway. Figure 5 shows a digital image showing the effect of ALAS 1 gene disruption on Magnaporthe grisea pathogenicity using whole plant infection assays. Rice variety CO39 was inoculated with wild-type and the transposon insertion strains, KOl-1 and KO1-106. Leaf segments were imaged at five days post-inoculation.

Figure 6A&B. Verification of Gene Function by Analysis of Nutritional Requirements. Wild-type and transposon insertion strains, KOl-1 and KO1-106, were grown in (A) minimal media and (B) minimal media with the addition of 5- aminolevulinate, respectively. The x-axis shows time in days and the y-axis shows turbidity measured at 490 nanometers and 750 nanometers. The symbols represent wildtype (--♦--), transposon strain KOl-1 (--■--), and transposon strain KO1-106 (-- A--

)•

Figure 7 shows the reaction performed by histidinol-phosphatase (HISP1) reaction. The Substrates/Products are L-histidinol phosphate and H₂O and the Products/Substrates are L-histidinol and orthophosphate. The function of the histidinol- phosphatase enzyme is the interconversion of L-histidinol phosphate and H O to L- histidinol and orthophosphate. This reaction is part of the L-histidine biosynthesis pathway.

Figure 8 shows a digital image showing the effect of HISP1 gene disruption on Magnaporthe grisea pathogenicity using whole plant infection assays. Rice variety CO39 was inoculated with wild-type and the transposon insertion strains, KOl-1 and KOI -3. Leaf segments were imaged at five days post-inoculation.

Figure 9A&B. Verification of Gene Function by Analysis of Nutritional Requirements. Wild-type and transposon insertion strains, KOl-1 and KOI -3, were grown in (A) minimal media and (B) minimal media with the addition of L-histidine, respectively. The x-axis shows time in days and the y-axis shows turbidity measured at 490 nanometers and 750 nanometers. The symbols represent wildtype (--♦-), transposon strain KOl-1 (--■--), and transposon strain KO1-3 (-- A-).

Figure 10 shows the reaction performed by 3-Isopropylmalate dehydratase (IPMDl) reaction. The Substrates/Products are 2-Isopropylmalate and H₂O and the Product/Substrate is 3-Isopropylmalate. The function of the 3-Isopropylmalate dehydratase enzyme is the interconversion of 2-Isopropylmalate and H₂O to 3- Isopropylmalate. This reaction is part of the L-leucine biosynthesis pathway.

Figure 11 shows a digital image showing the effect of IPMDl gene disruption on Magnaporthe grisea pathogenicity using whole plant infection assays. Rice variety CO39 was inoculated with wild-type and the transposon insertion strains, KO1-3 and KOI -7. Leaf segments were imaged at five days post-inoculation.

Figure 12A&B. Verification of Gene Function by Analysis of Nutritional Requirements. Wild-type and transposon insertion strains, KO1-3 and KO1-7, were grown in (A) minimal media and (B) minimal media with the addition of L-leucine, respectively. The x-axis shows time in days and the y-axis shows turbidity measured at 490 nanometers and 750 nanometers. The symbols represent wildtype (--♦-), transposon strain KO1-3 (TI) (--■-), and transposon strain KO1-7 (T2) (—A—).

Figure 13 shows the reaction performed by Threonine synthase (THR4) reaction. The Substrates/Products are O-phospho-L-homoserine and water and the Products/Substrates are 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 biosynthesis pathway.

Figure 14 shows a digital image showing the effect of THR4 gene disruption on Magnaporthe grisea pathogenicity using whole plant infection assays. Rice variety CO39 was inoculated with wild-type and the transposon insertion strains, KO1-3 and KOI -22. Leaf segments were imaged at five days post-inoculation.

Figure 15A&B. Verification of Gene Function by Analysis of Nutritional Requirements. Wild-type and transposon insertion strains, KOI -3 and KOI -22, were grown in (A) minimal media and (B) minimal media with the addition of L-threonine, respectively. The x-axis shows time in days and the y-axis shows turbidity measured at 490 nanometers and 750 nanometers. The symbols represent wildtype (--♦--), transposon strain KO1-3 (--■--), and transposon strain KO1-22 (- A-). 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 compounds, agents, or compositions having antibiotic activity indicates that the compound exerts an effect on a particular target or targets which is deleterious to the in vitro and/or in vivo growth of an organism having that target or targets. In particular, a compound active against a gene exerts an action on a target which affects an expression product of that gene. This does not necessarily mean that the compound acts directly on the expression product of the gene, but instead indicates that the compound affects the expression product in a deleterious manner. Thus, the direct target of the compound may be, for example, at an upstream component which reduces transcription from the gene, resulting in a lower level of expression. Likewise, the compound may affect the level of translation of a polypeptide expression product, or may act on a downstream component of a biochemical pathway in which the expression product of the gene has a major biological role. Consequently, such a compound can be said to be active against the gene, against the gene product, or against the related component either upstream or downstream of that gene or expression product. While the term "active against" encompasses a broad range of potential activities, it also implies some degree of specificity of target. Therefore, for example, a general protease is not "active against" a particular gene which produces a polypeptide product. In contrast, a compound which inhibits a particular enzyme is active against that enzyme and against the gene which codes for that enzyme.

As used herein, the term "allele" refers to any of the alternative forms of a gene ι that may occur at a given locus.

The term "antibiotic" refers to any substance or compound that when contacted with a living cell, organism, virus, or other entity capable of replication, results in a reduction of growth, viability, or pathogenicity of that entity.

As used herein, the term "ALAS1" means a gene encoding 5-Aminolevulinate synthase activity, referring to an enzyme that catalyses the interconversion of succinyl- CoA and glycine with 5-aminolevulinate, CoA, and CO₂, and may also be used to refer to the gene product.

As used herein, the terms "5-Aminolevulinate synthase" (EC 2.3.1.37) and "5- Aminolevulinate synthase polypeptide" are synonymous with "the ALAS1 gene product" and refer to an enzyme that catalyses the interconversion of succinyl-CoA and glycine with 5-aminolevulinate, CoA, and CO₂.

As used herein, the term "ASN1" means a gene encoding Asparagine Synthase activity, referring to an enzyme that catalyses the interconversion of L-aspartate, L- glutamine, and ATP with L-asparagine, L-glutamate, AMP, and pyrophosphate, and may also be used to refer to the gene product.

As used herein, the terms "Asparagine Synthase" (EC 6.3.5.4) and "Asparagine Synthase polypeptide" are synonymous with "the ASN1 gene product" and refer to an enzyme that catalyses the interconversion of L-aspartate, L-glutamine, and ATP with L- asparagine, L-glutamate, AMP, and pyrophosphate.

The term "binding" refers to a non-covalent or a covalent interaction, preferably non-covalent, that holds two molecules together. For example, two such molecules could be an enzyme and an inhibitor of that enzyme. Non-covalent interactions include hydrogen bonding, ionic interactions among charged groups, van der Waals interactions and hydrophobic interactions among 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 connected series of biochemical reactions normally occurring in a cell, or more broadly a cellular event such as cellular division or DNA replication. Typically, the steps in such a biochemical pathway act in a coordinated fashion to produce a specific product or products or to produce some other particular biochemical action. Such a biochemical pathway requires the expression product of a gene if the absence of that expression product either directly or indirectly prevents the completion of one or more steps in that pathway, thereby preventing or significantly reducing the production of one or more normal products or effects of that pathway. Thus, an agent specifically inhibits such a biochemical pathway requiring the expression product of a particular gene if the presence of the agent stops or substantially reduces the completion of the series of steps in that pathway. Such an agent, may, but does not necessarily, 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 permitting growth and/or survival only under special growth or environmental conditions.

As used herein, the term "cosmid" refers to a hybrid vector, used in gene cloning, that includes a cos site (from the lambda bacteriophage). It also contains drug resistance marker genes and other plasmid genes. Cosmids are especially suitable for cloning large genes or multigene fragments.

As used herein, the term "dominant allele" refers to a dominant mutant allele in which a discemable mutant phenotype can be detected when this mutation is present in an organism that also contains a wild type (non-mutant), recessive allele, or other dominant allele.

As used herein, the term "DNA" means deoxyribonucleic acid.

As used herein, the term "ELISA" means enzyme-linked immunosorbent assay.

"Fungi" (singular: fungus) refers to whole fungi, fungal organs and tissues (e.g., asci, hyphae, pseudohyphae, rhizoid, sclerotia, sterigmata, spores, sporodochia, sporangia, synnemata, conidia, ascostroma, cleistothecia, mycelia, perithecia, basidia and the like), spores, fungal cells and the progeny thereof. Fungi are a group of organisms (about 50,000 known species), including, but not limited to, mushrooms, mildews, moulds, yeasts, etc., comprising the kingdom Fungi. They can either exist as single cells or make up a multicellular body called a mycelium, which consists of filaments known as hyphae. Most fungal cells are multinucleate and have cell walls, composed chiefly of chitin. Fungi exist primarily in damp situations on land and, because of the absence of chlorophyll and thus the inability to manufacture their own food by photosynthesis, are either parasites on other organisms or saprotrophs feeding on dead organic matter. The principal criteria used in classification are the nature of the spores produced and the presence or absence of cross walls within the hyphae. Fungi are distributed worldwide in terrestrial, freshwater, and marine habitats. Some live in the soil. Many pathogenic fungi cause disease in animals and man or in plants, while some saprotrophs are destructive to timber, textiles, and other materials. Some fungi form associations with other organisms, most notably with algae to form lichens.

As used herein, the term "fungicide", "antifungal", or "antimycotic" refers to an antibiotic substance or compound that kills or suppresses the growth, viability, or pathogenicity of at least one fungus, fungal cell, fungal tissue or spore.

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 which can be referred to by the sequence of nucleotides forming the chain. Thus, "sequence" is used to indicate both the ordered listing of the nucleotides which form the chain, and the chain, itself, which has that sequence of nucleotides. ("Sequence" is used in the similar way in referring to RNA chains, linear chains made of ribonucleotides). The gene may include regulatory and control sequences, sequences which can be transcribed into an RNA molecule, and may contain sequences with unknown function. The majority of the RNA transcription products are messenger RNAs (mRNAs), which include sequences which are translated into polypeptides and may include sequences which are not translated. It should be recognized that small differences in nucleotide sequence for the same gene can exist between different fungal strains, or even within a particular fungal strain, without altering the identity of the gene.

As used in this disclosure, the terms "growth" or "cell growth" of an organism refers to an increase in mass, density, or number of cells of said organism. Some common methods for the measurement of growth include the determination of the optical density of a cell suspension, the counting of the number of cells in a fixed volume, the counting of the number of cells by measurement of cell division, the measurement of cellular mass or cellular volume, and the like.

As used in this disclosure, the term "growth conditional phenotype" indicates that a fungal strain having such a phenotype exhibits a significantly greater difference in growth rates in response to a change in one or more of the culture parameters than an otherwise similar strain not having a growth conditional phenotype. Typically, a growth conditional phenotype is described with respect to a single growth culture parameter, such as temperature. Thus, a temperature (or heat-sensitive) mutant (i.e., a fungal strain having a heat-sensitive phenotype) exhibits significantly different growth, and preferably no growth, under non-permissive temperature conditions as compared to growth under permissive conditions. In addition, such mutants preferably also show intermediate growth rates at intermediate, or semi-permissive, temperatures. Similar responses also result from the appropriate growth changes for other types of growth conditional phenotypes.

As used herein, the term "H₂O" means water.

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

As used herein, the term "heterologous ASN1 gene" means a gene, not derived from Magnaporthe grisea, and having: at least 50% sequence identity, preferably 60%, 10%, 80%, 90%), 95%), 99% sequence identity and each integer unit of sequence identity from 50-100% in ascending order to SEQ ID NO: 1 or SEQ ID NO: 2; or at least 10% of the activity of a Magnaporthe grisea Asparagine Synthase, preferably 25%, 50%, 75%>, 90%), 95%o, 99%) and each integer unit of activity from 10-100% in ascending order.

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

As used herein, the terms "histidinol-phosphatase" (EC 3.1.3.15) and "histidinol- phosphatase polypeptide" are synonymous with "the HISPl gene product" and refer to an enzyme that catalyses the interconversion of L-histidinol phosphate and H₂O with L- histidinol and orthophosphate.

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

As used herein, the term "heterologous THR4 gene" means a gene, not derived from Magnaporthe grisea, and having: at least 50%> sequence identity, preferably 60%, 70%), 80%, 90%), 95%, 99% sequence identity and each integer unit of sequence identity from 50-100% in ascending order to SEQ ID NO: 13 or SEQ ID NO: 14; or at least 10% of the activity oϊ a. Magnaporthe grisea Threonine synthase, preferably 25%, 50%, 75%, 90%, 95%, 99%o and each integer unit of activity from 10-100% in ascending order.

As used herein, the term "HISPl" means a gene encoding histidinol-phosphatase activity, referring to an enzyme that catalyses the interconversion of L-histidinol phosphate and H₂O with L-histidinol and orthophosphate, and may also be used to refer to the gene product.

As used herein, the term "His-Tag" refers to an encoded polypeptide consisting of multiple consecutive histidine amino acids.

As used herein, the term "HPLC" means 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 aminoglycosidic antibiotic, used for selection and maintenance of eukaryotic cells containing the E. coli hygromycin resistance gene.

"Hypersensitive" refers to a phenotype in which cells are more sensitive to antibiotic compounds than are wild-type cells of similar or identical genetic background.

"Hyposensitive" refers to a phenotype in which cells are less sensitive to antibiotic compounds than are wild-type cells of similar or identical genetic background.

As used herein, the term "imperfect state" refers to a classification of a fungal organism having no demonstrable sexual life stage.

The term "inhibitor", as used herein, refers to a chemical substance that inactivates the enzymatic activity or substantially reduces the level of enzymatic activity, of any one of Asparagine Synthase, 5-Aminolevulinate synthase, histidinol-phosphatase, 3-Isopropylmalate dehydratase or Threonine synthase wherein "substantially" means a reduction at least as great as the standard deviation for a measurement, preferably a reduction by 50%, more preferably a reduction of at least one magnitude, i.e. to 10%>. The inhibitor may function by interacting directly with the enzyme, a cofactor of the enzyme, the substrate of the enzyme, or any combination thereof.

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

As used herein, the term "TPMDl" means a gene encoding 3-Isopropylmalate dehydratase activity, referring to an enzyme that catalyses the interconversion of 2- Isopropylmalate and H₂O with 3-Isopropylmalate, and may also refer to fhe gene product.

As used herein, the terms "3-Isopropylmalate dehydratase" (EC 4.2.1.33), " - isopropylmalate isomerase" and "3-Isopropylmalate dehydratase polypeptide" are synonymous with "the IPMDl gene product" and refer to an enzyme that catalyses the interconversion of 2-Isopropylmalate and H₂O with 3-Isopropylmalate.

As used herein, the term "knockout" or "gene disruption" refers to the creation of organisms carrying a null mutation (a mutation in which there is no active gene product), a partial null mutation or mutations, or an alteration or alterations in gene regulation by interrupting a DNA sequence through insertion of a foreign piece of DNA. Usually the foreign DNA encodes a selectable marker.

As used herein, the term "LB agar" means Luria's Broth agar.

The term "method of screening" means that the method is suitable, and is typically used, for testing for a particular property or effect in a large number of compounds. Typically, more than one compound is tested simultaneously (as in a 96-well microtiter plate), and preferably significant portions of the procedure can be automated. "Method of screening" also refers to the determination of a set of different properties or effects of one compound simultaneously.

As used herein, the term "mRNA" means messenger ribonucleic acid.

As used herein, the term "mutant form" of a gene refers to a gene which has been altered, either naturally or artificially, changing the base sequence of the gene. The change in the base sequence may be of several different types, including changes of one or more bases for different bases, deletions, and/or insertions, such as by a transposon. By contrast, a normal form of a gene (wild type) is a form commonly found in natural populations of an organism. Commonly a single form of a gene will predominate in natural populations. In general, such a gene is suitable as a normal form of a gene, however, other forms which provide similar functional characteristics may also be used as a normal gene. In particular, a normal form of a gene does not confer a growth conditional phenotype on the strain having that gene, while a mutant form of a gene suitable for use in these methods does provide such a growth conditional 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 of a gene (wild type) is a form commonly found in natural populations of an organism. Commonly a single form of a gene will predominate in natural populations. In general, such a gene is suitable as a normal form of a gene, however, other forms which provide similar functional characteristics may also be used as a normal gene. In particular, a normal form of a gene does not confer a growth conditional phenotype on the strain having that gene, while a mutant form of a gene suitable for use in these methods does provide such a growth conditional phenotype.

As used herein, the term "one form" of a gene is synonymous with the term "gene", and a "different form" of a gene refers to a gene that has greater than 49% sequence identity and less than 100%> sequence identity with said first form.

As used herein, the term "pathogenicity" refers to a capability of causing disease. The term is applied to parasitic microorganisms in relation to their hosts.

As used herein, the term "PCR" means polymerase chain reaction. The "percent (%) sequence identity" between two polynucleotide or two polypeptide sequences is determined according to the either the BLAST program (Basic Local Alignment Search Tool; (Altschul, S.F., W. Gish, et al. (1990) J Mol Biol 215: 403 - 10 (PMID: 2231712)) at the National Center for Biotechnology or using Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147: 195 - 7 (PMID: 7265238)) as incorporated into GeneMatcher Plus™. It is understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymine nucleotide is equivalent to a uracil nucleotide.

By "polypeptide" is meant a chain of at least two amino acids joined by peptide bonds. The chain may be linear, branched, circular or combinations thereof. Preferably, polypeptides are from about 10 to about 1000 amino acids in length, more preferably 10- 50 amino acids in length. The polypeptides may contain amino acid analogs and other modifications, including, but not limited to glycosylated or phosphorylated residues.

As used herein, the term "proliferation" is synonymous to 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, "semi-permissive conditions" are conditions in which the relevant culture parameter for a particular growth conditional phenotype is intermediate between permissive conditions and non-permissive conditions. Consequently, in semi-permissive conditions an organism having a growth conditional phenotype will exhibit growth rates intermediate between those shown in permissive conditions and non-permissive conditions. In general, such intermediate growth rate may be due to a mutant cellular component which is partially functional under semi-permissive conditions, essentially fully functional under permissive conditions, and is non-functional or has very low function under non-permissive conditions, where the level of function of that component is related to the growth rate of the organism. An intermediate growth rate may also be a result of a nutrient substance or substances that are present in amounts not sufficient for optimal growth rates to be achieved.

"Sensitivity phenotype" refers to a phenotype that exhibits either hypersensitivity or hyposensitivity. 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, wherein the interaction is dependent upon the primary amino acid sequence and/or the conformation of the Asparagine Synthase, 5-Aminolevulinate synthase, histidinol- phosphatase, 3-Isopropylmalate dehydratase or Threonine synthase.

As used herein, the term "THR4" means a gene encoding Threonine, synthase activity, referring to an enzyme that catalyses the interconversion of O-phospho-L- homoserine and water with L-threonine and orthophosphate, and may 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 "the THR4 gene product" and refer to an enzyme that catalyses the interconversion of O-phospho-L-homoserine and water with L- threonine and orthophosphate.

As used herein, the term "TLC" means thin layer chromatography.

"Transform", as used herein, refers to the introduction of a polynucleotide (single or double stranded DNA, RNA, or a combination thereof) into a living cell by any means. Transformation may be accomplished by a variety of methods, including, but not limited to, elecfroporation, polyethylene glycol mediated uptake, particle bombardment, agrotransformation, and the like. This process may result in transient or stable expression of the transformed polynucleotide. By "stably transformed" is meant that the sequence of interest is integrated into a replicon in the cell, such as a chromosome or episome. Transformed cells encompass not only the end product of a transformation process, but also the progeny thereof which retain the polynucleotide of interest.

For the purposes of the invention, "transgenic" refers to any cell, spore, tissue or part, that contains all or part of at least one recombinant polynucleotide. In many cases, all or part of the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations.

As used herein, the term "transposase" refers to an enzyme that catalyzes transposition. Preferred transposons are described in WO 00/55346, PCT/US00/07317, and US 09/658859. As used herein, the term "transposition" refers to a complex genetic rearrangement process involving the movement or copying of a polynucleotide (transposon) from one location and insertion into another, often within or between a genome or genomes, or DNA constructs such as plasmids, bacmids, and cosmids.

As used herein, the term "transposon" (also known as a "transposable element", "transposable genetic element", "mobile element", or "jumping gene") refers to a mobile DNA element such as those, for example, described in WO 00/55346, PCT/USOO/07317, and US 09/658859. Transposons can disrupt gene expression or cause deletions and inversions, and hence affect both the genotype and phenotype of the organisms concerned. The mobility of transposable elements has long been used in genetic manipulation, to introduce genes or other information into the genome of certain model systems.

As used herein, the term "Tween 20" means sorbitan mono-9-octadecenoate poly(oxy- 1 , 1 -ethanediyl) .

As used in this disclosure, the term "viability" of an organism refers to the ability of an organism to demonstrate growth under conditions appropriate for said organism, or to demonstrate an active cellular function. Some examples of active cellular functions include respiration as measured by gas evolution, secretion of proteins and/or other compounds, dye exclusion, mobility, dye oxidation, dye reduction, pigment production, changes in medium acidity, and the like.

The present inventors have discovered that disruption of the ASNl gene and/or gene product inhibits the pathogenicity of Magnaporthe grisea. Thus, the inventors are the first to demonstrate that Asparagine Synthase is a target for antibiotics, preferably antifungals.

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

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting an Asparagine Synthase polypeptide with a test compound; and b) detecting the presence or absence of binding between said test compound and said Asparagine Synthase polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

The Asparagine Synthase protein may have the amino acid sequence of a naturally occurring Asparagine Synthase found in a fungus, animal, plant, or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence. Preferably the Asparagine Synthase is a fungal Asparagine Synthase. The cDNA (SEQ ID NO: 1) encoding the Asparagine Synthase protein, the genomic DNA (SEQ ID NO: 2) encoding the M. grisea protein, and the polypeptide (SEQ ID NO: 3) can be found herein.

In one aspect, the invention also provides for a polypeptide consisting essentially of SEQ ID NO: 3. For the purposes of the invention, a polypeptide consisting essentially of SEQ ID NO: 3 has at least 80% sequence identity with SEQ ID NO: 3 and catalyses the interconversion of L-aspartate, L-glutamine, and ATP with L-asparagine, L- glutamate, AMP, and pyrophosphate with at least 10%> of the activity of SEQ ID NO: 3. 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 is at least 95% or 97 or 99%, or any integer from 80-100%) sequence^* identity in ascending order. And, preferably, the polypeptide consisting essentially of SEQ ID NO: 3 has at least 25%, at least 50%, at least 75% or at least 90% of the activity of M. grisea Asparagine Synthase, or any integer from 60-100% activity in ascending order.

By "fungal Asparagine Synthase" is meant an enzyme that can be found in at least one fungus, and which catalyzes the interconversion of L-aspartate, L-glutamine, and ATP with L-asparagine, L-glutamate, AMP, and pyrophosphate. The Asparagine Synthase may be from any of the fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

In one embodiment, the Asparagine Synthase is a Magnaporthe Asparagine Synthase. Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae and the imperfect states of. Magnaporthe in the genus Pyricularia. Preferably, the Magnaporthe Asparagine Synthase is from Magnaporthe grisea.

In various embodiments, the Asparagine Synthase can be from Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armϊllaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armϊllaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Corn Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and the like.

Fragments of an Asparagine Synthase polypeptide may be used in the methods of the invention, preferably if the fragments include an intact or nearly intact epitope that occurs on the biologically active wildtype Asparagine Synthase. The fragments comprise at least 10 consecutive amino acids of an Asparagine Synthase. Preferably, the fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 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, or at least 580 consecutive amino acids residues of an Asparagine Synthase. In one embodiment, the fragment is from a Magnaporthe Asparagine Synthase. Preferably, the fragment contains an amino acid sequence conserved among fungal Asparagine Synthases.

Polypeptides having at least 50% sequence identity with a 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 any integer from 60-100% sequence identity in ascending order.

In addition, it is preferred that the polypeptide has at least 10% of the activity of a fungal Asparagine Synthase. More preferably, the polypeptide has at least 25%, at least 50%, at least 75% or at least 90% of the activity of a fungal Asparagine Synthase. Most preferably, the polypeptide has at least 10%, at least 25%>, at least 50%, at least 75%> or at least 90% of the activity of the M. grisea Asparagine Synthase protein.

Thus, in another embodiment, the invention provides a method for identifying a test compound as a candidate for a fungicide, comprising: a) contacting a test compound with at least one polypeptide selected from the group consisting of: a polypeptide having at least ten consecutive amino acids of a fungal Asparagine Synthase; a polypeptide having at least 50%> sequence identity with a fungal Asparagine Synthase; and a polypeptide having at least 10%> of the activity of a fungal Asparagine Synthase; and b) detecting the presence and/or absence of binding between said test compound and said polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

Any technique for detecting the binding of a ligand to its target may be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting the binding of a ligand to its target are known in the art, and include, but are not limited to the detection of an immobilized ligand-target complex or the detection of a change in the properties of a target when it is bound to a ligand. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with an 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, 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 ligands. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetric methods.

Once a compound is identified as a candidate for an antibiotic, it can be tested for the ability to inhibit Asparagine Synthase enzymatic activity. The compounds can be tested using either in vitro or cell based assays. Alternatively, a compound can be tested by applying it directly to a fungus or fungal cell, or expressing it therein, and monitoring the fungus or fungal cell for changes or decreases in growth, development, viability, pathogenicity, or alterations in gene expression. Thus, in one embodiment, the invention provides a method for determining whether a compound identified as an antibiotic candidate by an above method has antifungal activity, further comprising: contacting a fungus or fungal cells with said antifungal candidate and detecting a decrease in the growth, viability, or pathogenicity of said fungus or fungal cells.

By decrease in growth, is meant that the antifungal candidate causes at least a 10%) decrease in the growth of the fungus or fungal cells, as compared to the growth of the fungus or fungal cells in the absence of the antifungal candidate. By a decrease in viability is meant that at least 20% of the fungal cells, or portion of the fungus contacted with the antifungal candidate are nonviable. Preferably, the growth or viability will be decreased by at least 40%. More preferably, the growth or viability will be decreased 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. By decrease in pathogenicity, is meant that the antifungal candidate causes at least a 10%> decrease in the disease caused by contact of the fungal pathogen with its host, as compared to the disease caused in the absence of the antifungal candidate. Preferably, the disease will be decreased by at least 40%). More preferably, the disease will be decreased by at least 50%>, 75%> or at least 90%) or more. Methods for measuring fungal disease are well known to those skilled in the art, and include such metrics 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 in vitro enzymatic assays in which the disappearance of a substrate or the appearance of a product is directly or indirectly detected. Asparagine Synthase catalyzes the irreversible or reversible reaction L-aspartate, L-glutamine, and ATP = L-asparagine, L-glutamate, AMP, and pyrophosphate (see Figure 1). Methods for detection of L- aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, and/or pyrophosphate, include spectrophotometry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.

Thus, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-aspartate, L-glutamine, and ATP with an Asparagine Synthase; b) contacting L-aspartate, L-glutamine, and ATP with Asparagine Synthase and a test compound; and c) determining the change in concentration for at least one of the following: L- aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, and/or pyrophosphate, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with an Asparagine Synthase; b) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with an Asparagine Synthase and a test compound; and c) determining the change in concentration for at least one of the following: L- aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, and/or pyrophosphate, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

Enzymatically active fragments of a fungal Asparagine 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 a fungal Asparagine Synthase may be used in the methods of the invention. In addition, an enzymatically active polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity with a fungal Asparagine Synthase may be used in the methods of the invention. Most preferably, the polypeptide has at least 50% sequence identity with a fungal Asparagine Synthase and at least 10%, 25%, 75% or at least 90%> of the activity thereof.

Thus, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-aspartate, L-glutamine, and ATP with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with an Asparagine Synthase, a polypeptide having at least 50%> sequence identity with an Asparagine Synthase and having at least 10%) of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of an Asparagine Synthase; b) contacting L-aspartate, L-glutamine, and ATP with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: L- aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, and/or pyrophosphate, wherein a change in concentration for any of the above substances indicates that said, test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with a polypeptide selected from the group consisting of: a polypeptide having at least 50%> sequence identity with an Asparagine Synthase, a polypeptide having at least 50% sequence identity with an Asparagine Synthase and at least 10% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of an Asparagine Synthase; b) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate, with a polypeptide and said test compound; and c) determining the change in concentration for at least one of the following, L- aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, and/or pyrophosphate, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

For the in vitro enzymatic assays, Asparagine Synthase protein and derivatives thereof may be purified from a fungus or may be recombinantly produced in and purified from an archael, bacterial, fungal, or other eukaryotic cell culture. Preferably these proteins are produced using an E. coli, yeast, or filamentous fungal expression system. Methods for the purification of Asparagine Synthase may be described in Van Heeke and Schuster (1989) J Biol Chem 264: 5503 - 9 (PMID: 2564390). Other methods for the purification of Asparagine Synthase proteins and polypeptides are known to those skilled in the art.

As an alternative to in vitro assays, the invention also provides cell based assays. In one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) measuring the expression of an Asparagine Synthase in a cell, cells, tissue, or an organism in the absence of a test compound; b) contacting said cell, cells, tissue, or organism with said test compound and measuring the expression of said Asparagine Synthase in said cell, cells, tissue, or organism; and c) comparing the expression of Asparagine Synthase in steps (a) and (b), wherein a lower expression in the presence of said test compound indicates that said compound is a candidate for an antibiotic.

Expression of Asparagine Synthase can be measured by detecting the ASNl primary transcript or mRNA, Asparagine Synthase polypeptide, or Asparagine Synthase enzymatic activity. Methods for detecting the expression of RNA and proteins are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York, 1995. The method of detection is not critical to the invention. Methods for detecting ASNl RNA include, but are not limited to amplification assays such as quantitative reverse transcriptase-PCR, and/or hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using an ASNl 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 spectroscopy, and enzymatic assays. Also, any reporter gene system may be used to detect ASNl protein expression. For detection using gene reporter systems, a polynucleotide encoding a reporter protein is fused in frame with ASNl, so as to produce a chimeric polypeptide. Methods for using reporter systems are known to those skilled in the art.

Chemicals, compounds or compositions identified by the above methods as modulators, preferably inhibitors, of ASNl expression or activity can then be used to control fungal growth. Diseases such as rusts, mildews, and blights spread rapidly once established. Fungicides are thus routinely applied to growing and stored crops as a preventive measure, generally as foliar sprays or seed dressings. For example, compounds that inhibit fungal growth can be applied to a fungus or expressed in a fungus, in order to prevent fungal growth. Thus, the invention provides a method for inhibiting fungal growth, comprising contacting a fungus with a compound identified by the methods of the invention as having antifungal activity.

Antifungals and antifungal inhibitor candidates identified by the methods of the invention can be used to control the growth of undesired fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

Examples of undesired fungi include, but are not limited to Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculάtus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Corn Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), diseases of animals such as infections of lungs, blood, brain, skin, scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp. Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp., and the like).

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

In one particular embodiment, the method is performed by providing an organism having a first form of the gene corresponding to either SEQ ID NO: 1 or SEQ ID NO: 2, either a normal form, a mutant form, a homologue, or a heterologous ASNl gene that performs a similar function as ASNl. The first form of ASNl may or may not confer a growth conditional phenotype, i.e., a L-asparagine requiring phenotype, and/or a hypersensitivity or hyposensitivity phenotype on the organism having that altered form. In one particular embodiment a mutant form contains a transposon insertion. A comparison organism having a second form of an ASNl, different from the first form of the gene is also provided, and the two organisms are separately contacted with a test compound. The growth of the two organisms in the presence of the test compound is then compared.

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of an Asparagine Synthase gene, and providing comparison cells having a different form of an Asparagine Synthase gene; and b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and said comparison cells in the presence of the test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

It is recognized in the art that the optional determination of the growth of said first organism and said comparison second organism in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different genes. It is also recognized that any combination of two different forms of an ASNl gene, including normal genes, mutant genes, homologues, and functional homologues may be used in this method. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment the organism is Magnaporthe grisea.

Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics as inhibitors of the substrates, products and enzymes of the pathway. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lehninger, A., D. Nelson, et al. (1993) Principles of Biochemistry. New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which ASNl functions, comprising: > a) providing cells having one form of a gene in the L-asparagine biochemical and/or genetic pathway and providing comparison cells having a different form of said gene; b) contacting said cells and said comparison cells with a test compound; and ^λ c) determining the growth of said cells and said comparison cells in the presence of said test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

The use of multi-well plates for screening is a format that readily accommodates multiple different assays to characterize various compounds, concentrations of compounds, and fungal strains in varying combinations and formats. Certain testing parameters for the screening method can significantly affect the identification of growth inhibitors, and thus can be manipulated to optimize screening efficiency and/or reliability. Notable among these factors are variable sensitivities of different mutants, increasing hypersensitivity with increasingly less permissive conditions, an apparent increase in hypersensitivity with increasing compound concentration, and other factors known to those in the art.

Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lenninger et al. (1993) Principles of Biochemistry).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which ASNl functions, comprising:

(a) providing paired growth media comprising a first medium and a second medium, wherein said second medium contains a higher level of L-asparagine than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism, wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic. It is recognized in the art that determination of the growth of said organism in the paired media in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different media. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment, the organism is Magnaporthe grisea.

The present inventors have discovered that disruption of the ALAS1 gene and/or gene product inhibits the pathogenicity of Magnaporthe grisea. Thus, the inventors are the first to demonstrate that 5-Aminolevulinate synthase is a target for antibiotics, preferably antifungals.

Accordingly, the 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, assays for enzyme activity, cell-based assays, and assays for ALAS1 gene expression. Any compound that is a ligand for 5- Aminolevulinate synthase may have antibiotic activity. For the purposes of the invention, "ligand" refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the invention are useful as antibiotics.

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a 5-Aminolevulinate synthase polypeptide with a test compound; and b) detecting the presence or absence of binding between said test compound and said 5-Aminolevulinate synthase polypeptide; wherein binding indicates that said test compound is a candidate for an antibiotic.

The 5-Aminolevulinate synthase protein may have the amino acid sequence of a naturally occurring 5-Aminolevulinate synthase found in a fungus, animal, plant, or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence. Preferably the 5-Aminolevulinate synthase is a fungal 5-Aminolevulinate synthase. The cDNA (SEQ ID NO: 4) encoding the M. grisea 5-Aminolevulinate synthase protein, the genomic DNA (SEQ ID NO: 5) encoding the protein, and the polypeptide (SEQ ID NO: 6) can be found herein. In one aspect, the invention also provides for a polypeptide consisting essentially of SEQ ID NO: 6. For the purposes of the invention, a polypeptide consisting essentially of SEQ ID NO: 6 has at least 80% sequence identity with SEQ ID NO: 6 and catalyses the interconversion of succinyl-CoA and glycine with 5-aminolevulinate, CoA, and CO₂ with at least 10%> of the activity of SEQ ID NO: 6. 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 is at least 95% or 97 or 99%, or any integer from 80-100% sequence identity in ascending order. And, preferably, the polypeptide consisting essentially of SEQ ID NO: 6 has at least 25%o, at least 50%>, at least 75% or at least 90% of the activity of M. grisea 5- Aminolevulinate synthase, or any integer from 60-100% activity in ascending order.

By "fungal 5-Aminolevulinate synthase" is meant an enzyme that can be found in at least one fungus, and which catalyzes the interconversion of succinyl-CoA and glycine with 5-aminolevulinate, CoA, and CO₂. The 5-Aminolevulinate synthase may be from any of the fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

In one embodiment, the 5-Aminolevulinate synthase is a Magnaporthe 5- Aminolevulinate synthase. Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae and the imperfect states of Magnaporthe in the genus Pyricularia. Preferably, the Magnaporthe 5-Aminolevulinate synthase is from Magnaporthe grisea.

In various embodiments, the 5-Aminolevulinate synthase can be from Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Corn Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and the like.

Fragments of a 5-Aminolevulinate synthase polypeptide may be used in the methods of the invention, preferably if the fragments include an intact or nearly intact epitope that occurs on the biologically active wildtype 5-Aminolevulinate synthase. The fragments comprise at least 10 consecutive amino acids of a 5-Aminolevulinate synthase. Preferably, the fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 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, or at least 610 consecutive amino acids residues of a 5-Aminolevulinate synthase. In one embodiment, the fragment is from a Magnaporthe 5-Aminolevulinate synthase. Preferably, the fragment contains an amino acid sequence conserved among fungal 5-Aminolevulinate synthases.

Polypeptides having at least 50% sequence identity with a 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 any integer from 60-100%) sequence identity in ascending order.

In addition, it is preferred that the polypeptide has at least 10% of the activity of a fungal 5-Aminolevulinate synthase. More preferably, the polypeptide has at least 25%>, at least 50%, at least 75%> or at least 90%> of the activity of a fungal 5-Aminolevulinate synthase. Most preferably, the polypeptide has at least 10%, at least 25%, at least 50%, at least 75% or at least 90%> of the activity of the M. grisea 5-Aminolevulinate synthase protein.

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

Any technique for detecting the binding of a ligand to its target may be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting the binding of a ligand to its target are known in the art, and include, but are not limited to the detection of an immobilized ligand-target complex or the detection of a change in the properties of a target when it is bound to a ligand. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with a 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, bound 5- Aminolevulinate synthase is detected using a labeled binding partner, such as a labeled antibody. In a variation of this assay, 5-Aminolevulinate synthase is labeled prior to contacting the immobilized candidate ligands. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetric methods.

Once a compound is identified as a candidate for an antibiotic, it can be tested for the ability to inhibit 5-Aminolevulinate synthase enzymatic activity. The compounds can be tested using either in vitro or cell based assays. Alternatively, a compound can be tested by applying it directly to a fungus or fungal cell, or expressing it therein, and monitoring the fungus or fungal cell for changes or decreases in growth, development, viability, pathogenicity, or alterations in gene expression. Thus, in one embodiment, the invention provides a method for determining whether a compound identified as an antibiotic candidate by an above method has antifungal activity, further comprising: contacting a fungus or fungal cells with said antifungal candidate and detecting a decrease in the growth, viability, or pathogenicity of said fungus or fungal cells.

By decrease in growth, is meant that the antifungal candidate causes at least a 10%) decrease in the growth of the fungus or fungal cells, as compared to the growth of the fungus or fungal cells in the absence of the antifungal candidate. By a decrease in viability is meant that at least 20% of the fungal cells, or portion of the fungus contacted with the antifungal candidate are nonviable. Preferably, the growth or viability will be decreased by at least 40%. More preferably, the growth or viability will be decreased 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. By decrease in pathogenicity, is meant that the antifungal candidate causes at least a 10% decrease in the disease caused by contact of the fungal pathogen with its host, as compared to the disease caused in the absence of the antifungal candidate. Preferably, the disease will be decreased by at least 40%. More preferably, the disease will be decreased by at least 50%>, 75%> or at least 90%> or more. Methods for measuring fungal disease are well known to those skilled in the art, and include such metrics 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 in vitro enzymatic assays in which the disappearance of a substrate or the appearance of a product is directly or indirectly detected. 5-Aminolevulinate synthase catalyzes the irreversible or reversible reaction succinyl-CoA and glycine = 5- aminolevulinate, CoA, and CO₂ (see Figure 1). Methods for detection of succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂, include spectrophotometry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.

Thus, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting succinyl-CoA and glycine with a 5-Aminolevulinate synthase; b) contacting succinyl-CoA and glycine with 5-Aminolevulinate synthase and a test compound; and c) determining the change in concentration for at least one of the following: succinyl- CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 5-aminolevulinate, CoA, and CO with a 5-Aminolevulinate synthase; b) contacting 5-aminolevulinate, CoA, and CO₂ with a 5-Aminolevulinate synthase and a test compound; and c) determining the change in concentration for at least one of the following: succinyl- CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

Enzymatically active fragments of a fungal 5-Aminolevulinate 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 a fungal 5-Aminolevulinate synthase may be used in the methods of the invention. In addition, an enzymatically active polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity with a fungal 5-Aminolevulinate synthase may be used in the methods of the invention. Most preferably, the polypeptide has at least 50% sequence identity with a fungal 5-Aminolevulinate synthase and at least 10%, 25%>, 75% or at least 90% of the activity thereof.

Thus, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting succinyl-CoA and glycine with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with a 5- Aminolevulinate synthase; a polypeptide having at least 50%o sequence identity with a 5-Aminolevulinate synthase and having at least 10%> of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a 5- Aminolevulinate synthase; b) contacting succinyl-CoA and glycine with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: succinyl- CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂; wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 5-aminolevulinate, CoA, and CO₂ with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with a 5- Aminolevulinate synthase; a polypeptide having at least 50%> sequence identity with a 5-Aminolevulinate synthase and at least 10%> of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a 5- Aminolevulinate synthase; b) contacting 5-aminolevulinate, CoA, and CO , with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following, succinyl- CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂; wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

For the in vitro enzymatic assays, 5-Aminolevulinate synthase protein and derivatives thereof may be purified from a fungus or may be recombinantly produced in and purified from an archael, bacterial, fungal, or other eukaryotic cell culture. Preferably these proteins are produced using an E. coli, yeast, or filamentous fungal expression system. Methods for the purification of 5-Aminolevulinate synthase may be described in Volland and Felix (1984) Εur J Biochem 142: 551 - 7 (PMID: 6381051). Other methods for the purification of 5-Aminolevulinate synthase proteins and polypeptides are known to those skilled in the art. As an alternative to in vitro assays, the invention also provides cell based assays. In one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) measuring the expression of a 5-Aminolevulinate synthase in a cell, cells, tissue, or an organism in the absence of a test compound; b) contacting said cell, cells, tissue, or organism with said test compound and measuring the expression of said 5-Aminolevulinate synthase in said cell, cells, tissue, or organism; and c) comparing the expression of 5-Aminolevulinate synthase in steps (a) and (b); wherein a lower expression in the presence of said test compound indicates that said compound is a candidate for an antibiotic.

Expression of 5-Aminolevulinate synthase can be measured by detecting the ALASl primary transcript or mRNA, 5-Aminolevulinate synthase polypeptide, or 5- Aminolevulinate synthase enzymatic activity. Methods for detecting the expression of RNA and proteins are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al, eds., Greene Publishing and Wiley- Interscience, New York, 1995. The method of detection is not critical to the invention. Methods for detecting ALASl RNA include, but are not limited to amplification assays such as quantitative reverse transcriptase-PCR, and/or hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using an ALASl 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 elecfrophoresis, mass spectroscopy, and enzymatic assays. Also, any reporter gene system may be used to detect ALASl protein expression. For detection using gene reporter systems, a polynucleotide encoding a reporter protein is fused in frame with ALASl, so as to produce a chimeric polypeptide. Methods for using reporter systems are known to those skilled in the art.

Chemicals, compounds or compositions identified by the above methods as modulators, preferably inhibitors, of ALASl expression or activity can then be used to control fungal growth. Diseases such as rusts, mildews, and blights spread rapidly once established. Fungicides are thus routinely applied to growing and stored crops as a preventive measure, generally as foliar sprays or seed dressings. For example, compounds that inhibit fungal growth can be applied to a fungus or expressed in a fungus, in order to prevent fungal growth. Thus, the invention provides a method for inhibiting fungal growth, comprising contacting a fungus with a compound identified by the methods of the invention as having antifungal activity.

Antifungals and antifungal inhibitor candidates identified by the methods of the invention can be used to control the growth of undesired fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

Examples of undesired fungi include, but are not limited to Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Corn Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmor-um), Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), diseases of animals such as infections of lungs, blood, brain, skin, scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp. Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp., and the like). Also provided is a method of screening for an antibiotic by determining whether a test compound is active against the gene identified (SEQ JD NO: 4 or SEQ ID NO: 5), its gene product (SEQ ID NO: 6), or the biochemical pathway or pathways it functions on.

In one particular embodiment, the method is performed by providing an organism having a first form of the gene corresponding to either SEQ ID NO: 4 or SEQ ID NO: 5, either a normal form, a mutant form, a homologue, or a heterologous ALASl gene that performs a similar function as ALASl. The first form of ALASl may or may not confer a growth conditional phenotype, i.e., a 5-aminolevulinate requiring phenotype, and/or a hypersensitivity or hyposensitivity phenotype on the organism having that altered form. In one particular embodiment a mutant form contains a transposon insertion. A comparison organism having a second form of an ALASl, different from the first form of the gene is also provided, and the two organisms are separately contacted with a test compound. The growth of the two organisms in the presence of the test compound is then compared.

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a 5-Aminolevulinate synthase gene, and providing comparison cells having a different form of a 5-Aminolevulinate synthase gene; and b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and said comparison cells in the presence of the test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

It is recognized in the art that the optional determination of the growth of said first organism and said comparison second organism in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different genes. It is also recognized that any combination of two different forms of an ALASl gene, including normal genes, mutant genes, homologues, and functional homologues may be used in this method. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment the organism is Magnaporthe grisea.

Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics as inhibitors of the substrates, products and enzymes of the pathway. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lehninger, A., D. Nelson, et al. (1993) Principles of Biochemistry. New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which ALASl functions, comprising: d) providing cells having one form of a gene in the heme biochemical and/or genetic pathway and providing comparison cells having a different form of said gene; e) contacting said cells and said comparison cells with a test compound; and f) determining the growth of said cells and said comparison cells in the presence of said test compound; wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

The use of multi'-well plates for screening is a format that readily accommodates multiple different assays to characterize various compounds, concentrations of compounds, and fungal strains in varying combinations and formats. Certain testing parameters for the screening method can significantly affect the identification of growth inhibitors, and thus can be manipulated to optimize screening efficiency and/or reliability. Notable among these factors are variable sensitivities of different mutants, increasing hypersensitivity with increasingly less permissive conditions, an apparent increase in hypersensitivity with increasing compound concentration, and other factors known to those in the art. Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lehninger, A., D. Nelson, et al. (1993) Principles of Biochemistry. New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which ALASl functions, comprising:

(a) providing paired growth media comprising a first medium and a second medium, wherein said second medium contains a higher level of 5-aminolevulinate than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism; wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic.

It is recognized in the art that determination of the growth of said organism in the paired media in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different media. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment, the organism is Magnaporthe grisea. ,

The present inventors have discovered that disruption of the HISPl gene and/or gene product inhibits the pathogenicity of Magnaporthe grisea. Thus, the inventors are the first to demonstrate that histidinol-phosphatase is a target for antibiotics, preferably antifungals.

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

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

The histidinol-phosphatase protein may have the amino acid sequence of a naturally occurring histidinol-phosphatase found in a fungus, animal, plant, or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence. Preferably the histidinol-phosphatase is a fungal histidinol-phosphatase. The cDNA (SEQ ID NO: 7) encoding the histidinol-phosphatase protein, the genomic DNA (SEQ ED NO: 8) encoding the M. grisea protein, and the polypeptide (SEQ ED NO: 9) can be found herein.

In one aspect, the invention also provides for a polypeptide consisting essentially of SEQ ED NO: 9. For the purposes of the invention, a polypeptide consisting essentially of SEQ ID NO: 9 has at least 80% sequence identity with SEQ ID NO: 9 and catalyses the interconversion of L-histidinol phosphate and H₂O with L-histidinol and orthophosphate with at least 10% of the activity of SEQ ED NO: 9. 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%o, most preferably the sequence identity is at least 95% or 97 or 99%, or any integer from 80- 100% sequence identity in ascending order. And, preferably, the polypeptide consisting essentially of SEQ ID NO: 9 has at least 25%, at least 50%, at least 75% or at least 90% of the activity of M. grisea histidinol-phosphatase, or any integer from 60-100% activity in ascending order. By "fungal histidinol-phosphatase" is meant an enzyme that can be found in at least one fungus, and which catalyzes the interconversion of L-histidinol phosphate and H₂O with L-histidinol and orthophosphate. The histidinol-phosphatase may be from any of the fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

In one embodiment, the histidinol-phosphatase is a Magnaporthe histidinol- phosphatase. Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae and the imperfect states of Magnaporthe in the genus Pyricularia. Preferably, the Magnaporthe histidinol-phosphatase is from Magnaporthe grisea.

In various embodiments, the histidinol-phosphatase can be from Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Corn Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and the like.

Fragments of a histidinol-phosphatase polypeptide may be used in the methods of the invention, preferably if the fragments include an intact or nearly intact epitope that occurs on the biologically active wildtype histidinol-phosphatase. The fragments comprise at least 10 consecutive amino acids of a histidinol-phosphatase. Preferably, the fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, or at least 330 consecutive amino acids residues of a histidinol-phosphatase. In one embodiment, the fragment is from a Magnaporthe histidinol-phosphatase. Preferably, the fragment contains an amino acid sequence conserved among fungal histidinol- phosphatases.

Polypeptides having at least 50% sequence identity with a 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 any integer from 60-100%) sequence identity in ascending order.

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

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

Any technique for detecting the binding of a ligand to its target may be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting the binding of a ligand to its target are known in the art, and include, but are not limited to the detection of an immobilized ligand-target complex or the detection of a change in the properties of a target when it is bound to a ligand. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligands are 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, bound histidinol-phosphatase is detected using a labeled binding partner, such as a labeled antibody. In a variation of this assay, histidinol-phosphatase is labeled prior to contacting the immobilized candidate ligands. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetric methods.

Once a compound is identified as a candidate for an antibiotic, it can be tested for the ability to inhibit histidinol-phosphatase enzymatic activity. The compounds can be tested using either in vitro or cell based assays. Alternatively, a compound can be tested by applying it directly to a fungus or fungal cell, or expressing it therein, and monitoring the fungus or fungal cell for changes or decreases in growth, development, viability, pathogenicity, or alterations in gene expression. Thus, in one embodiment, the invention provides a method for determining whether a compound identified as an antibiotic candidate by an above method has antifungal activity, further comprising: contacting a fungus or fungal cells with said antifungal candidate and detecting a decrease in the growth, viability, or pathogenicity of said fungus or fungal cells.

By decrease in growth, is meant that the antifungal candidate causes at least a 10%) decrease in the growth of the fungus or fungal cells, as compared to the growth of the fungus or fungal cells in the absence of the antifungal candidate. By a decrease in viability is meant that at least 20% of the fungal cells, or portion of the fungus contacted with the antifungal candidate are nonviable. Preferably, the growth or viability will be decreased by at least 40%>. More preferably, the growth or viability will be decreased 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. By decrease in pathogenicity, is meant that the antifungal candidate causes at least a 10%> decrease in the disease caused by contact of the fungal pathogen with its host, as compared to the disease caused in the absence of the antifungal candidate. Preferably, the disease will be decreased by at least 40%». More preferably, the disease will be decreased by at least 50%>, 75% or at least 90% or more. Methods for measuring fungal disease are well known to those skilled in the art, and include such metrics 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 in vitro enzymatic assays in which the disappearance of a substrate or the appearance of a product is directly or indirectly detected. Histidinol-phosphatase catalyzes the irreversible or reversible reaction L-histidinol phosphate and H₂O = L- histidinol and orthophosphate (see Figure 1). Methods for detection of L-histidinol phosphate, H₂O, L-histidinol, and/or orthophosphate, include spectrophotometry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.

Thus, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-histidinol phosphate and H₂O with a histidinol-phosphatase; b) contacting L-histidinol phosphate and H₂O with histidinol-phosphatase and said test compound; and c) determining the change in concentration for at least one of the following: L- histidinol phosphate, H₂O, L-histidinol, and/or orthophosphate, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-histidinol and orthophosphate with a histidinol-phosphatase; b) contacting L-histidinol and orthophosphate with a histidinol-phosphatase and a test compound; and c) determining the change in concentration for at least one of the following: L- histidinol phosphate, H₂O, L-histidinol, and/or orthophosphate, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

Enzymatically active fragments of a fungal histidinol-phosphatase are also useful in the methods of the invention. For example, an enzymatically active polypeptide comprising at least 100 consecutive amino acid residues of a fungal histidinol- phosphatase may be used in the methods of the invention. In addition, an enzymatically active polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity with a fungal histidinol-phosphatase may be used in the methods of the invention. Most preferably, the polypeptide has at least 50% sequence identity with a fungal histidinol-phosphatase and at least 10%, 25%, 75%> or at least 90% of the activity thereof.

Thus, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-histidinol phosphate and H₂O with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with a histidinol-phosphatase; a polypeptide having at least 50% sequence identity with a histidinol-phosphatase and having at least 10%> of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a histidinol- phosphatase; b) contacting L-histidinol phosphate and H₂O with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: L- histidinol phosphate, H₂O, L-histidinol, and/or orthophosphate. wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-histidinol and orthophosphate with a polypeptide selected from the group consisting of: a polypeptide having at least 50%> sequence identity with a histidinol-phosphatase; a polypeptide having at least 50% sequence identity with a histidinol-phosphatase and at least 10% of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a histidinol-phosphatase; b) contacting L-histidinol and orthophosphate, with said polypeptide and a test compound; and • c) determining the change in concentration for at least one of the following, L- histidinol phosphate, H₂O, L-histidinol, and/or orthophosphate; wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

For the in vitro enzymatic assays, histidinol-phosphatase protein and derivatives thereof may be purified from a fungus or may be recombinantly produced in and purified from an archael, bacterial, fungal, or other eukaryotic cell culture. Preferably these proteins are produced using an E. coli, yeast, or filamentous fungal expression system. Methods for the purification of histidinol-phosphatase may be described in Millay and Houston (1973) Biochemistry 12: 2591 - 2596 (PMID: 4351203). Other methods for the purification of histidinol-phosphatase proteins and polypeptides are known to those skilled in the art.

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

Expression of histidinol-phosphatase can be measured by detecting the HISPl primary transcript or mRNA, histidinol-phosphatase polypeptide, or histidinol- phosphatase enzymatic activity. Methods for detecting the expression of RNA and proteins are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al, eds., Greene Publishing and Wiley-friterscience, New York, 1995. The method of detection is not critical to the invention. Methods for detecting HISPl RNA include, but are not limited to amplification assays such as quantitative reverse transcriptase-PCR, and/or hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using a HISPl 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 elecfrophoresis, mass spectroscopy, and enzymatic assays. Also, any reporter gene system may be used to detect HISPl protein expression. For detection using gene reporter systems, a polynucleotide encoding a reporter protein is fused in frame with HISPl, so as to produce a chimeric polypeptide. Methods for using reporter systems are known to those skilled in the art.

Chemicals, compounds or compositions identified by the above methods as modulators, preferably inhibitors, of HISPl expression or activity can then be used to control fungal growth. Diseases such as rusts, mildews, and blights spread rapidly once established. Fungicides are thus routinely applied to growing and stored crops as a preventive measure, generally as foliar sprays or seed dressings. For example, compounds that inhibit fungal growth can be applied to a fungus or expressed in a fungus, in order to prevent fungal growth. Thus, the invention provides a method for inhibiting fungal growth, comprising contacting a fungus with a compound identified by the methods of the invention as having antifungal activity.

Antifungals and antifungal inhibitor candidates identified by the methods of the invention can be used to control the growth of undesired fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

Examples of undesired fungi include, but are not limited to Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Moniliniafructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Com Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), diseases of animals such as infections of lungs, blood, brain, skin, scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp. Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp., and the like).

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

In one particular embodiment, the method is performed by providing an organism having a first form of the gene corresponding to either SEQ ED NO: 7 or SEQ ID NO: 8, either a normal form, a mutant form, a homologue, or a heterologous HISPl gene that performs a similar function as HISPl. The first form of HISPl may or may not confer a growth conditional phenotype, i.e., a L-histidine requiring phenotype, and/or a hypersensitivity or hyposensitivity phenotype on the organism having that altered form. In one particular embodiment a mutant form contains a transposon insertion. A comparison organism having a second form of a HISPl, different from the first form of the gene is also provided, and the two organisms are separately contacted with a test compound. The growthof the two organisms in the presence of the test compound is then compared.

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a histidinol-phosphatase gene, and providing comparison cells having a different form of a histidinol-phosphatase gene; and b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and said comparison cells in the presence of the test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

It is recognized in the art that the optional determination of the growth of said first organism and said comparison second organism in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different genes. It is also recognized that any combination of two different forms of a HISPl gene, including normal genes, mutant genes, homologues, and functional homologues may be used in this method. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like, hi a preferred embodiment the organism is Magnaporthe grisea.

Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics as inhibitors of the substrates, products and enzymes of the pathway. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lehninger, A., D. Nelson, et al. (1993) Principles of Biochemistry. New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which HISPl functions, comprising: g) providing cells having one form of a gene in the L-histidine biochemical and/or genetic pathway and providing comparison cells having a different form of said gene, h) contacting said cells and said comparison cells with a test compound; and i) determining the growth of said cells and said comparison cells in the presence of said test compound; wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

The use of multi-well plates for screening is a format that readily accommodates multiple different assays to characterize various compounds, concentrations of compounds, and fungal strains in varying combinations and formats. Certain testing parameters for the screening method can significantly affect the identification of growth inhibitors, and thus can be manipulated to optimize screening efficiency and or reliability. Notable among these factors are variable sensitivities of different mutants, increasing hypersensitivity with increasingly less permissive conditions, an apparent increase in hypersensitivity with increasing compound concentration, and other factors known to those in the art.

Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lehninger, A., D. Nelson, et al. (1993) Principles of Biochemistry. New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which HISPl functions, comprising:

(a) providing paired growth media comprising a first medium and a second medium, wherein said second medium contains a higher level of L-histidine than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism; wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic. It is recognized in the art that determination of the growth of said organism in the paired media in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different media. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment, the organism is Magnaporthe grisea.

The present inventors have discovered that disruption of the IPMDl gene and/or gene product inhibits the pathogenicity of Magnaporthe grisea. Thus, the inventors are the first to demonstrate that 3-Isopropylmalate dehydratase is a target for antibiotics, preferably antifungals.

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

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a 3-Isopropylmalate dehydratase polypeptide with a test compound; and b) detecting the presence or absence of binding between said test compound and said 3-Isopropylmalate dehydratase polypeptide; wherein binding indicates that said test compound is a candidate for an antibiotic.

The 3-Isopropylmalate dehydratase protein may have the amino acid sequence of a naturally occurring 3-Isopropylmalate dehydratase found in a fungus, animal, plant, or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence. Preferably the 3-Isopropylmalate dehydratase is a fungal 3-Isopropylmalate dehydratase. The cDNA (SEQ ID NO: 10) encoding the 3-Isopropylmalate dehydratase protein, the genomic DNA (SEQ DD NO: 11) encoding the M. grisea protein, and the polypeptide (SEQ ID NO: 12) can be found herein. In one aspect, the invention also provides for a polypeptide consisting essentially of SEQ ID NO: 12. For the purposes of the invention, a polypeptide consisting essentially of SEQ ID NO: 12 has at least 80% sequence identity with SEQ DD NO: 12 and catalyses the interconversion of 2-Isopropylmalate and H₂O with 3-Isopropylmalate with at least 10% of the activity of SEQ ED NO: 12. Preferably, the polypeptide consisting essentially of SEQ ED NO: 12 has at least 85% sequence identity with SEQ ED NO: 12, more preferably the sequence identity is at least 90%, most preferably the sequence identity is at least 95%> or 97 or 99%, or any integer from 80-100% sequence identity in ascending order. And, preferably, the polypeptide consisting essentially of SEQ DD NO: 12 has at least 25%, at least 50%, at least 75% or at least 90% of the activity of M. grisea 3-Isopropyhnalate dehydratase, or any integer from 60-100%) acti ity in ascending order.

By "fungal 3-Isopropylmalate dehydratase" is meant an enzyme that can be found in at least one fungus, and which catalyzes the interconversion of 2-,Isopropylmalate and H₂O with 3-Isopropylmalate. The 3-Isopropylmalate dehydratase may be from any of the fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

In one embodiment, the 3-Isopropylmalate dehydratase is a Magnaporthe 3- Isopropylmalate dehydratase. Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae and the imperfect states of Magnaporthe in the genus Pyricularia. Preferably, the Magnaporthe 3-Isopropylmalate dehydratase is from Magnaporthe grisea.

In various embodiments, the 3-Isopropylmalate dehydratase can be from Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armϊllaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallicά), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Moniliniafructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Com Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solanϊ), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and the like.

Fragments of a 3-Isopropylmalate dehydratase polypeptide may be used in the methods of the invention, preferably if the fragments include an intact or nearly intact epitope that occurs on the biologically active wildtype 3-Isopropylmalate dehydratase. The fragments comprise at least 10 consecutive amino acids of a 3-Isopropylmalate dehydratase. Preferably, the fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 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, 760, or at least 770 consecutive amino acids residues of a 3-Isopropylmalate dehydratase. In one embodiment, the fragment is from a Magnaporthe 3-Isopropylmalate dehydratase. Preferably, the fragment contains an amino acid sequence conserved among fungal 3- Isopropylmalate dehydratases.

Polypeptides having at least 50% sequence identity with a 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%o or 90 or 95 or 99%, or any integer from 60-100% sequence identity in ascending order.

In addition, it is preferred that the polypeptide has at least 10% of the activity of a fungal 3-Isopropylmalate dehydratase. More preferably, the polypeptide has at least 25%, at least 50%, at least 75% or at least 90% of the activity of a fungal 3- Isopropylmalate dehydratase. Most preferably, the polypeptide has at least 10%, at least 25%, at least 50%, at least 75% or at least 90% of the activity of the M. grisea 3- Isopropylmalate dehydratase protein. Thus, in another embodiment, the invention provides a method for identifying a test compound as a candidate for a fungicide, comprising: a) contacting a test compound with at least one polypeptide selected from the group consisting of: a polypeptide having at least ten consecutive amino acids of a fungal 3-Isopropylmalate dehydratase; a polypeptide having at least 50% sequence identity with a fungal 3-Isopropylmalate dehydratase; and a polypeptide having at least 10% of the activity of a fungal 3-Isopropylmalate dehydratase; and b) detecting the presence and/or absence of binding between said test compound and said polypeptide; wherein binding indicates that said test compound is a candidate for an antibiotic.

Any technique for detecting the binding of a ligand to its target may be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting the binding of a ligand to its target are known in the art, and include, but are not limited to the detection of an immobilized ligand-target complex or the detection of a change in the properties of a target when it is bound to a ligand. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with a 3-Isopropylmalate dehydratase protein or a fragment or variant thereof, the unbound protein is removed and the bound 3- Isopropylmalate dehydratase is detected. In a preferred embodiment, bound 3- Isopropylmalate dehydratase is detected using a labeled binding partner, such as a labeled antibody. In a variation of this assay, 3-Isopropylmalate dehydratase is labeled prior to contacting the immobilized candidate ligands. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetric methods.

Once a compound is identified as a candidate for an antibiotic, it can be tested for the ability to inhibit 3-Isopropylmalate dehydratase enzymatic activity. The compounds can be tested using either in vitro or cell based assays. Alternatively, a compound can be tested by applying it directly to a fungus or fungal cell, or expressing it therein, and monitoring the fungus or fungal cell for changes or decreases in growth, development, viability, pathogenicity, or alterations in gene expression. Thus, in one embodiment, the invention provides a method for determining whether a compound identified as an antibiotic candidate by an above method has antifungal activity, further comprising: contacting a fungus or fungal cells with said antifungal candidate and detecting a decrease in the growth, viability, or pathogenicity of said fungus or fungal cells.

By decrease in growth, is meant that the antifungal candidate causes at least a 10% decrease in the growth of the fungus or fungal cells, as compared to the growth of the fungus or fungal cells in the absence of the antifungal candidate. By a decrease in viability is meant that at least 20% of the fungal cells, or portion of the fungus contacted with the antifungal candidate are nonviable. Preferably, the growth or viability will be decreased by at least 40%. More preferably, the growth or viability will be decreased 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. By decrease in pathogenicity, is meant that the antifungal candidate causes at least a 10% decrease in the disease caused by contact of the fungal pathogen with its host, as compared to the disease caused in the absence of the antifungal candidate. Preferably, the disease will be decreased by at least 40%. More preferably, the disease will be decreased by at least 50%, 75% or at least 90% or more. Methods for measuring fungal disease are well known to those skilled in the art, and include such metrics 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 in vitro enzymatic assays in which the disappearance of a substrate or the appearance of a product is directly or indirectly detected. 3-Isopropylmalate dehydratase catalyzes the irreversible or reversible reaction 2-Isopropylmalate and H₂O = 3-Isopropylmalate (see Figure 1). Methods for detection of 2-Isopropylmalate, H₂O, and/or 3-Isopropylmalate, include spectrophotometry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC. ι

Thus, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 2-Isopropylmalate and H₂O with a 3-Isopropylmalate dehydratase; b) contacting 2-Isopropylmalate and H₂O with 3-Isopropylmalate dehydratase and said test compound; and c) determining the change in concentration for at least one of the following: 2- Isopropylmalate, H₂O, and/or 3-Isopropylmalate, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 3-Isopropylmalate with a 3-Isopropylmalate dehydratase; b) contacting 3-Isopropylmalate with a 3-Isopropylmalate dehydratase and a test compound; and c) determining the change in concentration for at least one of the following: 2- Isopropylmalate, H₂O, and/or 3-Isopropylmalate, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

Enzymatically active fragments of a fungal 3-Isopropylmalate dehydratase are also useful in the methods of the invention. For example, an enzymatically active polypeptide comprising at least 100 consecutive amino acid residues of a fungal 3- Isopropylmalate dehydratase may be used in the methods of the invention. In addition, an enzymatically active polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity with a fungal 3-Isopropylmalate dehydratase maybe used in the methods of the invention. Most preferably, the polypeptide has at least 50% sequence identity with a fungal 3-Isopropylmalate dehydratase and at least 10%, 25%, 75% or at least 90% of the activity thereof.

Thus, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 2-Isopropylmalate and H₂O with a polypeptide selected from the group consisting of: a polypeptide having at least 50%> sequence identity with a 3- Isopropylmalate dehydratase; a polypeptide having at least 50% sequence identity with a 3-Isopropylmalate dehydratase and having at least 10% of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a 3- Isopropylmalate dehydratase; b) contacting 2-Isopropylmalate and H₂O with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: 2- Isopropylmalate, H₂O, and/or 3-Isopropylmalate; wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 3-Isopropylmalate with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with a 3-Isopropylmalate dehydratase; a polypeptide having at least 50%> sequence identity with a 3- Isopropylmalate dehydratase and at least 10% of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a 3- Isopropylmalate dehydratase; b) contacting 3-Isopropylmalate, with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following, 2- Isopropylmalate, H₂O, and/or 3-Isopropylmalate; wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

For the in vitro enzymatic assays, 3-Isopropylmalate dehydratase protein and derivatives thereof may be purified from a fungus or may be recombinantly produced in and purified from an archael, bacterial, fungal, or other eukaryotic cell culture. Preferably these proteins are produced using an E. coli, yeast, or filamentous fungal expression system. Methods for the purification of 3-Isopropylmalate dehydratase may be described in (Bigelis.and Umbarger (1975) J Biol Chem 250: 4315 - 21 (PMID: 1126953); Kohlhaw (1988) Meth Enzymol 166: 423 - 9 (PMDD: 3071717)). Other methods for the purification of 3-Isopropylmalate dehydratase proteins and polypeptides are known to those skilled in the art.

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

Expression of 3-Isopropylmalate dehydratase can be measured by detecting the IPMDl primary transcript or mRNA, 3-Isopropylmalate dehydratase polypeptide, or 3- Isopropylmalate dehydratase enzymatic activity. Methods for detecting the expression of RNA and proteins are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al, eds., Greene Publishing and Wiley- friterscience, New York,' 1995. The method of detection is not critical to the invention. Methods for detecting IPMDl RNA include, but are not limited to amplification assays such as quantitative reverse transcriptase-PCR, and/or hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using an IPMDl 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 elecfrophoresis, mass spectroscopy, and enzymatic assays. Also, any reporter gene system may be used to detect IPMDl protein expression. For detection using gene reporter systems, a polynucleotide encoding a reporter protein is fused in frame with EPMDl, so as to produce a chimeric polypeptide. Methods for using reporter systems are known to those skilled in the art. Chemicals, compounds or compositions identified by the above methods as modulators, preferably inhibitors, of EPMDl expression or activity can then be used to control fungal growth. Diseases such as rusts, mildews, and blights spread rapidly once established. Fungicides are thus routinely applied to growing and stored crops as a preventive measure, generally as foliar sprays or seed dressings. For example, compounds that inhibit fungal growth can be applied to a fungus or expressed in a fungus, in order to prevent fungal growth. Thus, the invention provides a method for inhibiting fungal growth, comprising contacting a fungus with a compound identified by the methods of the invention as having antifungal activity.

Antifungals and antifungal inhibitor candidates identified by the methods of the invention can be used to control the growth of undesired fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

Examples of undesired fungi include, but are not limited to Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Com Smut (Ustilago mdydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Moniliniafructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Corn Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solanϊ), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), diseases of animals such as infections of lungs, blood, brain, skin, scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp. Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp., and the like). Also provided is a method of screening for an antibiotic by determining whether a test compound is active against the gene identified (SEQ ED NO: 10 or SEQ ID NO: 11), its gene product (SEQ ED NO: 12), or the biochemical pathway or pathways it functions on.

In one particular embodiment, the method is performed by providing an organism having a first form of the gene corresponding to either SEQ ED NO: 10 or SEQ ED NO: 11, either a normal form, a mutant form, a homologue, or a heterologous EPMDl gene that performs a similar function as EPMDl. The first form of EPMDl may or may not confer a growth conditional phenotype, i.e., a L-leucine requiring phenotype, and/or a hypersensitivity or hyposensitivity phenotype on the organism having that altered form. In one particular embodiment a mutant form contains a transposon insertion. A comparison organism having a second form of an EPMDl, different from the first form of the gene is also provided, and the two organisms are separately contacted with a test compound. The growth of the two organisms in the presence of the test compound is then compared.

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a 3-Isopropylmalate dehydratase gene, and providing comparison cells having a different form of a 3-Isopropylmalate dehydratase gene; and b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and said comparison cells in the presence of the test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

It is recognized in the art that the optional determination of the growth of said first organism and said comparison second organism in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different genes. It is also recognized that any combination of two different forms of an EPMDl gene, including normal genes, mutant genes, homologues, and functional homologues may be used in this method. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment the organism is Magnaporthe grisea.

Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics as inhibitors of the substrates, products and enzymes of the pathway. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lehninger, A., D. Nelson, et al. (1993) Principles of Biochemistry. New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screemng for test compounds acting against the biochemical and/or genetic pathway or pathways in which EPMDl functions, comprising: j) providing cells having one form of a gene in the L-leucine biochemical and/or genetic pathway and providing comparison cells having a different form of said gene; k) contacting said cells and said comparison cells with a test compound; and 1) determining the growth of said cells and said comparison cells in the presence of said test compound; wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

The use of multi-well plates for screening is a format that readily accommodates multiple different assays to characterize various compounds, concentrations of compounds, and fungal strains in varying combinations and formats. Certain testing parameters for the screening method can significantly affect the identification of growth inhibitors, and thus can be manipulated to optimize screening efficiency and/or reliability. Notable among these factors are variable sensitivities of different mutants, increasing hypersensitivity with increasingly less permissive conditions, an apparent increase in hypersensitivity with increasing compound concentration, and other factors known to those in the art.

Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lehninger, A., D. Nelson, et al. (1993) Principles of Biochemistry. New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which EPMDl functions, comprising:

(a) providing paired growth media comprising a first medium and a second medium, wherein said second medium contains a higher level of L-leucine than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism; wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic.

It is recognized in the art that determination of the growth of said organism in fhe paired media in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different media. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment, the organism is Magnaporthe grisea.

The present inventors have discovered that disruption of the THR4 gene and/or gene product inhibits the pathogenicity of Magnaporthe grisea. Thus, the inventors are the first to demonstrate that Threonine synthase is a target for antibiotics, preferably antifungals. Accordingly, the invention provides methods for identifying compounds that inhibit THR4 gene expression or biological activity of its gene product(s). Such methods include ligand binding assays, assays for enzyme activity, cell-based assays, and assays for THR4 gene expression. Any compound that is a ligand for Threonine synthase may have antibiotic activity. For the purposes of the invention, "ligand" refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the invention are useful as antibiotics.

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a Threonine synthase polypeptide with a test compound; and b) detecting the presence or absence of binding between said test compound and said Threonine synthase polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

The Threonine synthase protein may have the amino acid sequence of a naturally occurring Threonine synthase found in a fungus, animal, plant, or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence. Preferably the Threonine synthase is a fungal Threonine synthase. The cDNA (SEQ DD NO: 13) encoding the Threonine synthase protein, the genomic DNA (SEQ DD NO: 14) encoding the M. grisea protein, and the polypeptide (SEQ ED NO: 15) can be found herein.

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

By "fungal Threonine synthase" is meant an enzyme that can be found in at least one fungus, and which catalyzes the interconversion of O-phospho-L-homoserine and water with L-threonine and orthophosphate. The Threonine synthase may be from any of the fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

In one embodiment, the Threonine synthase is a Magnaporthe Threonine synthase. Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae and the imperfect states of Magnaporthe in the genus Pyricularia. Preferably, the Magnaporthe Threonine synthase is from Magnaporthe grisea.

In various embodiments, the Threonine synthase can be from Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Com Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Com Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solan ), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and the like.

Fragments of a Threonine synthase polypeptide may be used in the methods of the invention, preferably if the fragments include an intact or nearly intact epitope that occurs on the biologically active wildtype Threonine synthase. The fragments comprise at least 10 consecutive amino acids of a Threonine synthase. Preferably, the fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 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, or at least 540 consecutive amino acids residues of a Threonine synthase. In one embodiment, the fragment is from a Magnaporthe Threonine synthase. Preferably, the fragment contains an amino acid sequence conserved among fungal Threonine synthases.

Polypeptides having at least 50%> sequence identity with a 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 any integer from 60-100% sequence identity in ascending order.

In addition, it is preferred that the polypeptide has at least 10% of the activity of a fungal Threonine synthase. More preferably, the polypeptide has at least 25%, at least 50%, at least 75% or at least 90% of the activity of a fungal Threonine synthase. Most preferably, the polypeptide has at least 10%, at least 25%, at least 50%, at least 75% or at least 90% of the activity of the M. grisea Threonine synthase protein.

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

Any technique for detecting the binding of a ligand to its target may be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting the binding of a ligand to its target are known in the art, and include, but are not limited to the detection of an immobilized ligand-target complex or the detection of a change in the properties of a target when it is bound to a ligand. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with a 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, bound Threonine synthase is detected using a labeled binding partner, such as a labeled antibody. In a variation of this assay, Threonine synthase is labeled prior to contacting the immobilized candidate ligands. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetric methods.

Once a compound is identified as a candidate for an antibiotic, it can be tested for the ability to inhibit Threonine synthase enzymatic activity. The compounds can be tested using either in vitro or cell based assays. Alternatively, a compound can be tested by applying it directly to a fungus or fungal cell, or expressing it therein, and monitoring the fungus or fungal cell for changes or decreases in growth, development, viability, pathogenicity, or alterations in gene expression. Thus, in one embodiment, the invention provides a method for determining whether a compound identified as an antibiotic candidate by an above method has antifungal activity, further comprising: contacting a fungus or fungal cells with said antifungal candidate and detecting a decrease in the growth, viability, or pathogenicity of said fungus or fungal cells.

By decrease in growth, is meant that the antifungal candidate causes at least a 10% decrease in the growth of the fungus or fungal cells, as compared to the growth of the fungus or fungal cells in the absence of the antifungal candidate. By a decrease in viability is meant that at least 20% of the fungal cells, or portion of the fungus contacted with the antifungal candidate are nonviable. Preferably, the growth or viability will be decreased by at least 40%. More preferably, the growth or viability will be decreased 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. By decrease in pathogenicity, is meant that the antifungal candidate causes at least a 10% decrease in the disease caused by contact of the fungal pathogen with its host, as compared to the disease caused in the absence of the antifungal candidate. Preferably, the disease will be decreased by at least 40%. More preferably, the disease will be decreased by at least 50%, 75% or at least 90% or more. Methods for measuring fungal disease are well known to those skilled in the art, and include such metrics 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 in vitro enzymatic assays in which the disappearance of a substrate or the appearance of a product is directly or indirectly detected. Threonine synthase catalyzes the irreversible or reversible reaction O-phospho-L-homoserine and water = L-threonine and orthophosphate (see Figure 1). Methods for detection of O-phospho-L-homoserine, L-threonine, orthophosphate, and water, include spectrophotomefry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.

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

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-threonine and orthophosphate with a Threonine synthase; b) contacting L-threonine and orthophosphate with a Threonine synthase and a test compound; and c) determining the change in concentration for at least one of the following: O- phospho-L-homoserine, L-threonine, orthophosphate, and water, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic. Enzymatically active fragments of a 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 a fungal Threonine synthase may be used in the methods of the invention. In addition, an enzymatically active polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity with a fungal Threonine synthase may be used in the methods of the invention. Most preferably, the polypeptide has at least 50% sequence identity with a fungal Threonine synthase and at least 10%, 25%, 75% or at least 90%> of the activity thereof.

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

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-threonine and orthophosphate with a polypeptide selected from the group consisting of: a polypeptide having at least 50%) sequence identity with a Threonine synthase, and a polypeptide having at least 50% sequence identity with a Threonine synthase and at least 10%> of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a Threonine synthase; b) contacting L-threonine and orthophosphate, with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following, O- phospho-L-homoserine, L-threonine, orthophosphate, and water, wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

For the in vitro enzymatic assays, Threonine synthase protein and derivatives thereof may be purified from a fungus or may be recombinantly produced in and purified from an archael, bacterial, fungal, or other eukaryotic cell culture. Preferably these proteins are produced using an E. coli, yeast, or filamentous fungal expression system. Methods for the purification of Threonine synthase may be described in Malumbres et al. (1994) Appl Environ Microbiol 60: 2209 - 19 (PMED: 8074505). Other methods for the purification of Threonine synthase proteins and polypeptides are known to those skilled in the art.

As an alternative to in vitro assays, the invention also provides cell based assays. In one embodiment, theάnvention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) measuring the expression of a Threonine synthase in a cell, cells, tissue, or an organism in the absence of a test compound; b) contacting said cell, cells, tissue, or organism with said test compound and measuring the expression of said Threonine synthase in said cell, cells, tissue, or organism; and c) comparing the expression of Threonine synthase in steps (a) and (b), wherein a lower expression in the presence of said test compound indicates that said compound is a candidate for an antibiotic.

Expression of Threonine synthase can be measured by detecting the THR4 primary transcript or mRNA, Threonine synthase polypeptide, or Threonine synthase enzymatic activity. Methods for detecting the expression of RNA and proteins are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al, eds., Greene Publishing and Wiley-Interscience, New York, 1995. The method of detection is not critical to the invention. Methods for detecting THR4 RNA include, but are not limited to amplification assays such as quantitative reverse franscriptase-PCR, and/or hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using a 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 elecfrophoresis, mass spectroscopy, and enzymatic assays. Also, any reporter gene system may be used to detect THR4 protein expression. For detection using gene reporter systems, a polynucleotide encoding a reporter protein is fused in frame with THR4, so as to produce a chimeric polypeptide. Methods for using reporter systems are known to those skilled in the art.

Chemicals, compounds or compositions identified by the above methods as modulators, preferably inhibitors, of THR4 expression or activity can then be used to control fungal growth. Diseases such as rusts, mildews, and blights spread rapidly once established. Fungicides are thus routinely applied to growing and stored crops as a preventive measure, generally as foliar sprays or seed dressings. For example, compounds that inhibit fungal growth can be applied to a fungus or expressed in a fungus, in order to prevent fungal growth. Thus, the invention provides a method for inhibiting fungal growth, comprising contacting a fungus with a compound identified by the methods of the invention as having antifungal activity.

Antifungals and antifungal inhibitor candidates identified by the methods of the invention can be used to control the growth of undesired fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

Examples of undesired fungi include, but are not limited to Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Com Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Moniliniafructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Com Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), diseases of animals such as infections of lungs, blood, brain, skin, scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp. Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp., and the like).

Also provided is a method of screening for an antibiotic by determining whether a test compound is active against the gene identified (SEQ ED NO: 13 or SEQ ID NO: 14), its gene product (SEQ ED NO: 15), or the biochemical pathway or pathways on which it functions.

In one particular embodiment, the method is performed by providing an organism having a first form of the gene corresponding to either SEQ ED NO: 13 or SEQ DD NO: 14, either a normal form, a mutant form, a homologue, or a heterologous THR4 gene that performs a similar function as THR4. The first form of THR4 may or may not confer a growth conditional phenotype, i.e., a L-threonine requiring phenotype, and/or a hypersensitivity or hyposensitivity phenotype on the organism having that altered form. In one particular embodiment a mutant form contains a transposon insertion. A comparison organism having a second form of a THR4, different from the first form of the gene is also provided, and the two organisms are separately contacted with a test compound. The growth of the two organisms in the presence of the test compound is then compared.

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a Threonine synthase gene, and providing comparison cells having a different form of a Threonine synthase gene; and b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and said comparison cells in the presence of the test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

It is recognized in the art that the optional determination of the growth of said first organism and said comparison second organism in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different genes. It is also recognized that any combination of two different forms of a THR4 gene, including normal genes, mutant genes, homologues, and functional homologues may be used in this method. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment the organism is Magnaporthe grisea.

Conditional lethal mutants may identify particular biochemical and or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics as inhibitors of the substrates, products and enzymes of the pathway. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lehninger, A., D. Nelson, et al. (1993) Principles of Biochemistry, New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which THR4 functions, comprising: m) providing cells having one form of a gene in the L-threonine biochemical and/or genetic pathway and providing comparison cells having a different form of said gene; n) contacting said cells and said comparison cells with a test compound; and o) determining the growth of said cells and said comparison cells in the presence of said test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

The use of multi-well plates for screening is a format that readily accommodates multiple different assays to characterize various compounds, concentrations of compounds, and fungal strains in varying combinations and formats. Certain testing parameters for the screening method can significantly affect the identification of growth inhibitors, and thus can be manipulated to optimize screening efficiency and/or reliability. Notable among these factors are variable sensitivities of different mutants, increasing hypersensitivity with increasingly less permissive conditions, an apparent increase in hypersensitivity with increasing compound concentration, and other factors known to those in the art.

I Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway.

Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics. Pathways known in the art may be found at the Kyoto Encyclopedia of

Genes and Genomes and in standard biochemistry texts (Lehninger et al. (1993)

Principles of Biochemistry).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which

THR4 functions, comprising:

(a) providing paired growth media comprising a first medium and a second medium, wherein said second medium contains a higher level of L-threonine than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism, wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic. It is recognized in the art that determination of the growth of said organism in the paired media in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different media. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment, the organism is Magnaporthe grisea.

EXPERIMENTAL

Example 1 Construction of Plasmids with a Transposon Containing a Selectable Marker. Construction of Sif transposon: Sif was constructed using the GPS3 vector from the GPS-M mutagenesis system from New England Biolabs, Inc. (Beverly, MA) as a backbone. This system is based on the bacterial transposon Tn7. The following manipulations were done to GPS3 according to Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press. The kanamycin resistance gene (npt) contained between the Tn7 arms was removed by EcoRV digestion. The bacterial hygromycin B phosphotransferase (hph) gene (Gritz and Davies (1983) Gene 25: 179 - 88 (PMED: 6319235)) under control of the Aspergillus nidulans trpC promoter and terminator (Mullaney et al. (1985) Mol Gen Genet 199: 37 - 45 (PM D: 3158796)) was cloned by a Hpal/EcoRV blunt ligation into the Tn7 arms of the GPS3 vector yielding pSifl . Excision of the ampicillin resistance gene (bla) from pSifl was achieved by cutting pSifl with Xmnl and Bgll followed by a T4 DNA polymerase treatment to remove the 3' overhangs left by the Bgll digestion and religation of the plasmid to yield pSifi Top 10F' electrocompetent E. coli cells (Invitrogen) were transformed with ligation mixture according to manufacturer's recommendations. Transformants containing the Sif transposon were selected on LB agar (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual) containing 50ug/ml of hygromycin B (Sigma Chem. Co., St. Louis, MO). Example 2 Construction of a Fungal Cosmid Library Cosmid libraries were constructed in the pcosKA5 vector (Hamer et al. (2001) Proc Natl Acad Sci USA PS: 5110 - 15 (PMED: 11296265)) as described in Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual. Cosmid libraries were quality checked by pulsed-field gel elecfrophoresis, restriction digestion analysis, and PCR identification of single genes.

Example 3 Construction ofCosmids with Transposon Insertion into Fungal Genes Sif Transposition into a Cosmid: Transposition of Sif into the cosmid framework was carried out as described by the GPS-M mutagenesis system (New England Biolabs, Inc.). Briefly, 2ul of the 10X GPS buffer, 70 ng of supercoiled pS F, 8-12 ug of target cosmid DNA were mixed and taken to a final volume of 20ul with water, lul of transposase (TnsABC) was added to the assembly reaction and incubated for 10 minutes at 37°C. After the assembly reaction, lul of start solution was added to the tube, mixed well and incubated for 1 hour at 37°C followed by heat inactivation of the proteins at 75°C for 10 min. Destruction of the remaining untransposed pSif was done by PIScel digestion at 37°C for 2 hours followed by 10 min incubation at 75°C to inactivate the proteins. Transformation of Topi OF' electrocompetent cells (Invitrogen) was done according to manufacturers recommendations. Sif-containing cosmid transformants were selected by growth on LB agar plates containing 50ug/ml of hygromycin B (Sigma Chem. Co.) and 100 ug/ml of Ampicillin (Sigma Chem. Co.).

Example 4

High Throughput Preparation and Verification of Transposon Insertion into the M. grisea ASNl Gene

E. coli strains containing cosmids with transposon insertions were picked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB (Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/ml of ampicillin. Blocks were incubated with shaking at 37°C overnight. 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 (PMΕD: 9371743)). DNA quality was checked by elecfrophoresis on agarose gels. Cosmids were sequenced using primers from the ends of each transposon and commercial dideoxy sequencing kits (Big Dye Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNA sequencer (Perkin Elmer Co.).

DNA sequences adjacent to the site of the insertion were collected and used to search DNA and protein databases using the BLAST algorithms (Altschul et al. (1997) Nucleic Acids Res 25: 3389 - 3402 (PMED: 9254694)). A single insertion of SEF into the Magnaporthe grisea ASNl gene was chosen for further analysis. This construct was designated cpgmraOOl 1008al0 and it contains the SEF transposon approximately between amino acids 345 and 346 relative to the Saccharomyces cerevisiae homologue (total length: 572 amino acids, GENBANK: 6325403).

Example 5 Preparation of ASNl Cosmid DNA and Transformation of Magnaporthe grisea Cosmid DNA from the ASNl transposon tagged cosmid clone was prepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-PspI (New England Biolabs, Inc.). Fungal electro-transformation was performed essentially as described (Wu et al. (1997) MPMI 10: 700 - 708). Briefly, M. grisea strain Guy 11 was grown in complete liquid media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) shaking at 120 φm for 3 days at 25°C in the dark. Mycelia was harvested and washed with sterile H₂O and digested with 4 mg/ml beta-glucanase (EnterSpex) for 4-6 hours to generate protoplasts. Protoplasts, were collected by centrifugation and resuspended in 20% sucrose at the concentration of 2x10⁸ protoplasts/ml. 50ul protoplast suspension was mixed with 10-20ug of the cosmid DNA and pulsed using Gene Pulser II (BioRad) set with the following parameters: resistance 200 ohm, capacitance 25uF, voltage 0.6kV. Transformed protoplasts were regenerated in complete agar media (CM, Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) with the addition of 20% sucrose for one day, then overlayed with CM agar media containing hygromycin B (250ug/ml) to select transformants. Transformants were screened for homologous recombination events in the target gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110 - 15 (PMED: 11296265)). Two independent strains were identified and are hereby referred to as KOI -2 and KOI -8, respectively.

Example 6 Effect of Transposon Insertion on Magnaporthe pathogenicity The target fungal strains, KO1-2 and KO1-8, obtained in Example 5 and the wild type strain, Guyl 1, were subjected to a pathogenicity assay to observe infection over a 1- week period. Rice infection assays were performed using Indian rice cultivar CO39 essentially as described in Valent et al. ((1991) Genetics 127: 87 - 101 (PMED: 2016048)). All three strains were grown for spore production on complete agar media. Spores were harvested and the concentration of spores adjusted for whole plant inoculations. Two-week-old seedlings of cultivar CO39 were sprayed with 12 ml of conidial suspension (5 x 10⁴ conidia per ml in 0.01%> Tween-20 (Polyoxyethylensorbitan monolaureate) solution). The inoculated plants were incubated in a dew chamber at 27°C in the dark for 36 hours, and transferred to a growth chamber (27 °C 12 hours/21 °C 12 hours 70% humidity) for an additional 5.5 days. Leaf samples were taken at 3, 5, and 7 days post-inoculation and examined for signs of successful infection (i.e. lesions). Figure 2 shows the effects of ASNl gene disruption on Magnaporthe infection at five days post- inoculation.

Example 7 Verification of ASNl Gene Function by Analysis of Nutritional Requirements The fungal strains, KO1-2 and KO1-8, containing the ASNl disrupted gene obtained in Example 5 were analyzed for their nutritional requirement for L-asparagine using the PM5 phenotype microarray from Biolog, Inc. (Hayward, CA). The PM5 plate tests for the auxotrophic requirement for 94 different metabolites. The innoculating fluid consists of 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5mM NaNO₃, 6.7mM KC1, 3.5mM Na₂SO₄, llmM KH₂PO₄, 0.01% -iodonitrotetrazolium violet, O.lmM MgCl₂, l.OmM CaCl₂ and trace elements, pH adjusted to 6.0 with NaOH. Final concentrations of trace elements are: 7.6μM ZnCl₂, 2.5μM MnCl₂'4H₂O, 1.8μM FeCl₂'4H₂O, 0.71μM CoCl₂6H₂O, 0.64μM CuCl₂2H₂O, 0.62μM Na₂MoO₄, 18μM H BO₃. Spores for each strain were harvested into the inoculating fluid. The spore concentrations were adjusted to 2xl0⁵ spores/ml. lOOμl of spore suspension were deposited into each well of the microtiter plates. The plates were incubated at 25°C for 7 days. Optical density (OD) measurements at 490nm and 750nm were taken daily. The OD ₉₀ measures the extent of tetrazolium dye reduction and the level of growth, and OD₇5o measures growth only. Turbidity = OD₄₉₀ + OD₇₅₀. Data confirming the annotated gene function is presented as a graph of Turbidity vs. Time showing both the mutant fungi and the wild-type control in the absence (Figure 3 A) and presence (Figure 3B) of L-asparagine.

Example 8 Cloning and Expression Strategies, Extraction and Purification of Asparagine Synthase

Protein. The following protocol may be employed to obtain a purified Asparagine Synthase protein.

Cloning and expression strategies:

An ASNl cDNA gene can be cloned into E. coli (pET vectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing His/fusion protein tags, and the expression of recombinant protein can be evaluated by SDS-PAGE and Western blot analysis.

Extraction:

Extract recombinant protein from 250 ml cell pellet in 3 ml of extraction buffer by sonicating 6 times, with 6 sec pulses at 4°C. Centrifuge extract at 15000xg for 10 min and collect supernatant. Assess biological activity of the recombinant protein by activity assay.

Purification:

Purify recombinant protein by Ni-NTA affinity chromatography (Qiagen). Purification protocol: perform all steps at 4°C:

• Use 3 ml Ni-beads (Qiagen)

• Equilibrate column with the buffer

• Load protein extract

• Wash with the equilibration buffer

• Elute bound protein with 0.5 M imidazole

Example 9 Assays for Testing Binding of Test Compounds to Asparagine Synthase The following protocol may be employed to identify test compounds that bind to the Asparagine Synthase protein.

• Purified full-length Asparagine Synthase polypeptide with a His/fusion protein tag (Example 8) is bound to a HisGrab™ Nickel Coated Plate (Pierce, Rockford, IL) following manufacturer's instructions.

• Buffer conditions are optimized (e.g. ionic strength or pH, as may be described in Luehr and Schuster (1980) J Biochem Biophys Methods 3: 151 - 61 (PMED: 6108975)) for binding of radiolabeled L-[4-¹⁴C]aspartate (Dealing and Walker (1960) Nature 185: 690 - 691) to the bound Asparagine Synthase.

• Screening of test compounds is performed by adding test compound and L-[4- ¹⁴C]aspartate (Dealing and Walker (1960) Nature 755: 690 - 691) to the wells of the HisGrab™ plate containing bound Asparagine Synthase.

• The wells are washed to remove excess labeled ligand and scintillation fluid (Scintiverse®, Fisher Scientific) is added to each well.

• The plates are read in a microplate scintillation counter.

• Candidate compounds are identified as wells with lower radioactivity as compared to control wells with no test compound added.

Additionally, a purified polypeptide comprising 10-50 amino acids from the M. grisea Asparagine Synthase is screened in the same way. A polypeptide comprising 10- 50 amino acids is generated by subcloning a portion of the ASNl 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 ASNl gene using the polymerase chain reaction amplification method. The DNA fragment encoding a polypeptide of 10-50 amino acids is cloned into an expression vector, expressed in a host organism and purified as described in Example 8 above.

Test compounds that bind ASNl are further tested for antibiotic activity. M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)). Spores are harvested into minimal media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) to a concentration of 2 x 10⁵ spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 μg/ml. Solvent only is added to the second culture. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The growth curves of the solvent control sample and the test compound sample are compared. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the confrol culture.

Example 10 Assays for Testing Inhibitors or Candidates for Inhibition of Asparagine Synthase

Activity

The enzymatic activity of Asparagine Synthase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Luehr and Schuster (1980) J Biochem Biophys Methods 3: 151 - 61 (PMED: 6108975). Candidate compounds are identified when a decrease in products or a lack of decrease in substrates is detected with the reaction proceeding in either direction.

Additionally, the enzymatic activity of a polypeptide comprising 10-50 amino acids from the M. grisea Asparagine Synthase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Luehr and Schuster (1980) J Biochem Biophys Methods 3: 151 - 61 (PMED: 6108975). A polypeptide comprising 10-50 amino acids is generated by subcloning a portion of the ASNl 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 ASNl gene using polymerase chain reaction amplification method. The DNA fragment encoding a polypeptide of 10-50 amino acids is cloned into an expression vector, expressed and purified as described in Example 8 above.

Test compounds identified as inhibitors of ASNl activity are further tested for antibiotic activity. Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMID: 8312740)). Spores are harvested into minimal media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) to a concentration of 2 x 10⁵ spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 μg/ml. Solvent only is added to the second culture. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The growth curves of the solvent control sample and the test compound sample are compared. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the control culture.

Example 11 Assays for Testing Compounds for Alteration of Asparagine Synthase Gene Expression Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. Wild-type M. grisea spores are harvested from cultures grown on complete agar or oatmeal agar media after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. 25 ml cultures are prepared to which test compounds will be added at various concentrations. A culture with no test compound present is included as a control. The cultures are incubated at 25°C for 3 days after which test compound or solvent only control is added. The cultures are incubated an additional 18 hours. Fungal mycelia is harvested by filtration through Miracloth (CalBiochem®, La Jolla, CA), washed with water and frozen in liquid nitrogen. Total RNA is extracted with TRIZOL® Reagent using the methods provided by the manufacturer (Life Technologies, Rockville, MD). Expression is analyzed by Northern analysis of the RNA samples as described (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press) using a radiolabeled fragment of the ASNl gene as a probe. Test compounds resulting in a reduced level of ASNl mRNA relative to the untreated control sample are identified as candidate antibiotic compounds.

Example 12 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of Asparagine Synthase with No Activity Magnaporthe grisea fungal cells containing a mutant form of the ASNl gene which abolishes enzyme activity, such as a gene containing a transposon insertion (see Examples 4 and 5), are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-asparagine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium containing 100 μM L-asparagine to a concentration of 2xl0⁵ spores per ml. Approximately 4xl0⁴ spores are added to each well of 96-well plates to which a test compound is added (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉₀ (fungal strain plus test compound) / ODs ₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177 - 221 (PMED: 7749303)).

Example 13 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of Asparagine Synthase with Reduced Activity Magnaporthe grisea fungal cells containing a mutant form of the ASNl gene, such as a promoter truncation that reduces expression, are grown under standard fungal growth conditions that are well known and described in the art. A promoter truncation is made by deleting a portion of the promoter upstream of the transcription start site using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-asparagine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores are added to each well of 96-well plates to which a test compound is added (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉o (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild- type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 11 - 221). Example 14 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of a L-asparagine Biosynthetic Gene with No Activity Magnaporthe grisea fungal cells containing a mutant form of a gene in the L- asparagine biosynthetic pathway (e.g. Formiminoaspartate deiminase (E.C. 3.5.3.5)) are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-asparagine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium containing 100 μM L-asparagine to a concentration of 2x10⁵ spores per ml. Approximately 4 l0⁴ spores or cells are harvested and added to each well of 96-well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉₀ (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild- type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221).

Example 15 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of a L-asparagine Biosynthetic Gene with Reduced Activity Magnaporthe grisea fungal cells containing a mutant form of a gene in the L- asparagine biosynthetic pathway (e.g. Formiminoaspartate deiminase (E.C. 3.5.3.5)), such as a promoter truncation that reduces expression, are grown under standard fungal growth conditions that are well known and described in the art. A promoter truncation is made by deleting a portion of the promoter upstream of the transcription start site using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea fungal cells containing a mutant form of are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-asparagine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96-well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OU₅₉o (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177 - 221).

Example 16 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Fungal ASNl and a Second Fungal Strain Containing a Heterologous ASNl Gene Wild-type Magnaporthe grisea fungal cells and M. grisea fungal cells lacking a functional ASNl gene and containing an asparagine synthetase B gene from Vibrio cholerae (Genbank 11272666, 50% sequence identity) are grown under standard fungal growth conditions that are well known and described in the art. A M. grisea strain carrying a heterologous ASNl gene is made as follows:

• A grisea strain is made with a nonfunctional ASNl gene, such as one containing a transposon insertion in the native gene (see Examples 4 and 5).

• A construct containing a heterologous ASNl gene is made by cloning the asparagine synthetase B gene from Vibrio cholerae into a fungal expression vector containing a trpC promoter and terminator (e.g. pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41: 22) using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual).

• The said construct is used to transform the M. grisea strain lacking a functional ASNl gene (see Example 5). Transformants are selected on minimal agar medium lacking L-asparagine. Only transformants carrying a functional ASNl gene will grow. Wild-type strains of Magnaporthe grisea and strains containing a heterologous form of ASNl are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96- well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The effect of each compound on the wild-type and heterologous fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD590 (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on the wild-type and heterologous fungal strains are compared. Compounds that show differential growth inhibition between the wild-type and heterologous strains are identified as potential antifungal compounds with specificity to the native or heterologous ASNl gene products. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221).

Example 17 Pathway Specific In Vivo Assay Screening Protocol Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. Wild-type M. grisea spores are harvested from cultures grown on oatmeal agar media after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemocytometer and spore suspensions are prepared in «v minimal growth medium and a mimmal growth medium containing 4 mM L-asparagine (Sigma-Aldrich Co.) to a concentration of 2x10⁵ spores per ml. The minimal growth media contains carbon, nitrogen, phosphate, and sulfate sources, and magnesium, calcium, and trace elements (for example, see innoculating fluid in Example 7). Spore suspensions are added to each well of a 96-well microtiter plate (approximately 4xl0⁴ spores/well). For each well containing a spore suspension in minimal media, an additional well is present containing a spore suspension in minimal medium containing 4 mM L-asparagine. Test compounds are added to wells containing spores in minimal media and minimal media containing L- asparagine. The total volume in each well is 200μl. Both minimal media and L- asparagine containing media wells with no test compound are provided as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. A compound is identified as a candidate for an antibiotic acting against the L-asparagine biosynthetic pathway when the observed growth in the well containing minimal media is less than the observed growth in the well containing L-asparagine as a result of the addition of the test compound. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 11 - 221).

Example 18 High Throughput Preparation and Verification of Transposon Insertion into the M. grisea ALASl Gene

E. coli strains containing cosmids with transposon insertions were picked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB (Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/ml of ampicillin. Blocks were incubated with shaking at 37 C overnight. 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 (PMDD: 9371743)). DNA quality was checked by elecfrophoresis on agarose gels. Cosmids were sequenced using primers from the ends of each transposon and commercial dideoxy sequencing kits (Big Dye Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNA sequencer (Perkin Elmer Co.).

DNA sequences adjacent to the site of the insertion were collected and used to search DNA and protein databases using the BLAST algorithms (Altschul et al (1997) Nucleic Acids Res 25: 3389 - 3402 (PMED: 9254694)). A single insertion of SEF into the Magnaporthe grisea ALASl gene was chosen for further analysis. This construct was designated cpgmraOOl 1005e01 and it contains the SEF transposon at approximately amino acid 100 relative to the Aspergillus nidulans homologue HEMA (total length: 648 amino acids, GENBANK: 585244 (SWISS-PROT: P38092)).

Example 19 Preparation of ALASl Cosmid DNA and Transformation of Magnaporthe grisea Cosmid DNA from the ALAS 1 transposon tagged cosmid clone was prepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-PspI (New England Biolabs, Inc.). Fungal electro-transformation was performed essentially as described (Wu et al. (1997) MPMI 10: 700 - 708). Briefly, M. grisea strain Guy 11 was grown in complete liquid media (Talbot et al. (1993) Plant Cell J: 1575 - 1590 (PMED: 8312740)) shaking at 120 rpm for 3 days at 25°C in the dark. Mycelia was harvested and washed with sterile H₂O and digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6 hours to generate protoplasts. Protoplasts were collected by centrifugation and resuspended in 20% sucrose at the concentration of 2xl0⁸ protoplasts/ml. 50ul protoplast suspension was mixed with 10-20ug of the cosmid DNA and pulsed using Gene Pulser II (BioRad) set with the following parameters: resistance 200 ohm, capacitance 25uF, voltage 0.6kV. Transformed protoplasts were regenerated in complete agar media (CM, Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) with the addition of 20% sucrose and 200 μM 5-aminolevulinic acid (Sigma-Aldritch Co.) for one day, then overlayed with CM agar media containing hygromycin B (250ug ml) to select transformants. Transformants were screened for homologous recombination events in the target gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110 - 15 (PMED: 11296265)). Two independent strains were identified and are hereby referred to as KOl-1 and KO1- 106, respectively.

Example 20 Effect of Transposon Insertion on Magnaporthe pathogenicity The target fungal strains, KOl-1 and KOI -106, obtained in Example 19 and the wild type strain, Guyl 1, were subjected to a pathogenicity assay to observe infection over a 1-week period. Rice infection assays were performed using Indian rice cultivar 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 media. Spores were harvested and the concentration of spores adjusted for whole plant inoculations. Two-week-old seedlings of cultivar CO39 were sprayed with 12 ml of conidial suspension (5 x 10⁴ conidia per ml in 0.01%> Tween-20 (Polyoxyethylensorbitan monolaureate) solution). The inoculated plants were incubated in a dew chamber at 27°C in the dark for 36 hours, and transferred to a growth chamber (27 °C 12 hours/21 °C 12 hours 70% humidity) for an additional 5.5 days. Leaf samples were taken at 3, 5, and 7 days post-inoculation and examined for signs of successful infection (i.e. lesions). Figure 2 shows the effects of ALASl gene disruption on Magnaporthe infection at five days post-inoculation. Example 21 Verification of ALASl Gene Function by Analysis of Nutritional Requirements The fungal strains, KOl-1 and KOI -106, containing the ALASl disrupted gene obtained in Example 19 were analyzed for their nutritional requirement for 5- aminolevulinic acid using the PM5 phenotype microarray from Biolog, Inc. (Hayward, CA). The PM5 plate tests for the auxotrophic requirement for 94 different metabolites. The innoculating fluid consists of 0.05% Phytagel, 0.03% Pluronic F68, 1%> glucose, 23.5mM NaNO₃, 6.7mM KC1, 3.5mM Na₂SO₄, 1 ImM KH₂PO₄, 0.01% p- iodomtrotetrazolium violet, O.lmM MgCl₂, LOmM CaCl and trace elements. Final concentrations of trace elements are: 7.6μM ZnCl₂, 2.5μM MnCl₂ ^'4H₂O, 1.8μM FeCl₂4H₂O, 0.7 lμM CoCl₂6H₂O, 0.64μM CuCl₂2H₂O, 0.62μM Na₂MoO₄, 18μM H₃BO . pH adjusted to 6.0 with NaOH. Spores for each strain were harvested into the inoculating fluid. The spore concentrations were adjusted to 2xl0⁵ spores/ml. lOOμl of spore suspension were deposited into each well of the microtiter plates. The plates were incubated at 25°C for 7 days. Optical density (OD) measurements at 490nm and 750nm were taken daily. The OD₄₉₀ measures the extent of tetrazolium dye reduction and the level of growth, and OD₇₅o measures growth only. Turbidity = OD₄₉₀ + OD-₇₅₀. Data confirming the annotated gene function is presented in Figure 3, and as a graph of Turbidity vs. Time showing both the mutant fungi and the wild-type control in the absence (Figure 3 A) and presence (Figure 3B) of 5-aminolevulinate.

Example 22 Cloning and Expression Strategies, Extraction and Purification of 5-Aminolevulinate

Synthase Protein. The following protocol may be employed to obtain a purified 5-Aminolevulinate synthase protein.

Cloning and expression strategies:

An ALASl cDNA gene can be cloned into E. coli (pET vectors-No vagen), Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing His/fusion protein tags, and the expression of recombinant protein can be evaluated by SDS-PAGE and Western blot analysis. Extraction:

Extract recombinant protein from 250 ml cell pellet in 3 ml of extraction buffer by sonicating 6 times, with 6 sec pulses at 4°C. Centrifuge extract at 15000xg for

10 min and collect supernatant. Assess biological activity of the recombinant protein by activity assay.

Purification:

Purify recombinant protein by Ni-NTA affinity chromatography (Qiagen).

Purification protocol: perform all steps at 4°C:

• Use 3 ml Ni-beads (Qiagen)

• Equilibrate column with the buffer

• Load protein extract

• Wash with the equilibration buffer

• Elute bound protein with 0.5 M imidazole

Example 23 Assays for Testing Binding of Test Compounds to 5-Aminolevulinate Synthase The following protocol may be employed to identify test compounds that bind to the 5-Aminolevulinate synthase protein.

• Purified full-length 5-Aminolevulinate synthase polypeptide with a His/fusion protein tag (Example 22) is bound to a HisGrab™ Nickel Coated Plate (Pierce, Rockford, IL) following manufacturer's instructions.

• Buffer conditions are optimized (e.g. ionic strength or pH, Shoolingin- Jordan et al. (1997) Methods Enzymol 281: 309 - 16 (PMDD: 9250995)) for binding of radiolabeled succinyl-CoA (custom made, PerkinElmer Life Sciences, Inc., Boston, MA) to the bound 5-Aminolevulinate synthase.

• Screening of test compounds is performed by adding test compound and radiolabeled succinyl-CoA (custom made, PerkinElmer Life Sciences, Inc., Boston, MA) to the wells of the HisGrab™ plate containing bound 5- Aminolevulinate synthase.

• The wells are washed to remove excess labeled ligand and scintillation fluid (Scintiverse®, Fisher Scientific) is added to each well. • The plates are read in a microplate scintillation counter.

• Candidate compounds are identified as wells with lower radioactivity as compared to control wells with no test compound added.

Additionally, a purified polypeptide comprising 10-50 amino acids from the M. grisea 5-Aminolevulinate synthase is screened in the same way. A polypeptide comprising 10-50 amino acids is generated by subcloning a portion of the ALASl 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 ALASl gene using the polymerase chain reaction amplification method. The DNA fragment encoding a polypeptide of 10-50 amino acids is cloned into an expression vector, expressed in a host organism and purified as described in Example 22 above.

Test compounds that bind ALASl are further tested for antibiotic activity. M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)). Spores are harvested into minimal media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMDD: 8312740)) to a concentration of 2 x 10⁵ spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 μg/ml. Solvent only is added to the second culture. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The growth curves of the solvent control sample and the test compound sample are compared. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the confrol culture.

Example 24 Assays for Testing Inhibitors or Candidates for Inhibition of 5-Aminolevulinate Synthase

Activity The enzymatic activity of 5-Aminolevulinate synthase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Shoolingin- Jordan et al. (1997) Methods Enzymol 281: 309 - 16 (PMDD: 9250995). Candidate compounds are identified when a decrease in products or a lack of decrease in substrates is detected with the reaction proceeding in either direction. Additionally, the enzymatic activity of a polypeptide comprising 10-50 amino acids from the M. grisea 5-Aminolevulinate synthase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Shoolingin-Jordan et al. (1997) Methods Enzymol 281: 309 - 16 (PMDD: 9250995). A polypeptide comprising 10-50 amino acids is generated by subcloning a portion of the ALASl 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 ALASl gene using polymerase chain reaction amplification method. The DNA fragment encoding a polypeptide of 10-50 amino acids is cloned into an expression vector, expressed and purified as described in Example 22 above.

Test compounds identified as inhibitors of ALASl activity are further tested for antibiotic activity. Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMDD: 8312740)). Spores are harvested into minimal media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMID: 8312740)) to a concentration of 2 x 10⁵ spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 μg/ml. Solvent only is added to the second culture. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The growth curves of the solvent control sample and the test compound sample are compared. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the control culture.

Example 25 Assays for Testing Compounds for Alteration of 5-Aminolevulinate Synthase Gene

Expression Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. Wild-type M. grisea spores are harvested from cultures grown on complete agar or oatmeal agar media after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. 25 ml cultures are prepared to which test compounds will be added at various concentrations. A culture with no test compound present is included as a control. The cultures are incubated at 25°C for 3 days after which test compound or solvent only control is added. The cultures are incubated an additional 18 hours. Fungal mycelia is harvested by filtration through Miracloth (CalBiochem®, La Jolla, CA), washed with water and frozen in liquid nitrogen. Total RNA is extracted with TREZOL® Reagent using the methods provided by the manufacturer (Life Technologies, Rockville, MD). Expression is analyzed by Northern analysis of the RNA samples as described (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press) using a radiolabeled fragment of the ALASl gene as a probe. Test compounds resulting in a reduced level of ALASl mRNA relative to the untreated control sample are identified as candidate antibiotic compounds.

Example 26 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of 5-Aminolevulinate Synthase with No Activity Magnaporthe grisea fungal cells containing a mutant form of the ALASl gene which abolishes enzyme activity, such as a gene containing a transposon insertion (see Examples 18 and 19), are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium containing 20 μM 5-aminolevulinate to a concentration of 2xl0⁵ spores per ml. Approximately 4xl0⁴ spores are added to each well of 96-well plates to which a test compound is added (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the ODs₉o (fungal strain plus test compound) / ODs₉o (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMDD: 7749303)).

Example 27 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of 5-Aminolevulinate Synthase with Reduced Activity Magnaporthe grisea fungal cells containing a mutant form of the ALASl gene, such as a promoter truncation that reduces expression, are grown under standard fungal growth conditions that are well known and described in the art. A promoter truncation is made by deleting a portion of the promoter upstream of the transcription start site using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press). Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores are added to each well of 96-well plates to which a test compound is added (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the ODsgo (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild-type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology : 177 - 221 (PMDD: 7749303)).

Example 28 In Vivo Cell Based Assay Screening Protocol wifh a Fungal Strain Containing a Mutant Form of a Heme Biosynthetic Gene with No Activity Magnaporthe grisea fungal cells containing a mutant form of a gene in the heme biosynthetic pathway (e.g. Aminolevulinate dehydratase (E.C. 4.2.1.24)) are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium containing 20 μM 5-aminolevulinate to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96- well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD590 (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild- type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMED: 7749303)). Example 29 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of a Heme Biosynthetic Gene with Reduced Activity Magnaporthe grisea fungal cells containing a mutant form of a gene in the heme biosynthetic pathway (e.g. Aminolevulinate dehydratase (E.C. 4.2.1.24)), such as a promoter truncation that reduces expression, are grown under standard fungal growth conditions that are well known and described in the art. A promoter truncation is made by deleting a portion of the promoter upstream of the transcription start site using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual Cold Spring Harbor Laboratory Press). Magnaporthe grisea fungal cells containing a mutant form of are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) after growth for 10- 13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96-well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅ o (fungal strain plus test compound) / OD5₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177 - 221 (PMED: 7749303)). Example 30 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing M. grisea ALASl and a Second Fungal Strain Containing a Heterologous ALASl Gene Wild-type Magnaporthe grisea fungal cells and M. grisea fungal cells lacking a functional ALASl gene and containing a 5-Aminolevulinic Acid Synthase gene from Candida albicans (Genbank: 10720014, SWISS-PROT: O94069, 54% sequence identity) are grown under standard fungal growth conditions that are well known and described in the art. A . grisea strain carrying a heterologous ALASl gene is made as follows:

• A . grisea strain is made with a nonfunctional ALASl gene, such as one containing a transposon insertion in the native gene (see Examples 18 and 19).

• A construct containing a heterologous ALASl gene is made by cloning the 5- Aminolevulinic Acid Synthase gene from Candida albicans into a fungal expression vector containing a trpC promoter and terminator (e.g. pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41 : 22) using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press).

• The said construct is used to transform the M. grisea strain lacking a functional ALASl gene (see Example 19). Transformants are selected on minimal agar medium lacking 5-aminolevulinate. Only transformants carrying a functional ALASl gene will grow.

Wild-type strains of Magnaporthe grisea and strains containing a heterologous form of ALASl are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96- well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The effect of each compound on the wild-type and heterologous fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉₀ (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on the wild-type and heterologous fungal strains are compared. Compounds that show differential growth inhibition between the wild-type and heterologous strains are identified as potential antifungal compounds with specificity to the native or heterologous ALASl gene products. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177 - 221 (PMED: 7749303)).

Example 31 Pathway Specific In Vivo Assay Screening Protocol Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. Wild-type M. grisea spores are harvested from cultures grown on oatmeal agar media after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemocytometer and spore suspensions are prepared in a minimal growth medium and a mimmal growth medium containing 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) to a concentration of 2xl0⁵ spores per ml. The minimal growth media contains carbon, nitrogen, phosphate, and sulfate sources, and magnesium, calcium, and trace elements (for example, see innoculating fluid in Example 21). Spore suspensions are added to each well of a 96-well microtiter plate (approximately 4x 10⁴ spores/well). For each well containing a spore suspension in minimal media, an additional well is present containing a spore suspension in minimal medium containing 200 μM 5-aminolevulinate. Test compounds are added to wells containing spores in minimal media and minimal media containing 5-aminolevulinate. The total volume in each well is 200μl. Both minimal media and 5-aminolevulinate containing media wells with no test compound are provided as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. A compound is identified as a candidate for an antibiotic acting against^'the heme biosynthetic pathway when the observed growth in the well containing minimal media is less than the observed growth in the well containing 5- aminolevulinate as a result of the addition of the test compound. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMDD: 7749303)).

Example 32

High Throughput Preparation and Verification of Transposon Insertion into the M. grisea HISPl Gene

E. coli strains containing cosmids with transposon insertions were picked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB (Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/ml of ampicillin. Blocks were incubated with shaking at 37 C overnight. 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 (PMDD: 9371743)). DNA quality was checked by elecfrophoresis on agarose gels. Cosmids were sequenced using primers from the ends of each transposon and commercial dideoxy sequencing kits (Big Dye Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNA sequencer (Perkin Elmer Co.).

DNA sequences adjacent to the site of the insertion were collected and used to search DNA and protein databases using the BLAST algorithms (Altschul et al. (1997) Nucleic Acids Res 25: 3389 - 3402 (PMDD: 9254694)). A single insertion of SEF into the Magnaporthe grisea HISPl gene was chosen for further analysis. This construct was designated cpgmra0012021b05 and it contains the SEF transposon approximately 100 nucleotides before the start codon relative to the Schizosaccharomyces pombe homologue (total length: 315 amino acids, GENBANK: 3183028, SWISS-PROT: O14059), and is predicted to eliminate or reduce gene function. Example 33 Preparation of HISPl Cosmid DNA and Transformation of Magnaporthe grisea Cosmid DNA from the HISPl transposon tagged cosmid clone was prepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-PspI (New England Biolabs, Inc.). Fungal electro-transformation was performed essentially as described (Wu et al (1997) MPMI 10: 700 - 708). Briefly, M. grisea strain Guy 11 was grown in complete liquid media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) shaking at 120 m for 3 days at 25°C in the dark. Mycelia was harvested and washed with sterile H₂O and digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6 hours to generate protoplasts. Protoplasts were collected by centrifugation and resuspended in 20% sucrose at the concentration of 2x10⁸ protoplasts/ml. 50ul protoplast suspension was mixed with 10-20ug of the cosmid DNA and pulsed using Gene Pulser II (BioRad) set with the following parameters: resistance 200 ohm, capacitance 25uF, voltage 0.6kV. Transformed protoplasts were regenerated in complete agar media (CM, Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMDD: 8312740)) with the addition of 20% sucrose for one day, then overlayed with CM agar media containing hygromycin B (250ug/ml) to select transformants. Transformants were screened for homologous recombination events in the target gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110 - 15 (PMDD: 11296265)). Two independent strains were identified and are hereby referred to as KOl-1 and KO1-3, respectively.

Example 34 Effect of Transposon Insertion on Magnaporthe pathogenicity The target fungal strains, KOl-1 and KOI -3, obtained in Example 33 and the wild type strain, Guyl 1, were subjected to a pathogenicity assay to observe infection over a 1- week period. Rice infection assays were performed using Indian rice cultivar CO39 essentially as described in Valent et al. ((1991) Genetics 127: 87 - 101 (PMED: 2016048)). All three strains were grown for spore production on complete agar media. Spores were harvested and the concentration of spores adjusted for whole plant inoculations. Two- week-old seedlings of cultivar CO39 were sprayed with 12 ml of conidial suspension (5 x 10 conidia per ml in 0.01% Tween-20 (Polyoxyethylensorbitan monolaureate) solution). The inoculated plants were incubated in a dew chamber at 27°C in the dark for 36 hours, and transferred to a growth chamber (27 °C 12 hours/21 °C 12 hours 70% humidity) for an additional 5.5 days. Leaf samples were taken at 3, 5, and 7 days post-inoculation and examined for signs of successful infection (i.e. lesions). Figure 2 shows the effects of HISPl gene disruption on Magnaporthe infection at five days post- inoculation.

Example 35 Verification of HISPl Gene Function by Analysis of Nutritional Requirements The fungal strains, KOl-1 and KOI -3, containing the HISPl disrupted gene obtained in Example 33 were analyzed for their nutritional requirement for histidine using the PM5 phenotype microarray from Biolog, Inc. (Hayward, CA). The PM5 plate tests for the auxotrophic requirement for 94 different metabolites. The innoculating fluid consists of 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5mM NaNO₃, 6.7mM KC1, 3.5mM Na₂SO₄, HmM KH₂PO , 0.01% -iodonitrotetrazolium violet, O.lmM MgCl₂, LOmM CaCl₂ and trace elements. Final concentrations of trace elements are: 7.6μM ZnCl₂, 2.5μM MnCl₂4H₂O, 1.8μM FeCl₂4H₂O, 0.71μM CoCl₂6H₂O, 0.64μM CuCl₂2H₂O, 0.62μM Na₂MoO₄, 18μM H₃BO₃. pH adjusted to 6.0 with NaOH. Spores for each strain were harvested into the inoculating fluid. The spore concentrations were adjusted to 2xl0⁵ spores/ml. lOOμl of spore suspension were deposited into each well of the microtiter plates. The plates were incubated at 25°C for 7 days. Optical density (OD) measurements at 490nm and 750nm were taken daily. The OD₄₉₀ measures the extent of tetrazolium dye reduction and the level of growth, and OD₇₅₀ measures growth only. Turbidity = OD₄₉₀ + OD ₅o. Data confirming the annotated gene function is presented as a graph of Turbidity vs. Time showing both the mutant fungi and the wild-type control in the absence (Figure 3 A) and presence (Figure 3B) of L-histidine.

Example 36 Cloning and Expression Strategies, Extraction and Purification of Histidinol- phosphatase Protein. The following protocol may be employed to obtain a purified histidinol- phosphatase protein.

Cloning and expression strategies:

A HISPl cDNA gene can be cloned into E. coli (pΕT vectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing His/fusion protein tags and the expression of recombinant protein can be evaluated by SDS-PAGΕ and Western blot analysis.

Extraction:

Extract recombinant protein from 250 ml cell pellet in 3 ml of extraction buffer by sonicating 6 times, with 6 sec pulses at 4°C. Centrifuge extract at 15000xg for 10 min and collect supernatant. Assess biological activity of the recombinant protein by activity assay.

Purification:

Purify recombinant protein by Ni-NTA affinity chromatography (Qiagen). Purification protocol: perform all steps at 4°C:

• Use 3 ml Ni-beads (Qiagen)

• Equilibrate column with the buffer

• Load protein extract

• Wash with the equilibration buffer

• Elute bound protein with 0.5 M imidazole

Example 37 Assays for Testing Binding of Test Compounds to Histidinol-phosphatase The following protocol may be employed to identify test compounds that bind to the histidinol-phosphatase protein. • Purified full-length histidinol-phosphatase polypeptide with a His/fusion protein tag (Example 36) is bound to a HisGrab™ Nickel Coated Plate (Pierce, Rockford, IL) following manufacturer's instructions.

• Buffer conditions are optimized (e.g. ionic strength or pH, Millay and Houston (1973) Biochemistry 12: 2591 - 2596 (PMDD: 4351203)) for binding of radiolabeled L-Histidinol phosphate (custom made, PerkinElmer Life Sciences, Inc., Boston, MA) to the bound histidinol-phosphatase.

• Screening of test compounds is performed by adding test compound and L- Histidinol phosphate (custom made, PerkinElmer Life Sciences, Inc., Boston, MA) to the wells of the HisGrab™ plate containing bound histidinol- phosphatase.

• The wells are washed to remove excess labeled ligand and scintillation fluid (Scintiverse®, Fisher Scientific) is added to each well.

• The plates are read in a microplate scintillation counter.

• Candidate compounds are identified as wells with lower radioactivity as compared to control wells with no test compound added.

Additionally, a purified polypeptide comprising 10-50 amino acids from the M. grisea histidinol-phosphatase is screened in the same way. A polypeptide comprising 10- 50 amino acids is generated by subcloning a portion of the HISPl 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 HISPl gene using the polymerase chain reaction amplification method. The DNA fragment encoding a polypeptide of 10-50 amino acids is cloned into an expression vector, expressed in a host organism and purified as described in Example 36 above.

Test compounds that bind HISPl are further tested for antibiotic activity. M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)). Spores are harvested into minimal media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) to a concentration of 2 x 10⁵ spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 μg/ml. Solvent only is added to the second culture. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The growth curves of the solvent control sample and the test compound sample are compared. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the control culture.

Example 38 Assays for Testing Inhibitors or Candidates for Inhibition of Histidinol-phosphatase

Activity

The enzymatic activity of histidinol-phosphatase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Millay and Houston (1973) Biochemistry 12: 2591 - 2596 (PMED: 4351203). Candidate compounds are identified when a decrease in products or a lack of decrease in substrates is detected with the reaction proceeding in either direction.

Additionally, the enzymatic activity of a polypeptide comprising 10-50 amino acids from the M. grisea histidinol-phosphatase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Millay and Houston (1973) Biochemistry 12: 2591 - 2596 (PMDD: 4351203). A polypeptide comprising 10-50 amino acids is generated by subcloning a portion of the HISPl 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 HISPl gene using polymerase chain reaction amplification method. The DNA fragment encoding a polypeptide of 10-50 amino acids is cloned into an expression vector, expressed and purified as described in Example 36 above.

Test compounds identified as inhibitors of HISPl activity are further tested for antibiotic activity. Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. M grisea is grown as described for spore production on oatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMDD: 8312740)). Spores are harvested into minimal media (Talbot et al (1993) Plant Cell 5: 1575 - 1590 (PMDD: 8312740)) to a concentration of 2 x 10⁵ spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 μg/ml. Solvent only is added to the second culture. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The growth curves of the solvent control sample and the test compound sample are compared. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the control culture.

Example 39 Assays for Testing Compounds for Alteration of Histidinol-phosphatase Gene Expression

Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. Wild-type M. grisea spores are harvested from cultures grown on complete agar or oatmeal agar media after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. 25 ml cultures are prepared to which test compounds will be added at various concentrations. A culture with no test compound present is included as a control. The cultures are incubated at 25°C for 3 days after which test compound or solvent only control is added. The cultures are incubated an additional 18 hours. Fungal mycelia is harvested by filtration through Miracloth (CalBiochem®, La Jolla, CA), washed with water and frozen in liquid nitrogen. Total RNA is extracted with TRIZOL® Reagent using the methods provided by the manufacturer (Life Technologies, Rockville, MD). Expression is analyzed by Northern analysis of the RNA samples as described (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press) using a radiolabeled fragment of the HISPl gene as a probe. Test compounds resulting in a reduced level of HISPl mRNA relative to the untreated control sample are identified as candidate antibiotic compounds.

Example 40 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of Histidinol-phosphatase with No Activity Magnaporthe grisea fungal cells containing a mutant form of the HISPl gene which abolishes enzyme activity, such as a gene containing a transposon insertion (see Examples 32 and 33), are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-histidine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium containing 100 μM L-histidine to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores are added to each well of 96-well plates to which a test compound is added (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉₀ (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMED: 7749303)).

Example 41 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of Histidinol-phosphatase with Reduced Activity Magnaporthe grisea fungal cells containing a mutant form of the HISPl gene, such as a promoter truncation that reduces expression, are grown under standard fungal growth conditions that are well known and described in the art. A promoter truncation is made by deleting a portion of the promoter upstream of the transcription start site using standard molecular biology techniques that are well known and described in the art (Sambrook et al (1989). Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-histidine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores are added to each well of 96-well plates to which a test compound is added (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD_{5 0} (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild- type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 177 - 221 (PMED: 7749303)).

Example 42 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of a L-histidine Biosynthetic Gene with No Activity Magnaporthe grisea fungal cells containing a mutant form of a gene in the L- histidine biosynthetic pathway (e.g. Histidinol dehydrogenase (E.C. 1.1.1.23)) are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-histidine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium containing 100 μM L-histidine to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96-well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉₀ (fungal strain plus test compound) / ODs₉₀ (growth confrol) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild-type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 11 - 221 (PMED: 7749303)).

Example 43 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of a L-histidine Biosynthetic Gene with Reduced Activity Magnaporthe grisea fungal cells containing a mutant form of a gene in the L- histidine biosynthetic pathway (e.g. Histidinol dehydrogenase (E.C. 1.1.1.23)), such as a promoter truncation that reduces expression, are grown under standard fungal growth conditions that are well known and described in the art. A promoter truncation is made by deleting a portion of the promoter upstream of the transcription start site using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989). Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea fungal cells containing a mutant form of are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-histidine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96-well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉₀ (fungal strain plus test compound) / ODs₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMED: 7749303)).

Example 44 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Fungal HISPl and a Second Fungal Strain Containing a Heterologous HISPl Gene Wild-type Magnaporthe grisea fungal cells and M. grisea fungal cells lacking a functional HISPl gene and containing a heterologous HISPl gene are grown under standard fungal growth conditions that are well known and described in the art. A M. grisea strain carrying a heterologous HISPl gene is made as follows:

• AM. grisea strain is made with a nonfunctional HISPl gene, such as one containing a transposon insertion in the native gene (see Examples 32 and 33).

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

• The said construct is used to transform the M. grisea strain lacking a functional HISPl gene (see Example 33). Transformants are selected on minimal agar medium lacking L-histidine. Only transformants carrying a functional HISPl gene will grow. Wild-type strains of Magnaporthe grisea and strains containing a heterologous form of HISPl are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96- well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The effect of each compound on the wild-type and heterologous fungal strains is measured against the growth confrol and the percent of inhibition is calculated as the OD₅₉o (fungal strain plus test compound) / OD_{5 0} (growth control) x 100. The percent of growth inhibition as a result of a test compound on the wild-type and heterologous fungal strains are compared. Compounds that show differential growth inhibition between the wild-type and heterologous strains are identified as potential antifungal compounds with specificity to the native or heterologous HISPl gene products. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMED: 7749303)).

Example 45 Pathway Specific In Vivo Assay Screening Protocol Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. Wild-type M grisea spores are harvested from cultures grown on oatmeal agar media after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemocytometer and spore suspensions are prepared in a minimal growth medium and a minimal growth medium containing 4 mM L-histidine (Sigma-Aldrich Co.) to a concentration of 2x10⁵ spores per ml. The minimal growth media contains carbon, nitrogen, phosphate, and sulfate sources, and magnesium, calcium, and trace elements (for example, see innoculating fluid in Example 35). Spore suspensions are added to each well of a 96-well microtiter plate (approximately 4 l0⁴ spores/well). For each well containing a spore suspension in minimal media, an additional well is present containing a spore suspension in minimal medium containing 4 mM L-histidine. Test compounds are added to wells containing spores in minimal media and minimal media containing L- histidine. The total volume in each well is 200μl. Both minimal media and L-histidine containing media wells with no test compound are provided as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. A compound is identified as a candidate for an antibiotic acting against the L- histidine biosynthetic pathway when the observed growth in the well containing minimal media is less than the observed growth in the well containing L-histidine as a result of the addition of the test compound. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology d: 177 - 221 (PMED: 7749303)).

Example 46

High Throughput Preparation and Verification of Transposon Insertion into the M. grisea IPMDl Gene

E. coli strains containing cosmids with transposon insertions were picked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB (Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/ml of ampicillin. Blocks were incubated with shaking at 37 C overnight. 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 (PMED: 9371743)). DNA quality was checked by elecfrophoresis on agarose gels. Cosmids were sequenced using primers from the ends of each transposon and commercial dideoxy sequencing kits (Big Dye Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNA sequencer (Perkin Elmer Co.).

DNA sequences adjacent to the site of the insertion were collected and used to search DNA and protein databases using the BLAST algorithms (Altschul et al. (1997) Nucleic Acids Res 25: 3389 - 3402 (PMED: 9254694)). A single insertion of SEF into the Magnaporthe grisea EPMDl gene was chosen for further analysis. This construct was designated cpgmraOOl 408 lf03 and it contains the SIF transposon approximately between amino acids 560 and 561 relative to the Rhizomucor pusillus homologue LeuA (total length: 755 amino acids, SWISS-PROT: P55251).

Example 47 Preparation of IPMDl Cosmid DNA and Transformation of Magnaporthe grisea Cosmid DNA from the EPMDl transposon tagged cosmid clone was prepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-PspI (New England Biolabs, Inc.). Fungal electro-transformation was performed essentially as described (Wu et al. (1997) MPMI 10: 700 - 708). Briefly, M grisea strain Guy 11 was grown in complete liquid media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) shaking at 120 φm for 3 days at 25°C in the dark. Mycelia was harvested and washed with sterile H₂O and digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6 hours to generate protoplasts. Protoplasts were collected by centrifugation and resuspended in 20% sucrose at the concentration of 2x10^s protoplasts/ml. 50ul protoplast suspension was mixed with 10-20ug of the cosmid DNA and pulsed using Gene Pulser II (BioRad) set with the following parameters: resistance 200 ohm, capacitance 25uF, voltage 0.6kV. Transformed protoplasts were regenerated in complete agar media (CM, Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) with the addition of 20% sucrose for one day, then overlayed with CM agar media containing hygromycin B (250ug/ml) to select transformants. Transformants were screened for homologous recombination events in the target gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110 - 15 (PMED: 11296265)). Two independent strains were identified and are hereby refeπed to as KOI -3 and KOI -7, respectively.

Example 48 Effect of Transposon Insertion on Magnaporthe pathogenicity The target fungal strains, KOI -3 and KOI -7, obtained in Example 47and the wild type strain, Guyl 1, were subjected to a pathogenicity assay to observe infection over a 1- week period. Rice infection assays were performed using Indian rice cultivar CO39 essentially as described in Valent et al. ((1991) Genetics 127: 87 - 101 (PMED: 2016048)). All three strains were grown for spore production on complete agar media. Spores were harvested and the concentration of spores adjusted for whole plant inoculations. Two-week-old seedlings of cultivar CO39 were sprayed with 12 ml of conidial suspension (5 x 10⁴ conidia per ml in 0.01%» Tween-20 (Polyoxyethylensorbitan monolaureate) solution). The inoculated plants were incubated in a dew chamber at 27°C in the dark for 36 hours, and transferred to a growth chamber (27 °C 12 hours/21 °C 12 hours 70% humidity) for an additional 5.5 days. Leaf samples were taken at 3, 5, and 7 days post-inoculation and examined for signs of successful infection (i.e. lesions). Figure 2 shows the effects of IPMDl gene disruption on Magnaporthe infection at five days post-inoculation.

Example 49 Verification of IPMDl Gene Function by Analysis of Nutritional Requirements The fungal strains, KOI -3 and KOI -7, containing the EPMDl disrupted gene obtained in Example 47 were analyzed for their nutritional requirement for L-leucine using the PM5 phenotype microarray from Biolog, Inc. (Hayward, CA). The PM5 plate tests for the auxotrophic requirement for 94 different metabolites. The innoculating fluid consists of 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5mM NaNO₃, 6.7mM KC1, 3.5mM Na₂SO₄, llmM KH₂PO₄, 0.01%p-iodonitrotetrazolium violet, O.lmM MgCl₂, LOmM CaCl₂ and trace elements. Final concentrations of trace elements are: 7.6μM ZnCl₂, 2.5μM MnCl₂4H₂O, 1.8μM FeCl₂4H₂O, 0.71μM CoCl₂6H₂O, 0.64μM CuCl₂2H₂O, 0.62μM Na₂MoO₄, 18μM H₃BO₃. pH adjusted to 6.0 with NaOH. Spores for each strain were harvested into the inoculating fluid. The spore concentrations were adjusted to 2xl0⁵ spores/ml. lOOμl of spore suspension were deposited into each well of the microtiter plates. The plates were incubated at 25°C for 7 days. Optical density (OD) measurements at 490nm and 750nm were taken daily. The OD₄₉₀ measures the extent of tetrazolium dye reduction and the level of growth, and OD₇₅₀ measures growth only. Turbidity = OD₄₉₀ + OD₇s₀. Data confirming the annotated gene function is presented as a graph of Turbidity vs. Time showing both the mutant fungi and the wild-type control in the absence (Figure 3 A) and presence (Figure 3B) of L-leucine.

Example 50 Cloning and Expression Strategies, Extraction and Purification of 3-Isopropylmalate

Dehydratase Protein.

The following protocol may be employed to obtain a purified 3-Isopropylmalate dehydratase protein.

Cloning and expression strategies:

An EPMDl cDNA gene can be cloned into E. coli (pET vectors-No vagen), Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing His/fusion protein tags, and the expression of recombinant protein can be evaluated by SDS-PAGE and Western blot analysis.

Extraction:

Extract recombinant protein from 250 ml cell pellet in 3 ml of extraction buffer by sonicating 6 times, with 6 sec pulses at 4°C. Centrifuge extract at 15000xg for

10 min and collect supernatant. Assess biological activity of the recombinant protein by activity assay.

Purification:

Purify recombinant protein by Ni-NTA affinity chromatography (Qiagen).

Purification protocol: perform all steps at 4°C:

• Use 3 ml Ni-beads (Qiagen)

• Equilibrate column with the buffer

• Load protein extract

• Wash with the equilibration buffer

• Elute bound protein with 0.5 M imidazole

Example 51 Assays for Testing Binding of Test Compounds to 3-Isopropylmalate Dehydratase The following protocol may be employed to identify test compounds that bind to the 3-Isopropylmalate dehydratase protein. • Purified full-length 3-Isopropylmalate dehydratase polypeptide with a His/fusion protein tag (Example 50) is bound to a HisGrab™ Nickel Coated Plate (Pierce, Rockford, EL) following manufacturer's instructions.

• Buffer conditions are optimized (e.g. ionic strength or pH, as may be described in Satyanarayana et al. ((1968B) J Bacteriol 96: 2018 - 24 (PMED: 5724970)) and/or Kohlhaw ((1988) Methods Enzymol 166: 423 - 9 (PMED: 3071717))) for binding of radiolabeled 2-Isopropylmalate (custom made PerkinElmer Life Sciences, Inc., Boston, MA) to the bound 3-Isopropylmalate dehydratase.

• Screening of test compounds is performed by adding test compound and 2- Isopropylmalate (custom made PerkinElmer Life Sciences, Inc., Boston, MA) to the wells of the HisGrab™ plate containing bound 3-Isopropylmalate dehydratase.

• The wells are washed to remove excess labeled ligand and scintillation fluid (Scintiverse®, Fisher Scientific) is added to each well.

• The plates are read in a microplate scintillation counter.

• Candidate compounds are identified as wells with lower radioactivity as compared to control wells with no test compound added.

Additionally, a purified polypeptide comprising 10-50 amino acids from the M. grisea 3-Isopropylmalate dehydratase is screened in the same way. A polypeptide comprising 10-50 amino acids is generated by subcloning a portion of the EPMDl 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 EPMDl gene using the polymerase chain reaction amplification method. The DNA fragment encoding a polypeptide of 10-50 amino acids is cloned into an expression vector, expressed in a host organism and purified as described in Example 50 above.

Test compounds that bind EPMDl are further tested for antibiotic activity. M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)). Spores are harvested into minimal media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) to a concentration of 2 x 10⁵ spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 μg/ml. Solvent only is added to the second culture. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The growth curves of the solvent control sample and the test compound sample are compared. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the control culture.

Example 52 Assays for Testing Inhibitors or Candidates for Inhibition of 3-Isopropylmalate

Dehydratase Activity

The enzymatic activity of 3-Isopropyhnalate dehydratase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Satyanarayana et al. ((1968B) J Bacteriol 96: 2018 - 24 (PMED: 5724970)) and/or Kohlhaw ((1988) Methods Enzymol 166: 423 - 9 (PMED: 3071717)). Candidate compounds are identified when a decrease in products or a lack of decrease in substrates is detected with the reaction proceeding in either direction.

Additionally, the enzymatic activity of a polypeptide comprising 10-50 amino acids from the M. grisea 3-Isopropylmalate dehydratase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Satyanarayana et al. ((1968B) J Bacteriol 96: 2018 - 24 (PMED: 5724970)) and/or Kohihaw ((1988) Methods Enzymol 166: 423 - 9 (PMED: 3071717)). A polypeptide comprising 10-50 amino acids is generated by subcloning a portion of the IPMDl 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 IPMDl gene using polymerase chain reaction amplification method. The DNA fragment encoding a polypeptide of 10-50 amino acids is cloned into an expression vector, expressed and purified as described in Example 50 above.

Test compounds identified as inhibitors of EPMDl activity are further tested for antibiotic activity. Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)). Spores are harvested into minimal media (Talbot et al (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) to a concentration of 2 x 10⁵ spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 μg/ml. Solvent only is added to the second culture. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The growth curves of the solvent control sample and the test compound sample are compared. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the control culture.

Example 53 Assays for Testing Compounds for Alteration of 3-Isopropylmalate Dehydratase Gene

Expression Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. Wild-type M grisea spores are harvested from cultures grown on complete agar or oatmeal agar media after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. 25 ml cultures are prepared to which test compounds will be added at various concentrations. A culture with no test compound present is included as a control. The cultures are incubated at 25°C for 3 days after which test compound or solvent only control is added. The cultures are incubated an additional 18 hours. Fungal mycelia is harvested by filtration through Miracloth (CalBiochem®, La Jolla, CA), washed with water and frozen in liquid nitrogen. Total RNA is extracted with TRIZOL® Reagent using the methods provided by the manufacturer (Life Technologies, Rockville, MD). Expression is analyzed by Northern analysis of the RNA samples as described (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press) using a radiolabeled fragment of the EPMDl gene as a probe. Test compounds resulting in a reduced level of EPMDl mRNA relative to the untreated control sample are identified as candidate antibiotic compounds. Example 54 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of 3-Isopropylmalate dehydratase with No Activity Magnaporthe grisea fungal cells containing a mutant form of the EPMDl gene which abolishes enzyme activity, such as a gene containing a transposon insertion (see Examples 46 and 47), are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-leucine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium containing 100 μM L-leucine to a concenfration of 2xl0⁵ spores per ml. Approximately 4xl0⁴ spores are added to each well of 96-well plates to which a test compound is added (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉o (fungal strain plus test compound) / OD₅₉₀ (growth confrol) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMED: 7749303)).

Example 55 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of 3-Isopropylmalate dehydratase with Reduced Activity Magnaporthe grisea fungal cells containing a mutant form of the EPMDl gene, such as a promoter truncation that reduces expression, are grown under standard fungal growth conditions that are well known and described in the art. A promoter truncation is made by deleting a portion of the promoter upstream of the transcription start site using standard molecular biology techniques that are well known and described in the art (Sambrook et al (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-leucine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores are added to each well of 96-well plates to which a test compound is added (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉₀ (fungal strain plus test compound) / ODs₉o (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild- type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMED: 7749303)).

Example 56 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of a L-leucine Biosynthetic Gene with No Activity Magnaporthe grisea fungal cells containing a mutant form of a gene in the L- leucine biosynthetic pathway (e.g. a 3-Isopropylmalate dehydrogenase (E.C. 1.1.1.85)) are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-leucine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concenfration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium containing 100 μM L-leucine to a concenfration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96-well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉o (fimgal strain plus test compound) / ODs₉o (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild- type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMDD: 7749303)).

Example 57 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of a L-leucine Biosynthetic Gene with Reduced Activity Magnaporthe grisea fungal cells containing a mutant form of a gene in the L- leucine biosynthetic pathway (e.g. a 3-Isopropylmalate dehydrogenase (E.C. 1.1.1.85)), such as a promoter truncation that reduces expression, are grown under standard fungal growth conditions that are well known and described in the art. A promoter truncation is made by deleting a portion of the promoter upstream of the transcription start site using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press). Magnaporthe grisea fungal cells containing a mutant form of are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-leucine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x 10⁵ spores per ml. Approximately 4x 10 spores or cells are harvested and added to each well of 96-well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉₀ (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMED: 7749303)).

Example 58 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Fungal IPMDl and a Second Fungal Strain Containing a Heterologous IPMDl Gene Wild-type Magnaporthe grisea fungal cells and M. grisea fungal cells lacking a functional EPMDl gene and containing a 3-isopropylmalate dehydratase large subunit gene from Xylella fastidiosa (Genbank accession number H82564, 63% sequence identity) are grown under standard fungal growth conditions that are well known and described in the art. A M. grisea strain carrying a heterologous EPMDl gene is made as follows:

• AM. grisea strain is made with a nonfunctional EPMDl gene, such as one containing a transposon insertion in the native gene (see Examples 46 and 47).

• A construct containing a heterologous EPMDl gene is made by cloning the 3- isopropylmalate dehydratase large subunit gene from Xylella fastidiosa into a fungal expression vector containing a trp C promoter and terminator (e.g. pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41: 22) using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press). • The said construct is used to transform the M grisea strain lacking a functional

EPMDl gene (see Example 47). Transformants are selected on minimal agar medium lacking L-leucine. Only transformants carrying a functional IPMDl gene will grow. Wild-type strains of Magnaporthe grisea and strains containing a heterologous form of IPMDl are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96- well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The effect of each compound on the wild-type and heterologous fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD5₉₀ (fungal strain plus test compound) / OD₅₉o (growth control) x 100. The percent of growth inhibition as a result of a test compound on the wild-type and heterologous fungal strains are compared. Compounds that show differential growth inhibition between the wild-type and heterologous strains are identified as potential antifungal compounds with specificity to the native or heterologous IPMDl gene products. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 11 - 221 (PMDD: 7749303)).

Example 59 Pathway Specific In Vivo Assay Screening Protocol Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. Wild-type M. grisea spores are harvested from cultures grown on oatmeal agar media after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemocytometer and spore suspensions are prepared in a minimal growth medium and a minimal growth medium containing 4 mM L-leucine (Sigma-Aldrich Co.) to a concentration of 2x10⁵ spores per ml. The minimal growth media contains carbon, nitrogen, phosphate, and sulfate sources, and magnesium, calcium, and trace elements (for example, see innoculating fluid in Example 49). Spore suspensions are added to each well of a 96-well microtiter plate (approximately 4xl0⁴ spores/well). For each well containing a spore suspension in minimal media, an additional well is present containing a spore suspension in mimmal medium containing 4 mM L-leucine. Test compounds are added to wells containing spores in minimal media and minimal media containing L- leucine. The total volume in each well is 200μl. Both minimal media and L-leucine containing media wells with no test compound are provided as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. A compound is identified as a candidate for an antibiotic acting against the L- leucine biosynthetic pathway when the observed growth in the well containing minimal media is less than the observed growth in the well containing L-leucine as a result of the addition of the test compound. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMED: 7749303)).

Example 60 High Throughput Preparation and Verification of Transposon Insertion into the M. grisea THR4 Gene E. coli strains containing cosmids with transposon insertions were picked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB (Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/ml of ampicillin. Blocks were incubated with shaking at 37°C overnight. E. coli veils were pelleted by centrifugation and cosmids were isolated by a modified alkaline lysis method (Marra et al. (1997) Genome Res 7: 1072 - 84 (PMED: 9371743)). DNA quality was checked by elecfrophoresis on agarose gels. Cosmids were sequenced using primers from the ends of each transposon and commercial dideoxy sequencing kits (Big Dye Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNA sequencer (Perkin Elmer Co.).

DNA sequences adjacent to the site of the insertion were collected and used to search DNA and protein databases using the BLAST algorithms (Altschul et al. (1997) Nucleic Acids Res 25: 3389 - 3402 (PMED: 9254694)). A single insertion of SEF into the Magnaporthe grisea THR4 gene was chosen for further analysis. This construct was designated cpgmra0012020a04 and it contains the SEF transposon approximately between amino acids 314 and 315 relative to the Schizosaccharomyces pombe homologue ThrC (total length: 514 amino acids, GENBANK: 2501152).

Example 61 Preparation ofTHR4 Cosmid DNA and Transformation of Magnaporthe grisea Cosmid DNA from the THR4 fransposon tagged cosmid clone was prepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-PspI (New England Biolabs, Inc.). Fungal electro-transformation was performed essentially as described (Wu et al. (1997) MPMI 10: 700 - 708). Briefly, M. grisea strain Guy 11 was grown in complete liquid media (Talbot et al (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) shaking at 120 φm for 3 days at 25°C in the dark. Mycelia was harvested and washed with sterile H₂O and digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6 hours to generate protoplasts. Protoplasts were collected by centrifugation and resuspended in 20% sucrose at the concentration of 2x10⁸ protoplasts/ml. 50ul protoplast suspension was mixed with 10-20ug of the cosmid DNA and pulsed using Gene Pulser π (BioRad) set with the following parameters: resistance 200 ohm, capacitance 25uF, voltage 0.6kV. Transformed protoplasts were regenerated in complete agar media (CM, Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) with the addition of 20% sucrose for one day, then overlayed with CM agar media containing hygromycin B (250ug/ml) to select transformants. Transformants were screened for homologous recombination events in the target gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110 - 15 (PMED: 11296265)). Two independent strains were identified and are hereby referred to as KOI -3 and KOI -22, respectively. Example 62 Effect of Transposon Insertion on Magnaporthe pathogenicity The target fungal strains, KOI -3 and KOI -22, obtained in Example 61 and the wild type strain, Guyl 1, were subjected to a pathogenicity assay to observe infection over a 1-week period. Rice infection assays were performed using Indian rice cultivar CO39 essentially as described in Valent etal. ((1991) Genetics 127: 87 - 101 (PMED: 2016048)). All three strains were grown for spore production on complete agar media. Spores were harvested and the concentration of spores adjusted for whole plant inoculations. Two-week-old seedlings of cultivar CO39 were sprayed with 12 ml of conidial suspension (5 x 10⁴ conidia per ml in 0.01% Tween-20 (Polyoxyethylensorbitan monolaureate) solution). The inoculated plants were incubated in a dew chamber at 27°C in the dark for 36 hours, and transferred to a growth chamber (27 °C 12 hours/21 °C 12 hours 70% humidity) for an additional 5.5 days. Leaf samples were taken at 3, 5, and 7 days post-inoculation and examined for signs of successful infection (i.e. lesions). Figure 2 shows the effects of THR4 gene disruption on Magnaporthe infection at five days post- inoculation.

Example 63 Verification of THR4 Gene Function by Analysis of Nutritional Requirements The fungal strains, KOI -3 and KOI -22, containing the THR4 disrupted gene obtained in Example 61 were analyzed for their nutritional requirement for L-threonine using the PM5 phenotype microarray from Biolog, Inc. (Hayward, CA). The PM5 plate tests for the auxotrophic requirement for 94 different metabolites. The innoculating fluid consists of 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5mM NaNO₃, 6.7mM KC1, 3.5mM Na₂SO₄, 1 ImM KH₂PO₄, 0.01%/ odonitrotetrazolium violet, O.lmM MgCl₂, LOmM CaCl₂ and trace elements, pH adjusted to 6.0 with NaOH. Final concentrations of trace elements are: 7.6μM ZnCl₂, 2.5μM MnCl₂4H₂O, 1.8μM FeCl₂4H₂O, 0.71μM CoCl₂6H₂O, 0.64μM CuCl₂2H₂O, 0.62μM Na₂MoO₄, 18μM H₃BO₃. Spores for each strain were harvested into the inoculating fluid. The spore concentrations were adjusted to 2xl0⁵ spores/ml. lOOμl of spore suspension were deposited into each well of the microtiter plates. The plates were incubated at 25°C for 7 days. Optical density (OD) measurements at 490nm and 750nm were taken daily. The OD₄₉₀ measures the extent of tetrazolium dye reduction and the level of growth, and OD₇₅o measures growth only. Turbidity = OD₄₉₀ + OD₇5₀. Data confirming the annotated gene function is presented as a graph of Turbidity vs. Time showing both the mutant fungi and the wild-type control in the absence (Figure 3 A) and presence (Figure 3B) of L-threonine.

Example 64 Cloning and Expression Strategies, Extraction and Purification of Threonine Synthase

Protein. The following protocol may be employed to obtain a purified Threonine synthase protein.

Cloning and expression strategies:

A THR4 cDNA gene can be cloned into E. coli (pET vectors-Novagen),

Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing

His/fusion protein tags, and the expression of recombinant protein can be evaluated by SDS-PAGE and Western blot analysis.

Extraction:

Extract recombinant protein from 250 ml cell pellet in 3 ml of extraction buffer by sonicating 6 times, with 6 sec pulses at 4°C. Centrifuge extract at 15000xg for

10 min and collect supernatant. Assess biological activity of the recombinant protein by activity assay.

Purification:

Purify recombinant protein by Ni-NTA affinity chromatography (Qiagen).

Purification protocol: perform all steps at 4°C:

• Use 3 ml Ni-beads (Qiagen)

• Equilibrate column with the buffer

• Load protein extract

• Wash with the equilibration buffer

• Elute bound protein with 0.5 M imidazole Example 65 Assays for Testing Binding of Test Compounds to Threonine Synthase The following protocol may be employed to identify test compounds that bind to the Threonine synthase protein.

• Purified full-length Threonine synthase polypeptide with a His/fusion protein tag (Example 64) is bound to a HisGrab™ Nickel Coated Plate (Pierce, Rockford, IL) following manufacturer's instructions.

• Buffer conditions are optimized (e.g. ionic strength or pH, Ramos and Calderon (1994) FEBS Lett 351: 357 - 9 (PMDD: 8082795)) for binding of radiolabeled O-phospho-L-homoserine (Gening et al. (1994) Biokhimiia 59: 1238 - 44 (PMDD: 7819407)) to the bound Threonine synthase.

• Screening of test compounds is performed by adding test compound and radiolabeled O-phospho-L-homoserine (Gening et al. (1994) Biokhimiia 59: 1238 - 44 (PMDD: 7819407)) to the wells of the HisGrab™ plate containing bound Threonine synthase.

• The wells are washed to remove excess labeled ligand and scintillation fluid (Scintiverse®, Fisher Scientific) is added to each well.

• The plates are read in a microplate scintillation counter.

• Candidate compounds are identified as wells with lower radioactivity as compared to control wells with no test compound added.

Additionally, a purified polypeptide comprising 10-50 amino acids from the M. grisea Threonine synthase is screened in the same way. A polypeptide comprising 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. The DNA fragment encoding a polypeptide of 10-50 amino acids is cloned into an expression vector, expressed in a host organism and purified as described in Example 64 above.

Test compounds that bind THR4 are further tested for antibiotic activity. M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)). Spores are harvested into minimal media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMID: 8312740)) to a concentration of 2 x 10⁵ spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 μg/ml. Solvent only is added to the second culture. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The growth curves of the solvent control sample and the test compound sample are compared. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the control culture.

Example 66 Assays for Testing Inhibitors or Candidates for Inhibition of Threonine Synthase Activity

The enzymatic activity of Threonine synthase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Ramos and Calderon (1994) FEBS Lett 351: 357 - 9 (PMID: 8082795). Candidate compounds are identified when a decrease in products or a lack of decrease in substrates is detected with the reaction proceeding in either direction.

Additionally, the enzymatic activity of a polypeptide comprising 10-50 amino acids from the M. grisea Threonine synthase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Ramos and Calderon (1994) FEBS Lett 351: 357 - 9 (PMDD: 8082795). A polypeptide comprising 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 polymerase chain reaction amplification method. The DNA fragment encoding a polypeptide of 10-50 amino acids is cloned into an expression vector, expressed and purified as described in Example 64 above.

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 that are well known and described in the art. M grisea is grown as described for spore production on oatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)). Spores are harvested into minimal media (Talbot et al. (1993) Plant Cell 5: 1575 - 1590 (PMED: 8312740)) to a concentration of 2 x 10⁵ spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 μg/ml. Solvent only is added to the second culture. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The growth curves of the solvent control sample and the test compound sample are compared. A test compound is an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the control culture.

Example 67 Assays for Testing Compounds for Alteration of Threonine Synthase Gene Expression Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. Wild-type M grisea spores are harvested from cultures grown on complete agar or oatmeal agar media after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. 25 ml cultures are prepared to which test compounds will be added at various concentrations. A culture with no test compound present is included as a control. The cultures are incubated at 25°C for 3 days after which test compound or solvent only control is added. The cultures are incubated an additional 18 hours. Fungal mycelia is harvested by filtration through Miracloth (CalBiochem®, La Jolla, CA), washed with water and frozen in liquid nitrogen. Total RNA is extracted with TREZOL® Reagent using the methods provided by the manufacturer (Life Technologies, Rockville, MD). Expression is analyzed by Northern analysis of the 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 resulting in a reduced level of THR4 mRNA relative to the untreated control sample are identified as candidate antibiotic compounds. Example 68 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form ofTlireonine Synthase with No Activity Magnaporthe grisea fungal cells containing a mutant form of the THR4 gene which abolishes enzyme activity, such as a gene containing a transposon insertion (see Examples 60 and 61), are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-threonine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium containing 100 μM L-threonine to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores are added to each well of 96-well plates to which a test compound is added (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉o (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221 (PMDD: 7749303)).

Example 69

In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant

Form of Threonine Synthase with Reduced Activity

Magnaporthe grisea fungal cells containing a mutant form of the THR4 gene, such as a promoter truncation that reduces expression, are grown under standard fungal growth conditions that are well known and described in the art. A promoter truncation is made by deleting a portion of the promoter upstream of the transcription start site using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-threonine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores are added to each well of 96-well plates to which a test compound is added (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates are incubated at 25°C for seven days and opcical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉₀ (fungal strain plus test compound) / ODs₉o (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild- type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221).

Example 70 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of a L-threonine Biosynthetic Gene with No Activity Magnaporthe grisea fungal cells containing a mutant form of a gene in the L- threonine biosynthetic pathway (e.g. Homoserine kinase (E.C. 2.7.1.39)) are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-threonine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium containing 100 μM L-threonine to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96-well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth confrol and the percent of inhibition is calculated as the OD₅₉₀ (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild- type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221).

Example 71 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of a L-threonine Biosynthetic Gene with Reduced Activity Magnaporthe grisea fungal cells containing a mutant form of a gene in the L- threonine biosynthetic pathway (e.g. Homoserine kinase (E.C. 2.7.1.39)), such as a promoter truncation that reduces expression, are grown under standard fungal growth conditions that are well known and described in the art. A promoter truncation is made by deleting a portion of the promoter upstream of the transcription start site using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea fungal cells containing a mutant form of are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium containing 4 mM L-threonine (Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96-well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. Wild type cells are screened under the same conditions. The effect of each compound on the mutant and wild-type fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD5₉0 (fungal strain plus test compound) / OD₅₉₀ (growth control) x 100. The percent of growth inhibition as a result of a test compound on a fungal strain and that on the wild type cells are compared. Compounds that show differential growth inhibition between the mutant and the wild type are identified as potential antifungal compounds. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221).

Example 72 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Fungal THR4 and a Second Fungal Strain Containing a Heterologous THR4 Gene Wild-type Magnaporthe grisea fungal cells and M grisea fungal cells lacking a functional THR4 gene and containing a Thr4 gene from Saccharomyces cerevisiae (Genbank: 6319901, 50% sequence identity) are grown under standard fungal growth conditions that are well known and described in the art. A M. grisea strain carrying a heterologous THR4 gene is made as follows:

• AM. grisea strain is made with a nonfunctional THR4 gene, such as one containing a transposon insertion in the native gene (see Examples 60 and 61).

• A construct containing a heterologous THR4 gene is made by cloning the Thr4 gene from Saccharomyces cerevisiae into a fungal expression vector containing a trpC promoter and terminator (e.g. pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41: 22) using standard molecular biology techniques that are well known and described in the art (Sambrook et al. (1989) Molecular Cloning, a Laboratory

Manual). • The said construct is used to transform the M. grisea strain lacking a functional THR4 gene (see Example 61). Transformants are selected on mimmal agar medium lacking

L-threonine. Only transformants carrying a functional THR4 gene will grow.

Wild-type strains of Magnaporthe grisea and strains containing a heterologous form of THR4 are grown under standard fungal growth conditions that are well known and described in the art. Magnaporthe grisea spores are harvested from cultures grown on complete agar medium after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemacytometer and spore suspensions are prepared in a minimal growth medium to a concentration of 2x10⁵ spores per ml. Approximately 4xl0⁴ spores or cells are harvested and added to each well of 96- well plates to which growth media is added in addition to an amount of test compound (at varying concentrations). The total volume in each well is 200μl. Wells with no test compound present, and wells without cells are included as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. The effect of each compound on the wild-type and heterologous fungal strains is measured against the growth control and the percent of inhibition is calculated as the OD₅₉o (fungal strain plus test compound) / OD_{5 0} (growth control) x 100. The percent of growth inhibition as a result of a test compound on the wild-type and heterologous fungal strains are compared. Compounds that show differential growth inhibition between the wild-type and heterologous strains are identified as potential antifungal compounds with specificity to the native or heterologous THR4 gene products. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 11 - 221).

Example 73

Pathway Specific In Vivo Assay Screening Protocol

Magnaporthe grisea fungal cells are grown under standard fungal growth conditions that are well known and described in the art. Wild-type M. grisea spores are harvested from cultures .grown on oatmeal agar media after growth for 10-13 days in the light at 25°C using a moistened cotton swab. The concentration of spores is determined using a hemocytometer and spore suspensions are prepared in a minimal growth medium and a minimal growth medium containing 4 mM L-threonine (Sigma-Aldrich Co.) to a concenfration of 2x10⁵ spores per ml. The minimal growth media contains carbon, nitrogen, phosphate, and sulfate sources, and magnesium, calcium, and trace elements (for example, see innoculating fluid in Example 63). Spore suspensions are added to each well of a 96-well microtiter plate (approximately 4x 10 spores/well). For each well containing a spore suspension in minimal media, an additional well is present contaimng a spore suspension in minimal medium containing 4 mM L-threonine. Test compounds are added to wells containing spores in minimal media and minimal media containing L- threonine. The total volume in each well is 200μl. Both minimal media and L-threonine containing media wells with no test compound are provided as controls. The plates are incubated at 25°C for seven days and optical density measurements at 590nm are taken daily. A compound is identified as a candidate for an antibiotic acting against the L- threonine biosynthetic pathway when the observed growth in the well containing minimal media is less than the observed growth in the well containing L-threonine as a result of the addition of the test compound. Similar protocols may be found in Kirsch and DiDomenico ((1994) Biotechnology 26: 111 - 221).

While the foregoing describes certain embodiments of the invention, it will be understood by those skilled in the art that variations and modifications may be made and still fall within the scope of the invention. The foregoing examples are intended to exemplify various specific embodiments of the invention and do not limit its scope in any manner.

Claims

CLAIMS What is claimed is:

1. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting an Asparagine Synthase polypeptide with a test compound; and b) detecting the presence or absence of binding between said test compound and said Asparagine Synthase polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

2. The method of claim 1, wherein said Asparagine Synthase polypeptide is a fungal Asparagine Synthase polypeptide.

3. The method of claim 1, wherein said Asparagine Synthase polypeptide is a Magnaporthe Asparagine Synthase polypeptide.

4. The method of claim 1, wherein said Asparagine Synthase polypeptide is SEQ ED NO: 3.

5. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a test compound with at least one polypeptide selected from the group consisting of: a polypeptide having at least ten consecutive amino acids of a fungal Asparagine Synthase, a polypeptide having at least 50%> sequence identity with a fungal Asparagine Synthase, and a polypeptide having at least 10%> of the activity of a fungal Asparagine Synthase; and b) detecting the presence and/or absence of binding between said test compound and said polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

6. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-aspartate, L-glutamine, and ATP with an Asparagine Synthase; b) contacting L-aspartate, L-glutamine, and ATP with Asparagine Synthase and a test compound; and c) determining the change in concentration for at least one of the following: L- aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, and or pyrophosphate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

7. The method of claim 6, wherein said Asparagine Synthase is a fungal Asparagine Synthase.

8. The method of claim 6, wherein said Asparagine Synthase is a. Magnaporthe Asparagine Synthase.

9. The method of claim 6, wherein said Asparagine Synthase is SEQ DD NO: 3.

10. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with an Asparagine Synthase; b) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with an Asparagine Synthase and a test compound; and c) determining the change in concentration for at least one of the following: L- aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, and/or pyrophosphate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

11. The method of claim 10, wherein said Asparagine Synthase is a fungal Asparagine Synthase.

12. The method of claim 10, wherein said Asparagine Synthase is a. Magnaporthe Asparagine Synthase.

13. The method of claim 10, wherein said Asparagine Synthase is SEQ ED NO: 3.

14. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-aspartate, L-glutamine, and ATP with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with Asparagine Synthase, a polypeptide having at least 50% sequence identity with an Asparagine Synthase and having at least 10% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of an Asparagine Synthase; b) contacting L-aspartate, L-glutamine, and ATP with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: L- aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, and/or pyrophosphate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

15. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate with a polypeptide selected from the group consisting of: a polypeptide having at least 50%> sequence identity with an Asparagine Synthase, a polypeptide having at least 50% sequence identity with an Asparagine Synthase and at least 10% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of an Asparagine Synthase; b) contacting L-asparagine, L-glutamate, AMP, and pyrophosphate, with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: L- aspartate, L-glutamine, L-asparagine, L-glutamate, ATP, AMP, and/or pyrophosphate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

16. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) measuring the expression of an Asparagine Synthase in a cell, cells, tissue, or an organism in the absence of a test compound; b) contacting said cell, cells, tissue, or organism with said test compound and measuring the expression of said Asparagine Synthase in said cell, cells, tissue, or organism; and c) comparing the expression of Asparagine Synthase in steps (a) and (b), wherein a lower expression in the presence of said test compound indicates that said compound is a candidate for an antibiotic.

17. The method of claim 16, wherein said cell, cells, tissue, or organism is, or is derived from a fungus.

18. The method of claim 16, wherein said cell, cells, tissue, or organism is, or is derived from a Magnaporthe fungus or fungal cell.

19. The method of claim 16, wherein said Asparagine Synthase is SEQ TD NO: 3.

20. The method of claim 16, wherein the expression of Asparagine Synthase is measured by detecting ASNl mRNA.

21. The method of claim 16, wherein the expression of Asparagine Synthase is measured by detecting Asparagine Synthase polypeptide.

22. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of an Asparagine Synthase gene, and providing comparison cells having a different form of an Asparagine Synthase gene; and b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and comparison cells in the presence of the test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said compound indicates that said compound is a candidate for an antibiotic.

23. The method of claim 22 wherein the cells and the comparison cells are fungal cells.

24. The method of claim 22, wherein the cells and the comparison cells are Magnaporthe cells.

25. The method of claim 22, wherein said form and said different form of the Asparagine Synthase are fungal Asparagine Synthases.

26. The method of claim 22, wherein at least one of the forms is a Magnaporthe Asparagine Synthase.

27. The method of claim 22, wherein said form and said different form of the Asparagine Synthase are non-fungal Asparagine Synthases.

28. The method of claim 22, wherem one form of the Asparagine Synthase is a fungal Asparagine Synthase, and the different form is a non-fungal Asparagine Synthase.

29. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a gene in the L-asparagine biochemical and/or genetic pathway and providing comparison cells having a different form of said gene; b) contacting said cells and said comparison cells with a said test compound; and c) determining the growth of said cells and said comparison cells in the presence of said test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

30. The method of claim 29, wherein the cells and the comparison cells are fungal cells.

31. The method of claim 29, wherein the cells and the comparison cells are Magnaporthe cells.

32. The method of claim 29, wherein said form and said different form of the L- asparagine biosynthesis gene are fungal L-asparagine biosynthesis genes.

33. The method of claim 29, wherein at least one of the forms is a Magnaporthe L- asparagine biosynthesis gene.

34. The method of claim 29, wherein said form and said different form of the L- asparagine biosynthesis genes are non-fungal L-asparagine biosynthesis genes.

35. The method of claim 29, wherein one form of the L-asparagine biosynthesis gene is a fungal L-asparagine biosynthesis gene, and the different form is a non-fungal L- asparagine biosynthesis gene.

36. A method for determining whether the antibiotic candidate of claim 29 has antifungal activity, further comprising: contacting a fungus or fungal cells with said antibiotic candidate and detecting a decrease in growth, viability, or pathogenicity of said fungus or fungal cells, wherein a decrease in growth, viability, or pathogenicity of said fungus or fungal cells indicates that the antibiotic candidate has antifungal activity.

37. A method for identifying a test compound as a candidate for an antibiotic, comprising:

(a) providing paired growth media comprising^'a first medium and a second medium, wherein said second medium contains a higher level of L-asparagine than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism, wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic.

38. The method of claim 37, wherein said organism is a fungus.

39. The method of claim 37, wherein said organism is Magnaporthe.

40. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide ofSEQ ED NO: 3.

41. The nucleic acid of claim 40 comprising the nucleotide sequence of SEQ DD NO: 1.

42. An expression cassette comprising the nucleic acid of claim 40.

43. The isolated nucleic acid of claim 40 comprising a nucleotide sequence with at least 50 to at least 95% sequence identity to SEQ DD NO: 1.

44. A polypeptide consisting essentially of the amino acid sequence of SEQ ED NO: 3.

45. A polypeptide comprising the amino acid sequence of SEQ DD NO: 3.

46. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a 5-Aminolevulinate synthase polypeptide with a test compound; and b) detecting the presence or absence of binding between said test compound and said 5-Aminolevulinate synthase polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

47. The method of claim 46, wherein said 5-Aminolevulinate synthase polypeptide is a fungal 5-Aminolevulinate synthase polypeptide.

48. The method of claim 46, wherein said 5-Aminolevulinate synthase polypeptide is a Magnaporthe 5-Aminolevulinate synthase polypeptide.

49. The method of claim 46, wherein said 5-Aminolevulinate synthase polypeptide is SEQ ED NO: 6.

50. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a test compound with at least one polypeptide selected from the group consisting of: a polypeptide having at least ten consecutive amino acids of a fungal 5-Aminolevulinate synthase, a polypeptide having at least 50% sequence identity with a fungal 5-Aminolevulinate synthase, and a polypeptide having at least 10% of the activity of a fungal 5-Aminolevulinate synthase; and b) detecting the presence and/or absence of binding between said test compound and said polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

51. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting succinyl-CoA and glycine with a 5-Aminolevulinate synthase; b) contacting succinyl-CoA and glycine with 5-Aminolevulinate synthase and a test compound; and c) determining the change in concentration for at least one of the following: succinyl- CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

51. The method of claim 51, wherein said 5-Aminolevulinate synthase is a fungal 5- Aminolevulinate synthase.

52. The method of claim 51, wherein said 5-Aminolevulinate synthase is a Magnaporthe 5-Aminolevulinate synthase.

53. The method of claim 51, wherein said 5-Aminolevulinate synthase is SEQ ED NO: 6.

54. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 5-aminolevulinate, CoA, and CO₂ with a 5-Aminolevulinate synthase; b) contacting 5-aminolevulinate, CoA, and CO₂ with a 5-Aminolevulinate synthase and a test compound; and c) determining the change in concentration for at least one of the following: succinyl- CoA, glycine, 5-aminolevulinate, CoA, and/or CO , wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

55. The method of claim 54, wherein said 5-Aminolevulinate synthase is a fungal 5- Aminolevulinate synthase.

56. The method of claim 54, wherein said 5-Aminolevulinate synthase is a. Magnaporthe 5-Aminolevulinate synthase.

57. The method of claim 54, wherein said 5-Aminolevulinate synthase is SEQ ED NO: 6.

58. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting succinyl-CoA and glycine with a polypeptide selected from the group consisting of: a polypeptide having at least 50%> sequence identity with 5- Aminolevulinate synthase; a polypeptide having at least 50% sequence identity with a 5-Aminolevulinate synthase and having at least 10% of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a 5- Aminolevulinate synthase; b) contacting succinyl-CoA and glycine with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: succinyl- CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

59. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 5-aminolevulinate, CoA, and CO₂ with a polypeptide selected from the group consisting of: a polypeptide having at least 50%> sequence identity with a 5- Aminolevulinate synthase; a polypeptide having at least 50% sequence identity with a 5-Aminolevulinate synthase and at least 10% of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a 5- Aminolevulinate synthase; b) contacting 5-aminolevulinate, CoA, and CO₂, with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: succinyl- CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂, wherein a change in concenfration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

60. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) measuring the expression of a 5-Aminolevulinate synthase in a cell, cells, tissue, or an organism in the absence of a test compound; b) contacting said cell, cells, tissue, or organism with said test compound and measuring the expression of said 5-Aminolevulinate synthase in said cell, cells, tissue, or organism; and c) comparing the expression of 5-Aminolevulinate synthase in steps (a) and (b), wherein a lower expression in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

61. The method of claim 60 wherein said cell, cells, tissue, or organism is, or is derived from a fungus.

62. The method of claim 60 wherein said cell, cells, tissue, or organism is, or is derived from a Magnaporthe fungus or fungal cell.

63. The method of claim 60, wherein said 5-Aminolevulinate synthase is SEQ DD NO: 6.

64. The method of claim 60, wherein the expression of 5-Aminolevulinate synthase is measured by detecting ALASl mRNA.

65. The method of claim 60, wherein the expression of 5-Aminolevulinate synthase is measured by detecting 5-Aminolevulinate synthase polypeptide.

66. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a 5-Aminolevulinate synthase gene, and providing comparison cells having a different form of a 5-Aminolevulinate synthase gene; and b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and comparison cells in the presence of the test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said compound indicates that said compound is a candidate for an antibiotic.

67. The method of claim 66 wherein the cells and the comparison cells are fungal cells.

68. The method of claim 66 wherein the cells and the comparison cells are Magnaporthe cells.

69. The method of claim 66 wherem said form and said comparison form of the 5- Aminolevulinate synthase are fungal 5-Aminolevulinate synthases.

70. The method of claim 66, wherein at least one of the forms is a Magnaporthe 5- Aminolevulinate synthase.

71. The method of claim 66 wherein said form and said comparison form of the 5- Aminolevulinate synthase are non-fungal 5-Aminolevulinate synthases.

72. The method of claim 66 wherein one form of the 5-Aminolevulinate synthase is a fungal 5-Aminolevulinate synthase, and the other form is a non-fungal 5- Aminolevulinate synthase.

73. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a gene in the heme biochemical and/or genetic pathway and providing comparison cells having a different form of said gene. b) contacting said cells and said comparison cells with a test compound, c) determining the growth of said cells and said comparison cells in the presence of said test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

74. The method of claim 73 wherein the cells and the comparison cells are fungal cells.

75. The method of claim 73 wherein the cells and the comparison cells are Magnaporthe cells.

76. The method of claim 73 wherein said form and said different form of the heme biosynthesis gene are fungal heme biosynthesis genes.

77. The method of claim 73, wherein at least one form is a Magnaporthe heme biosynthesis gene.

78. The method of claim 73 wherein said form and said different form of the heme biosynthesis genes are non-fungal heme biosynthesis genes.

79. The method of claim73 wherein one form of the heme biosynthesis gene is a fungal heme biosynthesis gene, and the different form is a non-fungal heme biosynthesis gene.

80. A method for identifying a test compound as a candidate for an antibiotic, comprising:

(a) providing paired growth media; comprising a first medium and a second medium, wherein said second medium contains a higher level of 5-aminolevulinate than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism, wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic.

81. The method of claim 80, wherein said organism is a fungus.

82. The method of claim 80, wherein said organism is Magnaporthe.

83. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide ofSEQ DD NO: 6.

84. The nucleic acid of claim 83 comprising the nucleotide sequence of SEQ ED NO: 4.

85. An expression cassette comprising the nucleic acid of claim 83.

86. The isolated nucleic acid of claim 83 comprising a nucleotide sequence with at least 50 to at least 95% sequence identity to SEQ ID NO: 4.

87. A polypeptide consisting essentially of the amino acid sequence of SEQ ED NO: 6.

88. A polypeptide comprising the amino acid sequence of SEQ ED NO: 6.

89. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a histidinol-phosphatase polypeptide with a test compound; and b) detecting the presence or absence of binding between said test compound and said histidinol-phosphatase polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

90. . The method of claim 89, wherein said histidinol-phosphatase polypeptide is a fungal histidinol-phosphatase polypeptide.

91. The method of claim 89, wherein said histidinol-phosphatase polypeptide is a Magnaporthe histidinol-phosphatase polypeptide.

92. The method of claini 89, wherein said histidinol-phosphatase polypeptide is SEQ ED NO: 9.

93. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a test compound with at least one polypeptide selected from the group consisting of: a polypeptide having at least ten consecutive amino acids of a fungal histidinol-phosphatase; a polypeptide having at least 50%ι sequence identity with; and a polypeptide having at least 10% of the activity of a fungal histidinol- phosphatase; and b) detecting the presence and or absence of binding between said test compound and said polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

94. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-histidinol phosphate and H₂O with a histidinol-phosphatase; b) contacting L-histidinol phosphate and H O with histidinol-phosphatase and a test compound; and c) determining the change in concentration for at least one of the following: L- histidinol phosphate, H₂O, L-histidinol, and/or orthophosphate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

95. The method of claim 94, wherein said histidinol-phosphatase is a fungal histidinol- phosphatase.

96. The method of claim 94, wherein said histidinol-phosphatase is a Magnaporthe histidinol-phosphatase.

97. The method of claim 94, wherein said histidinol-phosphatase is SEQ D NO: 9.

98. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-histidinol and orthophosphate with a histidinol-phosphatase; b) contacting L-histidinol and orthophosphate with a histidinol-phosphatase and a test compound; and c) determining the change in concentration for at least one of the following: L- histidinol phosphate, H₂O, L-histidinol, and/or orthophosphate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

99. The method of claim 98, wherein said histidinol-phosphatase is a fungal histidinol- phosphatase.

100. The method of claim 98, wherein said histidinol-phosphatase is a Magnaporthe histidinol-phosphatase.

101. The method of claim 98, wherein said histidinol-phosphatase is SEQ ED NO: 9.

102. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-histidinol phosphate and H₂O with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with histidinol-phosphatase; a polypeptide having at least 50% sequence identity with a histidinol-phosphatase and having at least 10%o of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a histidinol- phosphatase; b) contacting L-histidinol phosphate and H O with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: L- histidinol phosphate, H₂O, L-histidinol, and/or orthophosphate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

103. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-histidinol and orthophosphate with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with a histidinol-phosphatase; a polypeptide having at least 50%> sequence identity with a histidinol-phosphatase and at least 10% of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a histidinol-phosphatase; b) contacting L-histidinol and orthophosphate, with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: L- histidinol phosphate, H₂O, L-histidinol, and/or orthophosphate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

104. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) measuring the expression of a histidinol-phosphatase in a cell, cells, tissue, or an organism in the absence of a test compound; b) contacting said cell, cells, tissue, or organism with said test compound and measuring the expression of said histidinol-phosphatase in said cell, cells, tissue, or organism; and c) comparing the expression of histidinol-phosphatase in steps (a) and (b), wherein a lower expression in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

105. The method of claim 104 wherein said cell, cells, tissue, or organism is, or is derived from a fungus.

106. The method of claim 104 wherein said cell, cells, tissue, or organism is, or is derived from a Magnaporthe fungus or fungal cell.

107. The method of claim 104, wherein said histidinol-phosphatase is SEQ DD NO: 9.

108. The method of claim 104, wherein the expression of histidinol-phosphatase is measured by detecting HISPl mRNA.

109. The method of claim 104, wherein the expression of histidinol-phosphatase is measured by detecting histidinol-phosphatase polypeptide.

110. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a histidinol-phosphatase gene, and providing comparison cells having a different form of a histidinol-phosphatase gene; and b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and comparison cells in the presence of the test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said compound indicates that said compound is a candidate for an antibiotic.

111. The method of claim 110 wherein the cells and the comparison cells are fungal cells.

112. The method of claim 110 wherein the cells and the comparison cells are Magnaporthe cells.

113. The method of claim 110 wherein said form and said comparison form of the histidinol-phosphatase are fungal histidinol-phosphatases.

114. The method of claim 110, wherein at least one of the forms is a Magnaporthe histidinol-phosphatase.

115. The method of claim 110 wherein said form and said comparison form of the histidinol-phosphatase are non-fungal histidinol-phosphatases.

116. The method of claim 110 wherein one form of the histidinol-phosphatase is a fungal histidinol-phosphatase, and the other form is a non-fungal histidinol-phosphatase.

117. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a gene in the L-histidine biochemical and/or genetic pathway and providing comparison cells having a different form of said gene. b) contacting said cells and said comparison cells with a test compound, c) determining the growth of said cells and said comparison cells in the presence of said test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

118. The method of claim 117 wherein the cells and the comparison cells are fungal cells.

119. The method of claim 117 wherein the cells and the comparison cells are Magnaporthe cells.

120. The method of claim 117 wherein said form and said different form of the L- histidine biosynthesis gene are fungal L-histidine biosynthesis genes.

121. The method of claim 117, wherein at least one form is a Magnaporthe L-histidine biosynthesis gene.

122. The method of claim 117 wherein said form and said different form of the L- histidine biosynthesis genes are non-fungal L-histidine biosynthesis genes.

123. The method of claim 117 wherein one form of the L-histidine biosynthesis gene is a fungal L-histidine biosynthesis gene, and the different form is a non-fungal L-histidine biosynthesis gene.

124. A method for identifying a test compound as a candidate for an antibiotic, comprising:

(a) providing paired growth media; comprising a first medium and a second medium, wherein said second medium contains a higher level of L-histidine than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism, wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic.

125. The method of claim 124, wherein said organism is a fungus.

126. The method of claim 124, wherein said organism is Magnaporthe.

127. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide of SEQ DD 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.

130. The isolated nucleic acid of claim 127 comprising a nucleotide sequence with at least 50 to at least 95% sequence identity to SEQ DD NO: 7.

131. A polypeptide consisting essentially of the amino acid sequence of SEQ DD NO: 9

132. A polypeptide comprising the amino acid sequence of SEQ DD NO: 9.

133. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a 3-Isopropylmalate dehydratase polypeptide with said test compound; and b) detecting the presence or absence of binding between a test compound and said 3- Isopropylmalate dehydratase polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

134. The method of claim 133, wherein said 3-Isopropylmalate dehydratase polypeptide is a fungal 3-Isopropylmalate dehydratase polypeptide.

135. The method of claim 133, wherein said 3-Isopropylmalate dehydratase polypeptide is a Magnaporthe 3-Isopropylmalate dehydratase polypeptide.

136. The method of claim 133, wherein said 3-Isopropylmalate dehydratase polypeptide is SEQ DD NO: 12.

137. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a test compound with at least one polypeptide selected from the group consisting of: a polypeptide having at least ten consecutive amino acids of a fungal 3-Isopropylmalate dehydratase; a polypeptide having at least 50% sequence identity with a fungal 3-Isopropylmalate dehydratase; and a polypeptide having at least 10% of the activity of a fungal 3-Isopropylmalate dehydratase; and b) detecting the presence and/or absence of binding between said test compound and said polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

138. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 2-Isopropylmalate and H₂O with a 3-Isopropylmalate dehydratase; b) contacting 2-Isopropylmalate and H₂O with 3-Isopropylmalate dehydratase and a test compound; and c) determining the change in concentration for at least one of the following: 2- Isopropylmalate, H₂O, and/or 3-Isopropylmalate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

139. The method of claim 138, wherein said 3-Isopropylmalate dehydratase is a fungal 3- Isopropylmalate dehydratase.

140. The method of claim 138, wherein said 3-Isopropylmalate dehydratase is a Magnaporthe 3-Isopropylmalate dehydratase.

141. The method of claim 138, wherein said 3-Isopropylmalate dehydratase is SEQ DD NO: 12.

142. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 3-Isopropylmalate with a 3-Isopropylmalate dehydratase; b) contacting 3-Isopropylmalate with a 3-Isopropylmalate dehydratase and a test compound; and c) determining the change in concenfration for at least one of the following: 2- Isopropylmalate, H₂O, and or 3-Isopropylmalate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

143. The method of claim 142, wherein said 3-Isopropylmalate dehydratase is a fungal 3- Isopropylmalate dehydratase.

144. The method of claim 142, wherein said 3-Isopropylmalate dehydratase is a Magnaporthe 3-Isopropylmalate dehydratase.

145. The method of claim 142, wherein said 3-Isopropylmalate dehydratase is SEQ DD NO: 12.

146. A method for determining whether the antibiotic candidate of claim 142 has antifungal activity, further comprising: contacting a fungus or fungal cells with said antibiotic candidate and detecting a decrease in growth, viability, or pathogenicity of said fungus or fungal cells.

147. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 2-Isopropylmalate and H₂O with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with 3- Isopropylmalate dehydratase; a polypeptide having at least 50% sequence identity with a 3-Isopropylmalate dehydratase and having at least 10% of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a 3-

, Isopropylmalate dehydratase; b) contacting 2-Isopropylmalate and H₂O with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: 2- Isopropylmalate, H₂O, and/or 3-Isopropylmalate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

148. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting 3-Isopropylmalate with a polypeptide selected from the group consisting of: a polypeptide having at least 50%> sequence identity with a 3-Isopropylmalate dehydratase; a polypeptide having at least 50% sequence identity with a 3- Isopropylmalate dehydratase and at least 10% of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a 3- Isopropylmalate dehydratase; b) contacting 3-Isopropylmalate, with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: 2- Isopropylmalate, H₂O, and/or 3-Isopropylmalate, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

149. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) measuring the expression of a 3-Isopropylmalate dehydratase in a cell, cells, tissue, or an organism in the absence of a test compound; b) contacting said cell, cells, tissue, or organism with said test compound and measuring the expression of said 3-Isopropylmalate dehydratase in said cell, cells, tissue, or organism; and c) comparing the expression of 3-Isopropylmalate dehydratase in steps (a) and (b), wherein a lower expression in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

150. The method of claim 149 wherein said cell, cells, tissue, or organism is, or is derived from a fungus.

151. The method of claim 149 wherein said cell, cells, tissue, or organism is, or is derived from a Magnaporthe fungus or fungal cell.

152. The method of claim 149, wherein said 3-Isopropylmalate dehydratase is SEQ DD NO: 12.

153. The method of claim 149, wherein the expression of 3-Isopropylmalate dehydratase is measured by detecting IPMDl mRNA.

154. The method of claim 149, wherein the expression of 3-Isopropylmalate dehydratase is measured by detecting 3-Isopropylmalate dehydratase polypeptide.

155. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a 3-Isopropylmalate dehydratase gene, and providing comparison cells having a different form of a 3-Isopropylmalate dehydratase gene; and b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and comparison cells in the presence of the test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said compound indicates that said compound is a candidate for an antibiotic.

156. The method of claim 155 wherein the cells and the comparison cells are fungal cells.

157. The method of claim 155 wherein the cells and the comparison cells are Magnaporthe cells.

158. The method of claim 155 wherein said form and said comparison form of the 3- Isopropylmalate dehydratase are fungal 3-Isopropylmalate dehydratases.

159. The method of claim 155, wherein at least one of the forms is a Magnaporthe 3- Isopropylmalate dehydratase.

160. The method of claim 155 wherein said form and said comparison form of the 3- Isopropylmalate dehydratase are non-fungal 3-Isopropylmalate dehydratases.

161. The method of claim 155 wherein one form of the 3-Isopropylmalate dehydratase is a fungal 3-Isopropylmalate dehydratase, and the other form is a non-fungal 3- Isopropylmalate dehydratase.

162. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a gene in the L-leucine biochemical and/or genetic pathway and providing comparison cells having a different form of said gene. b) contacting said cells and said comparison cells with a said test compound, c) determining the growth of said cells and said comparison cells in the presence of said test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

163. The method of claim 162 wherein the cells and the comparison cells are fungal cells.

164. The method of claim 162 wherein the cells and the comparison cells are Magnaporthe cells.

165. The method of claim 162 wherein said form and said different form of the L-leucine biosynthesis gene are fungal L-leucine biosynthesis genes.

166. The method of claim 162, wherein at least one form is a Magnaporthe L-leucine biosynthesis gene.

167. The method of claim 162 wherein said form and said different form of the L-leucine biosynthesis genes are non-fungal L-leucine biosynthesis genes.

168. The method of claim 162 wherein one form of the L-leucine biosynthesis gene is a fungal L-leucine biosynthesis gene, and the different form is a non-fungal L-leucine biosynthesis gene.

169. A method for identifying a test compound as a candidate for an antibiotic, comprising:

(a) providing paired growth media; comprising a first medium and a second medium, wherein said second medium contains a higher level of L-leucine than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism, wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic.

170. The method of claim 169, wherein said organism is a fungus.

171. The method of claim 169, wherein said organism is Magnaporthe.

172. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide of SEQ DD NO: 12.

173. The nucleic acid of claim 172 comprising the nucleotide sequence of SEQ DD NO: 10.

174. An expression cassette comprising the nucleic acid of claim 173.

175. The isolated nucleic acid of claim 172 comprising a nucleotide sequence with at least 50 to at least 95% sequence identity to SEQ ED NO: 10.

176. A polypeptide consisting essentially of the amino acid sequence of SEQ ED NO: 12

177. A polypeptide comprising the amino acid sequence of SEQ ED NO: 12.

178. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a Threonine synthase polypeptide with said test compound; and b) detecting the presence or absence of binding between a test compound and said Threonine synthase polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

179. The method of claim 178, wherein said Threonine synthase polypeptide is a fungal Threonine synthase polypeptide.

180. The method of claim 178, wherein said Threonine synthase polypeptide is a Magnaporthe Threonine synthase polypeptide.

181. The method of claim 178, wherein said Threonine synthase polypeptide is SEQ ED NO: 15.

182. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting a test compound with at least one polypeptide selected from the group consisting of: a polypeptide having at least ten consecutive amino acids of a fungal Threonine synthase, and a polypeptide having at least 50% sequence identity with a fungal Threonine synthase, and a polypeptide having at least 10% of the activity of a fungal Threonine synthase; and b) detecting the presence and or absence of binding between said test compound and said polypeptide, wherein binding indicates that said test compound is a candidate for an antibiotic.

183. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting O-phospho-L-homoserine and water with a Threonine synthase; b) contacting O-phospho-L-homoserine and water with Threonine synthase and a test compound; and c) determining the change in concentration for at least one of the following: O- phospho-L-homoserine, L-threonine, orthophosphate, and water, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

184. The method of claim 183, wherein said Threonine synthase is a fungal Threonine synthase.

185. The method of claim 183, wherein said Threonine synthase is a Magnaporthe Threonine synthase.

186. The method of claim 183, wherein said Threonine synthase is SEQ TD NO: 15.

187. A method for determining whether the antibiotic candidate of claim 8 has antifungal activity, further comprising contacting a fungus or fungal cells with said antibiotic candidate and detecting a decrease in growth, viability, or pathogenicity of said fungus or fungal cells.

188. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-threonine and orthophosphate with a Threonine synthase; b) contacting L-threonine and orthophosphate with a Threonine synthase and a test compound; and c) determining the change in concenfration for at least one of the following: O- phospho-L-homoserine, L-threonine, orthophosphate, and water, wherein a change in concentration for any of the above substances between 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.

190. The method of claim 188, wherein said Threonine synthase is a Magnaporthe Threonine synthase.

191. The method of claim 188, wherein said Threonine synthase is SEQ ED NO: 15.

192. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting O-phospho-L-homoserine and water with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with Threonine synthase, and a polypeptide having at least 50% sequence identity with a Threonine synthase and having at least 10%> of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a Threonine synthase; b) contacting O-phospho-L-homoserine and water with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: O- phospho-L-homoserine, L-threonine, orthophosphate, and water, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

193. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) contacting L-threonine and orthophosphate with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with a Threonine synthase, and a polypeptide having at least 50% sequence identity with a Threonine synthase and at least 10% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a Threonine synthase; b) contacting L-threonine and orthophosphate, with said polypeptide and a test compound; and c) determining the change in concentration for at least one of the following: O- phospho-L-homoserine, L-threonine, orthophosphate, and water, wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

194. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) measuring the expression of a Threonine synthase in a cell, cells, tissue, or an organism in the absence of a test compound; b) contacting said cell, cells, tissue, or organism with said test compound and measuring the expression of said Threonine synthase in said cell, cells, tissue, or orgamsm; and c) comparing the expression of Threonine synthase in steps (a) and (b), wherein a lower expression in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

195. The method of claim 194, wherein said cell, cells, tissue, or organism is, or is derived from a fungus.

196. The method of claim 194, wherein said cell, cells, tissue, or organism is, or is derived from a Magnaporthe fungus or fungal cell.

197. The method of claim 194, wherein said Threonine synthase is SEQ D NO: 15.

198. The method of claim 194, wherein the expression of Threonine synthase is measured by detecting THR4 mRNA.

199. The method of claim 194, wherein the expression of Threonine synthase is measured by detecting Threonine synthase polypeptide.

200. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a Threonine synthase gene, and providing comparison cells having a different form of a Threonine synthase gene; and b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and comparison cells in the presence of the test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said compound indicates that said compound is a candidate for an antibiotic.

201. The method of claim 200 wherein the cells and the comparison cells are fungal cells.

202. The method of claim 200 wherein the cells and the comparison cells are Magnaporthe cells.

203. The method of claim 200 wherein said form and said different form of the Threonine synthase are fungal Threonine synthases.

204. The method of claim 200, wherein at least one of the forms is a Magnaporthe Threonine synthase.

205. The method of claim 200, wherein said form and said different form of the Threonine synthase are non-fungal Threonine synthases.

206. The method of claim 200, wherein one form of the Threonine synthase is a fungal Threonine synthase, and the different form is a non-fungal Threonine synthase.

207. A method for identifying a test compound as a candidate for an antibiotic, comprising: a) providing cells having one form of a gene in the L-threonine biochemical and/or genetic pathway and providing comparison cells having a different form of said gene; b) contacting said cells and said comparison cells with a said test compound; and c) determining the growth of said cells and said comparison cells in the presence of said test compound, wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

208. The method of claim 207, wherein the cells and the comparison cells are fungal cells.

209. The method of claim 207, wherein the cells and the comparison cells are Magnaporthe cells.

210. The method of claim 207, wherein said form and said different form of the L- threonine biosynthesis gene are fungal L-threonine biosynthesis genes.

211. The method of claim 207, wherein at least one of the forms is a Magnaporthe L- threonine biosynthesis gene.

212. The method of claim 207, wherein said form and said different form of the L- threonine biosynthesis genes are non-fungal L-threonine biosynthesis genes.

213. The method of claim 207, wherein one form of the L-threonine biosynthesis gene is a fungal L-threonine biosynthesis gene, and the different form is a non- fungal L- threonine biosynthesis gene.

214. A method for identifying a test compound as a candidate for an antibiotic, comprising:

(a) providing paired growth media comprising a first medium and a second medium, wherein said second medium contains a higher level of L-threonine than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism, wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic.

215. The method of claim 214, wherein said organism is a fungus.

216. The method of claim 214, wherein said organism is Magnaporthe.

217. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide of SEQ DD NO: 15.

218. The nucleic acid of claim 217 comprising the nucleotide sequence of SEQ DD NO: 13.

219. An expression cassette comprising the nucleic acid of claim 218.

220. The isolated nucleic acid of claim 217 comprising a nucleotide sequence with at least 50 to at least 95% sequence identity to SEQ DD NO: 13.

221. A polypeptide consisting essentially of the amino acid sequence of SEQ ED NO: 15.

222. A polypeptide comprising the amino acid sequence of SEQ ED NO: 15.