WO2005005602A2 - Procedes d'identification d'inhibiteurs de fumarate reductase et proteine associee au tri des proteines cellulaires en tant qu'antibiotiques - Google Patents

Procedes d'identification d'inhibiteurs de fumarate reductase et proteine associee au tri des proteines cellulaires en tant qu'antibiotiques Download PDF

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WO2005005602A2
WO2005005602A2 PCT/US2004/018361 US2004018361W WO2005005602A2 WO 2005005602 A2 WO2005005602 A2 WO 2005005602A2 US 2004018361 W US2004018361 W US 2004018361W WO 2005005602 A2 WO2005005602 A2 WO 2005005602A2
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seq
test compound
vps
fumarate reductase
polypeptide
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PCT/US2004/018361
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WO2005005602A3 (fr
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Matthew M. Tanzer
Jeffrey Shuster
Lisbeth Hamer
Kiichi Adachi
Todd M. Dezwaan
Sze-Chung C. Lo
Maria V. Montenegro-Chamorro
Blaise A. Darveaux
Sheryl A. Frank
Ryan W. Heiniger
Sanjoy K. Mahanty
Huaqin Pan
Amy S. Covington
Rex Tarpey
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Paradigm Genetics, Inc.
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Publication of WO2005005602A2 publication Critical patent/WO2005005602A2/fr
Publication of WO2005005602A3 publication Critical patent/WO2005005602A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • the invention relates generally to methods for the identification of antibiotics.
  • Filamentous fungi are 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 Magnaporthe grisea, the fungus that causes rice blast disease, a significant threat to food supplies worldwide.
  • plant pathogens of economic importance include the pathogens in the genera Agaricus, Alterna ⁇ a, 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, Dip
  • Plasmodiophora Plasmopara, Pseudoperonospora, Pythium, Sclerophthora, and others. Oomycetes 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 Aspergilli, and others.
  • Control of fungal diseases in plants and animals is usually mediated by chemicals that inhibit growth, proliferation, and/or pathogenicity of fungal organisms.
  • 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, 27 Microb. Pathog.
  • Hensel et al. Hensel, M. et al, 258 Mol. Gen. Genet. 553 (1998) (PMID: 9669338) showed only moderate effects of the deletion of the are A transcriptional activator on the pathogenicity of
  • the present invention discloses fumarate reductase and vacuolar protein sorting- associated protein as targets for the identification of antifungal, biocide, and biostatic materials.
  • the present inventors have discovered that in vivo disruption of the gene encoding fumarate reductase in Magnaporthe grisea severely reduces the pathogenicity of the fungus.
  • the present inventors have discovered that in vivo disruption of the gene encoding vacuolar protein sorting-associated protein (VPS 17) in Magnaporthe grisea severely reduces the pathogenicity of the fungus.
  • VPS 17 vacuolar protein sorting-associated protein
  • fumarate reductase and vacuolar protein sorting-associated protein are useful as targets for the identification of antibiotics, preferably fungicides.
  • the present invention provides methods for the identification of compounds that inhibit fumarate reductase or vacuolar protein sorting-associated protein expression or activity. Methods of the invention are useful for the identification of antibiotics, preferably fungicides.
  • FIGURES Figure 1. Diagram of the reversible reaction catalyzed by fumarate reductase. The enzyme catalyzes the reversible interconversion of fumarate and ubiquinol to succinate and ubiquinone.
  • CO39 was inoculated with wild-type strain Guyl 1 and transposon insertion strains Kl-12 and Kl-19. Leaf segments were imaged at five days post-inoculation.
  • Figure 3 Digital image showing the effect of VPS 17 gene disruption on Magnaporthe grisea pathogenicity using whole plant infection assays.
  • Rice variety CO39 was inoculated with wild-type strain Guyl 1 and transposon insertion strains KO1- 22 and KO1-28. Leaf segments were imaged at five days post-inoculation.
  • 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.
  • antipathogenic refers to a mutant form of a gene that inactivates a pathogenic activity of an organism on its host organism or substantially reduces the level of pathogenic activity, wherein “substantially” means a reduction at least as great as the standard deviation for a measurement, preferably a reduction to 50% activity, more preferably a reduction of at least one magnitude, i.e.
  • the pathogenic activity affected may be an aspect of pathogenic activity governed by the normal form of the gene, or the pathway the normal form of the gene functions on, or the pathogenic activity of the organism in general.
  • Antipathogenic may also refer to a cell, cells, tissue, or organism that contains the mutant form of a gene; a phenotype associated with the mutant form of a gene, and/or associated with a cell, cells, tissue, or organism that contain the mutant form of a gene.
  • 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.
  • 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.
  • 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.
  • the term "conditional lethal" refers to a mutation permitting growth and/or survival only under special growth or environmental conditions.
  • the term "cosmid” refers to a hybrid vector used in gene cloning that includes a cos site (from the lambda bacteriophage).
  • the cosmids of the invention comprise drug resistance marker genes and other plasmid genes. Cosmids are especially suitable for cloning large genes or multigene fragments.
  • the terms “fumarate reductase” and “fumarate reductase polypeptide” refer to an enzyme that catalyzes the reversible interconversion of succinate and ubiquinone to fumarate and ubiquinol.
  • the terms “fumarate reductase,” “FRD1” and “FRD1 gene product” are intended to encompass any polypeptide that catalyzes the reversible interconversion of succinate and ubiquinone to fumarate and ubiquinol.
  • the phrase “fumarate reductase gene” includes the FRDl gene from M. grisea as well as genes from other organisms that encode a polypeptide that catalyzes the reversible interconversion of succinate and ubiquinone to fumarate and ubiquinol.
  • Fungi 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.
  • Fungi exist as single cells or 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 lacking the ability to manufacture their own food by photosynthesis due to the absence of chlorophyll, are either parasites on other organisms or saprotrophs feeding on dead organic matter. 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 fungi 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.
  • fungicide 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.
  • gene should be understood to refer to a unit of heredity. Each gene is composed of a linear chain of deoxyribonucleotides that can be referred to by the sequence of nucleotides forming the chain. Thus, “sequence” is used to indicate both the ordered listing of the nucleotides which form the chain, and the chain having that sequence of nucleotides.
  • 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.
  • growth or “cell growth” of an organism refer to an increase in mass, density, or number of cells of the organism.
  • 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.
  • a growth conditional phenotype is described with respect to a single growth culture parameter, such as temperature.
  • a temperature (or heat-sensitive) mutant i.e., a fungal strain having a heat-sensitive phenotype
  • 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.
  • heterologous fumarate reductase means either a nucleic acid encoding a polypeptide or a polypeptide, wherein the polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, or 99% sequence identity or each integer unit of sequence identity from 50-100% in ascending order to M.
  • grisea fumarate reductase protein SEQ ID NO:3 and at least 10%, 25%, 50%, 75%, 80%), 90%, 95%, or 99% activity or each integer unit of activity from 10-100% in ascending order of the activity of M.
  • grisea fumarate reductase protein SEQ ID NO:3
  • heterologous fumarate reductases include, but are not limited to, putative flavoprotein subunit from Schizosaccharomyces pombe and hypothetical FAD-dependent oxidoreductase FRDl from Saccharomyces cerevisiae.
  • heterologous VPS 17 means either a nucleic acid encoding a polypeptide or a polypeptide, wherein the polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%o sequence identity or each integer unit of sequence identity from 50-100% in ascending order to M. grisea VPS17 (SEQ ID NO:6) and at least 10%, 25%, 50%, 75%, 80%, 90%, 95%o, or 99% activity or each integer unit of activity from 10-100% in ascending order of the activity of M. grisea VPS 17 (SEQ ID NO:6).
  • heterologous VPS17s include, but are not limited to, VPS17 protein from
  • His-Tag refers to an encoded polypeptide consisting of multiple consecutive histidine amino acids.
  • hph hygromycin B phosphotransferase
  • hygromycin resistance gene refer to a hygromycin phosphotransferase gene or gene product.
  • imperfect state refers to a classification of a fungal organism having no demonstrable sexual life stage.
  • inhibitor refers to a chemical substance that inactivates the enzymatic activity of fumarate reductase or substantially reduces the level of enzymatic activity, wherein “substantially” means a reduction at least as great as the standard deviation for a measurement, preferably a reduction to 50% activity, more preferably a reduction of at least one magnitude, i.e to 10% activity.
  • 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.
  • the introduced polynucleotide may be present on an extra-chromosomal non-replicating vector and be transiently expressed or transiently active.
  • 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.
  • the term “mutant form” of a gene refers to a gene which has been altered, either naturally or artificially, by 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.
  • a normal form of a gene 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-NTA" refers to nickel sepharose.
  • a "normal" form of a gene 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.
  • the term "pathogenicity” refers to a capability of causing disease and/or degree of capacity to cause disease.
  • pathogenicity encompass the general capability of causing disease as well as various mechanisms and structural and/or functional deviations from normal used in the art to describe the causative factors and/or mechanisms, presence, pathology, and/or progress of disease, such as virulence, host recognition, cell wall degradation, toxin production, infection hyphae, penetration peg production, appressorium production, lesion formation, sporulation, and the like.
  • the "percent (%) sequence identity" between two polynucleotide or two polypeptide sequences is determined according to either the BLAST program (Basic Local Alignment Search Tool, (Altschul, S.F.
  • 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.
  • polypeptides may contain amino acid analogs and other modifications, including, but not limited to glycosylated or phosphorylated residues.
  • proliferation is synonymous to the term “growth.”
  • 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.
  • Such intermediate growth rate may be due to a mutant cellular component that 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.
  • Stress phenotype refers to a phenotype that exhibits either hypersensitivity or hyposensitivity.
  • specific binding refers to an interaction between fumarate reductase and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence and/or the tertiary conformation of fumarate reductase.
  • a “fumarate reductase ligand” is an example of specific binding.
  • specific binding also refers to an interaction between VPS 17 and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence and/or the tertiary conformation of VPS 17.
  • a “VPS 17 ligand” is an example of specific binding.
  • Transform 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, electroporation, polyethylene glycol mediated uptake, particle bombardment, agrotransformation, and the like. The transformation 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.
  • 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.
  • Teween 20 means sorbitan mono-9-octadecenoate poly(oxy-l , 1 -ethanediyl).
  • the terms “vacuolar protein sorting-associated protein (VPS 17),” “VPS 17,” and “vacuolar protein sorting-associated protein (VPS 17) polypeptide” refer to a protein that is required for sorting and delivery of vacuolar hydrolases. Although the protein and/or the name of the gene that encodes the protein may differ between species, the terms “vacuolar protein sorting-associated protein (VPS 17),” “VPS 17,” and “VPS 17 gene product” are intended to encompass any polypeptide that sorts and delivers vacuolar hydrolases.
  • the phrase “VPS 17 gene” includes the VPS 17 gene from M. grisea as wen as genes trom other organisms that encode a polypeptide that sorts and delivers vacuolar hydrolases.
  • the term "viability" of an organism refers to the ability of an organism to demonstrate growth under conditions appropriate for the organism, or to demonstrate an active cellular function.
  • 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 FRDl gene and/or gene product reduces the pathogenicity o ⁇ Magnaporthe grisea.
  • the inventors are the first to demonstrate that fumarate reductase is a target for antibiotics, preferably antifungals.
  • the invention provides methods for identifying compounds that inhibit fumarate reductase 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 FRDl gene expression.
  • the compounds identified by the methods of the invention are useful as antibiotics.
  • the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising contacting a fu arate reductase polypeptide with a test compound and detecting the presence or absence of binding between the test compound and the fumarate reductase polypeptide, wherein binding indicates that the test compound is a candidate for an antibiotic.
  • Fumarate reductase polypeptides of the invention have the amino acid sequence of a naturally occurring fumarate reductase found in a fungus, animal, plant, or microorganism, or have an amino acid sequence derived from a naturally occurring sequence.
  • the fumarate reductase is a fungal fumarate reductase.
  • a cDNA encoding M. grisea fumarate reductase protein is set forth in SEQ ID NO:l
  • an M. grisea fumarate reductase genomic DNA is set forth in SEQ ED NO:2
  • an M. grisea fumarate reductase polypeptide is set forth in SEQ ID NO:3.
  • the fumarate reductase is a Magnaporthe fumarate reductase.
  • Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe oryzae, Magnaporthe grisea and Magnaporthe poae and the imperfect states of Magnaporthe in the genus Pyricularia.
  • the Magnaporthe fumarate reductase is from Magnaporthe grisea.
  • the invention provides a polypeptide consisting essentially of SEQ ED NO:3.
  • a polypeptide consisting essentially of SEQ ED NO:3 has at least 90% sequence identity with M.
  • grisea fumarate reductase (SEQ ED NO:3) and at least 10% of the activity of SEQ ED NO:3.
  • a polypeptide consisting essentially of SEQ ID NO:3 has at least 90%, 91%, 92%, 93%, 94%,, 95%, 96%>, 97%, 98%, or 99% sequence identity with SEQ ID NO:3 and at least 25%>, 50%), 75%>, or 90% of the activity of M. grisea fumarate reductase.
  • polypeptides consisting essentially of SEQ ED NO: 3 include, but are not limited to, polypeptides having the amino acid sequence of SEQ ED NO:3 with the exception that one or more of the amino acids are substituted with structurally similar amino acids providing a conservative amino acid substitution. Conservative amino acid substitutions are well known to those of skill in the art. Examples of polypeptides consisting essentially of SEQ ID NO: 3 include polypeptides having 1, 2, or 3 conservative amino acid substitutions relative to SEQ ED NO:3. Other examples of polypeptides consisting essentially of SEQ ED NO:3 include polypeptides having the sequence of SEQ ED NO:3, but with truncations at either or both the 3' and the 5' end.
  • polypeptides consisting essentially of SEQ ED NO:3 include polypeptides having 1, 2, or 3 amino acids residues removed from either or both 3' and 5' ends relative to SEQ ID NO:3.
  • the fumarate reductase 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 (Moni
  • fragments of a fumarate reductase polypeptide are useful in the methods of the invention.
  • the fumarate reductase fragments include an intact or nearly intact epitope that occurs on the biologically active wild-type fumarate reductase.
  • the fragments comprise at least 10 consecutive amino acids of fumarate reductase set forth in SEQ ED NO:3.
  • the fragments comprise at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 300, 325, 350, 375, 400, 425, 450, 475, 500,525, 550, 575, 600, 610 or at least 615 consecutive amino acids residues of fumarate reductase set forth in SEQ ED NO:3. Fragments of heterologous fumarate reductases are also useful in the methods of the invention.
  • polypeptides having at least 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%o, 96%o, 97%), 98%) or 99%> sequence identity with at least 50 consecutive amino acid residues of SEQ ED NO: 3 are useful in the methods of the invention.
  • the fragment is from a Magnaporthe fumarate reductase.
  • the fragment contains an amino acid sequence conserved among fungal fumarate reductases.
  • Polypeptides having at least 35% sequence identity with M. grisea fumarate reductase (SEQ ED NO:3) protein are also useful in the methods of the invention.
  • sequence identity is at least 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%) or 99%, or any integer from 35-100% sequence identity in ascending order with M. grisea fumarate reductase (SEQ ID NO:3) protein.
  • polypeptides of the invention have at least 10% of the activity of M. grisea fumarate reductase (SEQ ED NO:3) protein.
  • Fumarate reductase polypeptides of the invention have at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%>, 80%, 85%> or at least 90% of the activity of M. grisea fumarate reductase (SEQ ED « NO:3) protein.
  • the invention provides a method for identifying a test compound as a candidate for a fungicide, comprising: contacting a test compound with at least one polypeptide selected from the group consisting of: a polypeptide consisting essentially of SEQ ED NO:3, a polypeptide having at least ten consecutive amino acids of an grisea fumarate reductase (SEQ ED NO:3) protein, a polypeptide having at least 35% sequence identity with an M. grisea fumarate reductase (SEQ ID NO:3) protein and at least 10%> of the activity of an M.
  • a polypeptide selected from the group consisting of: a polypeptide consisting essentially of SEQ ED NO:3, a polypeptide having at least ten consecutive amino acids of an grisea fumarate reductase (SEQ ED NO:3) protein, a polypeptide having at least 35% sequence identity with an M. grisea fumarate reductase (SEQ ID NO:3) protein and at least
  • grisea fumarate reductase (SEQ ED NO:3) protein, and a polypeptide consisting of at least 50 amino acids having at least 35% sequence identity with an M. grisea fumarate reductase (SEQ ED NO: 3) protein and at least 10%> of the activity of an M. grisea fumarate reductase (SEQ ED NO:3) protein, and detecting the presence and/or absence of binding between the test compound and the polypeptide, wherein binding indicates that the 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.
  • detecting the binding of a ligand to its target 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.
  • an array of immobilized candidate ligands is provided.
  • the immobilized ligands are contacted with a fumarate reductase protein or a fragment or variant thereof, the unbound protein is removed, and the bound fumarate reductase is detected.
  • bound fumarate reductase is detected using a labeled binding partner, such as a labeled antibody.
  • fumarate reductase 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.
  • 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.
  • 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 the antifungal candidate and detecting a decrease in the growth, viability, or pathogenicity of the fungus or fungal cells.
  • 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.
  • 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.
  • 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%o or at least 90%> or more. Methods for measuring fungal growth and cell viability are known to those skilled in the art.
  • the antifungal candidate causes at least a ⁇ 0% 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.
  • the disease will be decreased by at least 40%o. 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 fumarate reductase 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.
  • Fumarate reductase catalyzes the reversible interconversion of succinate and ubiquinone to fiimarate and ubiquinol (see Figure 1).
  • Methods for measuring the progression of the fumarate reductase enzymatic reaction and/or a change in the concentration of the individual reactants succinate, ubiquinone, fumarate, and ubiquinol, include spectrophotometry, fluorimetry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.
  • the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: contacting succinate and ubiquinone with a fumarate reductase in the presence and absence of a test compound or contacting fumarate and ubiquinol with a fumarate reductase in the presence and absence of a test compound; and determining a change in concentration for at least one of succinate, ubiquinone, fumarate, and/or ubiquinol in the presence and absence of the test compound, wherein a change in the concentration for any of the above reactants indicates that the test compound is a candidate for an antibiotic.
  • Enzymatically active fragments of M Enzymatically active fragments of M.
  • grisea fumarate reductase set forth in SEQ ID NO:3 are also useful in the methods of the invention.
  • an enzymatically active polypeptide comprising at least 50 consecutive amino acid residues and at least 10% of the activity of M. grisea fumarate reductase set forth in SEQ ED NO:3 are useful in the methods of the invention.
  • fragments of heterologous fumarate reductases are also useful in the methods of the invention.
  • Enzymatically active polypeptides having at least 10%> of the activity of SEQ ED NO:3 and at least 35%, 36%>, 37%, 38%, 39%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with at least 50 consecutive amino acid residues of SEQ ED NO:3 are useful in the methods of the invention. Most preferably, the enzymatically active polypeptide has at least 50% sequence identity with at least 50 consecutive amino acid residues of SEQ ID NO:3 and at least 25%,, 75% or at least 90%> of the activity thereof.
  • the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: contacting fumarate and ubiquinol or succinate and ubiquinone with a polypeptide selected from the group consisting of: a polypeptide consisting essentially of SEQ ED NO:3, a polypeptide having at least 35%> sequence identity with the M. grisea fumarate reductase set forth in SEQ ED NO: 3 and having at least 10%) of the activity thereof, a polypeptide comprising at least 50 consecutive amino acids of M.
  • fumarate reductase protein and derivatives thereof may be isolated from a fungus or may be recombinantly produced in and isolated 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.
  • An example of a method for the purification of a fumarate reductase polypeptide is described in Muratsubaki et al. Prep Biochem. 24:289-96 (1994). Other methods for the purification of fumarate reductase proteins and polypeptides are known to those skilled in the art.
  • the invention also provides cell based assays.
  • the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) measuring the expression or activity of a fumarate reductase in a cell, cells, tissue, or an organism in the absence of a test compound; b) contacting the cell, cells, tissue, or organism with the test compound and measuring the expression or activity of the fumarate reductase in the cell, cells, tissue, or organism; and c) comparing the expression or activity of the fumarate reductase in steps (a) and (b), wherein an altered expression or activity in the presence of the test compound indicates that the compound is a candidate for an antibiotic.
  • RNA and proteins are known to those skilled in the art. (Current Protocols in Molecular Biology, Ausubel et al, eds., Greene Publishing & Wiley- Interscience, New York, (1995)). The method of detection is not critical to the present invention.
  • Methods for detecting fumarate reductase 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 fumarate reductase 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.
  • any reporter gene system may be used to detect fumarate reductase protein expression.
  • a polynucleotide encoding a reporter protein is fused in frame with fumarate reductase 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 of fumarate reductase 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.
  • compounds that inhibit fungal growth can be applied to a fungus or expressed in a fungus to prevent fungal growth.
  • 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.
  • 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 gallic ⁇ ), 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
  • the fungal organisms comprise a first form of a fumarate reductase and a second form of the fumarate reductase, respectively.
  • at least one of the two forms of the fumarate reductase has at least 10% of the activity of the polypeptide set forth in SEQ ID NO:3.
  • the methods comprise comparing the growth of the two organisms in the presence of the test compound relative to their respective controls without the test compound.
  • a fumarate reductase useful in the methods of the invention are selected from the group consisting of: a nucleic acid encoding SEQ ID NO:3, a nucleic acid encoding a polypeptide consisting essentially of SEQ ED NO:3, SEQ ED NO:l or SEQ ID NO:2, SEQ ED NO:l or SEQ ED NO:2 comprising a mutation either reducing or abolishing fumarate reductase protein activity, a heterologous fumarate reductase, and a heterologous fumarate reductase comprising a mutation either reducing or abolishing fumarate reductase protein activity.
  • any combination of two different forms of the fumarate reductase genes listed above are useful in the methods of the invention, with the caveat that at least one of the forms of the fumarate reductase has at least 10% of the activity of the polypeptide set forth in SEQ ED NO:3.
  • the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: providing an organism having a first form of a fumarate reductase; providing an organism having a second form of the fumarate reductase; and determining the growth of the organism having the first form of the fumarate reductase and the growth of the organism having the second form of the fumarate reductase in the presence of the test compound, wherein a difference in growth between the two organisms in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.
  • the optional determination of the growth of the organism having the first form of the fumarate reductase and the growth of the organism having the second form of the fumarate reductase in the absence of any test compounds is performed to control for any inherent differences in growth as a result of the different genes. Growth and/or proliferation of an organism are measured by methods well known in the art such as optical density measurements, and the like.
  • the organism is Magnaporthe grisea.
  • the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: providing an organism having a first form of a fumarate reductase; providing a comparison organism having a second form of the fumarate reductase; and determining the pathogenicity of the organism having the first form of the fumarate reductase and the organism having the second form of the fumarate reductase in the presence of the test compound, wherein a difference in pathogenicity between the two organisms in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.
  • the pathogenicity of the organism having the first form of the fumarate reductase and the organism having the second form of the fumarate reductase in the absence of any test compounds is determined to control for any inherent differences in pathogenicity as a result of the different genes.
  • Pathogenicity of an organism is measured by methods well known in the art such as lesion number, lesion size, spomlation, and the like.
  • the organism is Magnaporthe grisea.
  • One embodiment of the invention is directed to the use of multi-well plates for screening of antibiotic compounds. The use of multi-well plates is a format that readily accommodates multiple different assays to characterize various compounds, concentrations of compounds, and fungal organisms 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.
  • the present inventors have discovered that disruption of the VPS 17 gene and/or gene product reduces the pathogenicity o ⁇ Magnaporthe grisea.
  • the inventors are the first to demonstrate that VPS 17 is a target for antibiotics, preferably antifungals. Accordingly, the invention provides methods for identifying compounds that inhibit VPS 17 gene expression or biological activity of its gene product(s).
  • the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising contacting a VPS 17 polypeptide with a test compound and detecting the presence or absence of binding between the test compound and the VPS 17 polypeptide, wherein binding indicates that the test compound is a candidate for an antibiotic.
  • VPS 17 polypeptides of the invention have the amino acid sequence of a naturally occurring VPS 17 found in a fungus, animal, plant, or microorganism, or have an amino acid sequence derived from a naturally occurring sequence.
  • the VPS 17 is a fungal VPS 17.
  • a cDNA encoding M. grisea VPS 17 protein is set forth in SEQ ED NO:4, an M. grisea VPS17 genomic DNA is set forth in SEQ ED NO:5, and an M. grisea VPS17 polypeptide is set forth in SEQ ED NO:6.
  • the VPS 17 is a Magnaporthe VPS 17.
  • Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae and the imperfect states o ⁇ Magnaporthe in the genus Pyricularia.
  • the Magnaporthe VPS 17 is from Magnaporthe grisea.
  • the invention provides a polypeptide consisting essentially of SEQ ED NO:6.
  • a polypeptide consisting essentially of SEQ ED NO:6 has at least 90%> sequence identity with M. grisea VPS 17 (SEQ ID NO:6) and at least 10% of the activity of SEQ ED NO:6.
  • a polypeptide consisting essentially of SEQ ED NO:6 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 91%, 98%, or 99% sequence identity with SEQ ED NO:6 and at least 25%, 50%, 75%, or 90%) of the activity of M.
  • polypeptides consisting essentially of SEQ ID NO:6 include, but are not limited to, polypeptides having the amino acid sequence of SEQ ED NO:6 with the exception that one or more of the amino acids are substituted with structurally similar amino acids providing a conservative amino ' acid substitution. Conservative amino acid substitutions are well known to those of skill in the art. Examples of polypeptides consisting essentially of SEQ ED NO:6 include polypeptides having 1, 2, or 3 conservative amino acid substitutions relative to SEQ ED NO:6.
  • polypeptides consisting essentially of SEQ ED NO:6 include polypeptides having the sequence of SEQ ED NO:6, but with truncations at either or both the 3' and the 5' end.
  • polypeptides consisting essentially of SEQ ID NO:6 include polypeptides having 1, 2, or 3 amino acids residues removed from either or both 3' and 5' ends relative to SEQ ID NO:6.
  • the VPS 17 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 (Heter
  • VPS 17 fragments include an intact or nearly intact epitope that occurs on the biologically active wild-type VPS17.
  • the fragments comprise at least 10 consecutive amino acids of VPS 17 set forth in SEQ ID NO:6.
  • the fragments comprise at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 300, 325, 350, 375, 400, 425, 450, 475, 500,525, 550, 575, 600 or at least 610 consecutive amino acids residues of VPS 17 set forth in SEQ ED NO:6.
  • Fragments of heterologous VPS17s are also useful in the methods of the invention.
  • polypeptides having at least 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%,, 96%, 97%, 98% or 99% sequence identity with at least 50 consecutive amino acid residues of SEQ ED NO:6 are useful in the methods of the invention.
  • the fragment is from a Magnaporthe VPS 17.
  • the fragment contains an amino acid sequence conserved among fungal VPS 17s.
  • Polypeptides having at least 35% sequence identity with M. grisea VPS 17 (SEQ ID NO: 6) protein are also useful in the methods of the invention.
  • sequence identity is at least 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or any integer from 35-100% sequence identity in ascending order with M. grisea VPS 17 (SEQ ED NO:6) protein.
  • polypeptides of the invention have at least 10%) of the activity of M grisea VPS17 (SEQ ID NO:6) protein.
  • VPS17 polypeptides of the invention have at least 10%o, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or at least 90% of the activity of M. grisea VPS 17 (SEQ ED NO:6) protein.
  • the invention provides a method for identifying a test compound as a candidate for a fungicide, comprising: contacting a test compound with at least one polypeptide selected from the group consisting of: a polypeptide consisting essentially of SEQ ED NO:6, a polypeptide having at least ten consecutive amino acids of an M.
  • grisea VPS 17 (SEQ ED NO:6) protein, a polypeptide having at least 35% sequence identity with an M. grisea VPS17 (SEQ ED NO:6) protein and at least 10% of the activity of an M. grisea VPS17 (SEQ ED NO:6) protein, and a polypeptide consisting of at least 50 amino acids having at least 35% sequence identity with an M. grisea VPS 17 (SEQ ED NO:6) protein and at least 10% of the activity of an M. grisea VPS17 (SEQ ID NO:6) protein, and detecting the presence and/or absence of binding between the test compound and the polypeptide, wherein binding indicates that the 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.
  • 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.
  • an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with a VPS 17 protein or a fragment or variant thereof, the unbound protein is removed, and the bound VPS 17 is detected.
  • bound VPS 17 is detected using a labeled binding partner, such as a labeled antibody.
  • VPS 1 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.
  • 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.
  • 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 the antifungal candidate and detecting a decrease in the growth, viability, or pathogenicity of the fungus or fungal cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, spomlation, respiratory failure, and/or death.
  • the present invention provides cell based assays.
  • the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: a) measuring the expression or activity of a VPS 17 in a cell, cells, tissue, or an organism in the absence of a test compound; b) contacting the cell, cells, tissue, or organism with the test compound and measuring the expression or activity of the VPS 17 in the cell, cells, tissue, or organism; and c) comparing the expression or activity of the VPS 17 in steps (a) and (b), wherein an altered expression or activity in the presence of the test compound indicates that the compound is a candidate for an antibiotic.
  • Expression of VPS 17 can be measured by detecting the VPS 17 primary transcript or mRNA, VPS 17 polypeptide, or VPS 17 activity.
  • Methods for detecting the expression of RNA and proteins are known to those skilled in the art. (Current Protocols in Molecular Biology, Ausubel et al, eds., Greene Publishing & Wiley- Interscience, New York, (1995)). The method of detection is not critical to the present invention.
  • Methods for detecting VPS 17 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 VPS 17 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.
  • any reporter gene system may be used to detect VPS 17 protein expression.
  • a polynucleotide encoding a reporter protein is fused in frame with VPS 17 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 of VPS 17 expression or activity can then be used to control fungal growth.
  • Fungicides are thus routinely applied to growing and stored crops as a preventive measure, generally as foliar sprays or seed dressings.
  • foliar sprays or seed dressings For example, compounds that inhibit fungal growth can be applied to a fungus or expressed in a fungus to prevent fungal growth.
  • 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.
  • 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), Co Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallic ⁇ ), 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
  • the fungal organisms comprise a first form of a VPS 17 and a second form of the VPS 17, respectively.
  • at least one of the two forms of the VPS 17 has at least 10%, of the activity of the polypeptide set forth in SEQ ED NO:6.
  • the methods comprise comparing the growth of the two organisms in the presence of the test compound relative to their respective controls without the test compound. A difference in growth between the two organisms in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.
  • VPS 17 useful in the methods of the invention are selected from the group consisting of: a nucleic acid encoding SEQ ED NO: 6, a nucleic acid encoding a polypeptide consisting essentially of SEQ ED NO:6, SEQ ED NO:4 or SEQ ED NO:5, SEQ ED NO:4 or SEQ ID NO:5 comprising a mutation either reducing or abolishing VPS 17 protein activity, a heterologous VPS 17, and a heterologous VPS 17 comprising a mutation either reducing or abolishing VPS 17 protein activity.
  • the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: providing an organism having a first form of a VPS17; providing an organism having a second form of the VPS17; and determining the growth of the organism having the first form of the VPS 17 and the growth of the organism having the second form of the VPS 17 in the presence of the test compound, wherein a difference in growth between the two organisms in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.
  • the optional determination of the growth of the organism having the first form of the VPS 17 and the growth of the organism having the second form of the VPS 17 in the absence of any test compounds is performed to control for any inherent differences in growth as a result of the different genes. Growth and/or proliferation of an organism are 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 invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising: providing an organism having a first form of a VPS 17; providing a comparison organism having a second form of the VPS 17; and determining the pathogenicity of the organism having the first form of the VPS 17 and the organism having the second form of the VPS 17 in the presence of the test compound, wherein a difference in pathogenicity between the two organisms in the presence of the test compound indicates that the test compound is a candidate for an antibiotic.
  • the pathogenicity of the organism having the first form of the VPS 17 and the organism having the second form of the VPS 17 in the absence of any test compounds is determined to control for any inherent differences in pathogenicity as a result of the different genes.
  • Pathogenicity of an organism is measured by methods well known in the art such as lesion number, lesion size, spomlation, and the like.
  • the organism is Magnaporthe grisea.
  • One embodiment of the invention is directed to the use of multi-well plates for screening of antibiotic compounds. The use of multi-well plates is a format that readily accommodates multiple different assays to characterize various compounds, concentrations of compounds, and fungal organisms 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.
  • hph The bacterial hygromycin B phosphotransferase (hph) gene (Gritz & Davies, 25 Gene 179 (1983) (PMID: 6319235)) under control of the Aspergillus nidulans trpC promoter and terminator (Mullaney et al, 199 Mol. Gen. Genet. 37 (1985) (PMID: 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 pSif.
  • 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, supra) containing 50 ⁇ g/ml of hygromycin B (Sigma Chem. Co., St. Louis, MO).
  • Cosmid libraries were constructed in the pcosKA5 vector (Hamer et al., 98 Proc. Nat'l. Acad. Sci. USA 5110 (2001) (PMED: 11296265)) as described in Sambrook et al. Cosmid libraries were quality checked by pulsed-field gel electrophoresis, restriction digestion analysis, and PCR identification of single genes.
  • Example 3 Construction of Cosmids 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, 2 ⁇ l of the 10X GPS buffer, 70 ng of supercoiled pSIF, 8-12 ⁇ g of target cosmid DNA were mixed and taken to a final volume of 20 ⁇ l with water. 1 ⁇ l of transposase (TnsABC) was added to the reaction and incubated for 10 minutes at 37°C to allow the assembly reaction to occur.
  • TnsABC transposase
  • DNA quality was checked by electrophoresis 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.). The DNA sequences adjacent to the site of the transposon insertion were used to search DNA and protein databases using the BLAST algorithms (Altschul et al, supra). A single insertion of SIF into the Magnaporthe grisea FRDl gene was chosen for further analysis.
  • This construct was designated cpgmra0050001bl2 and it contains the SIF transposon insertion approximately between amino acids 60 and 73 relative to the Schizosaccharomyces pombe putative flavoprotein subunit (513 amino acids total).
  • Example 5 Preparation of FRDl Cosmid DNA and Transformation o ⁇ Magnaporthe srisea Cosmid DNA from the FRDl 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, 10 MPMI 700 (1997)). Briefly, M.
  • grisea strain Guy 11 was grown in complete liquid media (Talbot et al, 5 Plant Cell 1575 (1993) (PMED: 8312740)) shaking at 120 rpm for 3 days at 25°C in the dark. Mycelia was harvested and washed with sterile H 2 O 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 a concentration of 2xl0 8 protoplasts/ml.
  • Example 6 Effect of Transposon Insertion on Masnavorthe pathogenicity
  • Rice infection assays were performed using Indica rice cultivar CO39 essentially as described in Valent et al. (Valent et al., 127 Genetics 87 (1991) (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.
  • Example 7 Verification of FRDl Gene Function by Analysis of Nutritional Requirements
  • the fungal strains, Kl-12 and Kl-19, containing the FRDl disrupted gene obtained in Example 5 are analyzed for their nutritional requirements using the PM5 phenotype microarray from Biolog, Inc. (Hayward, CA).
  • the PM5 plate tests for the auxotrophic requirement for 94 different metabolites.
  • the inoculating fluid consists of 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 M NaNO 3 , 6.7 mM KC1, 3.5 mM Na 2 SO 4 , 11.0 mM KH 2 PO 4 , 0.01%/7-iodonitrotetrazolium violet, 0.1 mM MgCl 2 , 1.0 mM CaCl 2 and trace elements, pH adjusted to 6.0 with NaOH.
  • Final concentrations of trace elements are: 7.6 ⁇ M ZnCl 2 , 2.5 ⁇ M MnCl 2 4H 2 O, 1.8 ⁇ M FeCl 2 4H 2 O, 0.71 ⁇ M CoCl 2 6H 2 O, 0.64 ⁇ M CuCl 2 2H 2 O, 0.62 ⁇ M Na 2 MoO 4 , 18 ⁇ M H 3 BO 3 .
  • Spores for each strain are harvested into the inoculating fluid. The spore concentrations are adjusted to 2xl0 5 spores/ml. 100 ⁇ l of spore suspension are deposited into each well of the microtiter plates. The plates are incubated at 25°C for 7 days.
  • Optical density (OD) measurements at 490nm and 750nm are taken daily.
  • Example 8 Cloning, Expression, and Purification of Fumarate Reductase The following is a protocol to obtain an isolated fumarate reductase protein. Cloning and expression strategies: A fumarate reductase encoding nucleic acid is cloned into E.
  • coli pET vectors- Novagen
  • Baculovirus Pharmingen
  • Yeast Invitrogen expression vectors containing His/fusion protein tags
  • Extraction Extract recombinant protein from 250 ml cell pellet in 3 ml of extraction buffer by sonicating 6 times, with 6 second pulses at 4°C. Centrifuge extract at 15000xg for 10 minutes 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 • Equilibrate column with the buffer • Load protein extract • Wash with the equilibration buffer • Elute bound protein with 0.5 M imidazole Another method for purifying fumarate reductase protein is described in
  • Example 9 Assays for Measuring Binding of Test Compounds to Fumarate Reductase The following is a protocol to identify test compounds that bind to the fumarate reductase protein. • Isolated full-length fumarate reductase 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, Shoolingin- Jordan et al.
  • Candidate compounds are identified as wells with lower radioactivity as compared to control wells with no test compound added. Additionally, an isolated polypeptide comprising 10-50 amino acids from the M. grisea fumarate reductase is screened in the same way. A polypeptide comprising 10-50 amino acids is generated by subcloning a portion of the fumarate reductase encoding nucleic acid 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 fumarate reductase coding region 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 isolated as described in Example 8 above.
  • Test compounds that bind fumarate reductase polypeptide are further tested for antibiotic activity.
  • M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al, supra). Spores are harvested into minimal media to a concentration of 2 x 10 5 spores/ml and the culture is divided. Id. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture.
  • test compounds are an antibiotic candidate if the growth of the culture containing the test compound is less than the growth of the control culture.
  • Test compounds that bind fumarate reductase polypeptide are further tested for antipathogenic activity.
  • M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al, supra). Spores are harvested into water with 0.01% Tween 20 to a concentration of 5x10 4 spores/ml and the culture is divided. Id.
  • test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml.
  • Solvent only is added to the second culture.
  • Rice infection assays are performed using Indica rice cultivar CO39 essentially as described in Valent et al, supra). Two-week-old seedlings of cultivar CO39 are sprayed with 12 ml of conidial suspension. The inoculated plants are 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 at 70% humidity) for an additional 5.5 days. Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity as compared to the control samples.
  • antipathogenic activity can be assessed using an excised leaf pathogenicity assay.
  • Spore suspensions are prepared in water only to a concentration of 5xl0 4 spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture.
  • Detached leaf assays are performed by excising 1 cm segments of rice leaves from Indica rice cultivar CO39 and placing them on 1%> agarose in water. 10 ⁇ l of each spore suspension is place on the leaf segments and the samples are incubated at 25°C for 5 days in the dark. Leaf samples are examined at 5 days post- inoculation to determine the extent of pathogenicity as compared to the control samples.
  • Example 10 Assays for Testing Inhibitors or Candidates for Inhibition of Fumarate Reductase Activity
  • the enzymatic activity of fumarate reductase is determined in the presence and absence of candidate compounds in a suitable reaction mixture, such as described by Chen et al. (2001) APMIS. 109:801-8.
  • Candidate compounds are identified by a decrease in products or a lack of a decrease in substrates in the presence of the compound, with the reaction proceeding in either direction.
  • Candidate compounds are additionally determined in the same manner using a polypeptide comprising a fragment of the M. grisea fumarate reductase .
  • the fumarate reductase polypeptide fragment is generated by subcloning a portion of the fumarate reductase encoding nucleic acid 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 fumarate reductase coding region using polymerase chain reaction amplification method. The DNA fragment encoding the fumarate reductase polypeptide fragment is cloned into an expression vector, expressed and isolated as described in Example 8 above. Test compounds identified as inhibitors of fumarate reductase 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., supra). Spores are harvested into minimal media to a concentration of 2 x 10 5 spores/ml and the culture is divided. Id.
  • the test compound is added to one culture to a final concentration of 20-100 ⁇ g/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.
  • Test compounds identified as inhibitors of fumarate reductase activity are further tested for antipathogenic activity.
  • M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al, supra). Spores are harvested into water with 0.01% Tween 20 to a concentration of 5x10 4 spores/ml and the culture is divided. Id. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture.
  • Rice infection assays are performed using Indica rice cultivar CO39 essentially as described in Valent et al, supra.
  • Two-week-old seedlings of cultivar CO39 are sprayed with 12 ml of conidial suspension.
  • the inoculated plants are 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 hoursat 70% humidity) for an additional 5.5 days.
  • Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity as compared to the control samples.
  • antipathogenic activity is assessed using an excised leaf pathogenicity assay.
  • Spore suspensions are prepared in water only to a concentration of 5xl0 4 spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml.
  • 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 2x10 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.
  • RNA 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, supra) using a radiolabeled fragment of the fumarate reductase encoding nucleic acid as a probe.
  • Test compounds resulting in an altered level of fumarate reductase mRNA relative to the untreated control sample are identified as candidate antibiotic compounds.
  • Test compounds identified as inhibitors of fumarate reductase expression 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, supra). Spores are harvested into minimal media to a concentration of 2 x 10 5 spores/ml and the culture is divided. Id.
  • the test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml.
  • Solvent only is added to the second culmre.
  • 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.
  • Test compounds identified as inhibitors of FRDl gene expression are further tested for antipathogenic activity.
  • M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al, supra). Spores are harvested into water with 0.01 %> Tween 20 to a concentration of 5xl0 4 spores/ml and the culture is divided. Id. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture.
  • Rice infection assays are performed using Indica rice cultivar CO39 essentially as described in Valent et al, supra.
  • Two-week-old seedlings of cultivar CO39 are sprayed with 12 ml of conidial suspension.
  • the inoculated plants are 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 at 70%, humidity) for an additional 5.5 days.
  • Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity as compared to the control samples.
  • antipathogenic activity is assessed using an excised leaf pathogenicity assay.
  • Spore suspensions are prepared in water only to a concentration of 5xl0 4 spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml.
  • Solvent only is added to the second culture. Detached leaf assays are performed by excising 1 cm segments of rice leaves from Indica rice cultivar CO39 and placing them on 1%> agarose in water. 10 ⁇ l of each spore suspension is place on the leaf segments and the samples are incubated at 25°C for 5 days in the dark. Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity as compared to the control samples.
  • Example 12 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of Fumarate Reductase with Reduced or No Activity
  • the effect of test compounds on the growth of wild-type fungal cells and mutant fungal cells having a mutant FRDl gene is measured and compared as follows.
  • Magnaporthe grisea fungal cells containing a mutant form of the FRDl gene that lacks activity 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 2x10 5 spores per ml.
  • Approximately 4xl0 4 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 200 ⁇ l. 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 of the test compounds on the mutant and wild-type fungal cells is measured against the growth control and the percent of inhibition is calculated as the OD 590 (fungal strain plus test compound)/OD 590 (growth control) x 100.
  • test compounds that show differential growth inhibition between the mutant and the wild-type cells are identified as potential antifungal compounds. Similar protocols may be found in Kirsch & DiDomenico, 26 Biotechnology 177 (1994) (PMID: 7749303)). Test compounds that produce a differential growth response between the mutant and wild-type fungal strains are further tested for antipathogenic activity. Alternatively, any test compound can be tested for antipathogenic activity with the following protocol. Each M. grisea strain is grown as described for spore production on oatmeal agar media (Talbot et al, supra).
  • the inoculated plants are 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 are examined at 5 days post-inoculation to determine the extent of pathogenicity of the mutant and wild-type fungal strains as compared to their untreated control samples.
  • antipathogenic activity can be assessed using an excised leaf pathogenicity assay.
  • Spore suspensions are prepared in water only to a concentration of 5xl0 4 spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture.
  • Detached leaf assays are performed by excising 1 cm segments of rice leaves from Indica rice cultivar CO39 and placing them on 1%> agarose in water. 10 ⁇ l of each spore suspension is place on the leaf segments and the samples are incubated at 25°C for 5 days in the dark. Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity of the mutant and wild-type fungal strains as compared to their untreated control samples.
  • Example 13 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., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press (1989).
  • the kanamycin resistance gene (npt) contained between the Tn7 arms was removed by EcoRV digestion.
  • the bacterial hygromycin B phosphotransferase (hph) gene (Gritz & Davies, 25 Gene 179 (1983) (PMID: 6319235)) under control of the Aspergillus nidulans trpC promoter and terminator (Mullaney et al, 199 Mol. Gen. Genet.
  • Transformants containing the Sif transposon were selected on LB agar (Sambrook et al, supra) containing 50 ⁇ g/ml of hygromycin B (Sigma Chem. Co., ' St. Louis, MO).
  • Example 14 Construction of a Fungal Cosmid Library Cosmid libraries were constructed in the pcosKA5 vector (Hamer et al, 98 Proc. Nat'l. Acad. Sci. USA 5110 (2001) (PMED: 11296265)) as described in Sambrook et al. Cosmid libraries were quality checked by pulsed-field gel electrophoresis, restriction digestion analysis, and PCR identification of single genes.
  • Example 15 Construction of Cosmids 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, 2 ⁇ l of the 10X GPS buffer, 70 ng of supercoiled pSEF, 8-12 ⁇ g of target cosmid DNA were mixed and taken to a final volume of 20 ⁇ l with water. 1 ⁇ l of transposase (TnsABC) was added to the reaction and incubated for 10 minutes at 37°C to allow the assembly reaction to occur.
  • TnsABC transposase
  • Example 16 High Throughput Preparation and Verification of Transposon Insertion into the M. grisea VPS 17
  • 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, supra) supplemented with 50 ⁇ g/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, 1 Genome Res. 1072 (1997) (PMID: 9371743)). DNA quality was checked by electrophoresis 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.). The DNA sequences adjacent to the site of the transposon insertion were used to search DNA and protein databases using the BLAST algorithms (Altschul et al, supra). A single insertion of SIF into the Magnaporthe grisea VPS 17 gene was chosen for further analysis.
  • This construct was designated cpgmraOOl 1038bl2 and it contains the SIF transposon insertion approximately between amino acids 474 and 475 relative to the Schizosaccharomyces pombe probable vacuolar protein sorting-associated protein (549 amino acids total).
  • Example 17 Preparation of VPS 17 Cosmid DNA and Transformation o ⁇
  • Mycelia was harvested and washed with sterile H 2 O 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 a concentration of 2x10 8 protoplasts/ml.
  • 50 ⁇ l of protoplast suspension was mixed with 10-20 ⁇ g of the cosmid DNA and pulsed using a Gene Pulser II instrument (BioRad) set with the following parameters: 200 ohm, 25 ⁇ F, and 0.6kV.
  • Transformed protoplasts were regenerated in complete agar media (Talbot et al, supra) with the addition of 20%> sucrose for one day, then overlayed with CM agar media containing hygromycin B (250 ug/ml) to select transformants. Transformants were screened for homologous recombination events in the target gene by PCR (Hamer et al, supra). Two independent strains were identified and are hereby referred to as KO1-22 and KOl-28.
  • Example 18 Effect of Transposon Insertion on Magnaporthe pathogenicity
  • Rice infection assays were performed using Indica rice cultivar CO39 essentially as described in Valent et al. (Valent et al, 127 Genetics 87 (1991) (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.
  • Example 1 Verification of VPS 17 Gene Function by Analysis of Nutritional Requirements
  • the fungal strains, KO1-22 and KOl-28, containing the VPS17 disrupted gene obtained in Example 17 are analyzed for their nutritional requirements using the PM3 and PM5 phenotype microarray from Biolog, Inc. (Hayward, CA).
  • the inoculating fluid consists of 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO 3 (omitted for PM3A), 6.7 mM KC1, 3.5 mM Na 2 SO 4 , 11.0 mM KH 2 PO 4 , 0.01%/?-iodonitrotetrazolium violet, 0.1 mM MgCl 2 , 1.0 mM CaCl 2 and trace elements, pH adjusted to 6.0 with NaOH.
  • Final concentrations of trace elements are: 7.6 ⁇ M ZnCl 2 , 2.5 ⁇ M MnCl 2 4H 2 O, 1.8 ⁇ M FeCl 2 4H 2 O, 0.71 ⁇ M CoCl 2 6H 2 O, 0.64 ⁇ M CuCl 2 2H 2 O, 0.62 ⁇ M Na 2 MoO 4 , 18 ⁇ M H BO 3 .
  • Spores for each strain are harvested into the inoculating fluid. The spore concentrations are adjusted to 2xl0 5 spores/ml. 100 ⁇ l of spore suspension are deposited into each well of the microtiter plates. The plates are incubated at 25°C for 7 days.
  • Optical density (OD) measurements at 490nm and 750nm are taken daily.
  • Example 20 Cloning, Expression, and Purification of VPS 17 The following is a protocol to obtain an isolated VPS 17 protein.
  • VPS17 encoding nucleic acid is 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 is 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 second pulses at 4°C. Centrifuge extract at 15000xg for 10 minutes 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 • Equilibrate column with the buffer • Load protein extract • Wash with the equilibration buffer • Elute bound protein with 0.5 M imidazole
  • Example 21 Assays for Measuring Binding of Test Compounds to VPS 17 The following is a protocol to identify test compounds that bind to the VPS 17 protein.
  • Isolated full-length VPS 17 polypeptide with a His/fusion protein tag (Example 20) 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 (PMID: 9250995)) for binding of radiolabeled vacuolar hydrolases to the bound VPS 17.
  • test compounds Screening of test compounds is performed by adding test compound and radioactive vacuolar hydrolases to the wells of the HISGRAB plate containing bound VPS 17. • 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, an isolated polypeptide comprising 10-50 amino acids from the M. grisea VPS 17 is screened in the same way. A polypeptide comprising 10-50 amino acids is generated by subcloning a portion of the VPS 17 encoding nucleic acid into a protein expression vector that adds a His-Tag when expressed (see Example 20).
  • Oligonucleotide primers are designed to amplify a portion of the VPS 17 coding region 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 isolated as described in Example 20 above.
  • Test compounds that bind VPS 17 polypeptide are further tested for antibiotic activity.
  • M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al, supra). Spores are harvested into minimal media to a concentration of 2 x 10 5 spores/ml and the culture is divided. Id. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml.
  • Solvent only is added to the second culture.
  • the growth of the solvent containing culture and the test compound containing culture 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.
  • Test compounds that bind VPS 17 polypeptide are further tested for antipathogenic activity.
  • M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al, supra). Spores are harvested into water with 0.01 % Tween 20 to a concentration of 5x10 4 spores/ml and the culture is divided. Id. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture.
  • Rice infection assays are performed using Lndica rice cultivar CO39 essentially as described in Valent et al, supra). Two-week-old seedlings of cultivar CO39 are sprayed with 12 ml of conidial suspension. The inoculated plants are 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 at 70% humidity) for an additional 5.5 days. Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity as compared to the control samples. Alternatively, antipathogenic activity can be assessed using an excised leaf pathogenicity assay.
  • Spore suspensions are prepared in water only to a concentration of 5xl0 4 spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture. Detached leaf assays are performed by excising 1 cm segments of rice leaves from Indica rice cultivar CO39 and placing them on 1%> agarose in water. 10 ⁇ l of each spore suspension is place on the leaf segments and the samples are incubated at 25°C for 5 days in the dark. Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity as compared to the control samples.
  • Example 22 Assays for Testing Inhibitors or Candidates for Inhibition of VPS 17 Activity Test compounds which may be inhibitors of VPS 17 activity are 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, supra). Spores are harvested into minimal media to a concentration of 2 x 10 5 spores/ml and the culture is divided. Id.
  • the test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture. The growth of the solvent containing culture and the test compound containing culture 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.
  • Test compounds identified as inhibitors of VPS 17 activity are further tested for antipathogenic activity.
  • M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al. , supra). Spores are harvested into water with 0.01 % Tween 20 to a concentration of 5x10 4 spores/ml and the culture is divided. Id. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture.
  • Rice infection assays are performed using Indica rice cultivar CO39 essentially as described in Valent et al, supra.
  • Two-week-old seedlings of cultivar CO39 are sprayed with 12 ml of conidial suspension.
  • the inoculated plants are 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 hoursat 70%> humidity) for an additional 5.5 days.
  • Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity as compared to the control samples.
  • antipathogenic activity is assessed using an excised leaf pathogenicity assay.
  • Spore suspensions are prepared in water only to a concentration of 5xl0 4 spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml.
  • Solvent only is added to the second culture. Detached leaf assays are performed by excising 1 cm segments of rice leaves from Indica rice cultivar CO39 and placing them on 1%> agarose in water. 10 ⁇ l of each spore suspension is place on the leaf segments and the samples are incubated at 25°C for 5 days in the dark. Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity as compared to the control samples.
  • Example 23 Assays for Testing Compounds for Alteration of VPS 17 Gene Expression
  • 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 2xl0 5 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.
  • 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, supra) using a radiolabeled fragment of the VPS 17 encoding nucleic acid as a probe. Test compounds resulting in an altered level of VPS 17 mRNA relative to frte untreated control sample are identified as candidate antibiotic compounds.
  • Test compounds identified as inhibitors of VPS 17 expression 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, supra). Spores are harvested into minimal media to a concentration of 2 x 10 5 spores/ml and the culture is divided. Id.
  • the test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture. The growth of the solvent containing culture and the test compound containing culture 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.
  • Test compounds identified as inhibitors of VPS 17 gene expression are further tested for antipathogenic activity.
  • M. grisea is grown as described for spore production on oatmeal agar media (Talbot et al, supra). Spores are harvested into water with 0.01% Tween 20 to a concentration of 5x10 4 spores/ml and the culture is divided. Id. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture.
  • Rice infection assays are performed using Indica rice cultivar CO39 essentially as described in Valent et al, supra.
  • Two-week-old seedlings of cultivar CO39 are sprayed with 12 ml of conidial suspension.
  • the inoculated plants are 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 at 70% humidity) for an additional 5.5 days.
  • Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity as compared to the control samples.
  • antipathogenic activity is assessed using an excised leaf pathogenicity assay.
  • Spore suspensions are prepared in water only to a concentration of 5xl0 4 spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml.
  • Solvent only is added to the second culture. Detached leaf assays are performed by excising 1 cm segments of rice leaves from Indica rice cultivar CO39 and placing them on 1% agarose in water. 10 ⁇ l of each spore suspension is place on the leaf segments and the samples are incubated at 25°C for 5 days in the dark. Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity as compared to the control samples.
  • Example 24 In Vivo Cell Based Assay Screening Protocol with a Fungal Strain Containing a Mutant Form of VPS 17 with Reduced or No Activity
  • the effect of test compounds on the growth of wild-type fungal cells and mutant fungal cells having a mutant VPS 17 gene is measured and compared as follows.
  • Magnaporthe grisea fungal cells containing a mutant form of the VPS 17 gene that lacks activity, for example a VPS 17 gene containing a transposon insertion 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 2x10 spores per ml. Approximately 4xl0 4 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 200 ⁇ l. Wells with no test compound present (growth control), and wells without cells are included as controls (negative control). The plates arc 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.
  • test compounds The effect of each of the test compounds on the mutant and wild-type fungal cells is measured against the growth control and the percent of inhibition is calculated as the OD59 0 (fungal strain plus test compound)/OD 59 o (growth control) x 100.
  • the percent of growth inhibition in the presence of the test compound on the mutant and wild-type fungal strains are compared.
  • Compounds that show differential growth inhibition between the mutant and the wild-type cells are identified as potential antifungal compounds. Similar protocols may be found in Kirsch & DiDomenico, 26 Biotechnology 177 (1994) (PMID: 7749303)). Test compounds that produce a differential growth response between the mutant and wild-type fungal strains are further tested for antipathogenic activity.
  • any test compound can be tested for antipathogenic activity with the following protocol.
  • Each M. grisea strain is grown as described for spore production on oatmeal agar media (Talbot et al, supra). Spores for each strain are harvested into water with 0.01% Tween 20 to a concentration of 5x10 4 spores/ml and the culture is divided. Id. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture.
  • Rice infection assays are performed using Indica rice cultivar CO39 essentially as described in Valent et al, supra. Two-week-old seedlings of cultivar CO39 are sprayed with 12 ml of conidial suspension.
  • the inoculated plants are 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 are examined at 5 days post-inoculation to determine the extent of pathogenicity of the mutant and wild-type fungal strains as compared to their untreated control samples.
  • antipathogenic activity can be assessed using an excised leaf pathogenicity assay.
  • Spore suspensions are prepared in water only to a concentration of 5xl0 4 spores/ml and the culture is divided. The test compound is added to one culture to a final concentration of 20-100 ⁇ g/ml. Solvent only is added to the second culture.
  • Detached leaf assays are performed by excising 1 cm segments of rice leaves from Indica rice cultivar CO39 and placing them on 1%> agarose in water. 10 ⁇ l of each spore suspension is place on the leaf segments and the samples are incubated at 25°C for 5 days in the dark. Leaf samples are examined at 5 days post-inoculation to determine the extent of pathogenicity of the mutant and wild-type fungal strains as compared to their untreated control samples.

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Abstract

La présente invention a trait à la découverte que la fumarate réductase et la protéine associée au tri de protéines cellulaires sont toutes deux essentielles au pouvoir pathogène fongique normal. De manière spécifique, l'inhibition de l'expression du gène de fumarate réductase chez des champignons entraîne une nette réduction du pouvoir pathogène et l'inhibition de l'expression du gène de la protéine associée au tri des protéines cellulaires chez des champignons entraîne une nette réduction du pouvoir pathogène. Ainsi, la fumarate réductase et la protéine associée au tri des protéines cellulaires sont utiles en tant que cibles pour l'identification d'antibiotiques, de préférence d'antifongiques. Par conséquent, la présente invention fournit des procédés pour l'identification de composés inhibiteurs de l'expression ou de l'activité de la fumarate réductase et fournit des procédés pour l'identification de composés inhibiteurs de l'expression ou de l'activité de la protéine associée au tri des protéines cellulaires. Les procédés de l'invention sont utiles pour l'identification d'antibiotiques, de préférence d'antifongiques.
PCT/US2004/018361 2003-06-13 2004-06-09 Procedes d'identification d'inhibiteurs de fumarate reductase et proteine associee au tri des proteines cellulaires en tant qu'antibiotiques WO2005005602A2 (fr)

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* Cited by examiner, † Cited by third party
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WO2008091485A2 (fr) 2007-01-23 2008-07-31 Euclid Discoveries, Llc Systèmes et procédés permettant de fournir des services vidéo personnels

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* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] EBBOLE ET AL. Database accession no. (CD029487) *
OMURA ET AL.: 'AN anthelmintic compound, nafuredin, shows selective inhibition of complex I n helminth mitochondria' PROC. NATL. ACAD. SCI. U.S.A. vol. 98, no. 1, 02 January 2001, pages 60 - 62, XP002278617 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008091485A2 (fr) 2007-01-23 2008-07-31 Euclid Discoveries, Llc Systèmes et procédés permettant de fournir des services vidéo personnels

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