The present invention relates to methods of identifying and isolating genes which are involved in the regulation of fungal gene expression. The invention also relates to methods useful for identifying fungal virulence factors. It further relates to a method of identifying agents which increase or decrease the expression or activity of a gene that regulates or is required for fungal pathogenesis. The invention also relates to the use of such agents as fungicides or fungistats.
Fungi are a large and diverse group of organisms with enormous importance to humans. Pathogenic fungi are a significant cause of human disease, particularly in the rapidly increasing proportion of the population whose immune system has been compromised by disease, chemotherapy, or immunosuppressive drugs. A wide variety of plant-pathogenic fungi (e.g., blights, rusts, molds, smuts, mildews) cause huge food crop loss and damage to ornamental plants. Plant diseases are caused by a myriad of invasive fungal pathogens falling into many genera, for example: soft rot (e.g., Rhizopus), leaf curl (e.g., Taphrina), powdery mildew (e.g., Sphaerotheca), leaf spots (e.g., Fulvia), blight (e.g., Alternaria), blast (e.g., Magnaporthe), black rot (e.g., Guignardia), scab (e.g., Venturia), wilts (e.g., Fusarium), rusts (e.g., Puccinia), smuts (e.g., Ustilago), and cankers (e.g., Rhizoctonia). In addition, fungal species are the commercial source of a great many medicinally useful products, such as antibiotics (e.g., beta-lactam antibiotics such as penicillin, cephalosporin, and their derivatives), anti-hypercholesterolemic agents (e.g., lovastatin and compactin), immunosuppressives (e.g., cyclosporin), and antifungal drugs (e.g., pneumocandin and echinocandin). All of these drugs are fungal secondary metabolites, small secreted molecules that fungi utilize against competitors in their microbial environment. Finally, fungi also produce commercially important enzymes (e.g., cellulases, proteases, and lipases) as well as other products (e.g., citric acid, gibberellic acid, natural pigments, and flavorings).
The specifics by which fungi invade their growth substrate are not understood in detail. However, two important themes regarding the fungal invasion process have emerged in recent years. First, important human fungal pathogens, such as Candida sp., Aspergillus sp., Mucor sp., Rhizopus sp., Fusarium sp, Penicillium marneffei, Microsporum sp. and Trichophyton sp. invade through host tissues as filamentous hyphae. The virulence of Candida (C.) albicans has been shown to be dependent upon invasion of host tissues; mutations in any of several genes required for invasive growth substantially reduce virulence in a mouse model of systemic infection. Pathogenesis of the plant fungal pathogen Ustilago (U.) maydis also requires invasion. Second, there is a correlation between genes that regulate agar invasion in Saccharomyces (S.) cerevisiae and genes that control invasion in pathogenic yeast. As S. cerevisiae is amenable to genetic studies, it can be utilized to molecularly dissect the genetics of fungal invasion.
Homologs of certain S. cerevisiae genes required for invasion also regulate the production of secondary metabolites and secreted catabolic enzymes in other fungi. For example, activating mutations in Aspergillus homologs of the S. cerevisiae INV genes cause increased production of the secondary metabolite penicillin and a secreted alkaline phosphatase (Orejas et al., Genes Dev. 1995, 9:1622).
In one aspect, the invention features a method for determining whether a candidate compound decreases the expression of a gene operably linked to a fungal invasin gene promoter. The method generally includes the steps of (a) providing a fungus expressing the gene operably linked to a fungal invasin gene promoter; (b) contacting the fungus with the candidate compound; and (c) detecting or measuring expression of the gene following contact of the fungus with the candidate compound. In preferred embodiments, the fungus is a wild-type strain (e.g., Saccharomyces cerevisiae, Candida albicans, or Aspergillus nidulans); a mutant strain; or a transgenic fungus. In other preferred embodiments, the gene used in the method of the invention is a fungal invasin gene. Exemplary fungal invasin genes include, without limitation, AFL1, DHH1, INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13, INV14, INV15, BEM2, CDC25, FLO11, IRA1, MCM1, MGA1, MUC1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SW14, TPK2, TPK3, RIM1, or YPR1.
In still other preferred embodiments, the gene used in the method of the invention is a reporter gene. Exemplary reporter genes useful in the methods of the invention include, without limitation, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), xcex2-galactosidase (lacZ), luciferase, URA3, or HIS3.
In preferred embodiments, the fungal invasin gene promoter utilized in the methods of the invention is derived from the FLO11, MUC1, STA1, ST2, or STA3 gene promoter. In other preferred embodiments, the fungal invasin gene promoter includes a promoter sequence derived from an AFL1, DHH1, INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13, RIM1, INV14, INV15, BEM2, CDC25, HOG1, IRA1, MCM1, MGA1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4, TPK2, TPK3, or YPR1 gene promoter. Preferably, the fungal invasin gene promoter is a fragment or a deletion of the fungal invasin gene promoter (e.g., a fragment of the FLO11 gene promoter); and, if desired, the fragment is fused to a basal promoter (e.g., a basal promoter from a PGK1, ADH1, GAL1-10, tet-R, MET25, CYC1 or CUP1 gene).
Typically, the expression of the gene (e.g., the endogenous FLO11 or a recombinant reporter gene expressed under the control of the FLO11 gene promoter or fragment thereof) is measured by assaying the RNA or protein levels or both of the expressed gene. For example, the polypeptide expressed by the fungal invasin gene or by the reporter gene produces a detectable signal under conditions such that the compound causes a measurable signal to be produced. Quantitatively determining the amount of signal produced requires comparing the amount of signal produced to the amount of signal detected in the absence of any compound being tested or upon contacting the cell with any other compound as is described herein. The comparison permits the identification of the compound as one which causes a change in the detectable signal produced by the expressed gene (e.g., at the RNA or protein level) and thus identifies a compound that is capable of inhibiting fungal invasion. A decrease in the expression of the fungal invasin gene is generally accompanied by an inhibition of fungal invasion or an inhibition of the developmental switch from yeast form to pseudohyphal growth or both.
In related aspects, the invention also features a method for determining whether a candidate compound increases the expression of a gene operably linked to a fungal invasin gene promoter. The method generally includes the steps of (a) providing a fungus expressing the gene operably linked to a fungal invasin gene promoter; (b) contacting the fungus with the candidate compound; and (c) detecting or measuring expression of the gene following contact of the fungus with the candidate compound. In preferred embodiments, the method further includes determining whether the candidate compound increases the production of a secondary metabolite in the fungus.
In another aspect, the invention features a method for determining whether a candidate compound inhibits fungal invasion. The method generally includes the steps of (a) contacting a fungus with a candidate compound under conditions suitable for invasion and (b) measuring or detecting invasion by the fungus following contact with the candidate compound. In preferred embodiments, the fungus is Candida albicans or Saccharomyces cerevisiae. 
In another aspect, the invention features a method for determining whether a candidate compound promotes fungal invasion. The method generally includes the steps of (a) contacting a fungus with a candidate compound under conditions suitable for invasion and (b) measuring or detecting invasion by the fungus following contact with the candidate compound. In preferred embodiments, the fungus is Candida albicans, Saccharomyces cerevisiae, or Aspergillus nidulans. 
In still another aspect, the invention features a method for identifying a fungal invasion-promoting gene. The method generally includes the steps of (a) expressing in a fungus (i) a first gene operably linked to a fungal invasin gene promoter and (ii) a second candidate gene or a fragment thereof and (b) monitoring the expression of the first gene, wherein an increase in the expression of the first gene identifies the second candidate gene as a fungal invasion-promoting gene. In preferred embodiments, the fungus is a wild-type strain (e.g., Saccharomyces cerevisiae, Aspergillus nidulans, Penicillium chrysogenum, or Acremonium chrysogenum); is a mutant strain; or is a transgenic fungus.
Preferably, the first gene includes a fungal invasin gene (e.g., FLO11 or MUC1). In yet other preferred embodiments, the first gene includes a fungal invasin gene derived from AFL1, DHH1, INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13, INV14, INV15, BEM2, CDC25, HOG1, IRA1, RIM1, MCM1, MGA1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4, TPK2, TPK3, or YPR1 gene; or the first gene includes a reporter gene (e.g., lacZ URA3, or HIS3).
In preferred embodiments, the fungal invasin gene promoter is derived from the FLO11, MUC1, STA1, STA2, or STA3 gene promoter. In other preferred embodiments, the fungal invasin gene promoter is derived from the AFL1, DHH1, INV1, RIM1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13, INV14, INV 15, BEM2, CDC25, HOG1, IRA1, MCM1, MGA1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4, TPK2, TPK3, or YPR1 gene promoter; or is a fragment or a deletion of the above-mentioned fungal invasin gene promoters. Preferably, the fragment of a fungal invasin gene promoter is fused to a basal promoter (e.g., a basal promoter of a PGK1, ADH1, GAL1-10, tet-R, MET25, CYC1, or CUP1 gene).
Preferably, the expression of the gene utilized in the method of the invention is measured by assaying the protein level of the expressed first gene or by assaying the RNA level of the expressed first gene.
In related aspects, the invention also features a method for identifying a fungal invasion-inhibiting gene. The method generally includes the steps of (a) expressing in a fungus (i) a first gene operably linked to a fungal invasin gene promoter and (ii) a second candidate gene or fragment thereof and (b) monitoring the expression of the first gene, wherein a decrease in the expression of the first gene identifies the second candidate gene as a fungal invasion-inhibiting gene.
In yet another aspect, the invention features a method for increasing production of a secondary metabolite in a fungal cell; the method generally includes the step of contacting the fungal cell with a fungal invasion-promoting compound and culturing the cells under conditions which promote the increased synthesis of a secondary metabolite.
In another aspect, the invention features a method for increasing production of a secondary metabolite in a fungal cell. The method generally includes the step of decreasing the expression of a fungal invasion-inhibiting gene. In preferred embodiments, the decreased expression of the fungal invasion-inhibiting gene (e.g., HOG1, BEM2, RIM15, SFL1, IRA1, SSD1, SRB11, SWI4, or TPK3) results from an inactivation of the fungal invasion-inhibiting gene. In other preferred embodiments, the increased production of a secondary metabolite results from the expression of a mutated fungal invasion-inhibiting gene.
In still another aspect, the invention features a method for increasing production of a fungal secondary metabolite. The method generally includes the step of increasing the expression of a fungal invasion-promoting gene. In preferred embodiments, the fungal invasion-promoting gene is AFL1, DHH1, INV7, INV8, STE21, PET9, MEP2, INV1, INV5, INV6, INV9, INV10, INV11, INV12, INV13, INV14, INV15, CDC25, MCM1, MGA1, PHD2, PHO23, PTC1, RIM1, STP22, TPK2, or YPR1. In other preferred embodiments, the increased expression of the fungal invasion-promoting gene is achieved by constitutively expressing the fungal invasion-promoting gene or by overexpressing such a gene. In yet other preferred embodiments, the fungal invasion-promoting gene is mutated.
In another aspect, the invention features a method for increasing production of a fungal secondary metabolite. The method generally includes the step of expressing a gene or fragment thereof that encodes an activated form of a invasion-promoting polypeptide. In preferred embodiments, the activated form of the invasion-promoting polypeptide includes a fusion between the invasion-promoting polypeptide and a second polypeptide that further enhances the activity of the invasion-promoting polypeptide. In other preferred embodiments, the gene has a mutation.
In another aspect, the invention features a method for increasing production of a secondary metabolite in a fungal cell. The method generally includes the step of decreasing the activity of a fungal invasion-inhibiting polypeptide. In preferred embodiments, the fungal invasion-inhibiting polypeptide has a mutation (e.g., a dominant-inactive polypeptide). In other preferred embodiments, the fungal invasion-inhibiting polypeptide is Hog1, Bem2, Rim15, Ira1, Sfl1, Ssd1, Srb11, Swi4, or Tpk3.
In another aspect, the invention features a method for increasing production of a fungal secondary metabolite. The method generally includes the steps of increasing the activity of a fungal invasion-promoting polypeptide. In preferred embodiments, the fungal invasion-promoting polypeptide has a mutation (e.g., a dominant-active polypeptide). In other preferred embodiments, the fungal invasion-promoting polypeptide is Afl1, Dhh1, Inv1, Inv5, Inv6, Inv9, Inv10, Inv11, Inv12, Inv13, Inv14, Rim1,Inv15, Cdc25, Inv7, Mcm1, Mga1, Phd2, Pho23, Ptc1, Inv8, Ste2, Pet9, Mep2, Stp22, Ypr1.
In another aspect, the invention features a method of isolating a fungal invasin gene. The method generally involves the steps of (a) providing a fungus expressing a gene operably linked to a fungal invasin gene promoter; (b) mutagenizing the fungus; (c) measuring expression of the gene, wherein an increase or decrease in the expression of said gene identifies a mutation in said invasin gene; and (d) using said mutation as a marker for isolating said invasin gene. In preferred embodiments, the fungus is a wild-type strain (e.g., Saccharomyces cerevisiae) or is a mutant strain. In preferred embodiments, the gene utilized in the method includes a fungal invasin gene (e.g., FLO11, MUC1, AFL1, DHH1, INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13, INV14, INV15, BEM2, CDC25, HOG1, IRA1, MCM1, MGA1, PET9, PHD2, PHO23, RIM1, PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4, TPK2, TPK3, or YPR1). In other preferred embodiments, the gene includes a reporter gene (e.g., lacZ, URA3, or HIS3). In still other preferred, the fungal invasin gene promoter is from FLO11, MUC1, STA1, STA2, or STA3 gene promoter; or is from the AFL1, DHH1, INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13, INV14, INV15, BEM2, CDC25, RIM1, HOG1, IRA1, MCM1, MGA1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4, TPK2, TPK3, or YPR1 gene promoter. In other preferred embodiments, the fungal invasin promoter is a fragment or deletion of the above-mentioned fungal invasin gene promoters. Preferably, the fragment of a fungal invasin gene promoter is fused to a basal promoter (e.g., a basal promoter of a PGK1, ADH1, GAL1-10, tet-R, MET25, CYC1 or CUP1 gene).
In another aspect, the invention features a method of using a cell (e.g., a fungal cell) for identifying a gene which regulates the expression from a Candida albicans gene promoter. The method generally includes the steps of (a) providing a cell expressing a reporter gene operably linked to a Candida albicans gene promoter; (b) expressing a candidate gene in the cell; and (c) detecting or measuring the expression of the reporter gene. In preferred embodiments, the fungal cell is Saccharomyces cerevisiae. 
In another aspect, the invention features a method for preparing a transgenic fungal cell having increased secondary metabolite production. The method generally includes the steps of (a) introducing a transgene (e.g., a transgene encoding an invasin gene such AFL1, DHH1, INV7, INV8, STE21, PET9, MEP2, INV1, INV5, INV6, INV9, INV10, INV11, RIM1, INV12, INV13, INV14, INV15, CDC25, MCM1, MGA1, PHD2, PHO23, PTC1, STP22, TPK2, YPR1 or a fragment thereof, which is positioned for expression in a fungal cell) and (b) selecting a cell that expresses the transgene. In preferred embodiments, the transgene has a mutation (e.g., a dominant-active mutation or a dominant-inactive mutation).
In another aspect, the invention features a method for increasing a secondary metabolite in a fungus. The method generally includes the steps of (a) culturing the fungus in culture with conditions allowing for secondary metabolite production; (b) adding to the culture a fungal invasion-promoting compound; and (c) isolating the metabolite from the culture. In preferred embodiments, the fungus has a mutation. In other preferred embodiments, the fungus is a wild-type strain.
In still another aspect, the invention features a transgenic fungus (e.g., a filamentous fungus) which includes a mutation in an invasin gene that inhibits its activity. Preferably, the invention features a transgenic filamentous fungus having a mutation in a HOG1, SWI4, BEM2, SRB11, SSD1, TPK3, SFL1, or an IRA1 gene or any combination thereof. Preferably, such mutations inhibit the activity of the expressed protein (e.g., Hog1, Swi4, Bem2, Srb 11, Ssd1, Tpk3, Sfl1, or Ira1 or any combination thereof). In other preferred embodiments, the transgenic fungus has increase secondary metabolite production (e.g., increased production of antibiotics).
In another aspect, the invention features a substantially pure Inv9 polypeptide. Preferably, the Inv9 polypeptide is at least 55% identical to the amino acid sequence of FIG. 6B (SEQ ID NO: 6). In preferred embodiments, the Inv9 polypeptide is from a fungus (e.g., a yeast such as Saccharomyces). In other preferred embodiments, the Inv9 has invasion promoting activities.
In a related aspect, the invention features an isolated nucleic acid (e.g., DNA) encoding an Inv9 polypeptide. Preferably, such an isolated nucleic acid sequence includes the INV9 gene of FIG. 6A (SEQ ID NO: 5) and complements an INV9 mutation in Saccharomyces cerevisiae. 
In another aspect, the invention features a substantially pure Rim1 polypeptide. Preferably, the Rim1 polypeptide is at least 75% identical to the amino acid sequence of FIG. 5A (SEQ ID NO: 4) in the zinc-finger domain, and at least 25% identical throughout the entire length of the polypeptide amino acid sequence. In preferred embodiments, the Rim1 polypeptide is from a fungus (e.g., a yeast such as Saccharomyces). In other preferred embodiments, the Rim1 polypeptide has invasion promoting activities.
In a related aspect, the invention features an isolated nucleic acid (e.g., DNA) encoding a Rim1 polypeptide. Preferably, such an isolated nucleic includes the RIM1 gene of FIG. 5A (SEQ ID NO: 3) and complements a RIM1 mutation in Saccharomyces cerevisiae, as is described herein.
In related aspects, the invention further features a cell or a vector (for example, a fungal expression vector), each of which includes an isolated nucleic acid molecule of the invention. In preferred embodiments, the cell is a fungal cell (for example, S. cerevisiae). In yet another preferred embodiment, the isolated nucleic acid molecule of the invention is operably linked to a promoter that mediates expression of a polypeptide encoded by the nucleic acid molecule. The invention further features a cell (for example, a fungal cell) which contains the vector of the invention.
In still another aspect, the invention features a transgenic fungus including any of the above nucleic acid molecules of the invention, wherein the nucleic acid molecule is expressed in the transgenic fungus.
In related aspects, the invention also features a method of producing an Inv9 or Rim1 polypeptide. The method involves: (a) providing a cell transformed with a nucleic acid molecule of the invention positioned for expression in the cell; (b) culturing the transformed cell under conditions for expressing the nucleic acid molecule; and (c) recovering the Inv9 or Rim1 polypeptide. The invention further features a recombinant Inv9 or Rim1 polypeptide produced by such expression of an isolated nucleic acid molecule of the invention, and a substantially pure antibody that specifically recognizes and binds to each of these polypeptides or a portion thereof.
By xe2x80x9cfungal invasionxe2x80x9d is meant a process by which a fungus penetrates, digs, adheres to, or attaches to a substrate. Invasion of a substrate by a fungus may be measured according to standard methods as described herein.
By xe2x80x9cfungal invasinxe2x80x9d gene is meant a gene encoding a polypeptide capable of promoting or inhibiting the invasion by a fungus into a substrate. This response may occur at the transcriptional level or it may be enzymatic or structural in nature. Fungal invasin genes may be identified and isolated from any fungal species, using any of the sequences disclosed herein in combination with conventional methods known in the art.
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
By a xe2x80x9creporter genexe2x80x9d is meant a gene whose expression may be assayed; such genes include, without limitation, genes encoding xcex2-galactosidase, xcex2-glucosidase, xcex2-glucosidase, and invertase, amino acid biosynthetic genes, e.g., the yeast LEU2, HIS3, LYS2, TRP1 genes, nucleic acid biosynthetic genes, e.g., the yeast URA3 and ADE2 genes, the mammalian chloramphenicol transacetylase (CAT) gene, or any surface antigen gene for which specific antibodies are available. A reporter gene may encode a protein detectable by luminescence or fluorescence, such as green fluorescent protein (GFP). Reporter genes may encode also any protein that provides a phenotypic marker, for example, a protein that is necessary for cell growth or viability, or a toxic protein leading to cell death, or the reporter gene may encode a protein detectable by a color assay leading to the presence or absence of color. Alternatively, a reporter gene may encode a suppressor tRNA, the expression of which produces a phenotype that can be assayed. A reporter gene according to the invention includes elements (e.g., all promoter elements) necessary for reporter gene function.
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 25%, preferably 50%, more preferably 80%, and most preferably 90%, or even 95% identity to a reference amino acid sequence (for example, the amino acid sequence shown in FIG. 5A (SEQ ID NO: 4) or nucleic acid sequence (for example, the nucleic acid sequences shown in FIG. 5A; SEQ ID NO: 3). For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids or greater. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides or greater.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BEAUTY, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide (for example, an invasin polypeptide such as the Inv9 or Rim1 polypeptide) that has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, an invasin polypeptide. A substantially pure invasin polypeptide may be obtained, for example, by extraction from a natural source (for example, a fungal cell); by expression of a recombinant nucleic acid encoding an invasin polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By xe2x80x9cderived fromxe2x80x9d is meant isolated from or having the sequence of a naturally-occurring sequence (e.g., a cDNA, genomic DNA, synthetic, or combination thereof).
By xe2x80x9cisolated DNAxe2x80x9d is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9ctransgenic fungal cellxe2x80x9d is meant a fungal cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) an invasin polypeptide.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, for example, an invasin polypeptide, a recombinant protein, or an RNA molecule).
By xe2x80x9cpromoter sequencexe2x80x9d is meant any minimal sequence sufficient to direct transcription. Included in the invention are promoter elements that are sufficient to render promoter-dependent gene expression controllable for gene expression, or elements that are inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene or engineered into a transgene construct.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s).
By xe2x80x9ccandidate genexe2x80x9d is meant any piece of DNA which is inserted by artifice into a cell and expressed in that cell. A candidate gene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic fungal cells. A transgenic fungal cell according to the invention may contain one or more invasin genes.
By xe2x80x9cincreasing production of a secondary metabolitexe2x80x9d is meant a greater level of production of a secondary metabolite in a transgenic fungus of the invention than the level of production relative to a control fungus (for example, a non-transgenic fungus). In preferred embodiments, the level of secondary metabolite production in a transgenic fungus of the invention is at least 10% greater (and preferably more than 30% or 50%) than the resistance of a control fungus. The level of secondary metabolite production is measured using conventional methods.
By xe2x80x9cdetectably-labelledxe2x80x9d is meant any direct or indirect means for marking and identifying the presence of a molecule, for example, an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule or a fragment thereof. Methods for detectably-labelling a molecule are well known in the art and include, without limitation, radioactive labelling (for example, with an isotope such as 32P or 35S) and nonradioactive labelling (for example, chemiluminescent labelling, for example, fluorescein labelling).
By xe2x80x9cpurified antibodyxe2x80x9d is meant antibody which is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, for example, an acquired resistance polypeptide-specific antibody. A purified invasin antibody (e.g. Inv9 or Rim1) may be obtained, for example, by affinity chromatography using a recombinantly-produced acquired resistance polypeptide and standard techniques.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody which recognizes and binds an invasin polypeptide but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes an invasin polypeptide such as Inv9 or Rim1.
By a xe2x80x9cmutationxe2x80x9d is meant an alteration in sequence, either by site-directed or random mutagenesis. A mutated form of a protein encompasses point mutations as well as insertions, deletions, or rearrangements. A mutant is an organism containing a mutation
The invention provides long awaited advantages over a wide variety of standard screening methods used for distinguishing and evaluating the efficacy of a compound in regulation of gene expression in fungal pathogens. For example, the methods allow for the identification, by genetic selection in a high throughput format, of peptides and compounds that specifically activate or inhibit fungal invasion. These methods also allow the mode of action for such agents to be rapidly delineated. Moreover, these methods are amenable to an iterative compound modification and retesting process to allow for the evolution of more effective compounds from initial hits and leads.
Compounds which inhibit fungal invasion will likely also prevent fungal virulence. These compounds may have therapeutic value in treating plant or animal fungal diseases. Compounds which promote fungal invasion may be useful in increasing yields of commercially important fungal secondary metabolites.
The invention also provides an approach to isolating novel fungal genes important for pathogenesis. These novel genes and the proteins they encode comprise additional targets for compounds.
In addition, the invention provides a means to increase the yield of commercially important secondary metabolites by genetic manipulation of the fungal organism itself. This facilitates the large scale production of fungal products which to date has not been possible. In addition, it allows for the facile identification of xe2x80x9cpotentiators,xe2x80x9d (i.e., compounds and peptides) that can activate secondary metabolite production when contacting, or expressed in, fungi. The ability to increase fungal secondary metabolite production has at least two important applications. First, increasing production of secondary metabolites will facilitate identification of new antimicrobial compounds in fungi that otherwise make undetectable levels of these compounds in the laboratory. Second, it will allow increased production of existing secondary metabolites which are useful clinically or for research.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.