WO1999013094A2

WO1999013094A2 - Fungal pathogenicity genes

Info

Publication number: WO1999013094A2
Application number: PCT/US1998/018730
Authority: WO
Inventors: James A. Sweigard
Original assignee: E.I. Du Pont De Nemours And Company
Priority date: 1997-09-10
Filing date: 1998-09-08
Publication date: 1999-03-18
Also published as: AU9567398A; WO1999013094A3

Abstract

This invention relates to isolated nucleic acid fragments encoding all or a substantial portion of genes that encode proteins important in effecting fungal pathogenicity in rice blast. The pathogenicity genes may be expressed as chimeric genes linked to suitable regulatory elements and are useful in the development of screens to identify inhibitors of the gene products.

Description

TITLE FUNGAL PATHOGENICITY GENES FIELD OF THE INVENTION This invention is in the field of molecular biology. More specifically, the invention pertains to nucleic acid fragments encoding key proteins that regulate fungal pathogenicity.

BACKGROUND OF THE INVENTION The parasitic interaction between pathogenic fungi and their plant hosts is a complex biological process. For Magnaporthe grisea, the causal agent of blast on many different grasses including rice, the pathogen cycle involves (minimally) the following developmental sequence: (1) deposition of a conidium on the plant surface, (2) germination of the conidium to form a germ tube, (3) differentiation of the germ tube into a specialized infection structure called an appressorium, (4) penetration of the plant leaf surface by the melanized appressorium via a penetration peg, (5) differentiation of the penetration peg into a secondary hypha that then grows throughout the invaded plant cell, (6) escape from the first infected plant cell and growth throughout the surrounding plant tissue, (7) production of aerial conidiophores, (8) production of conidia and, finally, (9) release of the conidia to complete the cycle (Howard and Valent, 1996, Annu. Rev. Microbiol. 50:491-512). In this developmental sequence, only the melanization and pressurization of the appressorium is understood at a detailed level (Howard and Valent, 1996, Annu. Rev. Microbiol. 50:491-512). Moreover, the entire interaction is apparently modified by pre-formed and active plant defenses.

Rice is a vital food crop in developing as well as industrial nations. Pathogenic rice blast fungi attack and destroy rice plants in the way described above. The effects of the fungi create an annual worldwide blast fungicide market of approximately $500 million.

Currently, the U.S. E.P.A. recommends only the fungicide benomyl for rice blast. Other compounds such as cymoxanil (E. I. du Pont de Nemours and Company, Wilmington, DE), oxadiargyl (Rhone-Poulenc, Collegeville, PA) propamopcarb hydrochloride (AgrEvo) and dimethomorph are being tested for fungal toxicity. Many of these compounds have general toxicity to fungi and are not specifically toxic to rice blast. Understanding the mode of action of blast pathogenicity and identifying pathways and targets that are susceptible to inhibition by selected compounds are necessary steps to more effectively control this important disease.

A greater understanding of the genes involved in pathways linked to pathogenicity will facilitate the development of alternate anti-fungals effective against rice blast. Several genes have been identified that play various roles in rice blast disease. Many of these genes are either necessary for the formation of the appressorium or are expressed at the time of appressorium formation. For example, Xu and Hamer (Genes Dev., 1996, 10(21), 2696-2706) report the isolation of the PMK1 gene encoding a MAP kinase, an enzyme essential for the development of the fungal appressorium. Talbot et al. (Plant Cell, 1996, 8(6), 985-999; Plant Cell, 1993, 5(11), 1575-90) and Beckerman et al. (Mol. Plant-Microbe Interact., 1996, 9(6), 450-456) teach the cloning of the MPG1 gene, expressed during appressoria formation and encoding a hydrophobic protein necessary for infectivity. Additionally, NPR1 and NPR2, genes involved in the regulation of MPG1 have also been identified. (Lau and Hamer, Plant Cell, 1996, 8(5), 771 -781 ). Other genes that play a role in appressorium development include APP1 (Zhu et al., Mol. Plant-Microbe Interact., 1996, 9(9), 161-114) and cpkA encoding a cAMP-dependant kinase subunit (Mitchell et al., Plant Cell, 1995, 7(11), 1869-78). Genes not involved in appressorium formation but implicated in rice blast pathogenicity include the PWL genes (PWL\, PWL2, PWL3, PWL4) involved in host specificity (Sweigard et al., Plant Cell 1995, 7(8), 1221-33; Kang et al, Mol. Plant-Microbe Interact., 1995, 8(6), 939-48).

Applicant's invention relates to new genes involved in rice blast pathogenicity that either encode enzymes or proteins not heretofore implicated in the pathogenic process. These novel genes expand the understanding of rice blast pathogenesis and will lead to the development of screens for compounds able to inhibit these newly discovered pathogenic targets.

SUMMARY OF THE INVENTION The instant invention relates to isolated genes encoding proteins implicated in the pathogenicity of rice blast. In addition, this invention relates to nucleic acid fragments that are complementary to the pathogenicity genes.

The invention further includes the gene products of isolated fungal pathogen genes.

An additional embodiment of the instant invention concerns a method for obtaining all or a portion of the instant fungal pathogenic genes by using the sequence of the genes to design oligonucleotide probes or PCR primers.

In an alternate embodiment the invention provides a method for evaluating at least one compound for its ability to inhibit the activity of a fungal pathogenicity gene product, the genes selected from the group consisting OΪPTH2, and PTH3, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a fungal pathogenicity gene selected from the group consisting of PTH2, and PTH3, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the protein encoded by the operably linked nucleic acid fragment in the transformed host cell; (c) optionally purifying the fungal pathogenicity protein expressed by the transformed host cell; (d) treating the fungal pathogenicity protein with a compound to be tested; and (e) comparing the activity of the protein that has been treated with a test compound to the activity of an untreated fungal pathogenicity protein, thereby selecting compounds with potential for inhibitory activity.

In an alternate embodiment, the invention provides a method for evaluating at least one compound for its ability to inhibit the activity of the PTH11 gene product by transforming a suitable host with the PTH11 gene wherein expression of the PTH11 gene permits the host to grow and inhibition of the gene will be lethal to the host; contacting the transformed host with a compound suspected of being a PTH11 inhibitor and monitoring the growth of the transformed host to measure the efficacy of the inhibitor compound.

In a further embodiment, the invention provides a method for evaluating at least one compound for its ability to inhibit the activity of the PTHI2 gene product by transforming a suitable host cell with the PTH12 gene such that the gene is expressed; further engineering the host cell such that the expression of a reporter gene is dependent on the binding of the PTH12 gene product to the reporter gene; contacting the transformed host with a compound suspected of inhibiting the activity of the PTH12 gene product and monitoring the expression of reporter gene to determine the efficacy of the inhibitory compound.

BRIEF DESCRIPTION OF THE DRAWINGS. SEQUENCE DESCRIPTIONS. AND BIOLOGICAL DEPOSITS The invention can be more fully understood from the following detailed description, the accompanying drawings, sequence descriptions, and biological deposits which form a part of this application.

Figs, la-e shows leaf sections from barley plants inoculated with wild-type strain 4091-5-8(2 x 10⁵ conidia/mL) and pth2. pth3. pthll and pth!2 strains (1 x IO⁶ conidia/mL). Wild type causes coalescing Type 5 lesions while pth2 and pthl 2 strains (Figs, lb and le) never cause disease lesions (Type 0). pthll (Fig. Id) strains cause rare lesions. pth3 (Fig. lc) strains cause numerous slowly expanding lesions (Type 2-3) that produce limited conidia compared to wild-type.

Figures la-e are a set of photographs showing leaf sections from barley plants with varying degrees of lesions cause by pathogenic strains of rice blast. Figure la shows type 5 lesion seen in wildtype plants. Figures lb and le show a type 0 lesion, indicating strong blast resistance. Figure lc shows a rare intermediate lesion of type 1 to type 2. Figure Id shows slowly expanding lesions of type 2 to 3. Figure 2 is a hydrophobicity plot illustrating the secondary structure of the protein encoded by pthll (SEQ ID NO:9).

The host strain Magnaporthe grisea 4091-5-8 was deposited on February 7, 1992 with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA (ATCC) under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purpose of Patent Procedure. The deposit is designated as ATCC 74134.

The following sequence descriptions and sequences listings attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. The Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IYUB standards described in Nucleic Acids Research 75:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2^:345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO:l is the full length nucleotide sequence comprising PTH1.

SEQ ID NO:2 is the nucleotide sequence for the coding region of PTH2.

SEQ ID NO: 3 is the deduced amino acid sequence encoded by the coding region ofPTHl demonstrating 41% sequence identity to yeast carnitine acetyl transferase.

SEQ ID NO:4 is the nucleotide sequence of the full length gene comprising PTH3.

SEQ ID NO:5 is the nucleotide sequence of the coding region for PTH3. SEQ ID NO:6 is the deduced amino acid sequence encoded by the coding region ofPTH3 demonstrating 63% sequence identity to a Saccharomyces imidazole glycerol phosphate dehydratase (IGPD).

SEQ ID NO: 7 is the nucleotide sequence of the full length gene comprising PTH11, a gene encoding a membrane associate protein. SEQ ID NO:8 is the nucleotide sequence of the coding region ofPTHll.

SEQ ID NO:9 is the deduced amino acid sequence encoded by the coding region of PTH11.

SEQ ID NO: 10 is the nucleotide sequence for the full length gene comprising PTH12, a gene encoding a homeodomain transcription factor. SEQ ID NO: 11 is the nucleotide sequence for the coding region of PTH12.

SEQ ID NO: 12 is the deduced amino acid sequence encoded by the coding region ofPTH12. DETAILED DESCRIPTION OF THE INVENTION Inhibition of any of the genes PTH2, PTH3, PTH1 and PTH12 results in the reduction or elimination of the pathogenic phenotype of the fungus. The isolated genes are useful in the design of screens to identify inhibitors of the fungal pathogenic gene products.

Isolation of the genes of the present invention proceeded by the transformation of pathogenic M. grisea strains with plasmids containing a gene for hygromycin resistance and the selection of hygromycin-resistant strains that demonstrated reduced or no pathogenicity. The plasmid inserts were recovered by plasmid rescue and confirmation of the gene's pathogenic function was obtained by complementation of the wildtype strain with the mutant gene. The genes have been sequenced and their function putatively identified on the basis of BLAST analysis of the genetic databases.

The following abbreviations and terms are relevant the interpretation of this subject matter: PTH refers to a wildtype gene. pth refers to a mutant gene. Pthp refers to the protein encoded by the gene. Thus, PTH2 is a wildtype gene encoding carnitine acetyl transferase; pth2 is the mutant gene encoding a mutation in that gene resulting in a phenotype of lessened pathogenicity; and Pth2p refers to the carnitine acetyl transferase protein. Similarly, PTH3 is a wildtype gene encoding imidazole glycerol phosphate dehydratase (IGPD); pth3 is the mutant gene encoding a mutation in that gene resulting in a phenotype of lessened pathogenicity; and Pth3p refers to the imidazole glycerol phosphate dehydratase protein. PTH 11 is a wildtype gene encoding a membrane associated protein involved in fungal pathogenicity; pthl 1 is the mutant gene encoding a mutation in that gene resulting in a phenotype of lessened pathogenicity; and Pthllp refers to the protein encoded by that gene. PTH12 is a wildtype gene encoding a homeodomain transcription factor involved in fungal pathogenicity; pthl 2 is the mutant gene encoding a mutation in that gene resulting in a phenotype of lessened pathogenicity; and Pthl2p refers to the protein encoded by that gene.

An "isolated nucleic acid fragment" is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term "fungal pathogenicity gene" refers to a gene which encodes a rice blast fungal protein necessary for the fungus to successfully penetrate a plant host cell and cause disease. The gene may be implicated in any system associated with pathogenicity, including but not limited to, formation of the appressorium, melanaization of the appressorium, formation or development of the penetration peg or development of secondary hypha.

The term "fungal pathogenicity protein" refers to any protein encoded by a fungal pathogenicity gene. Fungal pathogenicity proteins may have enzymatic function or be of unknown function but expression of the protein, either individually or in concert with the expression of other proteins, will be necessary to convey a pathogenic phenotype on the fungal organism.

The term "conidia" or "conidium" refers to a fungal asexual spore. The term "plasmid rescue" refers to a technique for circularizing restriction enzyme-digested fungal genomic DNA that carries DNA fragments bearing a bacterial origin of replication and antibiotic resistance such that this circularized fragment can be propagated as a plasmid in a bacterial host cell such as E. coli.

Substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but the functional properties of the protein encoded by the DNA sequence are not affected. "Substantially similar" also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript vis-a-vis the alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary sequences. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes which result in substitution of one negatively charged residue for another (such as aspartic acid for glutamic acid) or one positively charged residue for another (such as lysine for arginine) can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Moreover, the skilled artisan recognizes that substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65°C), with the sequences exemplified herein. Preferred substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are at least 90% identical to the identical to the DNA sequence of the nucleic acid fragments reported herein. Most preferred are nucleic acid fragments that are at least 95% identical to the DNA sequence of the nucleic acid fragments reported herein.

A "substantial portion" of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. 1993, J Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/).

In general, a sequence often or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches partial or complete amino acid and nucleotide sequences encoding one or more particular fungal proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk. A. M.. ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data. Part I (Griffin, A. M.. and Griffin, H. G., eds.) Humana Press. New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG Pileup program found in the GCG program package, as used in the instant invention, using the Needleman and Wunsch algorithm with their standard default values of gap creation penalty=12 and gap extension penalty=4 (Devereux et al., Nucleic Acids Res. 12:387-395 (1984)), BLASTP, BLASTN, and FASTA (Pearson et al, Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448 (1988). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual. Altschul et al., Natl. Cent. Biotechnol. Inf., Natl. Library Med. (NCBI NLM) NIH, Bethesda, Md. 20894; Altschul et al., J. Mol. Biol. 215:403-410 (1990)). Another preferred method to determine percent identity, is by the method of DNASTAR protein alignment protocol using the Jotun-Hein algorithm (Hein et al, Methods Enzymol. 183:626-645 (1990)). Default parameters for the Jotun-Hein method for alignments are: for multiple alignments, gap penalty=l 1, gap length penalty=3; for pairwise alignments ktuple=6. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO:l, for example, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1 , for example. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95%) identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO:3, for example, is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO:3, for example. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. "Codon degeneracy" refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the pathogenicity proteins as set forth in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO: 12. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell. "Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.

"Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence.

The term "suitable regulatory sequences" may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences as discussed infra.

"Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. "Overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.

"Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of fungal transformation are given in Lemke et al.. Genetic manipulation of fungi by DNA-mediated transformation. Mycota, 1995, Volume 2, 109-139. Editor(s): Kueck, Ulrich. Publisher: Springer, Berlin, Germany; Riach et al., Mycol. Ser., 1996, 13(Fungal Genetics), 209-233.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis").

Nucleic acid fragments have been isolated that encode all or a substantial portion of genes that encode proteins important in affecting fungal pathogenicity in rice blast disease. Fungal Strains

The present fungal pathogenicity genes were isolated from a wildtype strain (4091-5-8; Valent et al, 1986, Iowa State J. Res., 60:569-594) that can infect weeping lovegrass and barley. As a species Magnaporthe grisea infects many different grass hosts, though individual isolates have a limited host range. The present wildtype strains of M. grisea are particularly useful in the isolation of these pathogenicity genes since they are easily and efficiently transformed and may be used in high volume infectivity assays.

Transformation of Wildtype Rice Blast

Although a variety of methods of creating mutants are available, insertional mutagenesis was chosen in the present case. Insertional mutagenesis was originally employed in Aspergillus nidulans (Diallinas et al., 1989, Genetics, 122:341-350) and Neurospora crassa (Kang et al., 1993, Genetics, 133:193-202). Subsequently, methods for insertional mutagenesis were extended to include Restriction Enzyme- Mediated Integration (REMI). REMI transformations differ from normal transformations only in that restriction enzymes are added to the transformation milieu. The purpose of the restriction enzymes in REMI is to cut the genomic DNA of the transformation recipient to provide sites for the integration of the transforming DNA. Initially utilized in Saccharomyces cerevisiae (Schiestl et al., 1991, Proc. Natl. Acad. Sci. USA, 88:7585-7589), REMI has been extensively used in Dictyostelium discoideum (Kupsa et al., 1992, Proc. Natl. Acad. Sci. USA, 89:8803-8807). Among plant pathogens REMI has been used to tag the Toxl locus in Cochlibolous heterostrophus (Lu et al, 1994, Proc. Natl. Acad. Sci. USA, 91 :12649-12653), conidiation genes in M. grisea (Shi et al., 1995, Mol. Plant-Microbe Interact., 8:949-959)and genes required for pathogenicity in Ustilago maydis (Bolker et al., 1995, Mol. Gen. Genet., 248:547-552).

In the present method wildtype pathogenic strains of M. grisea were transformed by REMI using plasmids carrying a hygromycin resistance gene.

Transformants were selected for hygromycin resistance on a complete medium and single conidial isolates were tested for lessened pathogenicity. Screening For Hygromycin Resistance And Pathogenicity Infection Assays Methods of determining fungal pathogenicity are common (see, for example,

Howard et al., 1996, Annu. Rev. Microbiol., 50, 491-512). The assays used within the context of the present invention involved contacting an effective amount of fungal conidia with the host plant, either weeping lovegrass, barley, or rice. The exposed hosts were examined over time for the appearance of disease lesions as indicated in Figure 1. The degree of infectivity was determined visually as described in Example 2. Cloning and Sequencing

Methods of cloning mutated genes are common and a variety of methods are described in Maniatis. The preferred method in the present invention was plasmid rescue followed by isolation of the gene from the rescued plasmid. Plasmid rescue is a common technique and is employed in plant cell as well as fungal genetics (see, for example, Behringer et al., 1992, Plant Mol. Biol. Rep., 10, 190).

The vectors used to transform the fungal strains contain a gene for hygromycin resistance and carry sequences required for autonomous replication of DNA in bacteria. Once this DNA is inserted in the fungal genome, specific restriction endonuclease digests can be used to generate fragments that can be circularized, ligated, and transformed into E. coli. Circularized DNA from the T-DNA will generate functional plasmids that confer antibiotic resistance to their bacterial hosts such that they can be identified by growth on selective media.

Typically, the cloning of mutated genes proceeded by utilizing the insert flanking DNA of the rescued plasmids to obtain a plasmid or cosmid with the wildtype DNA sequence from wildtype strains. Wildtype cosmids or plasmids were then transfected into a non-pathogenic or lessened pathogenic mutant and infectivity was measured. Restoration of the pathogenic phenotype provided strong evidence that the isolated genes were implicated in fungal pathogenicity. Further confirmation was obtained by subcloning smaller pieces from the original complementing plasmid that could still complement the mutant and sequencing the smallest pieces that were still able to alter pathogenicity. In this fashion, putative open reading frames (ORF) were identified. Final confirmation was obtained by introducing mutations (by restriction digests) in the ORF of the pathogenic genes and assaying for lessened pathogenicity. The sequenced genes were compared with the GenΕMBL database using the

BLAST algorithm (Basic Local Alignment Search Tool; Altschul et al., 1993. J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) to determine and similarity to known sequences.

BLAST analysis revealed that PTH2 had on the order of 41% identity to a yeast carnitine acetyl transferase at the amino acid level and that PTH3 has about 63% identity to a Saccharomyces imidazole glycerol phosphate dehydratase, an enzyme implicated in histidine biosynthesis.

Although database comparison of the deduced amino acid sequence of PTH11 did not reveal any homology with a known protein, the secondary peptide structure in combination with the organization of hydrophilic and hydrophobic amino acids strongly suggests that this gene encodes a membrane protein.

The hydropathy plot of Pthl Ip of Figure 2 shows at least 7 trans-membrane domains in the N-terminal region of the protein. BLAST analysis of the genetic databases revealed a significant homology between 55 amino acids of PTH12 and the homeobox of homeodomain transcription factors. Microscopic examination of the development of PTH12 mutants on the leaf surface shows that these mutants form pseudo-appressoria (swellings that resemble appressoria which fail to mature into the full blown appressorial structure). Although PTH12 mutants give rise to a germ tube from the pseudo-appressoria, no other infectivity structures develop. This is in contrast to pathogenic wildtype strains where the appressoria mature, melanize, and develop a penetration peg at the bottom of the appressorium that breaches the plant cuticle. Thus, observation of the development of the PTH12 mutants suggest that PTH12 encodes a transcription factor that regulates genes required for appressorium maturation. Screens

The instant fungal pathogenicity genes can be used as targets to facilitate design and/or identification of inhibitors of the proteins that may be useful as fungicides. This is desirable because the proteins encoded by these genes are necessary for the fungus to attain pathogenic phenotype. Accordingly, inhibition of the activity of one or more of the proteins could lead to inhibition or eradication of pathogenicity.

Inhibitor screens are preferably facile and rapid. Screens that rely on whole plant systems, such as infectivity assays, are time consuming. Microbial-based expression systems that can over-express the desired gene products offer a more rapid method of designing gene inhibitor screens.

Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for the overexpression of the instant pathogenicity genes. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high level expression of the proteins.

For recombinant production of the proteins encoded by the present fungal pathogenicity genes in a host organism, the coding sequence may be inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced. The choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the chosen host is within the level of skill of the routineer in the art. The resultant molecule, containing the individual elements linked in proper reading frame, may be inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli (see, e.g., Studier and Moffatt, 1986. J. Mol. Biol. 189:113; Brosius, 1989, DNA 8:759), yeast (see, e.g., Schneider and Guarente, 1991, Meth. Enzymol. 194:373) and insect cells (see, e.g., Luckow and Summers, 1988, Bio/TechnoL, 6:47). Specific examples include plasmids such as pBluescript (Stratagene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New Haven, CT), pTrcHis (Invitrogen, La Jolla, CA), and baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV). A preferred baculovirus/insect system is pVl 11392/Sf21 cells (Invitrogen, La Jolla, CA).

Recombinantly produced fungal pathogenicity proteins can be isolated and purified using a variety of standard techniques. The specific techniques will vary depending upon the host organism used, whether the protein is designed for secretion, and other such factors familiar to the skilled artisan (see, e.g., chapter 16 of Ausubel, F. et al., "Current Protocols in Molecular Biology", pub. by John Wiley & Sons, Inc. (1994)). Where the fungal pathogenicity protein is an enzyme, as with PTH2 and

PTH3, standard enzyme assays may be used to screen for compounds that have fungicidal potential. A typical screen will proceed, for example, by the production and purification of the protein as described above, followed by contacting the enzyme with an appropriate substrate either in the presence or absence of a fungicide candidate. Comparison of the activity of the enzyme alone against the activity of the enzyme contacted with the fungicide candidate will indicate the candidate's efficacy. Enzyme assays for carnitine acetyl transferase (the enzyme encoded by PTH2) are known, (see, for example, Bergstrom et al., 1980, Arch. Biochem. Biophys., 204(1), 71-9; and Halperin et al., 1979, Methods Enzymol., 56(Biomembranes, Part G), 368-78)). Similarly methods for the assay of imidazole glycerol phosphate dehydratase (IGPD) (the enzyme encoded by PTH3) are also known (see, for example, Parker et al., 1994 Gene, 145(1), 135-8.

Where the pathogenicity protein is not an enzyme, screens must be developed around the characteristics of the protein. In the case of PTH11, for example, the characteristics of the protein suggest that it is membrane-associated and may be upstream of a cAMP-dependent step. These aspects of the protein must therefore form the basis for any assay.

It has been observed that mutants of the rice blast strain 4091-5-8 that lack a functional PTHI1 gene form few appressoria compared to wild-type strains. Addition of cAMP (10 mM) reverses the appressorial formation defect of pthl i⁽"⁾ mutants. This result suggests that PTH11 is upstream of cAMP in the signaling pathway that leads to appressorial formation and therefore also upstream of adenylyl cyclase, the enzyme that forms cAMP. It is known that in eukaryotes extracellular signals are often transmitted from a membrane-bound receptor via a GTP -binding protein to adenylyl cyclase, thereby stimulating adenylyl cyclase and increasing intracellular cAMP concentrations (e.g., the B-adrenergic receptor system; see, for example, Alberts et al., 1994, Molecular biology of the cell, Garland Publishing, Inc., New York, NY). The analogies between Pthllp and other membrane-associated cAMP-dependant proteins are three-fold. First, Pthllp is upstream of cAMP. Second, PTH11 has a hydropathy plot containing domains having characteristics of many membrane proteins. Finally, it appears the Pthllp is involved in signaling for appressorial formation as opposed to participating in appressoria formation proper. These observations suggest that Pthllp may perceive an extracellular signal which causes the release of GTP-binding protein that then activates adenylyl cyclase to produce cAMP. In yeast, for example, the GTP-binding protein that stimulates adenylyl cyclase are the ras proteins. In M. grisea ras- mutants fail to form appressoria and this defect is reversed by cAMP. This data is consistent with a model of M. grisea appressorial signaling where the GTP-binding protein ras transmits the signal from Pthllp to adenylyl cyclase. It is expected that inhibition of this signaling would yield to poor appressorial formation and, therefore, control of disease.

All of the above observations suggest that a screen could be developed to search for inhibitors of this M. grisea pathway in a single-celled organism like yeast. This could be accomplished by substituting M. grisea components for analogous components in yeast. In yeast, ras null mutants are not viable. If the M. grisea ras gene can be substituted for the yeast ras gene, then stimulation of the putative Pthllp-ras pathway should allow yeast ras null mutants to grow. Inhibition of this pathway would lead to yeast cell death.

As with PTH11, the protein encoded by PTH12 has no enzymatic function and a screen will be based on an alternate activity. PTH12 encodes a transcription factor, responsible for general fungal appressorial formation. It is contemplated that routine methods may be used to identify and isolate the genes that are the target of the transcription factor encoded by PTH12. For example, such an identification might be accomplished by differential expression of PTH 12 followed by subtraction. Two pools of mRNA could be prepared. One pool from a strain where PTH 12 is not present and the other from a strain where PTH12 expression is under the control of highly inductive promoter. By subtracting the mRNA from the pthl 2 null mutant from the pool of the strain where PTH 12 is being expressed to a high level should yield those messages that are turned on by PTH12. It can be appreciated that these genes are important in appressorium formation and will be critical for appressorium function. Thus, the genes turned on by PTH 12 will be good targets for antifungal targets as inhibition of the corresponding proteins would render the appressoria incapable of plant penetration. Examination of the promoters of the genes that are regulated by PTH 12 are expected to reveal a common potential binding site for the PTH 12 protein. The importance of this binding site could be evaluated in vivo and in vitro.

Once the binding site for the PTH 12 protein is determined, a screen could be designed to search for compounds that inhibit the binding of the PTH 12 protein to its DNA recognition site. For example, a strain of an organism suitable for high throughput screening (e.g., Saccharomyces cerevisiae) could be engineered to express the PTH12 protein. A gene required for growth by this organism could be engineered so that binding sites for the protein are upstream of the gene such that binding of the PTH12 protein is required for expression of this gene. Specifically, a leu2- auxotroph of S. cerevisiae could have the LEU2 gene engineered in such a fashion such that LEU2 expression was dependent on binding of the PTH12 protein to its DNA binding site. This strain could be screened for inhibition of growth in the presence of test compounds. General inhibitors of the organism could be distinguished from compounds that were directly interfering with the binding of Pthl 2p to its cognate binding site by reversal of inhibition by leucine. Mapping It is contemplated that the instant genes will also be useful in the mapping of the pathogenic fungal genome. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et at, 1987, Genomics 7:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein. D. et al, (l9S0)Am.J.Hum.Genet.32:314-331).

The production and use of fungal gene-derived probes for use in genetic mapping is described in Farman et al., 1995, Genetics, 140(2), 479-92. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof.

Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel, et al., In: Nonmammalian Genomic Analysis: A Practical Guide, Academic Press, 1996, pp. 319-346, and references cited therein).

In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping. Although current methods of FISH mapping favor use of large clones

(several to several hundred KB), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification, polymorphism of PCR-amplified fragments (CAPS), allele-specific ligation, nucleotide extension reactions, Radiation Hybrid Mappings and Happy Mapping. For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This is generally not necessary for mapping methods. Such information may be useful in plant breeding in order to develop lines with desired starch phenotypes. EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. GENERAL METHODS

Techniques suitable for use in the following examples may be found in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Restriction enzymes were obtained from Bethesda Research Labs, MD, and T4 DNA ligase was obtained from New England Biolabs, Boston, MA. These enzymes were used according to the respective manufacturers' instruction. Isolation of fungal genomic DNA and bacterial plasmids, oligolabeling of probes, gel electrophoresis, gel blotting, and blot hybridizations, and DNA sequencing were performed as described in Sweigard et al, 1992, Mol. Gen. Genet., 232:174-182. The meaning of abbreviations is as follows: "h" means hour(s), "min" means minue(s), "sec" means second(s), "d" means day(s), "mL" means milliliters, "L" means liters. Plasmids Plasmids pCB819 (Sweigard et al., 1995, Plant Cell 7:1221-1233) and pCB1004 (Carroll et al., 1995, Fungal Genet. Newsl. 41 :22) were used for fungal transformation and contained the following relevant characteristics: (i) hygromycin resistance gene, and (ii) chloramphenicol resistance gene.

EXAMPLE 1 Transformation of Pathogenic Masnaporthe Strains and Selection for Hygromycin Resistance The pathogenic Magnaportha strain 4091-5-8 (Valent et al, 1986, Iowa State J. Res. 60:569-594) was selected for transformation by plasmid pCB819 and pCB 1004 (both containing a hygromycin resistance marker) using Restriction Enzyme Mediated Integration (REMI). This strain was chosen because it has excellent and stable conidiation, transformability, fertility and pathogenicity compared to other strains of this fungus. Transformants were selected on the basis of their resistance to hygromycin coupled with a phenotype showing lessened pathogenicity. REMI Transformation was performed as described by Sweigard et. al., 7992, Mol.

Gen. Genet. 232:174-182, except that the polyethylene glycol solution was 40% PEG 8000, 20% sucrose, 50 mM KC1, 50 mM NaCl, 10 mM MgCl₂, 50 mM Tris-HCl, pH 8.0. This change was made to enhance the activity of restriction enzymes added to the transformation; i.e., to perform restriction enzyme-mediated integration (REMI, Schiestl and Petes, 1991, PNAS 88:7585-7589). A detailed protocol follows.

Mycelium for protoplast formation was produced by macerating about 25 cm² of mycelium from oatmeal agar plates in a blender containing about 50 mL of sterile complete medium. All subsequent manipulations were performed aseptically. This macerated mycelium was added to 100-200 mL of complete medium and grown with swirling at room temperature for 1-3 d with or without one or two additional cycles of blender maceration. The resulting mycelium was harvested by filtration, washed with distilled water, weighed, and resuspended in 30 mL of 1 M sorbitol. Novozym 234 (20 mg/mL in 1 M sorbitol) was then added at a rate of 1.75 mL of Novozym 234 solution per 3 grams of mycelium. The enzyme/mycelium mixture was gently swirled at room temperature. After 60-90 min, protoplasts were harvested by filtering sequentially through cheesecloth and a nylon membrane (Nytex, 25 μm pore size, from Tetko Co., Briarcliff Manor, NY). The protoplast suspension was centrifuged in a swinging bucket rotor (4100 X g, 10 min), and the pellet was resuspended in 10 mL of 1 M sorbitol. This step was repeated and the protoplasts were finally resuspended in 10 mL of STC (1 M sucrose, 50 mM Tris-Cl pH 8.0, 50 mM CaCl₂). The protoplasts were counted using a hemacytometer, centrifuged as before and resuspended at 5 X 10⁷/mL. Protoplasts (0.2 ml) were mixed with DNA (1-5 μg in 2-10 μL of 10 mM Tris-Cl, pH 8, 1 mM EDTA). After 15 min incubation at room temperature, restriction enzymes (10-50 units) were added to the transformation mix followed by the addition of 1.25 mL restriction PTC (40% PEG 8000, 20% sucrose, 50 mM KC1, 50 mM NaCl, 10 mM MgCl₂, 50 mM Tris-HCl, pH 8.0). After an additional 20 min, 3 mL of TB3 (complete medium with 1 M sorbitol) was added, and the protoplasts were gently swirled for 3-6 h. The protoplast suspension was then centrifuged as before, and the pellet was resuspended in 0.1 mL of STC. Molten regeneration medium (10-15 mL TB3 with 2%> low melting point agarose (Bethesda Research Labs, Gaithersburg, MD) at 50°C) was added, and the protoplasts were poured onto TB3 agar plates containing 200 μg hyg B/mL. After 5-7 d transformants were picked to oatmeal plates and allowed to sporulate. Single conidia were then isolated from each transformant to insure that the strain was derived from a single nucleus.

After transformation, hygromycin-resistant strains were selected on complete medium (Crawford et al, 1986, Genetics 114:1111-1129) with 200 ug hygromycin/mL. After 5 d hygromycin-resistant strains were transferred to oatmeal medium (Crawford et al., supra) and allowed to conidiate. Single conidial isolates were then picked for each transformant. Selection for hygromycin-resistance resulted in 4800 hygromycin resistant conidial isolates.

EXAMPLE 2 Selection of Transformants for Lessened Pathogenicity

Hygromycin resistance clones were selected for lessened pathogenicity according to a two part infectivity screen. All conidial isolates were first subjected to a rapid infectivity assay. Those isolates demonstrating reduced pathogenicity using the rapid screen were further tested using the standard infectivity assay. Rapid Assay

In the rapid assay a cotton swab was wetted with a 0.25% gelatin (Type B, Sigma) solution and rubbed on a conidiating culture grown on oatmeal medium containing 100 ug hygromycin/mL to collect conidia. The swab was then rubbed over 2-3 leaves of 7 d-old barley. Plants were examined 5 d after inoculation and those that showed reduced symptoms were tested in a standard infection assay. Standard Assay

The standard infection assay was performed essentially as described in Valent et al., (1991, Genetics 127:87-101). Conidial isolates are sprayed on weeping lovegrass (Eragrostis curvula) (2 x IO⁶ conidia total), barley (Hordeum vulgare) cultivar "Bonanza" (1 x IO⁶ conidia total), and rice (Oryza sativa) cultivar Yashiro- mochi (5 x 10⁵ conidia total). Lesions in these assays were scored according to the rating system described by Valent et al. (1991). This rating ranges from Type 0 (no visible symptoms) to Type 5 (large expanded lesions with a distinct center from conidia arise when the lesion are placed in a humid environment). Strains that produce Type 0 and Type 1 (small <1 mm brown flecks) symptoms are considered non-pathogenic. Symptoms produced by some mutants are shown in Figure 1.

Figs, la-e shows leaf sections from barley plants inoculated with wild-type strain 4091-5-8 (2 x IO⁵ conidia/mL) and pth2,pth3, pthll and pthl 2 strains (1 x IO⁶ conidia/mL). Wild type causes coalescing Type 5 lesions while pth2 andpth!2 strains (Figs, lb and le) never cause disease lesions (Type 0). pthll (Fig. Id) strains cause rare lesions. pth3 (Fig. lc) strains cause numerous slowly expanding lesions (Type 2-3) that produce limited conidia compared to wild-type. This rating system does not measure the potential of these mutants to cause epidemics. The system does not measure the ability of the mutants to produce inoculum and to initiate multiple sounds of infection.

Of the 4800 hygromycin-resistant transformants produced in Example 1. 4 of these were found to have lessened pathogenicity. These transformants were labeled PTH2, PTH3, PTH 11 and P7H72. Table 1 below lists the segregation data for the clones showing lessened pathogenicity coupled with hygromycin resistance.

TABLE 1

Segres jation Data

PTΗ¹ Disease² Vector³ Enzyme⁴ R+5 R- S+ S-

11 B pCB1004 Kpn l 0 7 15 0

12 A pCB1004 Sma l 0 26 22 0

2 B pCB819 Xho l 0 11 22 1

_. D pCB819 Hind III 1 14 30 0

!PTH = pathogenicity gene name

²Disease Phenotype: A = No symptoms, B = Rare lesions, C = Significantly reduced numbers of mainly type 1 and 2 lesions, D = Wild-type or near wild-type numbers of type 2 and type 3 lesions. E = Fully pathogenic, greatly reduced conidiation in culture. Lesion type described by Valent et al., 1991

³Vector used in transformation

Restriction enzyme used in transformation

Segregation data where R and S mean "resistant" and "sensitive" to hygromycin and + and - mean "wild-type" and "mutant" phenotype EXAMPLE 3 Isolation of Plasmids Containing Pathogenicity -Altering Integrations Plasmids from the transformants selected in Example 2 were isolated by the method of plasmid rescue and integration was confirmed by southern blot analysis. Genomic DNA (1-3 ug) was digested with a restriction enzyme (Hind III for pth2 and pth3; BamH I and Bgl II for pthll) that did not cut in the transforming vector or only cut the vector once in a region not required for replication in E. coli. The digested DNA was ethanol precipitated (Maniatis) and ligated in a 100 uL ligation reaction using a composition of ligation reaction solutions (Maniatis). Four uL of this ligation mix was transformed into E. coli strain DH5α (Hanahan, 19??, J Mol. Biol. 166:557-580). Chemical transformation was used for pth2 as described in Maniatis. Electroporation into DH10B (Bethesda Research Labs. Gaithersburg, MD) was used for pthll as described in Maniatis. Positive transformants were selected for chloramphenicol resistance. Genomic DNA from positive transformants was isolated and subjected to southern blots (Southern, A75, J. Mol. Biol. 98, 503) with genomic DNA from both mutant and wildtype strains which confirmed the existence of a single integration event in all transformants and confirmed that the DNA in the plasmid did indeed arise from a place in the fungal genome that had been modified by plasmid integration (i.e., that the rescued plasmid indeed contained genomic DNA corresponding to the DNA that had been modified by integration of the plasmid).

EXAMPLE 4 Identification of Wildtype Plasmid and Complementation of Mutant Strains Plasmids isolated in Example 3 were used to identify wildtype plasmids in strain 4091-8-5. The wildtype plasmids were then used to complement the DNA of the mutant strains, restoring pathogenicity.

Experiments demonstrating that non-pathogenic mutants could be restored to a pathogenic phenotype if complemented with the corresponding gene proceeded according to the following steps: 1) Plasmid rescued from mutant genomic DNA containing DNA flanking the transformation insertion that caused the mutation was used to identify a plasmid or cosmid with the wild-type DNA sequence.

2) The wild-type plasmid or cosmid was transformed into to the non- pathogenic mutant and a restoration of pathogenicity was seen as determined by infectivity assays (Example 2).

3) The gene was further localized by subcloning smaller pieces from the original complementing plasmid that could still complement the mutant. 4) The smallest complementing piece of the genomic DNA was sequenced to identify the putative open reading frame (ORF).

5) Hybridization with the smallest wild-type plasmid corresponding to the putative ORF was used to identify a cDNA. 6) Finally, mutations were produced within the smallest complementing piece by restriction site fill-in to demonstrate that these plasmids could not complement the mutant, thus confirming the position of the proper ORF.

In some cases a modification of this protocol was also used during subcloning. Plasmids were digested with restriction enzymes that cut within the genomic DNA fragment being tested. If the digested DNA could not complement the mutant this suggested that this restriction enzyme site was within the complementing ORF. Sequence from the rescued plasmid could also identify the place in wild-type genomic DNA where the initial insertion event occurred, thereby providing additional information about the location of the correct ORF. Cloning ofpth2

A plasmid was rescued from pth2 genomic DNA by digestion with Hindlll, ligation, transformation of the ligation into E. coli, and selection for chloramphenicol resistant plasmids. This plasmid was designated pCB880. This plasmid was used to probe a cosmid library (Sweigard et. al., 1995, Plant Cell 7:1221-1233, 1995) Cosmids homologous to pCB880 were transformed into pth2 protoplasts and the transformants were tested for pathogenicity. Cosmid A1F3 could transform the non- pathogenic pth2 mutant to pathogenicity. DNA fragments from this cosmid that were homologous to the rescued plasmid (pCB880) were subcloned and tested for the ability to complement the pth2 mutant. A 6 kb Hindlll fragment from this cosmid could also complement the mutant (pCB897). This plasmid was further subcloned to a 4 kb BamH I/SstII fragment (pCB913), and then a 3.2 kb EcoRI/Sstll fragment (pCB914). Genomic sequence from this region suggested that there was a large gene with homology to many members of a general class of enzymes called acyl transferases. A cDNA from this region (pCB1210) showed that this ORF indeed was transcribed. The sequence from the rescued plasmid indicated that the insertion event occurred in a Xhol site (the original mutant was obtained as part of a Xhol restriction enzyme-mediated transformation) that is in the middle of this ORF. Also, digestion of complementing plasmids with Bgl II (there is a Bgl II site in the middle of this ORF) destroyed the complementing activity. All of these data taken together prove that the gene mutated in the pth2 mutant is the gene encoded by the ORF identified in pCB1210. Cloning of pth3. pthll and pthl 2

The remaining genes corresponding to pth3, pthll and pthl 2 were cloned essentially as described above for pth2 with the exception that:

(i) pthll made use of a genomic plasmid library as opposed to a genomic cosmid library.

(ii) failure to restore a pathogenic phenotype by restriction digestion at a site in the ORF was accomplished by enzymes appropriate to each particular gene. Such sites are evident from the nucleotide sequences of the genes for pth3, pthll and pthl2 (SEQ ID NOS.:4, 7, and 10, respectively). EXAMPLE 5

Sequencing of Plasmid Insertions and BLAST Analysis The inserts in the wildtype clones identified in Example 4 were sequenced according to standard methods and activities were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The fungal pathogenicity nucleic acid sequences were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTX algorithm provided by the National Center for Biotechnology Information (NBC). Amino acid sequences were compared using the BLASTP program [Altschul, S.F., et al., Nucleic Acids Res. 25:3389-3402]. The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "NR" database using the BLAST algorithm (Gist et al., 1993, Nature Genetics 3:266-272) provided by the NBC or the Wisconsin Genetics Computer Group package (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI) represent homologous proteins. Amino acid sequences were compared against the SWISS-PROT protein sequence database.

The insert from clone PTH2 was sequenced. The BLAST analysis on the nucleic acid and deduced amino acid sequence similarity of the protein encoded by the cDNA to numerous acyltransferases including the choline acetyltransferase of mouse [Ishii,K., et al, Brain Res. Mol. Brain Res. 7 (2), 151-159 (1990)] (and carnitine acetyl transferase of rat [Ishii,K. et. al., supra] (Table 2). The strongest homology (41% identity, 52% similarity) was to carnitine acetyl transferase of S. cerevisiae (P32796) [Kispal,G. et al., J. Biol. Chem. 268 (3), 1824-1829 (1993)]. The insert from clone PTH3 was sequenced. The BLAST analysis using the nucleic acid and deduced amino acid sequence revealed 63%> identity to a Saccharomyces imidazole glycerol phosphate dehydratase(IGPD), the sixth step in histidine biosynthesis [Struhl.K. et al, Nucleic Acids Res. 13 (23), 8587-8601 (1985)]. PTH11 did not reveal any homology with a known protein, the secondary peptide structure in combination with the organization of hydrophilic and hydrophobic amino acids strongly suggests that this gene encodes a membrane protein.

BLAST analysis of the genetic databases revealed a significant homology between 55 amino acids of PTH12 and the homeobox of homeodomain transcription factors [Steelman,S. et al., Genome Res. 1 (2), 142-156 (1997)].

TABLE 2 BLAST Analysis

SEQ ID NO % Identity at % Similarity at pLog - DNA

Clone Similarity Identified Base Pep Blast Algorithm amino acid level amino acid level comparison

PTH2 carnitine acetyl transferase - Yeast 2 3 P-Swissprot Xnr 41% 52% e-1 12

PTH3 imidazole glycerol phosphate 5 6 P-Swissprot/Xnr 63% 72% 4e-81 dehydratase - histidine biosynthesis - Saccharomyces

PTHU None identified. 8 9 P-Swissprot/Xnr NA NA NA

PTH12* homeobox* of Xmeis 1-3 gene 1 1 12 P-Swissprot/Xnr 55% 78% 9e-20 Xanopus laeous

' Comparison is based on 55 conserved amino acids 142 through 196 of SEQ ID No 12

. t_/ι

EXAMPLE 6 Identification of Antifungal Compounds Inhibitory to the Gene Products of PTH2 Example 6 illustrates the use of the gene product of PTH2 in a method for the identification of antifungal compounds. The PTH2 gene may be first expressed recombinantly and the expressed gene may be purified or partially purified for use in the assay. Enzyme activity is measured both in the presence and the absence of an inhibitor candidate and a comparison of these activities indicates the efficacy of a particular candidate as an antifungal compound. Expression of Carnitine Acetyl Transferase PTH2 encodes for the enzyme carnitine acetyl transferase (CAT), PTH2, or any portion of PTH2 that contains the coding region of the gene may be inserted into an appropriate expression vector and used to transform a suitable expression host under the control of either an inducible or constitutive promoter. For example, plasmid DNA containing PTH2 may be purified QIAFilter cartridges (Qiagen, Inc., 9600 De Soto Ave, Chatsworth, CA) according to the manufacturer's instructions, and inserted into ligation independent cloning (LIC) pET30 vector (Novagen, Inc., 597 Science Dr., Madison, WI) under the control of the T7 promoter, according to the manufacturer's instructions (see Novagen publications "LIC Vector Kits", publication number TB163 and US 4952496, herein incorporated by reference). The vector may be used to transform competent E. coli hosts such as BL21(DE3).

Typically, in such an expression system, primers with a specific 3' extension designed for ligation-independent cloning are designed to amplify the PTH2 gene (Maniatis). Amplification products may be gel-purified and annealed into the LIC vector after treatment with T4 DNA polymerase (Novagen). Insert containing vectors are then used to transform NovaBlue competent E. coli cells and transformants may be screened for the presence of viable inserts. Clones in the correct orientation with respect to the T7 promoter are transformed into BL21(DE3) competent cells (Novagen) and selected on LB agar plates containing 50 ug/ml kanamycin. Colonies arising from this transformation are grown overnight at 37°C in Lauria Broth to OD 600=0.6 and induced with 1 mM IPTG and allowed to grow for an additional two h. The culture may be harvested, resuspended in binding buffer, lysed with a French Press and cleared by centrifugation. The expressed carnitine acetyl transferase can be purified or partially purified according to standard methods and used in an assay. Antifungal Screen The purified or partially purified CAT enzyme may be assayed in a 1 mL reaction solution that contains 900 uL of 0.1 M KHPO4, 0.1 mM 5,5'dithiobis-(2- nitrobenzoic acid), pH 8.0; 70 uL water; 10 uL 20 mM acetyl coenzyme A; 10 uL 80 mM L-carnitine; and 10 uL enzyme. Two reaction solutions are prepared, one with an inhibitor candidate and the other lacking the candidate. The reaction mixtures are incubated at about 30°C and absorbance is measured on a spectrophotometer where an increase in absorbance was measured at 412 nM and using an extinction coefficient of 13.6. These parameters measure the formation of 5-thio-2-nitro benzoate resulting from the nonenzymatic disulfide exchange between CoA and 5,5'dithiobis-(2- nitrobenzoic acid. A comparison of the absorbance measurements from the reaction solution containing the inhibitor candidate and the reaction solution lacking the candidate give an indication of the efficacy of the candidate as an antifungal compound. EXAMPLE 7

Identification of Antifungal Compounds Inhibitory to the Gene Products of PTH3 Example 7 illustrates the use of the gene product of PTH3 in a method for the identification of antifungal compounds. The PTH3 gene may be first expressed recombinantly and the expressed gene may be purified or partially purified for use in the assay, according to techniques essentially described in Example 6. As in

Example 6, enzyme activity may be measured both in the presence and the absence of an inhibitor candidate and a comparison of these activities indicates the efficacy of a particular candidate as an antifungal compound.

The gene product of PTH3 is Imidazoleglycerolphosphate dehydratase (IGPD). IGPD may be produced recombinantly using expression cassettes and hosts as described in Example 6. Expressed enzyme may be purified or partially purified according to standard methods.

The purified or partially purified IPGD enzyme may be assayed at 37°C in a 100 uL reaction consisting of 0.1 M triethalonamine hydrochloride (pH 7.2), 0.2 mM MnCl₂, 85 mM B-mercaptoethanol, 3 mM imidazolglycerol phosphate. Two reaction solutions are prepared, one with an inhibitor candidate and the other lacking the candidate. The reaction is initiated by addition of enzyme and terminated by addition of 175 uL of 1.43 N NaOH. After further incubation at 37°C for 30 min, the absorbance at 290 nm is determined using 5100 as the molar extinction coefficient for imidazolacetol phosphate, the product of the dehydratase reaction. A comparison of the absorbance measurements from the reaction solution containing the inhibitor candidate and the reaction solution lacking the candidate give an indication of the efficacy of the candidate as an antifungal compound.

Claims

What is claimed is:

1. An isolated nucleic acid fragment encoding a fungal carnitine acetyl transferase enzyme, selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence as set forth in SEQ ID NO:3;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence as set forth in SEQ ID NO:3;

(c) an isolated nucleic acid fragment encoding a polypeptide having at least 41 % identity with the amino acid sequence as set forth in

SEQ ID NO:3; and

(d) an isolated nucleic acid fragment that is complementary to (a), (b), or (c).

2. The isolated nucleic acid fragment of Claim 1 selected from the group consisting of SEQ ID NO:l and SEQ ID NO:2.

3. A polypeptide encoded by the isolated nucleic acid fragment of Claim 1.

4. A polypeptide as set forth in SEQ ID NO : 3.

5. A chimeric gene comprising the isolated nucleic acid fragment of Claim 1 operably linked to at least one suitable regulatory sequence. 6. A transformed host cell comprising a host cell and the chimeric gene of

Claim 5.

7. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a fungal carnitine acetyl transferase enzyme comprising: (a) probing a cDNA or genomic library with the nucleic acid fragment of Claim 1 ;

(b) identifying a DNA clone that hybridizes with the nucleic acid fragment of Claim 1 ; and

(c) sequencing the cDNA or genomic fragment that comprises the clone identified in step (b), wherein the sequenced cDNA or genomic fragment encodes all or substantially all of the amino acid sequence encoding a fungal carnitine acetyl transferase enzyme.

8. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a fungal carnitine acetyl transferase enzyme comprising:

(a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO:l and SEQ ID NO:2; and (b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector, wherein the amplified cDNA insert encodes a portion of an amino acid sequence encoding a fungal carnitine acetyl transferase enzyme.

9. The product of the method of Claims 7 or 8.

10. A method for identifying as an fungicide candidate a chemical compound that inhibits the activity of a fungal carnitine acetyl transferase enzyme, encoded by the isolated nucleic acid fragment of Claim 1 , the method comprising the steps of:

(a) transforming a host cell with a chimeric gene comprising the isolated nucleic acid fragment of Claim 1 encoding a fungal carnitine acetyl transferase enzyme the chimeric gene operably linked to at least one suitable regulatory sequence; (b) growing the transformed host cell of step (a) under conditions suitable for expression of the chimeric gene resulting in production of the fungal carnitine acetyl transferase;

(c) optionally purifying the fungal carnitine acetyl transferase enzyme expressed by the transformed host cell; (d) contacting the fungal carnitine acetyl transferase enzyme with a chemical compound of interest; and (e) identifying as an fungicide candidate the chemical compound of interest that reduces the activity of the fungal carnitine acetyl transferase enzyme relative to the activity of the fungal carnitine acetyl transferase enzyme in the absence of the chemical compound of interest.

11. The method of Claim 10 wherein the nucleic acid fragment is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:2 and the carnitine acetyl transferase enzyme as set forth in SEQ ID NO:3. 12. An isolated nucleic acid fragment encoding a fungal imidazole glycerol phosphate dehydratase enzyme, selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence as set forth in SEQ ID NO:6;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence as set forth in SEQ ID NO:6; (c) an isolated nucleic acid fragment encoding a polypeptide having at least 63%) identity with the amino acid sequence as set forth in SEQ ID NO:6; and

13. The isolated nucleic acid fragment of Claim 12 selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5.

14. A polypeptide encoded by the isolated nucleic acid fragment of Claim 12. 15. The polypeptide as set forth in SEQ ID NO:6.

16. A chimeric gene comprising the isolated nucleic acid fragment of Claim 12 operably linked to at least one suitable regulatory sequence.

17. A transformed host cell comprising a host cell and the chimeric gene of Claim 16. 18. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a fungal imidazole glycerol phosphate dehydratase enzyme comprising:

(a) probing a cDNA or genomic library with the nucleic acid fragment of Claim 12; (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of Claim 12; and (c) sequencing the cDNA or genomic fragment that comprises the clone identified in step (b), wherein the sequenced cDNA or genomic fragment encodes all or substantially all of the amino acid sequence encoding a fungal imidazole glycerol phosphate dehydratase enzyme.

19. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a fungal imidazole glycerol phosphate dehydratase enzyme comprising: (a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5; and

(b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector, wherein the amplified cDNA insert encodes a portion of an amino acid sequence encoding a fungal imidazole glycerol phosphate dehydratase enzyme.

20. The product of the method of Claims 18 or 19.

21. A method for identifying as an fungicide candidate a chemical compound that inhibits the activity of a fungal imidazole glycerol phosphate dehydratase enzyme encoded by the isolated nucleic acid fragment of Claim 12, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising the isolated nucleic acid fragment of Claim 12 encoding a fungal imidazole glycerol phosphate dehydratase enzyme, the chimeric gene operably linked to at least one suitable regulatory sequence;

(b) growing the transformed host cell of step (a) under conditions suitable for expression of the chimeric gene resulting in production of the fungal imidazole glycerol phosphate dehydratase;

(c) optionally purifying the fungal imidazole glycerol phosphate dehydratase enzyme expressed by the transformed host cell; (d) contacting the fungal imidazole glycerol phosphate dehydratase enzyme with a chemical compound of interest; and (e) identifying as an fungicide candidate the chemical compound of interest that reduces the activity of the fungal imidazole glycerol phosphate dehydratase enzyme relative to the activity of the fungal carnitine acetyl transferase enzyme in the absence of the chemical compound of interest.

22. The method of Claim 21 wherein the nucleic acid fragment is selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5 and the carnitine acetyl transferase enzyme as set forth in SEQ ID NO: 6. 23. An isolated nucleic acid fragment encoding a fungal membrane associated pathogenicity protein selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence as set forth in SEQ ID NO:9;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence as set forth in SEQ ID NO:9;

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

24. The isolated nucleic acid fragment of Claim 23 selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8.

25. A polypeptide encoded by the isolated nucleic acid fragment of Claim 23.

26. The polypeptide of Claim 25 as set forth in SEQ ID NO:9.

27. A chimeric gene comprising the isolated nucleic acid fragment of Claim 23 operably linked to at least one suitable regulatory sequences.

28. A transformed host cell comprising a host cell and the chimeric gene of Claim 27. 29. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a fungal membrane associated pathogenicity protein comprising:

(a) probing a cDNA or genomic library with the nucleic acid fragment of Claim 23; (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of Claim 23; and (c) sequencing the cDNA or genomic fragment that comprises the clone identified in step (b), wherein the sequenced cDNA or genomic fragment encodes all or substantially all of the amino acid sequence encoding a fungal membrane associated pathogenicity protein.

30. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a fungal membrane associated pathogenicity protein comprising: (a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of as SEQ ID NO:7 and SEQ ID NO:8; and

(b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector, wherein the amplified cDNA insert encodes a portion of an amino acid sequence for a fungal membrane associated pathogenicity protein.

31. The product of the method of Claim 29 or 30.

32. An isolated nucleic acid fragment encoding a fungal homeodomain transcription factor selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence as set forth in SEQ ID NO: 12;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence as set forth in SEQ ID NO: 12;

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

33. The isolated nucleic acid fragment of Claim 32 selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO.l l.

34. A polypeptide encoded by the isolated nucleic acid fragment of Claim 32. 35. The polypeptide of Claim 34 as set forth in SEQ ID NO: 12.

36. A chimeric gene comprising the isolated nucleic acid fragment of Claim 32 operably linked to at least one suitable regulatory sequence.

37. A transformed host cell comprising a host cell and the chimeric gene of Claim 36. 38. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a fungal homeodomain transcription factor comprising:

(a) probing a cDNA or genomic library with the nucleic acid fragment of Claim 32; (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of Claim 32; and (c) sequencing the cDNA or genomic fragment that comprises the clone identified in step (b), wherein the sequenced cDNA or genomic fragment encodes all or substantially all of the amino acid sequence encoding a fungal homeodomain transcription factor.

39. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a fungal homeodomain transcription factor comprising:

(a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of as

SEQ ID NO:10 and SEQ ID NO:l 1; and

(b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector, wherein the amplified cDNA insert encodes a portion of an amino acid sequence for a fungal homeodomain transcription factor.

40. The product of the method of Claims 38 or 39.

41. A method for evaluating at least one compound for its ability to inhibit the activity of the PTH 11 gene product as given by SEQ ID NO: 9 comprising: (i) transforming a suitable host with the PTH 11 gene as given by SEQ

ID NO: 8 wherein expression of said gene permits the host to grow and inhibition of said gene will be lethal to the host; (ii) contacting the transformed host with a compound suspected of being a PTH 11 inhibitor and monitoring the growth of the transformed host to measure the efficacy of the inhibitor compound.

42. A method for evaluating at least one compound for its ability to inhibit the activity of the PTH 12 gene product as given by SEQ ID NO: 12 comprising: (i) transforming a suitable host cell with: a) the PTH 12 gene as given by SEQ ID NO: 11 such that the gene is expressed; b) a reporter gene such that the expression of said reporter gene is dependent on the binding of the PTH12 gene product to said reporter gene;

(ii) contacting said transformed host with a compound suspected of inhibiting the activity of the PTH12 gene product and;

(iii) monitoring the expression of said reporter gene to determine the efficacy of the inhibitory compound.