WO2000022133A1

WO2000022133A1 - Fungal genes required for normal growth and development

Info

Publication number: WO2000022133A1
Application number: PCT/EP1999/007501
Authority: WO
Inventors: Thomas Deane Gaffney; Jürgen Wendland; Fred Dietrich; Peter Philippsen; Stephen Arthur Goff
Original assignee: Syngenta Participations Ag
Priority date: 1998-10-08
Filing date: 1999-10-06
Publication date: 2000-04-20
Also published as: AU6090299A

Abstract

The invention relates to nucleic acid sequences isolated from Ashbya gossypii that encode proteins essential for fungal growth. The invention also includes the methods of using these proteins pesticide targets, particularly fungicide targets, based on the essentiality of the gene for normal growth and development. The invention is also useful as a screening assay to identify inhibitors that are potential pesticides, particularly fungicides.

Description

FUNGAL GENES REQUIRED FOR NORMAL GROWTH AND DEVELOPMENT

The phytopathogenic fungus Ashbya gossypii is a f ilamentously growing ascomycete that was first isolated as a plant pathogen in tropical and sub-tropical regions. It infects the seed capsule of cotton plants and has also been isolated from tomatoes and citrus fruits. The infection of the seed capsule is caused by transmission of A. gossypii mycelium pieces or spores by stinging-sucking insects and causes a disease called stigmatomycosis. Presently, A. gossypii represents the most compact eukaryotic genome, compared to genome sizes of 12.5 Mb for S. cerevisiae (Chu et al., 1986), 31.0 Mb for Aspergillus nidulans (Brody and Carbon, 1989) and 47.0 Mb for Neurospora crassa (Orbach et al., 1988).

A. gossypii is systematically grouped to the endomycetales belonging to the family of spermophthoraceae. This classification is based on the observation that the spores that develop in hyphal compartments called sporangia look like ascospores, which are defined as endproducts of meiosis.

Since Ashbya gossypii is a filamentous ascomycete, and is capable of growing only by filamentous (hyphal) growth, fungal targets found in this model organism are predictive of targets which will be found in other pathogens, the vast majority of which grow in a filamentous fashion. .

DEFINITIONS

For clarity, certain terms used in the specification are defined and presented as follows: Chimeric: is used to indicate that a DNA sequence, such as a vector or a gene, is comprised of more than one DNA sequences of distinct origin which are fused together by recombinant DNA techniques resulting in a DNA sequence, which does not occur naturally, and which particularly does not occur in the plant to be transformed. Co-factor: natural reactant, such as an organic molecule or a metal ion, required in an enzyme-catalyzed reaction. A co-factor is e.g. NAD(P), riboflavin (including FAD and FMN), folate, molybdopterin, thiamin, biotin, lipoic acid, pantothenic acid and coenzyme A, S- adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone. Optionally, a co- factor can be regenerated and reused.

Enzyme activity: means herein the ability of an enzyme to catalyze the conversion of a substrate into a product. A substrate for the enzyme comprises the natural substrate of the enzyme but also comprises analogues of the natural substrate which can also be converted by the enzyme into a product or into an analogue of a product. The activity of the enzyme is measured for example by determining the amount of product in the reaction after a certain period of time, or by determining the amount of substrate remaining in the reaction mixture after a certain period of time. The activity of the enzyme is also measured by determining the amount of an unused co-factor of the reaction remaining in the reaction mixture after a certain period of time or by determining the amount of used co-factor in the reaction mixture after a certain period of time. The activity of the enzyme is also measured by determining the amount of a donor of free energy or energy-rich molecule (e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine) remaining in the reaction mixture after a certain period of time or by determining the amount of a used donor of free energy or energy-rich molecule (e.g. ADP, pyruvate, acetate or creatine) in the reaction mixture after a certain period of time.

Expression: refers to the transcription and/or translation of an endogenous gene or a transgene in plants. In the case of antisense constructs, for example, expression may refer to the transcription of the antisense DNA only.

Gene: refers to a coding sequence and associated regulatory sequences wherein the coding sequence is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Examples of regulatory sequences are promoter sequences, 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

Heterologous DNA Sequence: a DNA sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring DNA sequence.

Homologous DNA Sequence: a DNA sequence naturally associated with a host cell into which it is introduced. Isoαenic: plants which are genetically identical, except that they may differ by the presence or absence of a transgene.

Isolated: in the context of the present invention, an isolated DNA molecule or an isolated enzyme is a DNA molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.

Mature protein: protein which is normally targeted to a cellular organelle, such as a chloroplast, and from which the transit peptide has been removed.

Minimal Promoter: promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.

Modified Enzyme Activity: enzyme activity different from that which naturally occurs in a plant (i.e. enzyme activity that occurs naturally in the absence of direct or indirect manipulation of such activity by man), which is tolerant to inhibitors that inhibit the naturally occurring enzyme activity.

Recombinant DNA molecule: a combination of DNA sequences that are joined together using recombinant DNA technology

Recombinant DNA technology: procedures used to join together DNA sequences as described, for example, in Sambrook et al.,1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press

Significant Increase: an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater.

Significantly less: means that the amount of a product of an enzymatic reaction is larger than the margin of error inherent in the measurement technique, preferably a decrease by about 2-fold or greater of the activity of the wild-type enzyme in the absence of the inhibitor, more preferably an decrease by about 5-fold or greater, and most preferably an decrease by about 10-fold or greater.

In its broadest sense, the term "substantially similar", when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, e.g. where only changes in amino acids not affecting the polypeptide function occur. Desirably the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. The term "substantially similar" is specifically intended to include nucleotide sequences wherein the sequence has been modified to optimize expression in particular cells. The percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence desirably is at least 65%, more desirably at least 75%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, yet still more preferably at least 99%. Sequence comparisons are carried out using a Smith-Waterman sequence alignment algorithm (see e.g. Waterman, M.S. Introduction to Computational Biology: Maps, sequences and genomes. Chapman & Hall. London: 1995. ISBN 0-412-99391-0, or at _

HYPERLINK "http://www-hto.usc.edu/software/seqaln/index.html" http://www- hto.usc.edu/software/seqaln/index.html_). The locals program, version 1.16, is used with following parameters: match: 1 , mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2. A nucleotide sequence "substantially similar" to reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1% SDS at 65°C.

The term "substantially similar", when used herein with respect to a protein, means a protein corresponding to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, e.g. where only changes in amino acids sequence not affecting the polypeptide function occur. When used for a protein or an amino acid sequence the percentage of identity between the substantially similar and the reference protein or amino acid sequence desirably is at least 52%, more desirably 65%, more desirably at least 75%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, yet still more preferably at least 99%. Substrate: a substrate is the molecule that the enzyme naturally recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function, or is a modified version of the molecule, which is also recognized by the enzyme and is converted by the enzyme to a product in an enzymatic reaction similar to the naturally-occurring reaction.

Tolerance: the ability to continue normal growth or function when exposed to an inhibitor or herbicide in an amount sufficient to suppress the normal growth or function of native, unmodified plants.

Transformation: a process for introducing heterologous DNA into a cell, tissue, or plant. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.

Transgenic: stably transformed with a recombinant DNA molecule that preferably comprises a suitable promoter operatively linked to a DNA sequence of interest.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO:1 comprises a AG001 coding region

SEQ ID NO:2comprises an amino acid sequence encoded by the coding region of SEQ ID

NO:1

SEQ ID NO:3 comprises a AG002 coding region.

SEQ ID NO:4 comprises an amino acid sequence encoded by the coding region of SEQ ID

NO:3.

SEQ ID NO:5 comprises a AG003 coding region.

SEQ ID NO:6 comprises an amino acid sequence encoded by the coding region of SEQ ID

NO:5.

SEQ ID NO:7 comprises a AG004 coding region.

SEQ ID NO:8 comprises an amino acid sequence encoded by the coding region of SEQ ID

NO:7.

SEQ ID NO:9 comprises a AG005 coding region.

SEQ ID NO:10 comprises an amino acid sequence encoded by coding region of SEQ ID

NO:9.

SEQ ID NO:11 comprises a AG006 coding region.

SEQ ID NO:12 comprises an amino acid sequence encoded by coding region of SEQ ID

NO:11. It is an object of the invention to provide an effective and beneficial method to identify novel pesticides, particularly fungicides. A feature of the invention is the identification of genes having a putative activity based on their homology to yeast genes. Genes of the invention comprise a putative GTP binding protein genes (herein referred to as AG001 and AG002 genes), putative GTPase activating protein genes(AG003 and AG004), putative phosphatidylinositol-4 kinase protein gene (AG005) and putative cytokinesis gene (AG006). Another feature of the invention is the discovery that the genes of the invention, AG001 (SEQ ID. NO: 1), AG002 (SEQ Id. NO 3):, AG003 (SEQ ID. NO: 5), AG004 (SEQ ID. NO: 7), AG005 (SEQ Id. NO: 9) and AG006(SEQ ID. NO: 11) are essential for fungal growth and development. An advantage of the present invention is that the newly discovered essential genes containing a novel fungicidal mode of action enables one skilled in the art to easily and rapidly identify novel fungicides.

One object of the present invention is to provide essential genes in fungi for assay development to detect inhibitory compounds with pesticidal, particularly fungicidal activity. Genetic results show that when AG001 , AG002, AG003, AG004, AG005 and AG006 are mutated in Ashbya gossypii, the resulting phenotype is at best suppressed growth and at worst lethal. Suppressed growth as used herein results in a growth rate of half the growth rate observed in wild type or lower where 10% that of the wild-type growth rate was observed or no growth was macroscopically detected at all . Applicants further observed that when AG001 , AG002, AG003, AG004, AG005 and AG006 are mutated in Ashbya gossypii abnormal filament development was observed. This suggests a critical role for the gene products encoded by the mutated genes.

The inventors of the present invention have demonstrated that the gene products of the invention are essential in Ashbya gossypii. This implies that chemicals which inhibit the function of the protein in fungi, particularly, filamentous fungi, are likely to have detrimental effects on fungi and are potentially good fungicide candidates. The present invention therefore provides methods of using a purified protein encoded by the gene sequence described below to identify inhibitors thereof, which can then be used as fungicides to suppress the growth of pathogenic fungi.

Pathogenic fungi is defined as those capable of colonizing a host and causing disease. Examples of fungal pathogens include plant pathogens such as Septoria tritici, Stagnospora nodorum, Botrytis cinerea, Fusarium graminearum, Magnaporthe grisea, Cochliobolus heterostrophus, Colletotrichum heterostrophus, Ustilago maydis, Erisyphe graminis, plant pathogenic oomycetes such as Pythium ultimum and Phytophthora infestans, and human pathogens such as Candida albicans and Aspergillus fumigatus

The present invention discloses novel nucleotide sequences derived from Ashbya gossypii designated as the AGOOI gene, the AG002 gene, the AG003 gene, the AG004 gene, the AG005 gene and the AG006 gene. The nucleotide sequence of the genomic clones are set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 respectively. The amino acid sequence encoded by the above sequences are set forth in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12 . The present invention also includes nucleotide sequences substantially similar to those set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11 and amino acid sequences substantially similar to those set out in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12

The present invention also encompasses fungal proteins whose amino acid sequence are substantially similar to the amino acid sequences set forth in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12. Encompassed by the present invention is a nucleotide sequence having a 20 base pair nucleotide portion identical in sequence to a 20 consecutive base pair portion of a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11. Preferred is a nucleotide sequence having a base pair nucleotide portion identical in sequence to a 18 consecutive base pair portion of the sequence set forth in SEQ ID NO: 1. Preferred is a nucleotide sequence having a base pair nucleotide portion identical in to a consecutive 9 base pair portion of the sequence set forth in SEQ ID NO:3. . Preferred is a nucleotide sequence having a base pair nucleotide portion identical in sequence to a 15 consecutive base pair portion of the sequence set forth in SEQ ID NO:5. . Preferred is a nucleotide sequence having a base pair nucleotide portion identical in sequence to a 14 consecutive base pair portion of the sequence set forth in SEQ ID NO:7. . Preferred is a nucleotide sequence having a base pair nucleotide portion identical in sequence to a 12 consecutive base pair portion of the sequence set forth in SEQ ID NO:9. . Preferred is a nucleotide sequence having a base pair nucleotide portion identical in sequence to a consecutive 10 base pair portion of the sequence set forth in SEQ ID NO:11. In a particular embodiment, the present invention encompasses nucleic acid sequences and amino acid sequences of filamentous fungi. Preferred is a nucleotide sequence wherein the fungus is Ashbya gossypii.

Further encompassed by the invention is a chimeric gene comprising a promoter operably linked to a nucleotide sequence according to the invention. Preferred is a chimeric gene wherein the promoter is an inducible promoter. A further embodiment of the invention is a recombinant vector comprising a chimeric gene according to the invention wherein said vector is capable of being stably transformed into a host cell.

Also included in the invention is a host cell comprising the vector according to the invention, wherein the nucleotide sequence is expressible in the host cell. Preferred is a host cell , wherein the host cell is eukaryotic. Preferred is a host cell, wherein the host cell is selected from the group consisting of a yeast cell and a fungal cell. More preferred is a host cell, wherein the host cell is a filamentous fungal cell. Particularly preferred is a host cell according to claim 24, wherein the host cell is an Ashbya gossipii cell. Preferred is a host cell wherein the host cell is a prokaryotic cell. More preferred is a host cell wherein the host cell is a bacterial cell.

The present invention also includes methods of using the AG001 to AG006 gene products as fungicide targets, based on the essentiality of the genes for normal growth and development. Normal growth and development is defined as a growth rate substantially similar to that observed in wild type fungus, preferably greater than at least 50% the growth rate observed in wild type fungus and particularly greater than 10% the growth rate obeserved in wild type fungus. Normal growth and development may also be defined, when used in relation to filamentous fungi, as normal filament development (including normal septation and normal nuclear migration and distribution), normal sporulation, and normal production of any infection structures (e.g. appressoria). Conversely suppressed or inhibited growth as used herein is defined as less than half the growth rate observed in wild type or lower where 10% that of the wild-type growth rate was observed or no growth was macroscopically detected at all or abnormal filament development.

Furthermore, the invention can be used in screening assays to identify inhibitors that are potential pesticides, particularly fungicides. Encompassed by the present invention is the use of sequences selected from the attached Sequence Listing to identify substances having antifungal activity; the use of sequences selected from the attached Sequence Listing to identify substances having pesticidal, particularly fungicidal, activity. Further comprised is the use of an a DNA sequence selected from the Sequence Listing and variants thereof in a screening method for identifying compounds capable of inducing broad spectrum disease resistance in plants.

Encompassed by the invention is a process for identifying compounds having fungicidal activity comprising the steps of: a) combining a protein according to claim 16 and a compound to be tested for the ability to bind to the protein, under conditions having conducive binding, b) selecting a compound identified by step a) that is capable of binding the protein; c) applying the identified compound from step b) to a fungus to test for fungicidal activity; and d) selecting compounds having fungicidal activity.

Encompassed by the present invention is a process for identifying an inhibitor of a protein activity having an amino acid sequence according to claim 16 comprising: a) introducing SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 or nucleotide sequences substantially similar thereto into a host, such that the sequence is functionally expressible; b) combining the host cell of step a) with a compound to be tested for ability to inhibit the protein activity; c) over expressing the nucleotide sequence of step a); c) measuring the host cell growth in stepc); and d) selecting the compound that inhibits or suppresses host cell's normal growth or development in step c).

Further encompassed is a compound having fungicidal activity which compound can be identified by a process for identifying compounds having fungicidal activity according to the invention.

Further encompassed is a method of suppressing growth of a fungus comprising applying to the fungus a compound that inhibits the activity of a protein comprising the amino acid sequence according to the invention in an amount sufficient to suppress the growth of the fungus.

In a further embodiment according to the invention, a DNA sequence selected from the Sequence Listing may also be used for distinguishing among different species of plant pathogenic fungi and for distinguishing fungal pathogens from other pathogens such as bacteria. ln another preferred embodiment, the present invention describes a method for identifying chemicals having the ability to inhibit any one or more of AG001 , AG002, AG003, AG004, AG005 and AG006 activity in fungi preferably comprising the steps of: a) obtaining transgenic fungus and/or fungal cell, preferably stably transformed, comprising a non-native nucleotide sequence or an endogenous nucleotide sequences operably linked to non-native promoter, preferably an inducible promoter, encoding an enzyme having and activity and capable of overexpressing an enzymatically active AG001 , AG002, AG003, AG004, AG005 or AG006 gene product where overexpression of the gene product is suppresses or inhibits the normal growth and development of the fungus; b) applying a compound to the transgenic fungus and/or fungal cell c) determining the growth and/or development of the transgenic fungus and/or fungal cell after application of the compound; d) comparing the growth and/or development of the transgenic fungus and/or fungal cell after application of the chemical to the growth and/or development of the corresponding transgenic fungus and/or fungal cell to which the compound was not applied; and e) selecting compound that does not results in reduction of the suppressed or inhibited growth and/or development in the transgenic fungus and/or fungal cell in comparison to the untreated transgenic fungus and/or fungal cell.

In a preferred embodiment, the proteins having AG001 , AG002, AG003, AG004, AG005 or AG006 activities are encoded by nucleotide sequence derived from fungi, preferably filamentous fungi, particularly from Ashbya gossypii, desirably identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO:9 or SEQ ID NO:11. In another embodiment, the proteins having AG001 , AG002, AG003, AG004, AG005 or AG006 activity are encoded by nucleotide sequences capable of encoding the amino acid sequences of: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12. In yet another embodiment, the proteins having AG001 , AG002, AG003, AG004, AG005 or AG006 activity have amino acid sequences identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12 respectively.

The invention also provides a method for suppressing the growth of a fungus comprising the step of applying to the fungus a compound that inhibits the naturally occurring AG001 , AG002, AG003, AG004, AG005 and/or AG006 activity in the fungus. Other objects and advantages of the present invention will become apparent to those skilled in the art from a study of the following description of the invention and non-limiting examples.

Essentiality of the AG001 , AG002, AG003, AG004, AG005 and AG006 Genes in Ashbya gossypii Demonstrated by Gene Disruption

Owing to the provision within the scope of this invention of a novel and powerful gene disruption process, there is no longer a need to know the exact biological function of the protein product encoded by a gene comprising one of the A. gossypii DNA sequences provided herein.

As shown in the examples below, the identification of novel gene structures, as well as the essentiality of the AG001 , AG002, AG003, AG004, AG005 and AG006 genes for normal fungal growth and development, have been demonstrated for the first time in Ashbya gossypii using gene disruption techniques. Having established the essentiality of AG001 , AG002, AG003, AG004, AG005 and AG006 function in fungi and having identified the nucleic acid sequences encoding these essential activities, the inventors thereby provide an important and sought after tool for new pesticide, particularly fungicide, development.

Recombinant Production of and Uses Thereof

For recombinant production of AG001 , AG002, AG003, AG004, AG005 and AG006 in a host organism, a nucleotide sequence encoding AG001 , AG002, AG003, AG004, AG005 or AG006 protein is 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 operably 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, yeast, and insect cells (see, e.g., Luckow and Summers, Bio Technol. 6: 47 (1988), and baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV). A preferred baculovirus/insect system is pAcHLT (Pharmingen, San Diego, CA) used to transfect Spodoptera frugiperda Sf9 cells (ATCC) in the presence of linear Autographa californica baculovirus DNA (Pharmigen, San Diego, CA). The resulting virus is used to infect HighFive Tricoplusia ni cells (Invitrogen, La Jolla, CA). Further preferred expression systems are commercially available such as Baculovirus expression systems: MaxBac 2.0 kit; Invitrogen, Calsbad, CA;BacPAK Baculovirus Expression System; CLONTECH, Palo Alto, CA; for Yeast expression vectors: pYEUra3; CLONTECH, Palo Alto, CA; EasySelect Pichia expression kit; Invitrogen, Calsbad, CA;ESP Yeast Protein Expression and Purification System; Stratagene, La Jolla, CA; E. coli expression vectors: pKK233-2; CLONTECH, Palo Alto, CA; pET3 series vectors; Stratagene, La Jolla, CA.

In a preferred embodiment, the nucleotide sequence encoding a protein having AG001 , AG002, AG003, AG004, AG005 Or AG006 activity is derived from an eukaryote, such as a mammal, a fly or a yeast, but is preferably derived from a fungus, particularly a filamentous fungus. In a further preferred embodiment, the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 , or encodes a protein having AG001 , AG002, AG003, AG004, AG005 or AG006 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12 respectively. The nucleotide sequences set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11 encode the protein comprising amino acid sequence is set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 OR SEQ ID NO: 12. In another preferred embodiment, the nucleotide sequence is derived from a prokaryote, preferably a bacteria.

Recombinantly produced AG001 , AG002, AG003, AG004, AG005, or AG006 is isolated and purified using a variety of standard techniques. The actual techniques that may be used 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). Assays for Characterizing the AG001, AG002, AG003, AG004, AG005 and AG006 Proteins

Recombinantly produced AG001 , AG002, AG003, AG004, AG005 and AG006 proteins are useful for a variety of purposes. For example, they can be used in in vitro assays to screen known pestcidal, particularly fungicidal chemicals whose target has not been identified to determine if they inhibit AG001 , AG002, AG003, AG004, AG005 or AG006. Such in vitro assays may also be used as more general screens to identify chemicals that inhibit such enzymatic activities and that are therefore novel pesticide, particularly fungicide, candidates. Alternatively, recombinantly produced AG001 , AG002, AG003, AG004, AG005 or AG006 proteins may be used to elucidate the complex structure of these molecules and to further characterize their association with known inhibitors in order to rationally design new inhibitory pesticides, particularly fungicides. Nucleotide sequences substantially similar to SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11 and proteins substantially similar to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 OR SEQ ID NO: 12 from any source, including microbial sources, can be used in the assays exemplified herein. Desirably such nucleotide sequences and proteins are derived from fungi. More desirably, they are derived from filamentous fungi, particularly Ashbya gossypii. Alternatively, such nucleotide sequences and proteins are derived from non-yeast sources, alternatively from non-Saccharomyces cervisiae sources.

A simple assay can be developed to screen for compounds that affect normal functioning of the fungal-encoded activity. Such compounds are promising in vitro leads that can be tested for in vivo pesticidal, particularly fungicidal, activity. A nucleic acid sequence of the invention according to any one of the sequnces SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11 may be operably linked to a strong inducible promoter, such promoters being known in the art. The vector comprising the selected gene of the invention operably linked to the selected inducible promoter may be transformed into bacteria, such as E. coli. Transformed E. coli harboring and functionally overexpressing expressing a AG001 , AG002, AG003, AG004, AG005 or AG006 gene may be grown in a 96-well format for automated high-throughput screening where inducible over expression of the selected gene is lethal or suppresses growth of the host. Compounds that are effective in blocking function of the AG001 , AG002, AG003, AG004, AG005 or AG006 protein results in bacterial growth. This growth is measured by simple turbidometric means.

In another embodiment, an assay for inhibitors of the AG001 , AG002, AG003, AG004, AG005 or AG006 activities uses transgenic fungi or fungal cells capable of overexpressing a nucleotide sequence having AG001 , AG002, AG003, AG004, AG005 or AG006 activity respectively operably linked to a strong inducible promoter e.g. , wherein the selected gene product is enzymatically active in the transgenic fungi and/or fungal cells and inducible overexpression of the gene inhibits and/ or suppresses growth and/or development of the fungus. The nucleotide sequence is preferably derived from an eukaryote, such as a yeast, but is preferably derived from a fungus and more particularly from a filamentous fungus. In a further preferred embodiment, the nucleic acid sequences set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11 SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11 encode enzymes having AG001 , AG002, AG003, AG004.AG005 or AG006 activity respectivelyy, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 OR SEQ ID NO: 12. The transgenic fungus or fungal cells are grown in 96-well format microtiter dishes for high-throughput screening. Compounds that are effective in blocking function of the AG001 , AG002, AG003, AG004, AG005 or AG006 protein results in fungal growth. This growth is measured by methods known in the art. In a particular embodiment the transgenic fungus is Ashbya gossypii.

Similar assays based on expression of the fungal genes of the invention in yeast, using appropiate expression systems as described above may also be used.

In Vitro Inhibitor Assays: Discovery of Small Molecule Ligand that Interacts with Protein of Unknown Function

Novel technologies are being examined that can detect interactions between a protein and a ligand without knowing the biological function of the protein. A short description of three methods is presented, including fluorescence correlation spectroscopy, surface-enhanced laser desorption/ionization, and biacore technologies. Many more of these methods are currently being discovered, and some may be amenable to automated, large scale screening in light of this disclosure. Fluorescence Correlation Spectroscopy (FCS) theory was developed in 1972 but it is only in recent years that the technology to perform FCS became available (Madge et al. (1972) Phys. Rev. Lett., 29: 705-708; Maiti et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 11753-11757). FCS measures the average diffusion rate of a fluorescent molecule within a small sample volume. The sample size can be as low as 103 fluorescent molecules and the sample volume as low as a the cytoplasm of a single bacterium. The diffusion rate is a function of the mass of the molecule and decreases as the mass increases. FCS can therefore be applied to protein-ligand interaction analysis by measuring the change in mass and therefore in diffusion rate of a molecule upon binding.

Surface-Enhanced Laser Desorption/lonization (SELDI) was invented by Hutchens and Yip during the late 1980's (Hutchens and Yip (1993) Rapid Commun. Mass Spectrom. 7: 576-580). When coupled to a time-of-flight mass spectrometer (TOF), SELDI provides a mean to rapidly analyze molecules retained on a chip. It can be applied to ligand-protein interaction analysis by covalently binding the target protein on the chip and analyze by MS the small molecules retained by this protein (Worrall et al. (1998) Anal. Biochem. 70: 750- 756).

Biacore relies on changes in the refractive index at the surface layer upon binding of a ligand to a protein immobilized on the layer. In this system, a collection of small ligands is injected sequentially in a 2-5 ul cell with the immobilized protein. Binding is detected by surface plasmon resonance (SPR) by recording laser light refracting from the surface. In general, the refractive index change for a given change of mass concentration at the surface layer, is practically the same for all proteins and peptides, allowing a single method to be applicable for any protein (Liedberg et al. (1983) Sensors Actuators 4: 299-304; Malmquist (1993) Nature, 361 : 186-187).

IV. In Vivo Inhibitor Assay

In one embodiment, a suspected pesticide, particularly fungicide, for example identified by in vitro screening, is applied to fungi at various concentrations. After application of the suspected fungicide, its effect on the fungus, for example inhibition or suppression of growth and development is recorded. The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

EXAMPLES

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, et al., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and by T.J. Silhavy, M.L. Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and by Ausubel, F.M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-lnterscience (1987),

Construction and characterization of a Genomic Library of A. gossypii (strain ATCC10895), identification of ORF and promoters is described in U.S. Patent Application Ser. No.: 08/998,416 which is hereby incorporated by reference in its entirety.

Example 1 : Identification of Antifungal Drug Targets Represented in the Sequence Listing

Gene disruptions of Ashbya gossypii genes are generated by a method using short flanking homology regions to produce gene targeting events. The short flanking homology regions are included within polymerase chain reaction primers of 65 nucleotide overall sequence length. Each of these 65-mers contains approximately 45 nucleotides homology to the target gene locus the target gene locus being identified as described in U.S. Patent Application Ser. No. 08/998,416 incorporated above by reference, and 20 nucleotides homology (invariant) to a geneticin resistance gene module(also described in U.S. Patent Application Ser. No. 08/998,416 previously incorporated by reference) , with one primer (designated S1) anchored to the 5' end of the geneticin resistance module (using the invariant sequence 5'-GCTAGGGATAACAGGGTAAT-3') (SEQ ID NO:13)and the other primer of the pair (designated S2) anchored to the 3' end of the geneticin resistance module (using the invariant sequence 5'-AGGCATGCAAGCTTAGATCT-3') (SEQ ID NO:14). The PCR product resulting from the amplification of the geneticin resistance module with such an S1/S2 primer pair thus consists of the module flanked by short flanking homology regions of ca. 45 nucleotides specific to the chosen gene disruption site.

Once an S1/S2 primer pair is designed for a particular gene target, approximately 10 ug of the desired geneticin resistance module is obtained by linearizing a vector containing the geneticin resistance gene positioned behind the an appropriate fungal promoter (for example, the Saccharomyces cerevisiae TEF1 promoter) and subjecting the linearized template to approximately 35 rounds of a PCR reaction consisting of the following steps: Step 1 : Denaturation at 96 C for 30 seconds; Step 2: Primer annealing at 50 C for 30 seconds; Step 3: Elongation reaction at 72 C for 2.5 minutes. Following the 35th round of this protocol, a final elongation period of 5 minutes at 72 C is carried out.

Transformation of the PCR product resulting from amplification with the S1/S2 primer pair is done by electroporation as follows: 1) Inoculate 100 ml of AFM media (1% casein peptone, 2% glucose, 1% yeast extract, 0.1 % myo-inositol) with an Ashbya spore suspension of approximately 10⁷ spores. 2) Incubate at 30 C for a maximum of 18 hors at a shaker speed of 200 rpm. 3) Collect the resultant fungal mycelia by filtration and wash once with sterile water. 4) Resuspend 1 gram of mycelia (wet weight) in 40 ml of 50 mM potassium phosphate buffer, pH 7.5 containing 25mM DTT and incubate at 30 C for 30 minutes with gentle shaking. 5) Collect the mycelia by filtration and wash once with 50 ml of cold STM buffer (275 mM sucrose, 10 mM Tris-HCI, pH 7.5, 2 mM MgCI₂). 6) Resuspend the mycelia to a dense mixture in STM buffer. 7) Mix approximately 150 ul of the mycelial mixture with 10 ug of PCR product (in a maximum volume of 50 ul) in an Eppendorf tube and transfer the mixture to an electroporation cuvette with a 4mM gap distance. 8) Apply an electric field pulse of 1.5 kV, 100 ohms, 25 uF which will result in a pulse length of approximately 2.3 milliseconds. Add 1 ml of AFM media to the cuvette and spread equal amounts onto 3 pre-dried AFM agar plates. 9) Incubate plates for a minimum of 4 hours at 30 C. 10) Overlay the plates with 8 ml of a 0.5%agarose toplayer containing

Geneticin/G418 at a final concentration of 200 ug/ml. 11) Incubate at 30 C for approximately 3 days to allow sufficient growth of geneticin resistant transformants.

Verification of the desired transformation event resulting in homologous integration of the geneticin resistance module in the target of interest is achieved by PCR using verification primers designated G1 (positioned upstream of the S1 region) and G4 (positioned downstream of the S2 region) and template DNA purified from putative Ashbya transformants. Additional verification primers designated G2 (5'- GTTTAGTCTGACCATCTCATCTG-3') (SEQ ID N015)and G3 (5'- TCGCAGACCGATACCAGGATC-3') (SEQ ID NO:16) are derived from the open reading frame of the selectable geneticin resistance gene such that the detection of a G1/G2 PCR product and or a G3/G4 PCR product of a predictable size serves to verify the desired gene disruption event. Also, verification of the desired gene disruption can be determined by standard DNA hybridization experiments.

Determination of whether a gene is essential to growth of Ashbya can be achieved by the following analysis. The transformation of DNA fragments described above utilizes multinucleate Ashbya mycelia as recipients. Therefore a primary transformant able to grow on geneticin containing media originates as a mycelium containing cells at least one of which has at least one transformed nucleus, but usually containing non-transformed nuclei as well. Thus, if an essential gene is disrupted in the transformed nucleus, the essential gene product can, in many instances, still be supplied by the non-transformed nuclei within the same cell. Such primary transformants usually exhibit normal growth and sporulation, and spores are collected from primary transformants allowed to grow at 30 C for at least 5 days. Since spores are uninucleate, however, transformants which have an essential gene disrupted in nuclei containing the geneticin resistance cartridge will fail to yield spores which grow normally, if at all, on geneticin-containing media.

S1 and S2 primer pairs usable to generate disruptions of the indicated genes are as follows:

AG001 : S1 : 5'-AGGACCACTAGCTCGTTGCGCTGCAATATAATAATAAGAACGAGA GCTAGGGATAACAGGGTAAT-3' (SEQ ID NO:17)

S2: 5^,-AAGTATTCAATCAACTATGTGAGTAGTTTCTTGTAGGCAGTCTCC AGGCATGCAAGCTTAGATCT-3'(SEQ ID NO:18)

AG002: S1 : 5'-CTGGCATCAGAGGAAGCTCCCACCACCAAGCTCTACAAACACAAG GCTAGGGATAACAGGGTAAT-3'(SEQ ID NO:19)

S2: 5'-ATTATATTAGTATAGTCTAAAGTTGCAGGCAGTGGGTATTAAAGT AGGCATGCAAGCTTAGATCT-3'(SEQ ID NO:20)

AG003: S1 : δ'-ACTTGCGTACTCTTTCGCGTGCTCGTCAGCCACCGAACAACGCAG GCTAGGGATAACAGGGTAAT-3'(SEQ ID NO:21)

S2: δ'-TTAAAGAATGATAAAGAACCAAAAACACCACGAGCTTGCATAACA AGGCATGCAAGCTTAGATCT-3'(SEQ ID NO:22) AG004: S1 : 5'-GTGCGTGTCAGCGAGCATCTAATCAAGCTGCAAGGCGCCGGAAAT GCTAGGGATAACAGGGTAAT-3'(SEQ ID NO:23)

S2: 5'-TTATCACATATTTCTAAGTTAATAGATA I I I I I ACTTAGTATGAA AGGCATGCAAGCTTAGATCT-3'(SEQ ID NO:24)

AG006: S1 : 5'-GAGAGAGACGCTACGGTACTACGAATTTCTCTGTAGAGTTGGAGA GCTAGGGATAACAGGGTAAT-3'(SEQ ID NO:25)

S2: δ'-TACTATTGAGAATGTTCGCGACTGCATGTAAAGTCTCAAAAACTT AGGCATGCAAGCTTAGATCT-3'(SEQ ID NO:26)

AG005: S1 : 5'-AAATATAATAAAAATTGACAACTGGCTAGAAGTGATACCGCAGTT GCTAGGGATAACAGGGTAAT-3'(SEQ ID NO:27)

S2: 5'-CCTCTTATAGTTCATGACCCATTCATATGCGTCATTCAGGTCTCT AGGCATGCAAGCTTAGATCT-3'(SEQ ID NO:28)

The above disclosed embodiments are illustrative. This disclosure of the invention will place one skilled in the art in possession of many variations of the invention. All such obvious and foreseeable variations are intended to be encompassed by the appended claims.

Claims

What is claimed is:

1. A nucleotide sequence substantially similar to any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11.

2. The nucleotide sequence of claim 1 , wherein the nucleotide sequence is SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11.

3. The nucleotide sequence of claim 1 or 2, wherein the nucleotide sequence is a fungal nucleotide sequence.

4. The nucleotide sequence of claim 3, wherein the fungus is Ashbya gossypii.

5. The nucleotide sequence of claim 1 , wherein the nucleotide sequence encodes an amino acid sequence substantially similar to any one of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12 respectively.

6. A nucleotide sequence encoding an amino acid sequence according to any one of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12.

7. A nucleotide sequence having a 20 base pair nucleotide portion identical in sequence to a 20 consecutive base pair portion of a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11.

8. An amino acid sequence comprising an amino acid sequence encoded by a nucleic acid sequence substantially similar to any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11.

9. An amino acid sequence substantially similar to any one of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12.

10. A chimeric gene comprising a promoter operably linked to a nucleotide sequence according to claim 1 or 2.

11. The chimeric gene of claim 10'8 wherein the promoter is an inducible promoter.

12. A recombinant vector comprising a chimeric gene according to claim 11 wherein said vector is capable of being stably transformed into a host cell.

13. A host cell comprising the vector according to claim 12, wherein the nucleotide sequence is expressible in the host cell.

14. The host cell according to claim 13, wherein the host cell is an Ashbya gossipii cell.

15. A process for identifying compounds having fungicidal activity comprising the steps of: a) combining a protein according to claim 16 and a compound to be tested for the ability to bind to the protein, under conditions having conducive binding, b) selecting a compound identified by step a) that is capable of binding the protein; c) applying the identified compound from step b) to a fungus to test for fungicidal activity; and d) selecting compounds having fungicidal activity.

16. A compound having fungicidal activity identifiable according to claim 15.

17. A process for identifying an inhibitor of a protein activity having an amino acid sequence according to claim 9 comprising: a) introducing SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 or nucleotide sequences substantially similar thereto into a host, such that the sequence is functionally expressible; b) combining the host cell of step a) with a compound to be tested for ability to inhibit the protein activity; c) over expressing the nucleotide sequence of step a); c) measuring the host cell growth in stepc); and d) selecting the compound that inhibits or suppresses host cell's normal growth or development in step c).

18. A compound having fungicidal activity identifiable according to the process of claim 170.

19. A method of suppressing growth of a fungus comprsing applying to the fungus a compound that inhibits the activity of a protein comprising the amino acid sequence according to claim 9 in an amount sufficient to suppress the growth of the fungus.

20. The method of claim 19 wherein the compound is selected from a group consisting of the compounds of claims 16 and claim 18.