WO2003059936A2 - Phytophthora Infestans CDC 14 Phosphatase Gene, Protein and Methods of Use - Google Patents

Phytophthora Infestans CDC 14 Phosphatase Gene, Protein and Methods of Use Download PDF

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Publication number
WO2003059936A2
WO2003059936A2 PCT/US2003/000625 US0300625W WO03059936A2 WO 2003059936 A2 WO2003059936 A2 WO 2003059936A2 US 0300625 W US0300625 W US 0300625W WO 03059936 A2 WO03059936 A2 WO 03059936A2
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acid sequence
amino acid
protein
polypeptide
nos
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PCT/US2003/000625
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French (fr)
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WO2003059936A3 (en
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Howard S. Judelson
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Syngenta Participations Ag
The Regents Of The University Of California
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Priority to AU2003207496A priority Critical patent/AU2003207496A1/en
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Publication of WO2003059936A3 publication Critical patent/WO2003059936A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention pertains to nucleic acid molecules isolated from Phytophthora infestans comprising nucleotide sequences that encode proteins essential for plant growth and development.
  • the invention particularly relates to methods of using these proteins as fungicide targets, based on this essentiality.
  • P. infestans - Phytophthora infestans polyA polyadenosine RENT regulator of nucleolar silencing and telophase
  • fungicides to control undesirable fungal growth and pathogens in crop fields has become almost a universal practice. Despite this extensive use, fungal control remains a significant and costly problem for farmers.
  • fungicides require sound management. For instance, the time and method of application and stage of fungal development are critical to achieving good fungal control with fungicides. Because various fungal species are resistant to fungicides, the production of effective new fungicides becomes increasingly important. New fungicides can now be discovered using high-throughput screens that implement recombinant DNA technology. Metabolic enzymes found to be essential to fungal growth and development can be recombinantly produced through standard molecular biological techniques and utilized as fungicide targets in screens for novel inhibitors of the enzyme activity. More generally, any essential plant protein can be used to screen for inhibitors of its activity. The novel inhibitors discovered through such screens can then be used as fungicides to control undesirable fungal growth.
  • P. infestans is arguably the most important pathogen of the world's largest non-cereal crop, potato, and is also a significant tomato pathogen (Fry & Goodwin, 1997).
  • the late blight diseases have always been important and have been especially difficult over the past decade (Fry & Goodwin, 1997).
  • the worldwide cost of the potato disease alone exceeds $5 billion per year, including more than $1 billion spent on fungicides (Anonymous, 2000).
  • Oomycetes represent a large collection of important but poorly characterized species that include both saprophytes and significant parasites of plants (Pythium, Phytophthora, white rusts, downy mildews), animals, and insects (Alexopoulos et al., 1996). Oomycetes lack taxonomic affinity with the so-called true fungi (i.e. ascomycetes and basidiomycetes). Instead, oomycetes are properly classified with diatoms and brown algae (Baldauf et al., 2000; Gunderson et al., 1987).
  • Nuclear behavior during the life cycle has been characterized (Whittaker et al., 1991 ; Maltese et al., 1995; Laviola 1975; Sivak 1973).
  • vegetative hyphae which lack cross-walls or septa
  • nuclear division is asynchronous. Generally one nucleus moves into the sporangiophore, in which rapid synchronized mitoses occur coincident with the formation of a basal septum.
  • Sporangiophores extend to their full length over about 3 hours, generally forming several branches that develop terminal swellings. Nuclei and cytoplasm then move into the swellings, which become the asexual spores.
  • a septum forms at its base and a "cap” forms on each nucleus that does not appear to be a nucleolus (Sivak 1973; Marks 1965). Some nuclei may degenerate, possibly to maintain their proper ratio with cytoplasm (Maltese et al., 1995). The nuclei in the asexual spore, as well as zoospores, are at G1 (Whittaker et al., 1992). A different definition of the cell must be invoked for filamentous fungi, in which growth involves the expansion of hyphal tubes, as compared to syncytial species such as Physarum.
  • oomycete hyphae are aseptate and thus coenocytic; discrete cellular compartments only form when spores are made.
  • Cytoplasmic growth, nuclear division, and cytokinesis are tightly regulated by the cell cycle. This has been intensely studied in many organisms, particularly in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in mammals, and to a lesser extent in plants (McCollum & Gould 2001 ; Smits & Medema 2001 ; Stals et al., 2000; Heberle-Bors 2001.
  • cyclin B a G2/M cyclin
  • its interacting kinase have been studied in several species, with the latter named cdc28 in S. cerevisiae, cdc2 in S. pombe, NimX in Aspergillus, and p34 in metazoans (McCollum & Gould 2001 ; Osmani & Ye 1996; Bregman et al., 2000).
  • Cdc14 triggers cyclin B destruction by dephosphorylating Cdh1 , a component of the anaphase-promoting complex (APC) that accumulates when cyclin B- Cdk cdc28 activity is high.
  • APC anaphase-promoting complex
  • Cdc14 activates Sid , a cyclin- dependent kinase inhibitor, and Swi5, a transcriptional activator of the sid gene.
  • the combined effect of these activities is to destroy cyclin B and inhibit its associated kinase, which allows a cell to exit mitosis and begin a new cell cycle.
  • the cdc14 gene is essential in S. cerevisiae (Grandin et al., 1998; Sakumoto et al., 1999).
  • Cdc14 is regulated by its localization.
  • Cdc14 is sequestered in the nucleolus where it joins Net1 and Cfi1 in a complex called "regulator of nucleolar silencing and telophase" (RENT) (Cockell & Gasser 1999).
  • RENT releases Cdc14 and allows it to reach its nuclear targets.
  • Cdc14 There are several issues remaining regarding the role of Cdc14. Perhaps the most important is whether or not related proteins in other species function in the same way as Cdc14 has been shown to in S. cerevisiae. In humans, for example, two Cdc14-like proteins exist, only one of which behaves like the S. cerevisiae protein (Jaspersen et al., 2000; Bembenek & Yu, 2001).
  • hCdc14a affects cyclin B and its associated kinase, and also shows the same nucleolus/nucleus distribution pattern as its S. cerevisiae counterpart; hCdd 4a even complements a S. cerevisiae cdd 4- 1 ts mutant.
  • hCdc14b is present in the cytoplasm and does not complement cdd 4-1 ts .
  • both human proteins interact with the p53 tumor suppressor (Kroll et al., 1996).
  • the Cdd 4 orthologue (Flp1) might potentiate mitotic exit and septation, but is not required for inactivating the B-type cyclin (Cueille et al., 2001).
  • the subcellular distribution of Flp1 during the cell cycle does resemble that of Cdd 4 in S. cerevisiae, and Flp1 complements the cdd 4- 1 ts mutant, however (jd.).
  • nucleic acid molecules from Phytophthora infestans comprising nucleotide sequences that encode proteins essential for fungal growth and development. It is another object to provide the essential proteins encoded by these essential nucleotide sequences for assay development to identify inhibitory compounds with fungicide activity. It is still another object of the present invention to provide an effective and beneficial method for identifying new or improved fungicides using the essential proteins of the invention.
  • the present invention provides nucleic acid molecules isolated from P. infestans comprising nucleotide sequences that encode proteins essential for plant viability.
  • the activity of each protein of the present invention is essential for the growth of Phytophthora infestans, an aspect that is disclosed herein for the first time.
  • the critical role in fungal growth of the proteins encoded by each of the nucleotide sequences of the invention implies that chemicals that inhibit the function of any one of these proteins in fungi are likely to have detrimental effects on fungal growth and are thus potentially good fungicide candidates.
  • the proteins encoded by the essential nucleotide sequences provide the bases for assays designed to easily and rapidly identify novel fungicides.
  • the present invention therefore provides methods of using a purified protein encoded by any one of the nucleotide sequences described below to identify inhibitors thereof, which can then be used as fungicides to suppress the growth of undesirable fungi, for example, in fields where crops are grown, particularly agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
  • agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
  • nucleic acid molecules isolated from Phytophthora infestans in particular, nucleic acid molecules encoding cdc14.
  • the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence, the complement of which hybridizes under stringent conditions to a sequence selected from the group consisting of the odd-numbered SEQ ID NOs:1 -5.
  • the present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a protein comprising an amino acid sequence having in one embodiment at least 60%, in another embodiment at least 70%, in another embodiment at least 80%, in another embodiment at least 90%, in another embodiment at least 95%, and in yet another embodiment at least 99-100% sequence identity to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
  • the present invention also provides a chimeric construct comprising a promoter operatively linked to a nucleic acid molecule according to the present invention.
  • the promoter is functional in a eukaryote.
  • the promoter is heterologous to the nucleic acid molecule.
  • the present invention further provides a recombinant vector comprising a chimeric construct according to the present invention, wherein said vector is capable of being stably transformed into a host cell.
  • the present invention still further provides a host cell comprising a nucleic acid molecule according to the present invention.
  • the nucleic acid molecule is expressible in the cell.
  • the host cell can be selected from the group consisting of a plant cell, a yeast cell, an insect cell, and a prokaryotic cell.
  • the present invention additionally provides a plant or seed comprising a plant cell according to the present invention.
  • the present invention also provides proteins essential for fungal growth in Phytophthora infestans.
  • the present invention provides an isolated protein comprising an amino acid sequence having in one embodiment at least 60%, in another embodiment at least 70%, in another embodiment at least 80%, in another embodiment at least 90%, in another embodiment at least 95%, and in yet another embodiment at least 99-100% sequence identity to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
  • the present invention also relates to the recombinant production of proteins of the invention and methods of using the proteins of the invention in assays for identifying compounds that interact with the protein.
  • the present invention provides a method of identifying a fungicidal compound, comprising: (a) combining a polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the even- numbered SEQ ID NOs:2-6 with a compound to be tested for the ability to bind to said polypeptide, under conditions conducive to binding; (b) selecting a compound identified in (a) that binds to said polypeptide; (c) applying a compound selected in (b) to a plant to test for fungicidal activity; and (d) selecting a compound identified in (c) that has fungicidal activity.
  • the polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6. In another embodiment, the polypeptide comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of the even- numbered SEQ ID NOs:2-6. In still another embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
  • the present invention also provides a method for killing or inhibiting the growth or viability of a plant, comprising applying to the plant an herbicidal compound identified according to this method.
  • the present invention provides a method of identifying a fungicidal compound, comprising: (a) combining a polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the even- numbered SEQ ID NOs:2-6 with a compound to be tested for the ability to inhibit the activity of said polypeptide, under conditions conducive to inhibition; (b) selecting a compound identified in (a) that inhibits the activity of said polypeptide; (c) applying a compound selected in (b) to a plant to test for fungicidal activity; and (d) selecting a compound identified in (c) that has fungicidal activity.
  • the polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6. In another embodiment, the polypeptide comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6. In still another embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
  • the present invention also provides a method for killing or inhibiting the growth or viability of a fungus, comprising applying to the plant a fungicidal compound identified according to this method.
  • the present invention still further provides a method for killing or inhibiting the growth or viability of a fungus, comprising inhibiting expression in said fungus of a protein having in one embodiment at least 60%, in another embodiment at least 70%, in another embodiment at least 80%, in another embodiment at least 90%, in another embodiment at least 95%, and in yet another embodiment at least 99-100% sequence identity to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
  • Odd-numbered SEQ ID NOs:1 -5 are nucleotide sequences isolated from Phytophthora infestans that are more fully described below.
  • SEQ ID NOs:2-6 are protein sequences encoded by the immediately preceding nucleotide sequence, e.g., SEQ ID NO:2 is the protein encoded by the nucleotide sequence of SEQ ID NO:1 , SEQ ID NO:4 is the protein encoded by the nucleotide sequence of SEQ ID NO:3, etc.
  • the phrases "associated with” and “operatively linked” refer to two or more nucleic acid sequences that are related physically or functionally.
  • a promoter or regulatory DNA sequence is said to be “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked or are situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
  • chimeric construct refers to a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or is associated with, a nucleic acid sequence that codes for an mRNA or which is expressed as a protein, such that the regulatory nucleic acid sequence is able to regulate the transcription or expression of the associated nucleic acid sequence.
  • the regulatory nucleic acid sequence of the chimeric construct is not normally operatively linked to the associated nucleic acid sequence as found in nature.
  • co-factor refers to a natural reactant, such as an organic molecule or a metal ion, that is required in an enzyme- catalyzed reaction.
  • Co-factors include, but are not limited to NADP, riboflavin (including FAD and FMN), folate, molybdopterin, thiamin, biotin, lipoic acid, pantothenic acid, coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone, and menaquinone.
  • a co-factor can be regenerated and reused.
  • coding sequence refers to a nucleic acid sequence that is transcribed into RNA including, but not limited to mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA.
  • the RNA can then be translated in vitro or in vivo to produce a protein.
  • complementary refers to two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences.
  • enzyme activity refers to the ability of an enzyme to catalyze the conversion of a substrate into a product.
  • a substrate for an enzyme comprises the natural substrate of the enzyme but also comprises analogues of the natural substrate that can also be converted by the enzyme into a product or into an analogue of a product.
  • the activity of the enzyme can be measured, for example, by determining the amount of product in the reaction after a certain period of time, or alternatively, by determining the amount of substrate remaining in the reaction mixture after a certain period of time.
  • the activity of the enzyme can also be 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 can also be measured by determining the amount of a donor of free energy or energy-rich molecule (including, but not limited to ATP, phosphoenolpyruvate, acetyl phosphate, and 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 (including, but not limited to ADP, pyruvate, acetate, and creatine) in the reaction mixture after a certain period of time.
  • a donor of free energy or energy-rich molecule including, but not limited to ATP, phosphoenolpyruvate, acetyl phosphate, and phosphocreatine
  • essential refers to a nucleotide sequence encoding a protein that is essential to the growth and/or survival of the fungus. Proteins that are essential to the growth and/or survival of the fungus include, but are not limited to biosynthetic enzymes, receptors, signal transduction proteins, structural gene products, and transport proteins.
  • expression cassette refers to a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell. An expression cassette typically comprises a promoter operatively linked to a nucleotide sequence of interest, which is operatively linked to termination signals.
  • An expression cassette typically further comprises sequences required for proper translation of the nucleotide sequence.
  • the nucleotide sequence of interest usually encodes a protein of interest, but can also code for a functional RNA of interest, for example an antisense RNA or a non-translated RNA, in the sense or antisense direction.
  • the expression cassette comprising the nucleotide sequence of interest can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette can also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression cassette is heterologous with respect to the host, meaning that the particular DNA sequence of the expression cassette does not occur naturally in the host cell and thus is introduced into the host cell or an ancestor of the host cell by a transformation event.
  • the expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue, organ, or stage of development.
  • fungus refers to both true fungi and organisms traditionally classified in some schemes as fungi, for example oomycetes.
  • fungal refers to substances derived from species defined herein as fungi, including, but not limited to oomycetes.
  • fungicide refers to compounds capable of inhibiting or preventing the growth of a fungus as defined herein.
  • genes refers broadly to any segment of DNA associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. Genes also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and can include sequences designed to have desired parameters.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.
  • the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found.
  • Exogenous DNA segments can be expressed to yield exogenous polypeptides.
  • nucleic acid sequence for example, a DNA sequence
  • a nucleic acid sequence refers to a nucleic acid sequence naturally associated with a host cell into which it is introduced.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture including, but not limited to total cellular DNA and RNA.
  • bind(s) substantially refers to complementary hybridization between a nucleic acid probe and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization conditions to achieve the desired detection of the target nucleic acid sequence.
  • the term “inhibitor” refers to a chemical substance that inactivates the enzymatic activity of a protein such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein.
  • fungicide or “fungicidal compound” is used herein to define an inhibitor applied to a fungus or plant at any stage of development, whereby the fungicide inhibits the growth of the fungus or kills the fungus.
  • a nucleic acid sequence is "isocoding with" a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.
  • the term “isogenic” refers to plants that are genetically identical, except that they can differ by the presence or absence of a heterologous DNA sequence.
  • 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 can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell.
  • mature protein refers to a protein from which the transit peptide, signal peptide, and/or propeptide portions have been removed.
  • minimal promoter refers to the smallest fragment of a promoter, such as a TATA element, that can support any transcription.
  • a minimal promoter typically has greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, a minimal promoter functions to permit transcription.
  • modified enzyme activity refers to an enzyme activity that is different from an enzyme activity that 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.
  • native refers to a macromolecule that is normally present in an untransformed plant cell.
  • a native gene is a gene that is normally present in the genome of an untransformed plant.
  • naturally occurring refers to an object that can be found in nature as distinct from being artificially produced by man.
  • a protein or nucleotide sequence present in an organism including, but not limited to a virus, which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991 ; Ohtsuka et al., 1985; Rossolini et al., 1994).
  • the terms "nucleic acid” or “nucleic acid sequence” may also be used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • ORF open reading frame
  • ORF refers to a contiguous series of deoxyribonucleotides or ribonucleotides that encode a polypeptide or a fragment of a polypeptide.
  • the ORF will be discontinuous in the genome. Splicing produces a continuous ORF that can be translated to produce a polypeptide.
  • percent identity and “percent identical,” in the context of two nucleic acid or protein sequences, refer to two or more sequences or subsequences that have in one embodiment at least 60%, in another embodiment at least 70%, in another embodiment at least 80%, in another embodiment at least 90%, in another embodiment at least 95%, and in yet another embodiment at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the percent identity exists in one embodiment over a region of the sequences that is at least about 50 residues in length, in another embodiment over a region of at least about 100 residues, and in still another embodiment the percent identity exists over at least about 150 residues. In yet another embodiment, the percent identity exists over the entire length of the coding regions.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman (1981), by the homology alignment algorithm of Needleman & Wunsch (1970), by the search for similarity method of Pearson & Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection. See generally, Ausubel et al., 1994.
  • One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990).
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See e.g., Karlin & Altschul 1993.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in one embodiment less than about 0.1 , in another embodiment less than about 0.01 , and in still another embodiment less than about 0.001.
  • pre-protein refers to a protein that is normally targeted to a cellular organelle, such as a chloroplast, and still comprises its native transit peptide.
  • the term "purified”, when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be in a homogeneous state although it also can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.
  • the term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is in one embodiment at least about 50% pure, in another embodiment at least about 85% pure, and in still another embodiment at least about 99% pure.
  • Two nucleic acids are “recombined” when sequences from each of the two nucleic acids are combined in a progeny nucleic acid.
  • Two sequences are “directly” recombined when both of the nucleic acids are substrates for recombination.
  • Two sequences are "indirectly recombined” when the sequences are recombined using an intermediate such as a cross-over oligonucleotide.
  • no more than one of the sequences is an actual substrate for recombination, and in some cases, neither sequence is a substrate for recombination.
  • regulatory elements refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operatively linked to the nucleotide sequence of interest and termination ⁇ signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.
  • significant increase refers to an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique.
  • a significant increase is in one embodiment an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, in another embodiment an increase by about 5-fold or greater, and in still another embodiment an increase by about 10-fold or greater.
  • the phrase "significantly less” means that the amount of a product of an enzymatic reaction is reduced by more than the margin of error inherent in the measurement technique, in one embodiment a decrease by about 2-fold or greater of the activity of the wild-type enzyme in the absence of the inhibitor, in another embodiment an decrease by about 5-fold or greater, and in still another embodiment an decrease by about 10-fold or greater.
  • the phrase “specific binding” and “immunological cross-reactivity” refer to an indication that two nucleic acid sequences or proteins are substantially identical in that the protein encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the protein encoded by the second nucleic acid.
  • a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised to a protein with an amino acid sequence encoded by any of the nucleic acid sequences of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins except for polymorphic variants.
  • a variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase enzyme-linked immunosorbent assays (ELISA), Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize to its target subsequence, but not to other sequences.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes. See, Sambrook et al. 2001 , for a description of SSC buffer.
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An exemplary medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1x SSC at 45°C for 15 minutes.
  • An exemplary low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0 M Na + ion, typically about 0.01 -1.0 M Na + ion concentration (or other salts) at pH 7.0 to 8.3, and a temperature typically of at least about 30°C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still considered substantially identical if the proteins that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • a nucleotide sequence can hybridize to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C, with washing in 2X SSC, 0.1 % SDS at 50°C.
  • SDS sodium dodecyl sulfate
  • a nucleotide sequence can hybridize to the reference nucleotide sequence in 7% SDS, 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C. In another embodiment, a nucleotide sequence can hybridize to the reference nucleotide sequence in 7% SDS, 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1 % SDS at 50°C.
  • a nucleotide sequence can hybridize to the reference nucleotide sequence in 7% SDS, 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1 % SDS at 50°C.
  • a nucleotide sequence can hybridize to the reference nucleotide sequence in 7% SDS, 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1 % SDS at 65°C.
  • sequence refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., protein) respectively.
  • substrate refers to a molecule that an enzyme recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function, or to 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.
  • transformation refers to a process for introducing heterologous DNA into a cell, a tissue, or an entire organism including, but not limited to viruses, bacteria, plants, and animals.
  • Transformed plant cells, plant tissue, or plants are understood to encompass not only the cell, tissue, or plant that is the end product of a transformation process, but also transgenic progeny thereof.
  • transformed refers to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome of the host, but can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • a “non- transformed,” “non-transgenic”, or “non-recombinant” host refers to a wild- type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
  • viability refers to a fitness parameter of a plant. Plants are assayed for their homozygous performance of plant development, indicating which proteins are essential for plant growth. Detailed Description of the Invention L Identification of Essential Fungal Nucleotide Sequences and Encoded Proteins
  • the essentiality of the clones was determined by expressing sense and antisense copies of the gene using assays described below. Having established the essentiality of the function of the encoded protein(s) in Phytophthora infestans and having identified the nucleotide sequences encoding these essential proteins, the inventors thereby provide an important and sought after tool for new fungicide development.
  • cDNA and genes of the present invention were cloned and characterized as described in the Examples below.
  • a nucleotide sequence encoding the protein is inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced.
  • the polypeptides of the invention are recombinantly produced upon expression of respective heterologous DNA sequences introduced in the hosts.
  • nucleotide sequences selected from the group consisting of the odd-numbered SEQ ID NOs:1 -5 or nucleotide sequences substantially similar to those selected from the group consisting of the odd-numbered SEQ ID NOs:1 -5, or nucleotide sequences encoding polypeptides selected from the amino acid sequences of even-numbered SEQ ID NOs:2-6, are introduced into chosen hosts for the recombinant production of the polypeptides of the invention.
  • the nucleotide sequences of the invention are derived from a eukaryote, including, but not limited to a mammal, a fly, and a yeast.
  • the nucleotide sequences of the invention are derived from a fungus.
  • the nucleic acid molecules can also be produced using available synthetic methods known in the art.
  • the choice of the specific regulatory sequences such as promoters, signal sequences, 5' and 3' untranslated sequences, and enhancers appropriate for the chosen host is within the level of the skill of the routineer in the art.
  • the resultant molecule, containing the individual elements linked in the proper reading frame, is 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., Lucknow and Summers 1988.
  • baculovirus expression vectors include, but are not limited to baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV).
  • a representative baculovirus/insect system is PVL1392(3) used to transfect Spodoptera frugiperda SF9 cells (available from the American Type Culture Collection (ATCC), Manassas, Virginia, United States of America) in the presence of linear Autographica californica baculovirus DNA (Pharmingen, San Diego, California, United States of America). The resulting virus is used to infect HIGH FIVETM Tricoplusia ni cells (Invitrogen, Carlsbad, California, United States of America).
  • Recombinantly produced proteins are isolated and purified using a variety of standard techniques. The actual techniques used vary depending upon the host organism used, whether the protein is designed for secretion, and other such factors. Such techniques are well known to the skilled artisan. See e.g. chapter 16 of Ausubel et al., 1994. Recombinantly produced polypeptides of the invention are useful for a variety of purposes. For example, they can be used in in vitro assays to screen for known fungicidal chemicals, the target for which has not been identified, to determine if the chemicals inhibit expression of the nucleotide sequences of the invention.
  • Such in vitro assays can also be used as more general screens to identify chemicals that inhibit the biological activity of the polypeptides of the invention, and that are therefore novel fungicide candidates.
  • recombinantly produced polypeptides of the invention are used to elucidate the complex structure of these polypeptides and to further characterize their association with known fungicides in order to rationally design novel fungicides.
  • the recombinantly produced proteins described herein are useful for a variety of purposes. For example, they can be used in in vitro assays to screen known fungicidal chemicals the target for which has not been identified to determine if they inhibit protein activity. Such in vitro assays can also be used as more general screens to identify chemicals that inhibit such protein activity and that are therefore novel fungicide candidates. Recombinantly produced proteins can also 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 herbicides. Alternatively, the recombinant protein can be used to isolate antibodies or peptides that modulate the activity and are useful in transgenic solutions.
  • a simple assay is developed to screen for chemicals that affect normal functioning of the polypeptides of the invention. Such chemicals are promising in vitro leads that can be tested for in vivo fungicidal activity.
  • Any one of the nucleotide sequences of the invention is operatively linked to a strong inducible promoter, such promoters being known in the art.
  • the vector comprising the selected nucleotide of the invention operatively linked to the selected inducible promoter is transformed into a host, such as E. coli. Transformed E.
  • coli harboring and functionally over-expressing one of the nucleotide sequence of the invention are grown in a 96-well format for automated high-throughput screening where inducible over-expression of the nucleotide sequence of the invention is lethal or suppresses growth of the host.
  • Chemicals effective in blocking function of the polypeptides of the invention result in bacterial growth. This growth is measured by simple turbidometric means.
  • an assay for chemicals that block the functions of the polypeptides of the invention uses transgenic fungi or fungal cells capable of over-expressing the nucleotide sequences of the invention, operatively linked to a strong inducible promoter, e.g., wherein the selected polypeptide of the invention is biologically active in the transgenic fungi and/or fungal cells, and inducible over-expression of a nucleotide sequence of the invention inhibits and/ or suppresses growth and/or development of the fungus.
  • the transgenic fungus or transgenic fungal cells are grown in 96-well format microtiter dishes for high-throughput screening. Chemicals that are effective in blocking the functions of the polypeptides of the invention result in fungal growth.
  • a representative assay includes the following general steps: (a) obtaining transgenic fungus and/or fungal cell, in one embodiment stably transformed, comprising a non-native nucleotide sequence or an endogenous nucleotide sequence operatively linked to non- native promoter, in one embodiment an inducible promoter, encoding an enzyme having an activity and capable of over-expressing a polypeptide of the invention, where over-expression of the polypeptide 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
  • FCS Fluorescence Correlation Spectroscopy
  • the target to be analyzed is expressed as a recombinant protein with a sequence tag, such as a poly-histidine sequence, inserted at the N-terminus or C-terminus of the protein.
  • the expression takes place in E. coli, yeast, or insect cells.
  • the protein is purified by chromatography.
  • the poly-histidine tag can be used to bind the expressed protein to a metal chelate column such as Ni 2+ chelated on iminodiacetic acid agarose.
  • the protein is then labeled with a fluorescent tag such as carboxytetramethylrhodamine or BODIPY ® (Molecular Probes, Eugene, Oregon, United States of America).
  • the protein is then exposed in solution to the potential ligand, and its diffusion rate is determined by FCS using instrumentation available from Carl Zeiss, Inc. (Thornwood, New York, United States of America). Ligand binding is determined by changes in the diffusion rate of the protein.
  • SELDI Surface-Enhanced Laser Desorption/lonization
  • the SELDI chip it is bound to the SELDI chip either by utilizing the poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction.
  • the chip thus prepared is then exposed to the potential ligand via, for example, a delivery system capable to pipette the ligands in a sequential manner (autosampler).
  • the chip is then submitted to washes of increasing stringency, for example a series of washes with buffer solutions containing an increasing ionic strength. After each wash, the bound material is analyzed by submitting the chip to SELDI- TOF. Ligands that specifically bind the target will be identified by the stringency of the wash needed to elute them.
  • Biacore relies on changes in the refractive index at the surface layer upon binding of a ligand to a protein immobilized on the layer.
  • a collection of small ligands is injected sequentially in a 2-5 ⁇ l cell with the immobilized protein. Binding is detected by surface plasmon resonance (SPR) by recording laser light refracting from the surface.
  • SPR surface plasmon resonance
  • 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; Malmquist 1993).
  • the target to be analyzed is expressed as described for FCS.
  • the purified protein is then used in the assay without further preparation. It is bound to the Biacore chip either by utilizing the poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction.
  • the chip thus prepared is then exposed to the potential ligand via the delivery system incorporated in the instruments sold by Biacore AB (Uppsala, Sweden) to pipette the ligands in a sequential manner (autosampler).
  • the SPR signal on the chip is recorded and changes in the refractive index indicate an interaction between the immobilized target and the ligand. Analysis of the signal kinetics on rate and off rate allows the discrimination between non-specific and specific interaction.
  • an inhibitor assay useful for identifying inhibitors of the products of essential fungal nucleic acid sequences, such as the essential fungal proteins described herein, comprises the steps of: (a) reacting an essential fungal protein described herein and a substrate thereof in the presence of a suspected inhibitor of the protein's function; (b) comparing the rate of enzymatic activity of the protein in the presence of the suspected inhibitor to the rate of enzymatic activity under the same conditions in the absence of the suspected inhibitor; and (c) determining whether the suspected inhibitor inhibits the essential fungal protein.
  • the inhibitory effect on the activity of a herein described essential fungal protein can be determined by a reduction or complete inhibition of protein activity in the assay. Such a determination can be made by comparing, in the presence and absence of the candidate inhibitor, the amount of substrate used or intermediate or product made during the reaction.
  • Phage particles displaying diverse peptide libraries permit rapid library construction, affinity selection, amplification, and selection of ligands directed against an essential protein (Lowman 1997). Structural analysis of these selectants can provide new information about ligand-target molecule interactions and then in the process also provide a novel molecule that can enable the development of new herbicides based upon these peptides as leads.
  • VI. In vivo Inhibitor Assay In one embodiment, a suspected fungicide, for example identified by in vitro screening, is applied to a fungus or fungi at various concentrations. The suspected fungicide can be sprayed on the plants. After application of the suspected fungicide, its effect on the fungus/fungi, for example death or suppression of growth is recorded. VIL Method of Using Nucleotide Sequences of the Invention to Distinguish Fungal Species
  • a nucleotide sequence selected from the Sequence Listing can also be used for distinguishing among different species of plant pathogenic fungi and for distinguishing fungal pathogens from other pathogens such as bacteria (Weising et al., 1995).
  • a nucleotide sequence selected from the Sequence Listing can also be used for distinguishing among different species of plant pathogenic fungi and for distinguishing fungal pathogens from other pathogens such as bacteria using the polymerase chain reaction (PCR). See, U.S. Patent Nos. 5,800,997; 5,814,453; 5,827,695; 5,955,274; 6,221 ,595 and 6,319,673.
  • a nucleotide sequence of the present invention, or homologs thereof, can be incorporated in fungal or bacterial cells using conventional recombinant DNA technology. Generally, this involves inserting a nucleotide sequence into an expression system to which the sequence is heterologous (i.e., not normally present) using standard cloning procedures known in the art.
  • the vector contains the necessary elements for the transcription and translation of the inserted polypeptide-coding sequences in a fungal cell containing the vector.
  • a large number of vector systems known in the art can be used, including, but not limited to plasmids (van den Hondel & Punt 1990).
  • the components of the expression system can also be modified to increase expression.
  • truncated sequences for example, truncated sequences, nucleotide substitutions, nucleotide optimization, or other modifications can be employed.
  • Expression systems known in the art can be used to transform fungal cells under suitable conditions (Lemke & Peng 1997).
  • a heterologous DNA sequence comprising a gene, selected from the group consisting of the odd-numbered SEQ ID NOs:1 -5, can be stably transformed and integrated into the genome of the fungal host cells.
  • Nucleotide sequences intended for expression in transgenic fungi are first assembled in expression cassettes operatively linked to a suitable promoter capable of driving expression of genes in fungi (Lang-Hinrichs 1997; Jacobs & Stahl 1997).
  • the expression cassettes can also comprise any further sequences required or selected for the expression of the heterologous nucleotide sequence. Such sequences include, but are not restricted to transcription terminators, extraneous sequences to enhance expression such as introns, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the fungal transformation vectors as described (Lemke & Peng 1997).
  • PB mRNA from a sporulating culture; grown on polycarbonate membrane on top of rye agar. Inoculated by spreading sporangia. Culture was 1 1 days old
  • PE mRNA from carbon starvation, Chinese minimal medium, grown then transferred for 2 days to the same medium but lacking glucose.
  • Chinese minimal medium is a defined medium for Phytophthora, and contains the following: 1 M glucose; 10 mM (NH 4 ) 2 SO 4 ; 20 mM CaCI 2 ⁇ H 2 O; 4 mM MgSO 4 » 7 H 2 O; 5 mM KH 2 PO 4 ; 1 .7 mM K 2 HPO 4 ; 0.025 mM Fe 2 (SO 4 ) 3 ; 20 mM fumaric acid; 0.2 mM MnSO 4 ; 0.15 mM ZnSO 4 ; 0.14 mM thiamine; adjust pH to 4.6. See Xu et al., 1982
  • PJ mRNA from purified asexual sporangia
  • the assembled libraries are also referred to as the EST libraries J, A, B or E (spores; non-sporulating hyphae; sporulating, and carbon starved, respectively).
  • EST Expressed Sequence Tag
  • Frozen ligation mixes in water on dry ice were sent to Syngenta Biotechnology Inc. (Durham, North Carolina, United States of America; hereinafter "SBI") for library construction.
  • a fraction of the individual ligation mixes was electroporated into E. co/ DH10B, plated out on selection media, and 6144 clones from each library were arrayed into 96 well plates (64 plates for each library) using a robot (QBot, available from Genetix USA Inc., Boston, Massachusetts, United States of America).
  • a subset of these plates for each library was sent, as E. coli colonies in 96 well format, to Lark Technologies (Houston, Texas, United States of America) to be sequenced from the 5' end of the clone with primer T7.
  • the EST database was searched for sequences with matches to protein kinases and protein phosphatases, since these are known regulatory proteins. At this time the putative cdd 4 clone was identified as a phosphatase.
  • EST clones were then hybridized to Northern blots of RNA isolated from P. infestans at various developmental stages. The clones tested were selected based on the results described in sections 1 and 2 above. The cdd 4 phosphatase RNA was present in spores and sporulating hyphae, but not in non-sporulating hyphae (including non-sporulating hyphae from carbon-starved, nitrogen-starved, heat-treated, or mating cultures).
  • Cdd 4 was demonstrated to be involved in asexual sporulation from analysis of the expression of 4800 clones from a P. infestans EST project.
  • DNA from 4800 selected EST clones were spotted on a nylon membrane and hybridized with 32 P-cDNA made from sporulating or non-sporulating hyphae.
  • the cDNA probes were made using MMLV reverse transcriptase using 32 P-dCTP (internal labeling) and a mixture of oligo-dT and random hexamer primers. Hybridizations were performed in 7% SDS, 0.5 M Sodium phosphate pH 7.2, 0.25 mM EDTA at 65°C.
  • washes were performed at 65C in 0.2X SSPE, 0.2% SDS, 0.1 % sodium pyrophosphate. These hybridization and wash conditions are high stringency conditions. The cdd 4 clone was observed to be an up-regulated gene during sporulation.
  • the EST database was then searched for overlapping clones using the BLAST tool. Clones were present in libraries B (sporulating hyphae), J (spores), and F (cleaving spores). The overlapping sequences were assembled. The 5' end of the cDNA was identified since multiple EST sequences started at the same place. There was a small polyA tail in one of the sequences. The sequence was confirmed by sequencing the 3' ends of some of the cDNA clones.
  • SEQ ID NO:1 is a consensus sequence that ignores some sequence polymorphisms between alleles. Between alleles 1 and 2 (SEQ ID NOS:3 and 5, respectively) there are three nucleotide and three amino acid differences.
  • the protein product of 423 amino acids was predicted from the nucleotide sequence of SEQ ID NO:1 , and is set forth in SEQ ID NO:2.
  • the predicted protein amino acid sequences of the two alleles are set forth in SEQ ID NOs:4 and 6, respectively.
  • the genomic sequence was obtained by using primers mapping to the 5' and 3' ends of the cDNA to amplify genomic DNA.
  • the genomic amplicon was the same size as the cDNA, based on gel electrophoresis and sequencing. This indicated that there were no introns.
  • Southern blotting indicated that this was likely a single-copy gene, based on the analysis of four digests in two isolates of P. infestans.
  • infestans gene was single copy and lacked introns as demonstrated by a Southern blot analysis of the cdd 4 gene against DNA from isolates Ca65 and 216 digested with restriction endonucleases Hindlll, Pstl or Xhol.
  • the cdd 4 genomic sequence is the size of the full- length cDNA (about 1460 nucleotides) and is within 10 basepairs (bp) of the size of the complementary genomic region, based on PCR and "high- resolution" electrophoresis.
  • Cdd 4 is most similar to metazoan cdd 4 proteins as determined by phylogenetic comparison between cdc14-like proteins from P. infestans, human, Drosophila, C. elegans, S. pombe, S. cerevisiae, Candida albicans, and Neurospora crassa. Additional support for this conclusion came from searching for protein motifs, using the CDD database at NCBI. This showed that it had an apparently conserved protein phosphatase motif. The sequence contained a region homologous to the catalytic domain motif of dual specificity phosphatases, i.e. those acting at tyrosine and threonine residues (Pfam motif 00782).
  • the motif was between amino acids 214 and 318 of the P. infestans protein.
  • the amino acid sequence of the region matching the motif was NGTLVVRLNDKQYDEKKFLSAGIDHIDLIYPDGTNA PMPILMKFIEACEKTPGAVAVHCKAGLGRTGTCIGAYMMKHHLFSAHELIG WLRLCRPGSVIGPQQQFM (SEQ ID NO:8).
  • Within that motif was the actual active site: AVHCKAGLGRTG (SEQ ID NO:7).
  • the cdd 4 amino acid sequence obtained as disclosed herein was also aligned with known cdc14-like proteins.
  • the cdd 4 gene contained all of the conserved regions present in other members of the family.
  • the P. infestans protein is about 100 amino acids smaller than cdd 4 proteins from other species.
  • the missing region has been shown to not participate in cell cycle functions, however.
  • Example 2 Effects of Expressing Sense and Antisense Seguences in Transformants
  • sense SEQ ID NO:13
  • antisense cdd 4 SEQ ID NO:14
  • cdd 4 gene silencing or by cdd 4 overexpression, as described further herein below.
  • levels of cdd 4 transcript and/or protein can be assayed in transformants displaying a non-sporulation phenotype. Determination of cdd 4 expression levels can be accomplished using methods known to one of skill in the art including but not limited to in reverse transcriptase PCR, Northern blotting, and Western blotting.
  • infestans protoplasts obtained from germinated spores incubated with Novozyme 234 and cellulase
  • DNA DNA, calcium chloride, and polyethylene glycol
  • KC buffer 0.64 M KCI, 0.2 M CaCI
  • KC buffer containing 5 mg/ml Novozyme 234 and 2 mg/ml cellulase
  • protoplasts were recovered by filtration through 50 micron nylon mesh, washed once in KC buffer by centrifugation, once in 50% KC and 50% MT (1 M mannitol and 10 mM Tris pH 7.5), and then once in MT.
  • the protoplasts were resuspended in a small volume (about 0.8 ml) of MT plus 25 mM CaCI 2 , mixed with the DNA for transformation, incubated for 5 minutes, and then mixed with 0.8 ml of 50% polyethylene glycol. After 5 minutes, the protoplast-DNA mixture was diluted slowly into about 30 ml of rye media containing 1 M mannitol.
  • the regenerated protoplasts were pelleted by centrifugation and then plated on rye agar containing a selectable marker drug (for example, 7 //g/ml GENETICIN ® (G418) or 50 ⁇ g/ml hygromycin B. Transformed colonies, which appear after 7-12 days, were then transferred to fresh plates. Interestingly, the sporulation-deficient strains appeared to lack both mature asexual spores and progenitors such as terminal swellings. This suggested that Cdd 4 acts relatively early during spore development. Based on these experiments, modulation of cdd 4 activity is envisioned to be useful for controlling sporulation.
  • a selectable marker drug for example, 7 //g/ml GENETICIN ® (G418) or 50 ⁇ g/ml hygromycin B.
  • the term “modulate” means an increase, decrease, or other alteration of any or all chemical and biological activities or properties of a wild type fungal cdd 4 polypeptide, in one embodiment a cdd 4 polypeptide of any one of the even- numbered SEQ ID NOs:2-6.
  • a odd 4 modulator is an agonist of a fungal cdd 4 protein.
  • the term "agonist” means a substance that synergizes or potentiates the biological activity of a functional cdd 4 protein.
  • a cdd 4 modulator is an antagonist of a fungal cdd 4 protein.
  • the term “antagonist” or “inhibitor” refers to a substance that blocks or mitigates the biological activity of a fungal cdd 4 polypeptide.
  • the present invention further discloses a method for identifying a compound that modulates a fungal cdd 4 polypeptide.
  • the terms “candidate substance” and “candidate compound” are used interchangeably and refer to a substance that is believed to interact with another moiety, wherein a biological activity is modulated.
  • a representative candidate compound is believed to interact with a fungal cdd 4 polypeptide, or fragment thereof, and can be subsequently evaluated for such an interaction.
  • Exemplary candidate compounds that can be investigated using the methods of the present invention include, but are not restricted to, viral epitopes, peptides, enzymes, enzyme substrates, co- factors, lectins, sugars, oligonucleotides or nucleic acids, oligosaccharides, proteins, chemical compounds, small molecules, and antibodies.
  • a candidate compound to be tested can be a purified molecule, a homogenous sample, or a mixture of molecules or compounds.
  • Gene disruptions of Phytophthora infestans genes or nucleotide sequences 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 of homology to the target gene locus, the target gene locus being identified as described in Wendland et al., 2000, and 20 nucleotides of homology (invariant) to a GENETICIN ® resistance gene module also described in Wendland et al., 2000, 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:9) 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:10).
  • 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 about 45 nucle
  • the desired GENETICIN ® resistance module is obtained by linearizing a vector containing the GENETICIN ® resistance gene positioned behind an appropriate fungal (i.e. oomycete) promoter (for example, the Bremia lactucae HAM34 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 amplification, a final elongation period of 5 minutes at 72°C is carried out.
  • 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 Phytophthora transformants.
  • Additional verification primers designated G2 (5'- GTTTAGTCTGACCATCTC ATCTG-3'; SEQ ID NO:11) and G3 (5'- TCGCAGACCGATACCAGGATC-3'; SEQ ID NO:12) 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, the desired gene disruption can be verified by standard DNA hybridization experiments.
  • Determination of whether a gene is essential to the growth of Phytophthora can be achieved by the following analysis.
  • the transformation of DNA fragments described above utilizes multinucleate Phytophthora 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 contains a non-transformed nucleus as well.
  • 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 that are 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.
  • the coding region of the polypeptides of the invention are subcloned into previously described expression vectors, and transformed into E. coli using the manufacturer's conditions. Specific examples include plasmids such as pBLUESCRIPT ® (Stratagene, La Jolla, California, United States of
  • pFLAG International Biotechnologies, Inc., New Haven, Connecticut, United States of America
  • pTrcHis Invitrogen
  • E. coli is cultured, and expression of the polypeptides is confirmed.
  • eukaryotic expression systems such as cultured insect cells infected with specific viruses can be employed. Examples of vectors and insect cell lines are described previously.
  • the polypeptides of the present invention are isolated using standard techniques.
  • the nucleotide sequences of the invention are amplified by PCR.
  • the resulting DNA molecule is digested by DNasel treatment essentially as described (Stemmer et a , 1994) and the PCR primers are removed from the reaction mixture.
  • a PCR reaction is carried out without primers and is followed by a PCR reaction with the primers, both as described (Id.).
  • the resulting DNA molecules are cloned into pTRC99a (Amann & Abel, 1988) for use in bacteria, and transformed into a bacterial strain deficient in the biological activity of the polypeptides of the invention by electroporation using the Biorad Gene Pulser and the manufacturer's conditions (Biorad Laboratories, Hercules, California, United States of America).
  • the transformed bacteria are grown on medium that contains inhibitory concentrations of a potential inhibitor of the biological activity of the polypeptides of the invention. Those colonies that grow in the presence of the inhibitor are selected, and purified by repeated re-streaking. Plasmids from the purified colonies are purified and the DNA sequences of cDNA inserts are then determined.
  • the DNA fragments are cloned into expression vectors for transient or stable transformation into fungal cells, which are screened for differential survival and/or growth in the presence of an inhibitor of the biological activity of the polypeptides of the invention.
  • PCR-amplified DNA fragments comprising one of the Phytophthora nucleotide sequences of the invention and PCR- amplified DNA fragments derived from a different nucleotide sequence of the invention, are recombined in vitro and resulting variants with improved tolerance to the inhibitor are recovered as described above.
  • Recombinant polypeptides of the invention are obtained, for example, according to Example 2.
  • the polypeptides are immobilized on chips appropriate for ligand binding assays using techniques that are well known in the art.
  • the polypeptides immobilized on the chip are exposed to a chemical in solution according to methods well know in the art. While the sample chemical is in contact with the immobilized polypeptide, measurements capable of detecting polypeptide-ligand interactions are conducted. Methods used to make such measurements are SELDI, FCS, and SPR as described above. Chemicals found to bind the polypeptides are readily discovered in this fashion and are subjected to further characterization.
  • Nucleotide sequences of the invention are operatively linked to a strong inducible promoter, e.g. GAL1 promoter, GAL10 promoter, or other such promoters known in the art.
  • a strong inducible promoter e.g. GAL1 promoter, GAL10 promoter, or other such promoters known in the art.
  • overexpression of a nucleotide sequence of the invention confers upon the fungal cells a greater degree of resistance to an inhibitory chemical than is attainable in the wild type fungus. Wild type fungal cells are cultured in 96 well microtiter plates (e.g.
  • transgenic fungal cells overexpressing the essential fungal gene are challenged with the same set of chemical compounds at the same defined concentration.
  • Situations in which growth of the wild type fungus, but not the transgenic fungus, is inhibited by a given chemical are identified as prospective situations in which overexpression of the particular nucleotide sequence confers resistance to the inhibitory effect of the test compound.
  • induced overexpression of a nucleotide sequence of the invention has deleterious effects upon growth or viability of the fungal cells.
  • transgenic fungal cells in which the essential fungal gene is operatively linked to an inducible promoter are cultured in 96 well microtiter plates in the presence of a defined concentration of a different chemical test compound in each well. After a short incubation period, cells are shifted to full inducing conditions (for example by adding an inducing compound to each well). Normally this induced overexpression would lead to growth arrest of the culture, but, in wells containing inhibitors of the essential nucleotide sequence, growth would proceed and would be monitored via the increased turbidity within such wells.
  • Recombinant cdd 4 is isolated for these assays and to generate antigen for antibody production.
  • the protein is expressed in E. coli using the
  • IMPACTTM -TWIN system New England Biolabs, Beverley, Massachusetts, United States of America
  • IMPACTTM -TWIN system which involves linking cdd 4 via a labile intein linkage to a chitin-binding domain.
  • the released enzyme is tested for phosphatase activity against compounds previously shown to be non-specific substrates of Cdc14-like proteins from other species including p-nitrophenyl phosphate, Tyrosine-P-casein and Ser-P-casein (Jaspersen & Morgan 2000).
  • the open reading frame from the P. infestans cdc14-like gene was placed under the control of a galactose- inducible promoter and transformed into a yeast strain carrying the cdd 4-1 ts allele (Wan et al, 1992). Transformants were selected on uracil-lacking media at 27°C, and then grown on galactose-containing plates at the permissive and nonpermissive temperatures (30°C and 37°C, respectively). Growth was assessed to determine the effect of the P. infestans gene.
  • transformants were able to grow at the non- permissive temperature. Transformants did not grow in the absence of the galactose inducer. Yeast cells transformed with an "empty" vector (a vector lacking the P. infestans cdc14-like gene open reading frame) also did not grow at the non-permissive temperature. A parallel experiment was performed using the wild-type yeast cdd 4 allele as a control (a gift of M. Grunstein).
  • Example 1 Subcellular Localization of cdd 4 Protein Two approaches are used to determine intracellular location of the cdd 4 protein, immunofluorescence and a GFP fusion. These studies also address whether the protein resides in the sporangiophore or sporangia.
  • Immunolocalization Polyclonal antibodies are generated against the recombinant Cdd 4 using an external supplier and methods known to those skilled in the art. Rabbit antisera is tested for specificity in Western blots along with preimmune controls, and then used against whole mounts fixed in paraformaldehyde or paraffin-embedded 10 ⁇ m sections (Scanziani, 1998). Sectioning may be required due to the sporangial wall. After blocking and incubation with primary antibody, FITC-conjugated goat anti-rabbit IgG is added and visualized by confocal microscopy using 3-D image analysis. Dr.
  • Prue Talbot (Department of Cell Biology and Neuroscience, University of California of Riverside, California, United States of America) provides technical assistance in this regard.
  • the location of Cdd 4 is compared to that of nuclei, which is visualized using the DNA-binding dye DAPI.
  • GFP fusion The location of Cdd 4 is also determined by using a Cdc14::GUS fusion vector transformed into P. infestans. Ideally, the construct serves several purposes including localizing the protein, studying the stability of Cdd 4, and identifying a functional Cdd 4 promoter. In previous work, fusing GFP to the C-terminus of the S. pombe Cdd 4 homologue was shown to not impair its function in the cell cycle (Cueille et al., 2001). Also, GFP has proved to be a successful marker in several Phytophthora species (van West et a , 1999b).
  • the vector is constructed to include 2 kb of DNA 5' of the open reading frame, plus the ORF, which is isolated from a BAG library. This region is inserted upstream of GFP in the pGPF-NPT plasmid, and transformed into isolate 1306 of P. infestans. Next, the transformants are checked to determine if they shown normal growth and development, and induction of GFP in a pattern consistent with the expression of the normal cdd 4 gene. The transformants are then examined by confocal microscopy to determine where the cdc14::GFP fusion protein localizes.
  • Example 12 Pattern and Regulation of Cdd 4 Expression To learn how Cdd 4 is regulated, its spatial and temporal expression patterns during sporulation are analyzed using GFP; protein levels during sporulation and germination are measured; and promoter regions involved in regulation are identified. Experiments to identify transcription factors binding to the promoter are also performed.
  • RNA blot analyses indicated that cdd 4 RNA is first detected during sporulation, and disappears rapidly in encysted zoospores germinated in water or asexual sporangia producing direct germ tubes in rye media.
  • the RNA analyses do not address the stability or subcellular location of the protein.
  • the precise point at which RNA levels drop is difficult to measure since germination is not totally synchronous ( ⁇ 2 and ⁇ 12 hours for cyst and asexual spore germination, respectively). Ideally, these issues are studied by microscopic examination of the GFP-expressing transformants.
  • Expression is first measured during the development of sporangiophores. Observations are made determining whether expression occurs in sporangiophores, in sporangial initials, or in mature asexual spores. Next, GFP levels are measured in spores induced to release zoospores in water at 10 9 C, zoospores encysted by vortexing, zoospore cysts germinating in water, and asexual spores germinating in clarified rye media.
  • Example 13 Cyclin B-dependent Kinase Activity During Growth and Development Regulation of the cyclin B-cyclin dependent kinase (CDK) complex, which is the ultimate target of Cdd 4 activity in other systems, is studied during the life cycle of P. infestans. Experiments are focused on obtaining a broad understanding of how Cdd 4, cyclin B, and the latter's CDK (i.e. p34, p34 cdc2 , etc.) interact to regulate the transition from vegetative growth to spores. It is also important to see if regulation of this complex in oomycetes resembles that in other eukaryotes, where activity drops abruptly after mitosis due to cyclin B proteolysis and CDK inhibition.
  • CDK Cyclin B-dependent Kinase Activity During Growth and Development Regulation of the cyclin B-cyclin dependent kinase (CDK) complex, which is the ultimate target of Cdd 4 activity in other systems, is studied during the life cycle of P.
  • An anti-cyclin B antibody is used to immunoprecipitate cyclin B/CDK complexes, which are assayed in vitro for kinase activity against histone H1.
  • Initial experiments use a commercially available polyclonal antibody made against the entire human B1 cyclin protein (Rockland Immunochemicals). Once an effective antibody is obtained, the cyclin B-CDK complex is immunoprecipitated from vegetative hyphae, spores, zoospores, germinated zoospore cysts, and directly germinated sporangia.
  • Kinase assays are then performed using standard methodologies (Kottom et al., 2000).
  • a S/M DNA replication checkpoint prevents nuclear and cytoplasmic events of cell division including centrosomal axis alignment and inhibits activation of cyclin-dependent kinase-like proteins in fucoid zygotes, Development (Cambridge). 127:1651 -1660. Cueille N, Salimova E, Esteban V, Blanco M, Moreno S, Bueno A, & Simanis V (2001 ) Flp1 , a fission yeast orthologue of the S.
  • Li L, Ernsting BR, Wishart MJ, Lohse DL, & Dixon JE (1997) A family of putative tumor suppressors is structurally and functionally conserved in humans and yeast, J Biol Chem 272:29403-29406. Li L, Ljungman M, & Dixon JE (2000) The human Cdd 4 phosphatases interact with and dephosphorylate the tumor suppressor protein p53, J
  • McCollum D & Gould KL (2001) Timing is everything: Regulation of mitotic exit and cytokinesis by the MEN and SIN, Trends Cell Biol 1 1 :89-95.
  • Aspergillus fumigatus and Aspergillus nidulans Polarity, germ tube emergence and septation, Microbiology (Reading) 146:3279-3284.

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Abstract

Nucleotide sequences are isolated from Phytophthora infestans that code for proteins essential for fungal growth and development. In particular, the cdc14 phosphatase is described. The essentially of the proteins is exploited by recombinantly expressing the proteins and using them in screening assays to identify compounds that interact with or inhibit the proteins and are therefore fungicides.

Description

Description
NUCLEIC ACID MOLECULES ENCODING PROTEINS ESSENTIAL FOR
FUNGAL GROWTH AND DEVELOPMENT AND USES THEREOF
Cross Reference to Related Applications
This application is based on and claims priority to U.S. Provisional
Patent Application Serial No. 60/347,609, filed January 10, 2002 and entitled
"NUCLEIC ACID MOLECULES ENCODING PROTEINS ESSENTIAL FOR
FUNGAL GROWTH AND DEVELOPMENT AND USES THEREOF", herein incorporated by referenced in its entirety.
Technical Field of the Invention The present invention pertains to nucleic acid molecules isolated from Phytophthora infestans comprising nucleotide sequences that encode proteins essential for plant growth and development. The invention particularly relates to methods of using these proteins as fungicide targets, based on this essentiality.
Table of Abbreviations AcMNPV Autographies californica nuclear polyhedrosis virus
ADP adenosine diphosphate
APC anaphase-promoting complex
ATCC American Type Culture Collection
ATP adenosine triphosphate
BAC bacterial artificial chromosome
BLAST Basic Local Alignment Search Tool bp base pair(s)
C. elegans Caenorhabditis elegans
CDs compact discs
CDK cyclin-dependent kinase cDNA complementary DNA
DAPI 4,6-diamidino-2-phenylindole dCTP deoxycytosine triphosphate
DNA deoxyribonucleic acid- DNasel deoxyribonuclease I
DTT dithiothreitol
E. coli Escherichia coli
EDTA ethylenediamine tetra-acetic acid
ELISA enzyme-linked immunosorbent assay
EST expressed sequence tag FAD flavin adenine dinucleotide
FCS Fluorescence Correlation Spectroscopy
FITC fluorescein isothiocyanate
FMN flavin mononucleotide
GDP guanosine diphosphate
GFP green fluorescent protein
GTP guanosine triphosphate
GTPase GTP binding protein/hydrolase
GUS β-glucuronidase
HSPs high scoring sequence pairs
MEN mitotic exit network
MMLV Moloney murine leukemia virus mRNA messenger RNA
MS mass spectroscopy
Na+ sodium ion
NADP nicotinamide adenine dinucleotide phosphate
NCBI National Center for Biotechnology
Information
NCGR National Center for Genome Resources oligo-dT oligo-deoxythymidine
ORF open reading frame
32p phosphorus-32
32P-γ-ATP - phosphorus-32-gamma-ATP
PCR polymerase chain reaction
P. infestans - Phytophthora infestans polyA polyadenosine RENT regulator of nucleolar silencing and telophase
RNA ribonucleic acid rRNA ribosomal RNA
S. cerevisiae ■ Saccharomyces cerevisiae
S. pombe Schizosaccharomyces pombe
SBI Syngenta Biotechnology Inc.
SDS sodium dodecyl sulfate
SELDI Surface-Enhanced Laser Desorption/ lonization
SIN septation initiation network snRNA small nuclear RNA
SPR surface plasmon resonance
SSC standard saline citrate
SSPE saline-sodium phosphate-EDTA
Tm thermal melting point TOF time-of-flight mass spectrometer tRNA transfer RNA ts temperature sensitive Background of the Invention The use of fungicides to control undesirable fungal growth and pathogens in crop fields has become almost a universal practice. Despite this extensive use, fungal control remains a significant and costly problem for farmers.
Effective use of fungicides requires sound management. For instance, the time and method of application and stage of fungal development are critical to achieving good fungal control with fungicides. Because various fungal species are resistant to fungicides, the production of effective new fungicides becomes increasingly important. New fungicides can now be discovered using high-throughput screens that implement recombinant DNA technology. Metabolic enzymes found to be essential to fungal growth and development can be recombinantly produced through standard molecular biological techniques and utilized as fungicide targets in screens for novel inhibitors of the enzyme activity. More generally, any essential plant protein can be used to screen for inhibitors of its activity. The novel inhibitors discovered through such screens can then be used as fungicides to control undesirable fungal growth.
P. infestans is arguably the most important pathogen of the world's largest non-cereal crop, potato, and is also a significant tomato pathogen (Fry & Goodwin, 1997). The late blight diseases have always been important and have been especially difficult over the past decade (Fry & Goodwin, 1997). The worldwide cost of the potato disease alone exceeds $5 billion per year, including more than $1 billion spent on fungicides (Anonymous, 2000).
This is enough to purchase potatoes to fulfill the caloric needs of the entire world for 2.7 days (based on 2200 kilocalories/day and current U.S. prices; see Passmore 1974; Watt & Merrill 1975). The success of P. infestans as a pathogen is largely due to its ability to produce large amounts of asexual spores, which travel between plants to initiate new infections.
P. infestans is not just a pathogen, but also an experimentally accessible oomycete. Oomycetes represent a large collection of important but poorly characterized species that include both saprophytes and significant parasites of plants (Pythium, Phytophthora, white rusts, downy mildews), animals, and insects (Alexopoulos et al., 1996). Oomycetes lack taxonomic affinity with the so-called true fungi (i.e. ascomycetes and basidiomycetes). Instead, oomycetes are properly classified with diatoms and brown algae (Baldauf et al., 2000; Gunderson et al., 1987). Consequently, processes appearing similar between oomycetes and true fungi, such as sporulation, are likely quite different genetically and biochemically. Unfortunately, oomycetes have remained understudied compared to true fungi. P. infestans presents a good opportunity for remedying this deficiency since it is amenable to genetic and biochemical manipulation and easily grown. P. infestans has both sexual and asexual cycles. Growth usually starts from asexual spores that germinate to yield tubular hyphae. These ramify through plants or artificial media by polarized, linear expansion. Continued growth and branching results in a mycelial mass that eventually produces new asexual spores, which are multinucleate and form upon a branched specialized hypha called the sporangiophore. In cool and moist environments, 8-12 biflagellated and mononucleate zoospores are released which later encyst and germinate (Note: the term "asexual spore," as used herein, refers to a zoosporangium, since it releases zoospores, but the former terminology is used to avoid confusion). Asexual spores can also germinate directly.
Nuclear behavior during the life cycle has been characterized (Whittaker et al., 1991 ; Maltese et al., 1995; Laviola 1975; Sivak 1973). Within vegetative hyphae (which lack cross-walls or septa), nuclear division is asynchronous. Generally one nucleus moves into the sporangiophore, in which rapid synchronized mitoses occur coincident with the formation of a basal septum. Sporangiophores extend to their full length over about 3 hours, generally forming several branches that develop terminal swellings. Nuclei and cytoplasm then move into the swellings, which become the asexual spores. As the spore matures, a septum forms at its base and a "cap" forms on each nucleus that does not appear to be a nucleolus (Sivak 1973; Marks 1965). Some nuclei may degenerate, possibly to maintain their proper ratio with cytoplasm (Maltese et al., 1995). The nuclei in the asexual spore, as well as zoospores, are at G1 (Whittaker et al., 1992). A different definition of the cell must be invoked for filamentous fungi, in which growth involves the expansion of hyphal tubes, as compared to syncytial species such as Physarum. In true filamentous fungi such as Aspergillus, growth generally involves the extension of hyphal tubes in which cross-walls (septa) form. Septation generates discrete cell-like compartments and is linked to the nuclear division cycle although the relative timing of septation and mitosis varies in different life stages (Wolkow et al., 2000; Momany & Taylor 2000). Oomycete growth differs in several important aspects from that of true fungi. For example, oomycete hyphae are aseptate and thus coenocytic; discrete cellular compartments only form when spores are made. Also, growth and mitosis are not well coordinated even within a single vegetative hypha, where nuclei at different stages of division (G1 , G2, M) are observed (Whittaker et al., 1991). Another difference is that the asexual spores of oomycetes remain hydrated and metabolically active, unlike those of true fungi that generally become dormant and desiccated. A special mechanism may exist within Phytophthora to arrest growth and nuclear division within the spore.
Cytoplasmic growth, nuclear division, and cytokinesis are tightly regulated by the cell cycle. This has been intensely studied in many organisms, particularly in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in mammals, and to a lesser extent in plants (McCollum & Gould 2001 ; Smits & Medema 2001 ; Stals et al., 2000; Heberle-Bors 2001.
Progression through the cycle appears to be universally regulated by oscillating activities of cyclin-dependent kinases (CDK). Entry into mitosis (G2/M progression) has been subjected to the most biochemical and genetic analyses. For example, cyclin B (a G2/M cyclin) and its interacting kinase have been studied in several species, with the latter named cdc28 in S. cerevisiae, cdc2 in S. pombe, NimX in Aspergillus, and p34 in metazoans (McCollum & Gould 2001 ; Osmani & Ye 1996; Bregman et al., 2000).
Exit from mitosis is less understood, but clearly involves the destruction of mitotic cyclins by ubiquitin-mediated proteolysis and the accumulation of CDK inhibitors (McCollum & Gould 2001). The proteins that control the exit from mitosis in S. cerevisiae and S. pombe are termed the mitotic exit network (MEN) and the septation initiation network (SIN), respectively (McCollum & Gould 2001). In S. cerevisiae, the key protein involved in mitotic exit is Cdc14, a dual specificity (i.e. serine/threonine and tyrosine) phosphatase. Information about the involvement of Cdc14 in mitotic exit comes mostly from work with the S. cerevisiae protein and its human homologue, hCdc14a (Taylor et al., 1997). These studies showed that Cdc14 triggers cyclin B destruction by dephosphorylating Cdh1 , a component of the anaphase-promoting complex (APC) that accumulates when cyclin B- Cdkcdc28 activity is high. In addition, Cdc14 activates Sid , a cyclin- dependent kinase inhibitor, and Swi5, a transcriptional activator of the sid gene. The combined effect of these activities is to destroy cyclin B and inhibit its associated kinase, which allows a cell to exit mitosis and begin a new cell cycle. The cdc14 gene is essential in S. cerevisiae (Grandin et al., 1998; Sakumoto et al., 1999).
Transcriptional regulation plays a minor role in most systems, as the abundance of Cdc14 RNA varies only slightly during the cell cycle (Wan et al., 1992). Instead, Cdc14 is regulated by its localization. During interphase in budding yeast, Cdc14 is sequestered in the nucleolus where it joins Net1 and Cfi1 in a complex called "regulator of nucleolar silencing and telophase" (RENT) (Cockell & Gasser 1999). Upon cell cycle entry, RENT releases Cdc14 and allows it to reach its nuclear targets. Release occurs through a protein kinase cascade involving four kinases (Cdc5, Cdc15, Dbf2, Dbf20), a GTPase (Teιm1 ), a GDP-GTP exchange protein (Lte1), a Dbf2 binding factor (Mob1 ), plus Cdc14 (McCollum & Gould 2001 ).
There are several issues remaining regarding the role of Cdc14. Perhaps the most important is whether or not related proteins in other species function in the same way as Cdc14 has been shown to in S. cerevisiae. In humans, for example, two Cdc14-like proteins exist, only one of which behaves like the S. cerevisiae protein (Jaspersen et al., 2000; Bembenek & Yu, 2001). hCdc14a affects cyclin B and its associated kinase, and also shows the same nucleolus/nucleus distribution pattern as its S. cerevisiae counterpart; hCdd 4a even complements a S. cerevisiae cdd 4- 1ts mutant. However, hCdc14b, on the other hand, is present in the cytoplasm and does not complement cdd 4-1 ts. Interestingly, both human proteins interact with the p53 tumor suppressor (Kroll et al., 1996). In S. pombe, the Cdd 4 orthologue (Flp1) might potentiate mitotic exit and septation, but is not required for inactivating the B-type cyclin (Cueille et al., 2001). The subcellular distribution of Flp1 during the cell cycle does resemble that of Cdd 4 in S. cerevisiae, and Flp1 complements the cdd 4- 1ts mutant, however (jd.). There are also several studies that suggest that even in budding yeast Cdd 4 has functions besides controlling mitotic exit. These include promoting cytokinesis and DNA replication (Li et al., 1997; Li et al., 2000). A final intriguing issue is that cdc14-like genes are undetectable in any plant sequence in GenBank, which raises the question of whether cdd 4 originally evolved as a regulator of mitotic exit in eukaryotes.
In view of the above, there remain persistent and ongoing problems with unwanted or detrimental fungal growth (e.g. plant pathogens). Furthermore, as the population continues to grow there will be increasing food shortages. Therefore, there exists a long felt, yet unfulfilled, need to find new, effective, and economic fungicides.
Summary of the Invention In view of these needs, it is an object of the invention to provide nucleic acid molecules from Phytophthora infestans comprising nucleotide sequences that encode proteins essential for fungal growth and development. It is another object to provide the essential proteins encoded by these essential nucleotide sequences for assay development to identify inhibitory compounds with fungicide activity. It is still another object of the present invention to provide an effective and beneficial method for identifying new or improved fungicides using the essential proteins of the invention. These and other objects are achieved in whole or in part by the present invention.
In furtherance of these and other objects, the present invention provides nucleic acid molecules isolated from P. infestans comprising nucleotide sequences that encode proteins essential for plant viability. In particular, by disrupting gene function, the activity of each protein of the present invention is essential for the growth of Phytophthora infestans, an aspect that is disclosed herein for the first time.
This knowledge is exploited to provide novel fungicide modes of action. The critical role in fungal growth of the proteins encoded by each of the nucleotide sequences of the invention implies that chemicals that inhibit the function of any one of these proteins in fungi are likely to have detrimental effects on fungal growth and are thus potentially good fungicide candidates. Thus, the proteins encoded by the essential nucleotide sequences provide the bases for assays designed to easily and rapidly identify novel fungicides.
The present invention therefore provides methods of using a purified protein encoded by any one of the nucleotide sequences described below to identify inhibitors thereof, which can then be used as fungicides to suppress the growth of undesirable fungi, for example, in fields where crops are grown, particularly agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
Disclosed herein are nucleic acid molecules isolated from Phytophthora infestans, in particular, nucleic acid molecules encoding cdc14. In one embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence, the complement of which hybridizes under stringent conditions to a sequence selected from the group consisting of the odd-numbered SEQ ID NOs:1 -5. The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a protein comprising an amino acid sequence having in one embodiment at least 60%, in another embodiment at least 70%, in another embodiment at least 80%, in another embodiment at least 90%, in another embodiment at least 95%, and in yet another embodiment at least 99-100% sequence identity to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6. The present invention also provides a chimeric construct comprising a promoter operatively linked to a nucleic acid molecule according to the present invention. In one embodiment, the promoter is functional in a eukaryote. In another embodiment, the promoter is heterologous to the nucleic acid molecule. The present invention further provides a recombinant vector comprising a chimeric construct according to the present invention, wherein said vector is capable of being stably transformed into a host cell. The present invention still further provides a host cell comprising a nucleic acid molecule according to the present invention. In one embodiment, the nucleic acid molecule is expressible in the cell. The host cell can be selected from the group consisting of a plant cell, a yeast cell, an insect cell, and a prokaryotic cell. The present invention additionally provides a plant or seed comprising a plant cell according to the present invention.
The present invention also provides proteins essential for fungal growth in Phytophthora infestans. In one embodiment, the present invention provides an isolated protein comprising an amino acid sequence having in one embodiment at least 60%, in another embodiment at least 70%, in another embodiment at least 80%, in another embodiment at least 90%, in another embodiment at least 95%, and in yet another embodiment at least 99-100% sequence identity to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6. In accordance with another embodiment, the present invention also relates to the recombinant production of proteins of the invention and methods of using the proteins of the invention in assays for identifying compounds that interact with the protein.
According to another aspect, the present invention provides a method of identifying a fungicidal compound, comprising: (a) combining a polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the even- numbered SEQ ID NOs:2-6 with a compound to be tested for the ability to bind to said polypeptide, under conditions conducive to binding; (b) selecting a compound identified in (a) that binds to said polypeptide; (c) applying a compound selected in (b) to a plant to test for fungicidal activity; and (d) selecting a compound identified in (c) that has fungicidal activity. In one embodiment, the polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6. In another embodiment, the polypeptide comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of the even- numbered SEQ ID NOs:2-6. In still another embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6. The present invention also provides a method for killing or inhibiting the growth or viability of a plant, comprising applying to the plant an herbicidal compound identified according to this method.
According to yet another aspect, the present invention provides a method of identifying a fungicidal compound, comprising: (a) combining a polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the even- numbered SEQ ID NOs:2-6 with a compound to be tested for the ability to inhibit the activity of said polypeptide, under conditions conducive to inhibition; (b) selecting a compound identified in (a) that inhibits the activity of said polypeptide; (c) applying a compound selected in (b) to a plant to test for fungicidal activity; and (d) selecting a compound identified in (c) that has fungicidal activity. In one embodiment, the polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6. In another embodiment, the polypeptide comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6. In still another embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6. The present invention also provides a method for killing or inhibiting the growth or viability of a fungus, comprising applying to the plant a fungicidal compound identified according to this method.
The present invention still further provides a method for killing or inhibiting the growth or viability of a fungus, comprising inhibiting expression in said fungus of a protein having in one embodiment at least 60%, in another embodiment at least 70%, in another embodiment at least 80%, in another embodiment at least 90%, in another embodiment at least 95%, and in yet another embodiment at least 99-100% sequence identity to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
Other objects and advantages of the present invention will become apparent to those skilled in the art and from a study of the following description of the invention and non-limiting examples. The entire contents of all publications mentioned herein are hereby incorporated by reference. Brief Description of the Sequences in the Sequence Listing
Odd-numbered SEQ ID NOs:1 -5 are nucleotide sequences isolated from Phytophthora infestans that are more fully described below.
Even-numbered SEQ ID NOs:2-6 are protein sequences encoded by the immediately preceding nucleotide sequence, e.g., SEQ ID NO:2 is the protein encoded by the nucleotide sequence of SEQ ID NO:1 , SEQ ID NO:4 is the protein encoded by the nucleotide sequence of SEQ ID NO:3, etc.
SEQ ID NO. Description 1 full-length nucleotide consensus sequence of P. infestans cdd 4 cDNA 2 P. infestans cd 4 protein 3 P. infestans cdd 4 allele 1 cDNA
~] P. /nfesfanscdc14 protein encoded by aϊiele 1
P. infestans cdd 4 allele 2 cDNA
Figure imgf000013_0001
P. infestans cdc14 protein encoded by allele 2 _____ cdc14 active site
8 cdc14 conserved protein phosphatase motif _____ , primers
13 ϊ cdd 4 sense construct 14 cdd 4 antisense construct
Three nucleotide and three amino acid differences exist between the allele sequences.
Definitions For clarity, certain terms used in the specification are defined and presented as follows:
As used herein, the phrases "associated with" and "operatively linked" refer to two or more nucleic acid sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked or are situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
As used herein, the phrase "chimeric construct" refers to a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or is associated with, a nucleic acid sequence that codes for an mRNA or which is expressed as a protein, such that the regulatory nucleic acid sequence is able to regulate the transcription or expression of the associated nucleic acid sequence. The regulatory nucleic acid sequence of the chimeric construct is not normally operatively linked to the associated nucleic acid sequence as found in nature.
As used herein, the term "co-factor" refers to a natural reactant, such as an organic molecule or a metal ion, that is required in an enzyme- catalyzed reaction. Co-factors include, but are not limited to NADP, riboflavin (including FAD and FMN), folate, molybdopterin, thiamin, biotin, lipoic acid, pantothenic acid, coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone, and menaquinone. Optionally, a co-factor can be regenerated and reused.
As used herein, the phrase "coding sequence" refers to a nucleic acid sequence that is transcribed into RNA including, but not limited to mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. The RNA can then be translated in vitro or in vivo to produce a protein.
As used herein, the term "complementary" refers to two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences.
As used herein, the phrase "enzyme activity" refers to the ability of an enzyme to catalyze the conversion of a substrate into a product. A substrate for an enzyme comprises the natural substrate of the enzyme but also comprises analogues of the natural substrate that can also be converted by the enzyme into a product or into an analogue of a product. The activity of the enzyme can be measured, for example, by determining the amount of product in the reaction after a certain period of time, or alternatively, by determining the amount of substrate remaining in the reaction mixture after a certain period of time. The activity of the enzyme can also be 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 can also be measured by determining the amount of a donor of free energy or energy-rich molecule (including, but not limited to ATP, phosphoenolpyruvate, acetyl phosphate, and 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 (including, but not limited to ADP, pyruvate, acetate, and creatine) in the reaction mixture after a certain period of time.
As used herein, the term "essential" as in "essential fungal nucleotide sequence" refers to a nucleotide sequence encoding a protein that is essential to the growth and/or survival of the fungus. Proteins that are essential to the growth and/or survival of the fungus include, but are not limited to biosynthetic enzymes, receptors, signal transduction proteins, structural gene products, and transport proteins. As used herein, the phrase "expression cassette" refers to a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell. An expression cassette typically comprises a promoter operatively linked to a nucleotide sequence of interest, which is operatively linked to termination signals. An expression cassette typically further comprises sequences required for proper translation of the nucleotide sequence. The nucleotide sequence of interest usually encodes a protein of interest, but can also code for a functional RNA of interest, for example an antisense RNA or a non-translated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette can also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, meaning that the particular DNA sequence of the expression cassette does not occur naturally in the host cell and thus is introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, organ, or stage of development. The term "fungus" as used herein refers to both true fungi and organisms traditionally classified in some schemes as fungi, for example oomycetes. Similarly, "fungal" refers to substances derived from species defined herein as fungi, including, but not limited to oomycetes. As such, the term "fungicide" refers to compounds capable of inhibiting or preventing the growth of a fungus as defined herein.
The term "gene" as used herein refers broadly to any segment of DNA associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. Genes also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and can include sequences designed to have desired parameters.
The terms "heterologous" and "exogenous" when used herein to refer to a nucleic acid sequence (including, but not limited to a DNA sequence) or to a gene refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments can be expressed to yield exogenous polypeptides.
The term "homologous", as used herein to describe a nucleic acid sequence (for example, a DNA sequence), refers to a nucleic acid sequence naturally associated with a host cell into which it is introduced.
As used herein, the phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture including, but not limited to total cellular DNA and RNA. As used herein, the phrase "bind(s) substantially" refers to complementary hybridization between a nucleic acid probe and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization conditions to achieve the desired detection of the target nucleic acid sequence.
As used herein, the term "inhibitor" refers to a chemical substance that inactivates the enzymatic activity of a protein such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein. The term "fungicide" (or "fungicidal compound") is used herein to define an inhibitor applied to a fungus or plant at any stage of development, whereby the fungicide inhibits the growth of the fungus or kills the fungus.
As used herein, the term "interaction" refers to a quality or state of mutual action such that the effectiveness or toxicity of one protein or compound on another protein is inhibitory (as for antagonists) or enhancing (as for agonists). As used herein, a nucleic acid sequence is "isocoding with" a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.
As used herein, the term "isogenic" refers to plants that are genetically identical, except that they can differ by the presence or absence of a heterologous DNA sequence.
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 can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell.
As used herein, the phrase "mature protein" refers to a protein from which the transit peptide, signal peptide, and/or propeptide portions have been removed.
As used herein the phrase "minimal promoter" refers to the smallest fragment of a promoter, such as a TATA element, that can support any transcription. A minimal promoter typically has greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, a minimal promoter functions to permit transcription.
As used herein, the phrase "modified enzyme activity" refers to an enzyme activity that is different from an enzyme activity that 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.
As used herein, the term "native" refers to a macromolecule that is normally present in an untransformed plant cell. For example, a native gene is a gene that is normally present in the genome of an untransformed plant.
As used herein, the term "naturally occurring" refers to an object that can be found in nature as distinct from being artificially produced by man. For example, a protein or nucleotide sequence present in an organism (including, but not limited to a virus), which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.
As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991 ; Ohtsuka et al., 1985; Rossolini et al., 1994). The terms "nucleic acid" or "nucleic acid sequence" may also be used interchangeably with gene, cDNA, and mRNA encoded by a gene.
As used herein, the phrases "open reading frame" and "ORF" are given their common meaning and refer to a contiguous series of deoxyribonucleotides or ribonucleotides that encode a polypeptide or a fragment of a polypeptide. In an organism that splices precursor RNAs to form mRNAs, the ORF will be discontinuous in the genome. Splicing produces a continuous ORF that can be translated to produce a polypeptide. The phrases "percent identity" and "percent identical," in the context of two nucleic acid or protein sequences, refer to two or more sequences or subsequences that have in one embodiment at least 60%, in another embodiment at least 70%, in another embodiment at least 80%, in another embodiment at least 90%, in another embodiment at least 95%, and in yet another embodiment at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The percent identity exists in one embodiment over a region of the sequences that is at least about 50 residues in length, in another embodiment over a region of at least about 100 residues, and in still another embodiment the percent identity exists over at least about 150 residues. In yet another embodiment, the percent identity exists over the entire length of the coding regions.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman (1981), by the homology alignment algorithm of Needleman & Wunsch (1970), by the search for similarity method of Pearson & Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection. See generally, Ausubel et al., 1994. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See Henikoff & Henikoff 1989.
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See e.g., Karlin & Altschul 1993. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in one embodiment less than about 0.1 , in another embodiment less than about 0.01 , and in still another embodiment less than about 0.001.
As used herein, the term "pre-protein" refers to a protein that is normally targeted to a cellular organelle, such as a chloroplast, and still comprises its native transit peptide.
As used herein, the term "purified", when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be in a homogeneous state although it also can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is in one embodiment at least about 50% pure, in another embodiment at least about 85% pure, and in still another embodiment at least about 99% pure.
Two nucleic acids are "recombined" when sequences from each of the two nucleic acids are combined in a progeny nucleic acid. Two sequences are "directly" recombined when both of the nucleic acids are substrates for recombination. Two sequences are "indirectly recombined" when the sequences are recombined using an intermediate such as a cross-over oligonucleotide. For indirect recombination, no more than one of the sequences is an actual substrate for recombination, and in some cases, neither sequence is a substrate for recombination.
As used herein, the phrase "regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operatively linked to the nucleotide sequence of interest and termination^ signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.
As used herein, the phrase "significant increase" refers to an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique. A significant increase is in one embodiment an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, in another embodiment an increase by about 5-fold or greater, and in still another embodiment an increase by about 10-fold or greater.
As used herein, the phrase "significantly less" means that the amount of a product of an enzymatic reaction is reduced by more than the margin of error inherent in the measurement technique, in one embodiment a decrease by about 2-fold or greater of the activity of the wild-type enzyme in the absence of the inhibitor, in another embodiment an decrease by about 5-fold or greater, and in still another embodiment an decrease by about 10-fold or greater.
As used herein, the phrase "specific binding" and "immunological cross-reactivity" refer to an indication that two nucleic acid sequences or proteins are substantially identical in that the protein encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions. The phrase "specifically (or selectively) binds to an antibody," or "specifically (or selectively) immu no reactive with," when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to a protein with an amino acid sequence encoded by any of the nucleic acid sequences of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins except for polymorphic variants. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase enzyme-linked immunosorbent assays (ELISA), Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
"Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions" a probe will hybridize to its target subsequence, but not to other sequences.
The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes. See, Sambrook et al. 2001 , for a description of SSC buffer. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An exemplary medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1x SSC at 45°C for 15 minutes. An exemplary low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na+ ion, typically about 0.01 -1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3, and a temperature typically of at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still considered substantially identical if the proteins that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
The following are examples of sets of hybridization/wash conditions that can be used to identify and/or clone nucleotide sequences that are homologues of the reference nucleotide sequences of the present invention. In one embodiment, a nucleotide sequence can hybridize 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. In another embodiment, a nucleotide sequence can hybridize to the reference nucleotide sequence in 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C. In another embodiment, a nucleotide sequence can hybridize to the reference nucleotide sequence in 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1 % SDS at 50°C. In another embodiment, a nucleotide sequence can hybridize to the reference nucleotide sequence in 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1 % SDS at 50°C. In still another embodiment, a nucleotide sequence can hybridize to the reference nucleotide sequence in 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1 % SDS at 65°C.
As used herein, the term "subsequence" refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., protein) respectively.
As used herein the term "substrate" refers to a molecule that an enzyme recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function, or to 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.
As used herein, the term "transformation" refers to a process for introducing heterologous DNA into a cell, a tissue, or an entire organism including, but not limited to viruses, bacteria, plants, and animals. Transformed plant cells, plant tissue, or plants are understood to encompass not only the cell, tissue, or plant that is the end product of a transformation process, but also transgenic progeny thereof.
As used herein, the terms "transformed", "transgenic", and "recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host, but can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non- transformed," "non-transgenic", or "non-recombinant" host refers to a wild- type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule. As used herein, the term "viability" refers to a fitness parameter of a plant. Plants are assayed for their homozygous performance of plant development, indicating which proteins are essential for plant growth. Detailed Description of the Invention L Identification of Essential Fungal Nucleotide Sequences and Encoded Proteins
As shown in the examples below, the essentiality of the nucleotide sequences described herein for normal fungal growth and development have been demonstrated for the first time in Phytophthora infestans using three approaches: (1 ) comparison of ESTs in libraries constructed from cDNA from spores and non-sporulating hyphae; (2) searching an EST database for sequences with matches to protein kinases and protein phosphatases, and (3) hybridizing an array of EST clones from the spore library (J) with labeled cDNA from spores and non-sporulating hyphae. These methods are described in detail in the Examples below.
The essentiality of the clones was determined by expressing sense and antisense copies of the gene using assays described below. Having established the essentiality of the function of the encoded protein(s) in Phytophthora infestans and having identified the nucleotide sequences encoding these essential proteins, the inventors thereby provide an important and sought after tool for new fungicide development.
The cDNA and genes of the present invention were cloned and characterized as described in the Examples below.
Additional genomic and cDNA sequences for each gene are identified by standard molecular biology procedures. IL Recombinant Production Of Essential Proteins And Uses Thereof
For recombinant production of a protein of the invention in a host organism, a nucleotide sequence encoding the protein is inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced. The polypeptides of the invention are recombinantly produced upon expression of respective heterologous DNA sequences introduced in the hosts. For example, nucleotide sequences selected from the group consisting of the odd-numbered SEQ ID NOs:1 -5 or nucleotide sequences substantially similar to those selected from the group consisting of the odd-numbered SEQ ID NOs:1 -5, or nucleotide sequences encoding polypeptides selected from the amino acid sequences of even-numbered SEQ ID NOs:2-6, are introduced into chosen hosts for the recombinant production of the polypeptides of the invention. In one embodiment, the nucleotide sequences of the invention are derived from a eukaryote, including, but not limited to a mammal, a fly, and a yeast. In another embodiment, the nucleotide sequences of the invention are derived from a fungus. The nucleic acid molecules can also be produced using available synthetic methods known in the art.
The choice of the specific regulatory sequences such as promoters, signal sequences, 5' and 3' untranslated sequences, and enhancers appropriate for the chosen host is within the level of the skill of the routineer in the art. The resultant molecule, containing the individual elements linked in the proper reading frame, is 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., Lucknow and Summers 1988. Additional suitable expression vectors include, but are not limited to baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV). A representative baculovirus/insect system is PVL1392(3) used to transfect Spodoptera frugiperda SF9 cells (available from the American Type Culture Collection (ATCC), Manassas, Virginia, United States of America) in the presence of linear Autographica californica baculovirus DNA (Pharmingen, San Diego, California, United States of America). The resulting virus is used to infect HIGH FIVE™ Tricoplusia ni cells (Invitrogen, Carlsbad, California, United States of America).
Recombinantly produced proteins are isolated and purified using a variety of standard techniques. The actual techniques used vary depending upon the host organism used, whether the protein is designed for secretion, and other such factors. Such techniques are well known to the skilled artisan. See e.g. chapter 16 of Ausubel et al., 1994. Recombinantly produced polypeptides of the invention are useful for a variety of purposes. For example, they can be used in in vitro assays to screen for known fungicidal chemicals, the target for which has not been identified, to determine if the chemicals inhibit expression of the nucleotide sequences of the invention. Such in vitro assays can also be used as more general screens to identify chemicals that inhibit the biological activity of the polypeptides of the invention, and that are therefore novel fungicide candidates. Alternatively, recombinantly produced polypeptides of the invention are used to elucidate the complex structure of these polypeptides and to further characterize their association with known fungicides in order to rationally design novel fungicides. III. Assays For Characterizing the Essential Proteins
The recombinantly produced proteins described herein are useful for a variety of purposes. For example, they can be used in in vitro assays to screen known fungicidal chemicals the target for which has not been identified to determine if they inhibit protein activity. Such in vitro assays can also be used as more general screens to identify chemicals that inhibit such protein activity and that are therefore novel fungicide candidates. Recombinantly produced proteins can also 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 herbicides. Alternatively, the recombinant protein can be used to isolate antibodies or peptides that modulate the activity and are useful in transgenic solutions.
A simple assay is developed to screen for chemicals that affect normal functioning of the polypeptides of the invention. Such chemicals are promising in vitro leads that can be tested for in vivo fungicidal activity. Any one of the nucleotide sequences of the invention is operatively linked to a strong inducible promoter, such promoters being known in the art. The vector comprising the selected nucleotide of the invention operatively linked to the selected inducible promoter is transformed into a host, such as E. coli. Transformed E. coli harboring and functionally over-expressing one of the nucleotide sequence of the invention are grown in a 96-well format for automated high-throughput screening where inducible over-expression of the nucleotide sequence of the invention is lethal or suppresses growth of the host. Chemicals effective in blocking function of the polypeptides of the invention result in bacterial growth. This growth is measured by simple turbidometric means.
In another embodiment, an assay for chemicals that block the functions of the polypeptides of the invention uses transgenic fungi or fungal cells capable of over-expressing the nucleotide sequences of the invention, operatively linked to a strong inducible promoter, e.g., wherein the selected polypeptide of the invention is biologically active in the transgenic fungi and/or fungal cells, and inducible over-expression of a nucleotide sequence of the invention inhibits and/ or suppresses growth and/or development of the fungus. The transgenic fungus or transgenic fungal cells are grown in 96-well format microtiter dishes for high-throughput screening. Chemicals that are effective in blocking the functions of the polypeptides of the invention result in fungal growth. This growth is measured by methods known in the art. In more detail, a representative assay includes the following general steps: (a) obtaining transgenic fungus and/or fungal cell, in one embodiment stably transformed, comprising a non-native nucleotide sequence or an endogenous nucleotide sequence operatively linked to non- native promoter, in one embodiment an inducible promoter, encoding an enzyme having an activity and capable of over-expressing a polypeptide of the invention, where over-expression of the polypeptide 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 a compound that results in the growth and/or development of the transgenic fungus and/or fungal cell in comparison to the untreated transgenic fungus and/or fungal cell.
Similar assays, based on expression of the fungal genes of the invention in yeast, using appropriate expression systems, as described above, can also be used.
IV. In vitro Inhibitor Assay: Discovery of Small Molecule Ligands that Interact with Essential Proteins of Unknown Biochemical Function Once a protein has been identified as a potential fungicide target based on its essentiality for normal fungal growth and viability, a next step is to develop an assay that allows screening large number of chemicals to determine which ones interact with the protein. Although it is straightforward to develop assays for proteins of known function, developing assays with proteins of unknown functions can be more difficult.
To address this issue, novel technologies are used that can detect interactions between a protein and a compound 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.
Fluorescence Correlation Spectroscopy (FCS) theory was developed in 1972, but it is only in recent years that the technology necessary to perform FCS became available (Madge et al., 1972; Maiti et al., 1997). 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 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. In a typical experiment, the target to be analyzed is expressed as a recombinant protein with a sequence tag, such as a poly-histidine sequence, inserted at the N-terminus or C-terminus of the protein. The expression takes place in E. coli, yeast, or insect cells. The protein is purified by chromatography. For example, the poly-histidine tag can be used to bind the expressed protein to a metal chelate column such as Ni2+ chelated on iminodiacetic acid agarose. The protein is then labeled with a fluorescent tag such as carboxytetramethylrhodamine or BODIPY® (Molecular Probes, Eugene, Oregon, United States of America). The protein is then exposed in solution to the potential ligand, and its diffusion rate is determined by FCS using instrumentation available from Carl Zeiss, Inc. (Thornwood, New York, United States of America). Ligand binding is determined by changes in the diffusion rate of the protein.
Surface-Enhanced Laser Desorption/lonization (SELDI) was invented by Hutchens and Yip during the late 1980's (Hutchens and Yip 1993). When coupled to a time-of-flight (TOF) mass spectrometer, SELDI provides a tool 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 analyzed by mass spectroscopy (MS) the small molecules that bind to this protein (Worrall et al., 1998). In a typical experiment, the target to be analyzed is expressed as described for FCS. The purified protein is then used in the assay without further preparation. It is bound to the SELDI chip either by utilizing the poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction. The chip thus prepared is then exposed to the potential ligand via, for example, a delivery system capable to pipette the ligands in a sequential manner (autosampler). The chip is then submitted to washes of increasing stringency, for example a series of washes with buffer solutions containing an increasing ionic strength. After each wash, the bound material is analyzed by submitting the chip to SELDI- TOF. Ligands that specifically bind the target will be identified by the stringency of the wash needed to elute them.
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 μl 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; Malmquist 1993).
In a typical experiment, the target to be analyzed is expressed as described for FCS. The purified protein is then used in the assay without further preparation. It is bound to the Biacore chip either by utilizing the poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction. The chip thus prepared is then exposed to the potential ligand via the delivery system incorporated in the instruments sold by Biacore AB (Uppsala, Sweden) to pipette the ligands in a sequential manner (autosampler). The SPR signal on the chip is recorded and changes in the refractive index indicate an interaction between the immobilized target and the ligand. Analysis of the signal kinetics on rate and off rate allows the discrimination between non-specific and specific interaction. Another assay for small molecule ligands that interact with a polypeptide is an inhibitor assay. For example, such an inhibitor assay useful for identifying inhibitors of the products of essential fungal nucleic acid sequences, such as the essential fungal proteins described herein, comprises the steps of: (a) reacting an essential fungal protein described herein and a substrate thereof in the presence of a suspected inhibitor of the protein's function; (b) comparing the rate of enzymatic activity of the protein in the presence of the suspected inhibitor to the rate of enzymatic activity under the same conditions in the absence of the suspected inhibitor; and (c) determining whether the suspected inhibitor inhibits the essential fungal protein.
For example, the inhibitory effect on the activity of a herein described essential fungal protein can be determined by a reduction or complete inhibition of protein activity in the assay. Such a determination can be made by comparing, in the presence and absence of the candidate inhibitor, the amount of substrate used or intermediate or product made during the reaction. Y_ Production of Peptides
Phage particles displaying diverse peptide libraries permit rapid library construction, affinity selection, amplification, and selection of ligands directed against an essential protein (Lowman 1997). Structural analysis of these selectants can provide new information about ligand-target molecule interactions and then in the process also provide a novel molecule that can enable the development of new herbicides based upon these peptides as leads. VI. In vivo Inhibitor Assay In one embodiment, a suspected fungicide, for example identified by in vitro screening, is applied to a fungus or fungi at various concentrations. The suspected fungicide can be sprayed on the plants. After application of the suspected fungicide, its effect on the fungus/fungi, for example death or suppression of growth is recorded. VIL Method of Using Nucleotide Sequences of the Invention to Distinguish Fungal Species
In a further embodiment of the invention, a nucleotide sequence selected from the Sequence Listing can also be used for distinguishing among different species of plant pathogenic fungi and for distinguishing fungal pathogens from other pathogens such as bacteria (Weising et al., 1995). In another embodiment, a nucleotide sequence selected from the Sequence Listing can also be used for distinguishing among different species of plant pathogenic fungi and for distinguishing fungal pathogens from other pathogens such as bacteria using the polymerase chain reaction (PCR). See, U.S. Patent Nos. 5,800,997; 5,814,453; 5,827,695; 5,955,274; 6,221 ,595 and 6,319,673. VIII. Fungal Transformation Technology
A nucleotide sequence of the present invention, or homologs thereof, can be incorporated in fungal or bacterial cells using conventional recombinant DNA technology. Generally, this involves inserting a nucleotide sequence into an expression system to which the sequence is heterologous (i.e., not normally present) using standard cloning procedures known in the art. The vector contains the necessary elements for the transcription and translation of the inserted polypeptide-coding sequences in a fungal cell containing the vector. A large number of vector systems known in the art can be used, including, but not limited to plasmids (van den Hondel & Punt 1990). The components of the expression system can also be modified to increase expression. For example, truncated sequences, nucleotide substitutions, nucleotide optimization, or other modifications can be employed. Expression systems known in the art can be used to transform fungal cells under suitable conditions (Lemke & Peng 1997). A heterologous DNA sequence comprising a gene, selected from the group consisting of the odd-numbered SEQ ID NOs:1 -5, can be stably transformed and integrated into the genome of the fungal host cells.
Nucleotide sequences intended for expression in transgenic fungi are first assembled in expression cassettes operatively linked to a suitable promoter capable of driving expression of genes in fungi (Lang-Hinrichs 1997; Jacobs & Stahl 1997). The expression cassettes can also comprise any further sequences required or selected for the expression of the heterologous nucleotide sequence. Such sequences include, but are not restricted to transcription terminators, extraneous sequences to enhance expression such as introns, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the fungal transformation vectors as described (Lemke & Peng 1997).
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 herein are well known in the art and are described by Sambrook et al., 2001 ; Silhavy et al., 1984; Reiter et al., 1992; Ausubel et al., 1994; and Schultz et al., 1998. Example 1 Identification of Antifungal Targets Represented in the Sequence Listing The cdd 4 gene was identified using three simultaneous approaches, each described herein below.
_L Comparison of EST libraries
Libraries PA, PB, PE, and PJ were constructed from different mRNA populations (described below) from Phytophthora infestans strain 88069. Descriptions of the libraries: PA: mRNA from a non-sporulating, liquid culture in clarified rye, pre- sporulation (10 days after inoculation with a small amount of asexual sporangia)
PB: mRNA from a sporulating culture; grown on polycarbonate membrane on top of rye agar. Inoculated by spreading sporangia. Culture was 1 1 days old
PE: mRNA from carbon starvation, Chinese minimal medium, grown then transferred for 2 days to the same medium but lacking glucose. Chinese minimal medium is a defined medium for Phytophthora, and contains the following: 1 M glucose; 10 mM (NH4)2SO4; 20 mM CaCI2 βH2O; 4 mM MgSO4 »7 H2O; 5 mM KH2PO4; 1 .7 mM K2HPO4; 0.025 mM Fe2(SO4)3; 20 mM fumaric acid; 0.2 mM MnSO4; 0.15 mM ZnSO4; 0.14 mM thiamine; adjust pH to 4.6. See Xu et al., 1982
PJ: mRNA from purified asexual sporangia
The assembled libraries are also referred to as the EST libraries J, A, B or E (spores; non-sporulating hyphae; sporulating, and carbon starved, respectively).
Expressed Sequence Tag (EST) libraries were constructed from spores and sporulating hyphae, respectively, as follows. Total RNA was isolated from sporulating hyphae of isolate 88069 (spores were inoculated into liquid rye media, and by about 2 weeks the hyphal mat was sporulating profusely) and purified spores were isolated from rye-agar cultures by adding cold water to the plates and rubbing off the spores. mRNA was then purified by binding to oligo-dT paramagnetic beads. cDNA was then made using Superscript Plasmid system from Gibco/Life Technologies (now a part of Invitrogen, Inc., Carlsbad, California, United States of America). This involved reactions primed at the 3' ends of the mRNA with an Notl-oligo-dT primer, using Moloney murine leukemia virus (MMLV) reverse transcriptase. After second strand synthesis, the cDNAs were size-fractionated by gel-filtration to eliminate small molecules. A Sail linker was then added to the 5' end. The Sall-Notl cDNA fragments were then ligated into Sall-Notl digested pSPORTI (a bacterial plasmid available from Invitrogen, Inc., Carlsbad, California, United States of America).
Frozen ligation mixes in water on dry ice were sent to Syngenta Biotechnology Inc. (Durham, North Carolina, United States of America; hereinafter "SBI") for library construction. A fraction of the individual ligation mixes was electroporated into E. co/ DH10B, plated out on selection media, and 6144 clones from each library were arrayed into 96 well plates (64 plates for each library) using a robot (QBot, available from Genetix USA Inc., Boston, Massachusetts, United States of America). A subset of these plates for each library was sent, as E. coli colonies in 96 well format, to Lark Technologies (Houston, Texas, United States of America) to be sequenced from the 5' end of the clone with primer T7. After sequencing, Lark Technologies sent back compact discs (CDs) containing chromatographs of all of the sequencing reactions done from each of these plates. The sequences were uploaded onto a local server and used a base-calling program (Phred, available form the University of Washington, Seattle, Washington, United States of America) to turn them into sequence files. The output from the Phred analysis (sequence files in FASTA format and quality files) was then burned to a CD and sent to the National Center for Genome Resources (NCGR: Santa Fe, New Mexico, United States of America) for processing. ESTs from libraries A and J were compared to identify those unique to spores. The putative cdc14-like clone was on the list of J-specific clones. 2. EST Database Sequence Comparisons
The EST database was searched for sequences with matches to protein kinases and protein phosphatases, since these are known regulatory proteins. At this time the putative cdd 4 clone was identified as a phosphatase.
Selected EST clones were then hybridized to Northern blots of RNA isolated from P. infestans at various developmental stages. The clones tested were selected based on the results described in sections 1 and 2 above. The cdd 4 phosphatase RNA was present in spores and sporulating hyphae, but not in non-sporulating hyphae (including non-sporulating hyphae from carbon-starved, nitrogen-starved, heat-treated, or mating cultures).
3_ EST Array Hybridization
An array of EST clones from library J was hybridized with labeled cDNA from spores and non-sporulating hyphae. The cdc14-like clone hybridized only with the cDNA from the spores.
Cdd 4 was demonstrated to be involved in asexual sporulation from analysis of the expression of 4800 clones from a P. infestans EST project. DNA from 4800 selected EST clones were spotted on a nylon membrane and hybridized with 32P-cDNA made from sporulating or non-sporulating hyphae. The cDNA probes were made using MMLV reverse transcriptase using 32P-dCTP (internal labeling) and a mixture of oligo-dT and random hexamer primers. Hybridizations were performed in 7% SDS, 0.5 M Sodium phosphate pH 7.2, 0.25 mM EDTA at 65°C. Washes were performed at 65C in 0.2X SSPE, 0.2% SDS, 0.1 % sodium pyrophosphate. These hybridization and wash conditions are high stringency conditions. The cdd 4 clone was observed to be an up-regulated gene during sporulation.
This indicated that the abundance of mRNA from a putative protein phosphatase-encoding gene was >10 times higher in sporulating than non- sporulating hyphae. The transcript was specific to sporulating hyphae and spores, and disappeared upon germination.
To identify a full-length cdd 4 clone, the EST database was then searched for overlapping clones using the BLAST tool. Clones were present in libraries B (sporulating hyphae), J (spores), and F (cleaving spores). The overlapping sequences were assembled. The 5' end of the cDNA was identified since multiple EST sequences started at the same place. There was a small polyA tail in one of the sequences. The sequence was confirmed by sequencing the 3' ends of some of the cDNA clones. This identified the full-length cDNA sequence as having 1459 nucleotides as set forth in SEQ ID NO:1 (Note: SEQ ID NO:1 is a consensus sequence that ignores some sequence polymorphisms between alleles). Between alleles 1 and 2 (SEQ ID NOS:3 and 5, respectively) there are three nucleotide and three amino acid differences.
The protein product of 423 amino acids was predicted from the nucleotide sequence of SEQ ID NO:1 , and is set forth in SEQ ID NO:2. The predicted protein amino acid sequences of the two alleles are set forth in SEQ ID NOs:4 and 6, respectively. The genomic sequence was obtained by using primers mapping to the 5' and 3' ends of the cDNA to amplify genomic DNA. The genomic amplicon was the same size as the cDNA, based on gel electrophoresis and sequencing. This indicated that there were no introns.
Southern blotting indicated that this was likely a single-copy gene, based on the analysis of four digests in two isolates of P. infestans.
Information that the sequence was a member of the cdd 4 family came from BLAST analysis. It was most similar to the human cdd 4a, NP_201570.1 (blast score=316, E=3e-85). It was also similar to the S. cerevisiae cdd 4 protein (score=231 , E=5e-60). Once a full-length cDNA was obtained, its strong similarity to other
Cdc14-like proteins became clear (Blast E =10"60 vs. S. cerevisiae). The P. infestans protein was shorter than its relatives (418 amino acids vs. 551 amino acids in S. cerevisiae, for example) but contained an apparently functional active site (AVHCKAGLGRTG; SEQ ID NO:7). The S. cerevisiae protein has an additional 133 amino acids at the C-terminus, a domain that has been shown to not be required for cell cycle functions. The P. infestans gene was single copy and lacked introns as demonstrated by a Southern blot analysis of the cdd 4 gene against DNA from isolates Ca65 and 216 digested with restriction endonucleases Hindlll, Pstl or Xhol. Further, the cdd 4 genomic sequence is the size of the full- length cDNA (about 1460 nucleotides) and is within 10 basepairs (bp) of the size of the complementary genomic region, based on PCR and "high- resolution" electrophoresis.
Cdd 4 is most similar to metazoan cdd 4 proteins as determined by phylogenetic comparison between cdc14-like proteins from P. infestans, human, Drosophila, C. elegans, S. pombe, S. cerevisiae, Candida albicans, and Neurospora crassa. Additional support for this conclusion came from searching for protein motifs, using the CDD database at NCBI. This showed that it had an apparently conserved protein phosphatase motif. The sequence contained a region homologous to the catalytic domain motif of dual specificity phosphatases, i.e. those acting at tyrosine and threonine residues (Pfam motif 00782). The motif was between amino acids 214 and 318 of the P. infestans protein. The amino acid sequence of the region matching the motif was NGTLVVRLNDKQYDEKKFLSAGIDHIDLIYPDGTNA PMPILMKFIEACEKTPGAVAVHCKAGLGRTGTCIGAYMMKHHLFSAHELIG WLRLCRPGSVIGPQQQFM (SEQ ID NO:8). Within that motif was the actual active site: AVHCKAGLGRTG (SEQ ID NO:7).
The cdd 4 amino acid sequence obtained as disclosed herein was also aligned with known cdc14-like proteins. The cdd 4 gene contained all of the conserved regions present in other members of the family. The P. infestans protein is about 100 amino acids smaller than cdd 4 proteins from other species. The missing region has been shown to not participate in cell cycle functions, however. Within the regions of similarity:
Table 1 : Similarities Among cdd 4 of P. infestans and of Other Species
Figure imgf000040_0001
Figure imgf000041_0001
Example 2 Effects of Expressing Sense and Antisense Seguences in Transformants To disrupt cdd 4 activity, for example by homology-dependent gene silencing or overexpression, sense (SEQ ID NO:13) and antisense cdd 4 (SEQ ID NO:14) constructs were expressed in P. infestans transformants. Reduced sporulation was observed in transformants expressing both sense and antisense cdd 4 constructs, suggesting that regulation of cdd 4 activity is required for sporulation.
While applicants do not intend to be bound by any particular theory of operation, the observed non-sporulation phenotypes could be caused by cdd 4 gene silencing or by cdd 4 overexpression, as described further herein below. To distinguish among different possible mechanisms, levels of cdd 4 transcript and/or protein can be assayed in transformants displaying a non-sporulation phenotype. Determination of cdd 4 expression levels can be accomplished using methods known to one of skill in the art including but not limited to in reverse transcriptase PCR, Northern blotting, and Western blotting.
Gene silencing is becoming increasingly successful in P. infestans, and data so far indicate that silencing works best when sense or antisense sequences are expressed, yielding transcriptional silencing through a homology-dependent mechanism (Van West et al., 1999a; Muskens et al., 2000). Constructs expressing inverted repeats have not proved comparably successful in P. infestans, even though they achieve post-transcriptional silencing in animals and plants (Tuschl et al., 1999; Corellou et al., 2000). Expression of sense and antisense cdd 4 constructs in P. infestans yielded sporulation-deficient strains. These transformants were generated by treating P. infestans protoplasts (obtained from germinated spores incubated with Novozyme 234 and cellulase) with DNA, calcium chloride, and polyethylene glycol (Judelson et al, 1991). Briefly, asexual sporangia from rye agar cultures were placed in rich broth media for 24-48 hours to induce germination. The germinated material was harvested, washed in KC buffer (0.64 M KCI, 0.2 M CaCI ), and resuspended in KC buffer containing 5 mg/ml Novozyme 234 and 2 mg/ml cellulase. After gentle agitation for 30 minutes, protoplasts were recovered by filtration through 50 micron nylon mesh, washed once in KC buffer by centrifugation, once in 50% KC and 50% MT (1 M mannitol and 10 mM Tris pH 7.5), and then once in MT. The protoplasts were resuspended in a small volume (about 0.8 ml) of MT plus 25 mM CaCI2, mixed with the DNA for transformation, incubated for 5 minutes, and then mixed with 0.8 ml of 50% polyethylene glycol. After 5 minutes, the protoplast-DNA mixture was diluted slowly into about 30 ml of rye media containing 1 M mannitol. After 24 hours, the regenerated protoplasts were pelleted by centrifugation and then plated on rye agar containing a selectable marker drug (for example, 7 //g/ml GENETICIN® (G418) or 50 μg/ml hygromycin B. Transformed colonies, which appear after 7-12 days, were then transferred to fresh plates. Interestingly, the sporulation-deficient strains appeared to lack both mature asexual spores and progenitors such as terminal swellings. This suggested that Cdd 4 acts relatively early during spore development. Based on these experiments, modulation of cdd 4 activity is envisioned to be useful for controlling sporulation. As used herein, the term "modulate" means an increase, decrease, or other alteration of any or all chemical and biological activities or properties of a wild type fungal cdd 4 polypeptide, in one embodiment a cdd 4 polypeptide of any one of the even- numbered SEQ ID NOs:2-6. In one embodiment of the invention, a odd 4 modulator is an agonist of a fungal cdd 4 protein. As used herein, the term "agonist" means a substance that synergizes or potentiates the biological activity of a functional cdd 4 protein. In another embodiment of the invention, a cdd 4 modulator is an antagonist of a fungal cdd 4 protein. As used herein, the term "antagonist" or "inhibitor" refers to a substance that blocks or mitigates the biological activity of a fungal cdd 4 polypeptide. Thus, the present invention further discloses a method for identifying a compound that modulates a fungal cdd 4 polypeptide. As used herein, the terms "candidate substance" and "candidate compound" are used interchangeably and refer to a substance that is believed to interact with another moiety, wherein a biological activity is modulated. For example, a representative candidate compound is believed to interact with a fungal cdd 4 polypeptide, or fragment thereof, and can be subsequently evaluated for such an interaction. Exemplary candidate compounds that can be investigated using the methods of the present invention include, but are not restricted to, viral epitopes, peptides, enzymes, enzyme substrates, co- factors, lectins, sugars, oligonucleotides or nucleic acids, oligosaccharides, proteins, chemical compounds, small molecules, and antibodies. A candidate compound to be tested can be a purified molecule, a homogenous sample, or a mixture of molecules or compounds. Example 3
Gene Disruption Experiments Gene disruptions of Phytophthora infestans genes or nucleotide sequences 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 of homology to the target gene locus, the target gene locus being identified as described in Wendland et al., 2000, and 20 nucleotides of homology (invariant) to a GENETICIN® resistance gene module also described in Wendland et al., 2000, 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:9) 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:10). 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 about 45 nucleotides specific to the chosen gene disruption site.
Once an S1/S2 primer pair is designed for a particular gene target, approximately 10 μg of the desired GENETICIN® resistance module is obtained by linearizing a vector containing the GENETICIN® resistance gene positioned behind an appropriate fungal (i.e. oomycete) promoter (for example, the Bremia lactucae HAM34 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 amplification, a final elongation period of 5 minutes at 72°C is carried out. 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 Phytophthora transformants. Additional verification primers designated G2 (5'- GTTTAGTCTGACCATCTC ATCTG-3'; SEQ ID NO:11) and G3 (5'- TCGCAGACCGATACCAGGATC-3'; SEQ ID NO:12) 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, the desired gene disruption can be verified by standard DNA hybridization experiments.
Determination of whether a gene is essential to the growth of Phytophthora can be achieved by the following analysis. The transformation of DNA fragments described above utilizes multinucleate Phytophthora 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 contains a non-transformed nucleus 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 that are 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.
Example 4 Expression of the Polypeptides of the Invention in Heterologous
Expression Systems
The coding region of the polypeptides of the invention are subcloned into previously described expression vectors, and transformed into E. coli using the manufacturer's conditions. Specific examples include plasmids such as pBLUESCRIPT® (Stratagene, La Jolla, California, United States of
America), the pET vector system (Novagen, Inc., Madison, Wisconsin,
United States of America) pFLAG (International Biotechnologies, Inc., New Haven, Connecticut, United States of America), and pTrcHis (Invitrogen,
Carlsbad, California, United States of America). E. coli is cultured, and expression of the polypeptides is confirmed. Alternatively, eukaryotic expression systems such as cultured insect cells infected with specific viruses can be employed. Examples of vectors and insect cell lines are described previously. The polypeptides of the present invention are isolated using standard techniques.
Example 5 In vitro Recombination of the Nucleotide Sequences of the Invention by DNA
Shuffling The nucleotide sequences of the invention are amplified by PCR. The resulting DNA molecule is digested by DNasel treatment essentially as described (Stemmer et a , 1994) and the PCR primers are removed from the reaction mixture. A PCR reaction is carried out without primers and is followed by a PCR reaction with the primers, both as described (Id.). The resulting DNA molecules are cloned into pTRC99a (Amann & Abel, 1988) for use in bacteria, and transformed into a bacterial strain deficient in the biological activity of the polypeptides of the invention by electroporation using the Biorad Gene Pulser and the manufacturer's conditions (Biorad Laboratories, Hercules, California, United States of America). The transformed bacteria are grown on medium that contains inhibitory concentrations of a potential inhibitor of the biological activity of the polypeptides of the invention. Those colonies that grow in the presence of the inhibitor are selected, and purified by repeated re-streaking. Plasmids from the purified colonies are purified and the DNA sequences of cDNA inserts are then determined. Alternatively, the DNA fragments are cloned into expression vectors for transient or stable transformation into fungal cells, which are screened for differential survival and/or growth in the presence of an inhibitor of the biological activity of the polypeptides of the invention.
In a similar reaction, PCR-amplified DNA fragments comprising one of the Phytophthora nucleotide sequences of the invention and PCR- amplified DNA fragments derived from a different nucleotide sequence of the invention, are recombined in vitro and resulting variants with improved tolerance to the inhibitor are recovered as described above.
Example 6
In vitro Recombination of the Nucleotide Seguences of the Invention by Staggered Extension Process One of the nucleotide sequences of the invention, or a homolog or fragment thereof, and another such sequence, or a homolog or fragment thereof, are each cloned into the polylinker of a pBluescript vector. A PCR reaction is carried out essentially as described (Zhao et al., 1998) using the "reverse primer" and the "M13 -20 primer" (Stratagene, La Jolla, California, United States of America). Amplified PCR fragments are digested with appropriate restriction enzymes and cloned into pTRC99a and mutated genes, are screened as described in Example 4. Example 7
In vitro Binding Assays
Recombinant polypeptides of the invention are obtained, for example, according to Example 2. The polypeptides are immobilized on chips appropriate for ligand binding assays using techniques that are well known in the art. The polypeptides immobilized on the chip are exposed to a chemical in solution according to methods well know in the art. While the sample chemical is in contact with the immobilized polypeptide, measurements capable of detecting polypeptide-ligand interactions are conducted. Methods used to make such measurements are SELDI, FCS, and SPR as described above. Chemicals found to bind the polypeptides are readily discovered in this fashion and are subjected to further characterization.
Example 8 Cell-Based Assay
Simple cell-based assays are developed to screen for chemicals that affect normal biological functions of the polypeptides of the invention. Such chemicals are promising in vitro leads that can be tested for in vivo fungicidal activity. Nucleotide sequences of the invention are operatively linked to a strong inducible promoter, e.g. GAL1 promoter, GAL10 promoter, or other such promoters known in the art. In one embodiment, overexpression of a nucleotide sequence of the invention confers upon the fungal cells a greater degree of resistance to an inhibitory chemical than is attainable in the wild type fungus. Wild type fungal cells are cultured in 96 well microtiter plates (e.g. 100 μl volume per well) in the presence of a defined concentration of a different chemical in each well. Likewise, transgenic fungal cells overexpressing the essential fungal gene (e.g. under inducing conditions) are challenged with the same set of chemical compounds at the same defined concentration. Situations in which growth of the wild type fungus, but not the transgenic fungus, is inhibited by a given chemical are identified as prospective situations in which overexpression of the particular nucleotide sequence confers resistance to the inhibitory effect of the test compound. Follow up experiments are carried out to repeat this result with a variety of concentrations of the identified chemicals.
In another embodiment, induced overexpression of a nucleotide sequence of the invention has deleterious effects upon growth or viability of the fungal cells. In this instance, transgenic fungal cells in which the essential fungal gene is operatively linked to an inducible promoter are cultured in 96 well microtiter plates in the presence of a defined concentration of a different chemical test compound in each well. After a short incubation period, cells are shifted to full inducing conditions (for example by adding an inducing compound to each well). Normally this induced overexpression would lead to growth arrest of the culture, but, in wells containing inhibitors of the essential nucleotide sequence, growth would proceed and would be monitored via the increased turbidity within such wells. Example 9
Phosphatase Activity Assays
Recombinant cdd 4 is isolated for these assays and to generate antigen for antibody production. The protein is expressed in E. coli using the
IMPACT™ -TWIN system (New England Biolabs, Beverley, Massachusetts, United States of America), which involves linking cdd 4 via a labile intein linkage to a chitin-binding domain. After binding on a chitin column and cleavage from the intein, the released enzyme is tested for phosphatase activity against compounds previously shown to be non-specific substrates of Cdc14-like proteins from other species including p-nitrophenyl phosphate, Tyrosine-P-casein and Ser-P-casein (Jaspersen & Morgan 2000).
Example 10
Complementation of S. cerevisiae cdd 4 Mutant
In order to assess the ability of the P. infestans cdc14-like gene to complement a S. cerevisiae cdd 4 mutant, the open reading frame from the P. infestans cdc14-like gene was placed under the control of a galactose- inducible promoter and transformed into a yeast strain carrying the cdd 4-1 ts allele (Wan et al, 1992). Transformants were selected on uracil-lacking media at 27°C, and then grown on galactose-containing plates at the permissive and nonpermissive temperatures (30°C and 37°C, respectively). Growth was assessed to determine the effect of the P. infestans gene. In the presence of galactose, transformants were able to grow at the non- permissive temperature. Transformants did not grow in the absence of the galactose inducer. Yeast cells transformed with an "empty" vector (a vector lacking the P. infestans cdc14-like gene open reading frame) also did not grow at the non-permissive temperature. A parallel experiment was performed using the wild-type yeast cdd 4 allele as a control (a gift of M. Grunstein).
Example 1 1 Subcellular Localization of cdd 4 Protein Two approaches are used to determine intracellular location of the cdd 4 protein, immunofluorescence and a GFP fusion. These studies also address whether the protein resides in the sporangiophore or sporangia.
Immunolocalization: Polyclonal antibodies are generated against the recombinant Cdd 4 using an external supplier and methods known to those skilled in the art. Rabbit antisera is tested for specificity in Western blots along with preimmune controls, and then used against whole mounts fixed in paraformaldehyde or paraffin-embedded 10 μm sections (Scanziani, 1998). Sectioning may be required due to the sporangial wall. After blocking and incubation with primary antibody, FITC-conjugated goat anti-rabbit IgG is added and visualized by confocal microscopy using 3-D image analysis. Dr. Prue Talbot (Department of Cell Biology and Neuroscience, University of California of Riverside, California, United States of America) provides technical assistance in this regard. The location of Cdd 4 is compared to that of nuclei, which is visualized using the DNA-binding dye DAPI.
GFP fusion: The location of Cdd 4 is also determined by using a Cdc14::GUS fusion vector transformed into P. infestans. Ideally, the construct serves several purposes including localizing the protein, studying the stability of Cdd 4, and identifying a functional Cdd 4 promoter. In previous work, fusing GFP to the C-terminus of the S. pombe Cdd 4 homologue was shown to not impair its function in the cell cycle (Cueille et al., 2001). Also, GFP has proved to be a successful marker in several Phytophthora species (van West et a , 1999b).
The vector is constructed to include 2 kb of DNA 5' of the open reading frame, plus the ORF, which is isolated from a BAG library. This region is inserted upstream of GFP in the pGPF-NPT plasmid, and transformed into isolate 1306 of P. infestans. Next, the transformants are checked to determine if they shown normal growth and development, and induction of GFP in a pattern consistent with the expression of the normal cdd 4 gene. The transformants are then examined by confocal microscopy to determine where the cdc14::GFP fusion protein localizes.
Example 12 Pattern and Regulation of Cdd 4 Expression To learn how Cdd 4 is regulated, its spatial and temporal expression patterns during sporulation are analyzed using GFP; protein levels during sporulation and germination are measured; and promoter regions involved in regulation are identified. Experiments to identify transcription factors binding to the promoter are also performed.
Spatial and temporal pattern of expression. RNA blot analyses indicated that cdd 4 RNA is first detected during sporulation, and disappears rapidly in encysted zoospores germinated in water or asexual sporangia producing direct germ tubes in rye media. However, the RNA analyses do not address the stability or subcellular location of the protein. Also, the precise point at which RNA levels drop is difficult to measure since germination is not totally synchronous (±2 and ±12 hours for cyst and asexual spore germination, respectively). Ideally, these issues are studied by microscopic examination of the GFP-expressing transformants.
Expression is first measured during the development of sporangiophores. Observations are made determining whether expression occurs in sporangiophores, in sporangial initials, or in mature asexual spores. Next, GFP levels are measured in spores induced to release zoospores in water at 109C, zoospores encysted by vortexing, zoospore cysts germinating in water, and asexual spores germinating in clarified rye media.
Example 13 Cyclin B-dependent Kinase Activity During Growth and Development Regulation of the cyclin B-cyclin dependent kinase (CDK) complex, which is the ultimate target of Cdd 4 activity in other systems, is studied during the life cycle of P. infestans. Experiments are focused on obtaining a broad understanding of how Cdd 4, cyclin B, and the latter's CDK (i.e. p34, p34cdc2, etc.) interact to regulate the transition from vegetative growth to spores. It is also important to see if regulation of this complex in oomycetes resembles that in other eukaryotes, where activity drops abruptly after mitosis due to cyclin B proteolysis and CDK inhibition.
Methods. An anti-cyclin B antibody is used to immunoprecipitate cyclin B/CDK complexes, which are assayed in vitro for kinase activity against histone H1. Initial experiments use a commercially available polyclonal antibody made against the entire human B1 cyclin protein (Rockland Immunochemicals). Once an effective antibody is obtained, the cyclin B-CDK complex is immunoprecipitated from vegetative hyphae, spores, zoospores, germinated zoospore cysts, and directly germinated sporangia. Kinase assays are then performed using standard methodologies (Kottom et al., 2000). Briefly, this involves using a homogenizer to make extracts in kinase assay buffer, which after clarification and preabsorption to protein A-sepharose are mixed with the antibody and precipitated with protein A-sepharose. The precipitate is then mixed with histone H1 and 32P-γ-ATP, and labeled histones are visualized by SDS- PAGE followed by phosphorimager analysis.
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Claims

Claims What is claimed is:
1. A method of identifying a fungicidal compound, comprising:
(a) combining a polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6 with a compound to be tested for the ability to bind to said polypeptide, under conditions conducive to binding;
(b) selecting a compound identified in (a) that binds to said polypeptide;
(c) applying a compound selected in (b) to a plant to test for fungicidal activity; and
(d) selecting a compound identified in (c) that has fungicidal activity.
2. The method according to claim 1 , wherein said polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
3. The method according to claim 2, wherein said polypeptide comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
4. The method according to claim 3, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
5. A method of identifying a fungicide compound, comprising:
(a) combining a polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6 with a compound to be tested for the ability to inhibit the activity of said polypeptide, under conditions conducive to inhibition; (b) selecting a compound identified in (a) that inhibits the activity of said polypeptide;
(c) applying a compound selected in (b) to a plant to test for fungicidal activity; and (d) selecting a compound identified in (c) that has fungicidal activity.
6. The method according to claim 5, wherein said polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
7. The method according to claim 6, wherein said polypeptide comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
8. The method according to claim 7, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
9. A method for killing or inhibiting the growth or viability of a fungus, comprising applying to the fungus or a plant a fungicidal compound identified according to the method of claim 1.
10. A method for killing or inhibiting the growth or viability of a fungus, comprising applying to the fungus or a plant a fungicidal compound identified according to the method of claim 5.
1 1. An isolated polypeptide selected from the group consisting of: (a) an isolated polypeptide comprising an amino acid sequence of even-numbered sequences of SEQ ID NO: 2-6; and (b) a isolated polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6 with a compound.
12. The isolated polypeptide of claim 11 , wherein said polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
13. The isolated polypeptide of claim 11 , wherein said polypeptide comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of the even-numbered SEQ ID NOs:2-6.
14. An isolated nucleic acid molecule comprising a nucleic acid sequence of odd-numbered SEQ ID NO: 1-5.
15. An isolated nucleic acid molecule encoding an amino acid sequence of an even-numbered sequence of SEQ ID NO:2-6.
16. An isolated nucleic acid molecule comprising a nucleotide sequence, the complement of which hybridizes under stringent conditions to a sequence selected from the group consisting of the odd-numbered SEQ ID NOs:1-5.
17. A chimeric construct comprising a promoter operatively linked to a nucleic acid molecule according to one of claims 14, 15, or 16.
18. The chimeric construct of claim 17, wherein the promoter is functional in a eukaryote.
19. The chimeric construct of claim 17, wherein the promoter is heterologous to the nucleic acid molecule.
20. A recombinant vector comprising a chimeric construct according to claim 17, wherein said vector is capable of being stably transformed into a host cell.
21. A host cell comprising a nucleic acid molecule according to one of claims 14, 15, or 16.
22. The host cell of claim 21 , wherein the nucleic acid molecule is expressible in the cell.
23. The host cell of claim 21 , wherein the host is selected from the group consisting of a plant cell, a yeast cell, an insect cell, and a prokaryotic cell.
24. A plant or seed comprising a plant cell according to claim 23.
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CN108103001A (en) * 2018-02-08 2018-06-01 云南农业大学 A kind of method that phytophthora infestans is promoted to generate a large amount of sporangiums

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