Field Of The Invention The present invention pertains to nucleic acid molecules isolated from Phytophthora infestans comprising nucleotide sequences that encode proteins that play a role in plant growth and development. The invention particularly relates to methods of using these proteins as fungicide targets, based on this role.

Abbreviations AcMNPV Autographica californica nuclear polyhedrosis virus ADP adenosine diphosphate ASO allele-specific oligonucleotide ATCC American Type Culture Collection ATP adenosine triphosphate BAC bacterial artificial chromosome CaMK calcium/calmodulin-dependent protein kinase CCaMKs calcium and/or calcium/calmodulin regulated kinase cDNA complementary DNA CDPKs calcium-dependent protein kinases (with calmodulin-like domains) CRKs plant calcium-independent protein kinases DEAE diethyl aminoethyl

DNA deoxyribonucleic acid DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EF-1 elongation factor-1 ELISA enzyme-linked immunosorbent assay EST expressed sequence tag FAD flavin adenine dinucleotide FCS Fluorescence Correlation Spectroscopy FMN flavin mononucleotide HSPs high scoring sequence pairs IP3 inositol triphosphate Kcal kilocalories kV kilovolts p, F microfarads MALDI matrix assisted laser desorption ionization mRNA messenger RNA NAD nicotinamide adenine dinucleotide NADP nicotinamide adenine dinucleotide phosphate NCBI National Center for Biotechnology Information OLAs oligonucleotide ligation assays ORF open reading frame PCR polymerase chain reaction PEP phosphoenolpyruvate PEPRK PPCK-related kinase P. infestans Phytophthora infestans PPCKs PEP carboxylase kinases RNA ribonucleic acid rRNA ribosomal RNA

SABRI Syngenta Agricultural Biotechnology Research Institute SDS sodium dodecyl sulfate SELDI Surface-Enhanced Laser Desorption/lon ization SNRKs SNF1-like kinases snRNA small nuclear RNA SPR surface plasmon resonance SSC standard saline citrate (0.15 M NaCI/0. 015 M sodium citrate, pH 7) SSCP single-strand conformation polymorphism TAFs Transcription Associated Factors Tm thermal melting point TOF time-of-flight tRNA transfer RNA 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 (Id.). 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 (Kcal) per day and current U. S. prices (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 ; the present invention focuses on the latter. 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 ~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 nucleus (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 where growth involves the expansion of hyphal tubes, or 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 dessicated. 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 the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, mammals, and to a lesser extent in plants (McCollum & Gould 2001; Smits & Medema 2001; Stals et al., 2000; Heberle-Bors 2001).

Thus, the oomycetes include many important plant pathogens including Phytophthora, Pythium, and the downy mildews. Such species lack taxonomic affinity with the so-called true fungi (i. e. ascomycetes and basidiomycetes), instead being related to diatoms and brown algae (Baldauf et al., 2000; Gunderson et al., 1987). The asexual sporangia of oomycetes either germinate directly through a germ tube or indirectly by releasing biflagellate zoospores. Many species germinate through either pathway, while others produce only zoospores and some only germinate directly (Erwin & Ribeiro 1996; Michelmore et al., 1988). For those species displaying both modes of germination, zoospores are considered most important for dispersal and host infection.

One species exhibiting this duality in germination behavior is Phytophthora infestans, which causes the late blight diseases of potato and tomato. Sporangia form on sporangiophores that emerge from infected plant tissue, or from hyphae growing on artificial media. As in other oomycetes, the sporangia of P. infestans are multinucleate, non-desiccated, and metabolically active. Germination becomes possible once sporangia detached from sporangiophores encounter liquid. Indirect germination

predominates in the absence of nutrients and at cool temperatures, typically below 12°C (Ribeiro, 1983). In contrast, direct germination is favored by higher temperatures and nutrients. Indirect germination takes about one hour and involves the cleavage of the sporangial cytoplasm into 6-10 discrete zoospores. These emerge from the sporangial apex and swim, displaying several tactic behaviors (Deacon & Donaldson 1993; Hill et al., 1998), until encystment occurs in response to chemical or physical stimulation (Griffith et al., 1988). Encystment occurs within seconds, involving the detachment of flagella and deposition of a cell wall (Griffith et al., 1988). Cysts subsequently elaborate a germ tube that can penetrate and colonize a plant host, or grow into new mycelium in artificial culture.

Many elegant microscopic and physiological studies have been performed of zoosporogenesis, encystment, and cyst germination. Changes in cytoskeletal organization and vesicle distribution are well documented, as is the flux of ions, especially calcium (Griffith et al., 1988; Dearnaley et al., 1996; Hardham 1995; Marshall et al., 2001; Warburton & Deacon 1998; Jackson & Hardham 1996). In contrast, little is known of the molecular biology of zoospore development or how the various stages are regulated.

Cleavage, zoospore release, and encystment are reportedly insensitive to actinomycin D, which suggests the involvement of pre-existing mRNA or proteins (Penington et al., 1989; Clark et al., 1978). Later steps, such as cyst germination, are sensitive to such transcriptional inhibitors (Clark et al., 1978; Penington etal., 1986).

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 It is an object of the invention to provide nucleic acid molecules from Phytophthora infestans comprising nucleotide sequences that encode

proteins that play a role in fungal growth and development. It is another object to provide the proteins encoded by these 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 proteins of the invention. These and other objects are achieved in whole or in part by the present invention.

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, e. g. 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 is 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 of SEQ ID NO : 2 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. Optionally, the polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence of SEQ ID NO : 2. Optionally, the polypeptide comprises an amino acid sequence at least 99% identical to an amino acid sequence of SEQ ID NO : 2.

In one embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO : 2.

Also disclosed herein is 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 of SEQ ID

NO : 2 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.

Optionally, the polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence of SEQ ID NO : 2. Optionally, the polypeptide comprises an amino acid sequence at least 99% identical to an amino acid sequence of SEQ ID NO : 2. In one embodiment, the polypeptide comprises an amino acid sequence of SEQID NO : 2.

Also disclosed herein is 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 one of the above-disclosed methods Also disclosed herein is an isolated polypeptide selected from the group consisting of: (a) an isolated polypeptide comprising an amino acid sequence of SEQ) D N0 : 2; and (b) a isolated polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence of SEQ ID NO : 2. Optionally, the polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence of SEQ) D N0 : 2.

Optionally, the polypeptide comprises an amino acid sequence at least 99% identical to an amino acid sequence of SEQ) D N0 : 2. In one embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO : 2.

An isolated nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO : 1 is also disclosed. An isolated nucleic acid molecule encoding an amino acid sequence of SEQ ID NO : 2 is also disclosed. An isolated nucleic acid molecule comprising a nucleotide sequence, the complement of which hybridizes under stringent conditions to a sequence of SEQ) D N0 : 1 is also disclosed.

A chimeric construct comprising a promoter operatively linked to an above-disclosed nucleic acid molecule is also provided. Optionally, the promoter is functional in a eukaryote and/or the promoter is heterologous to

the nucleic acid molecule. A recombinant vector comprising the chimeric construct is also disclosed, wherein the vector is capable of being stably transformed into a host cell.

A host cell comprising the nucleic acid molecule is also disclosed.

Optionally, the nucleic acid molecule is expressible in a cell. Optionally, the host is selected from the group consisting of a plant cell, a yeast cell, an insect cell, and a prokaryotic cell. A plant or seed comprising the plant cell is also disclosed.

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 Drawings Figures 1A-1 D depict the expression of a calcium/calmodulin- dependent protein kinase (CaMK) in P. infestans (SEQ ID NO : 1). RNA was extracted from various tissues of P. infestans isolate 88069 and probed with a 32P-labeled fragment of SEQ ID NO : 1. In Figure 1, CaMK denotes the gene represented by SEQ ID NO : 1 and EF-1 denotes elongation factor 1, a housekeeping gene that is used as an internal control for RNA loading in the Northern blots depicted in Figures 1A-1D.

Figure 1A depicts a Northern blot showing that the expression of P. infestans CaMK is specific for zoospores (ZO). P. infestans CaMK expression was not detected in non-sporulating hyphae (HY), sporulating hyphae (SH), purified sporangia (SP), or directly germinating sporangia (DG). The mRNA detected using the P. infestans CaMK probe was about 1. 3 kilobases (kb).

Figure 1 B (left panel) depicts a Northern blot showing that P. infestans CaMK is expressed early in zoospore development. Cultures of growing P. infestans were flooded with cool water at t = 0 (0), the spores rubbed off and incubated at 10°C. At 30,60, 90, and 120 minutes, RNA was extracted. As shown in Figure 1 B, P. infestans CaMK was expressed within

30 minutes of incubation. As shown on the right panel, after 30 minutes P. infestans CaMK expression was already at 61% of its maximum relative to the EF-1 control (open circles). The graph in the right panel also depicts the percent of sporangia showing visible cytoplasmic cleavage but not having released zoospores (triangles); percent of empty sporangia (those having released zoospores; closed circles); and the percent of sporangia not showing any differentiation (squares).

Figure 1C depicts a Northern blot showing that the abundance of P. infestans CaMK peaked during cleavage (60 minutes in 10°C water; CL) and zoospore release (ZO), and fell afterwards. In cysts germinated in 10°C water for 8 hours (GC), the abundance of the transcript had dropped about 35% from its peak. The numbers at the bottom of the Figure indicate the ratio of P. infestans CaMK/EF-1, with the value at cleavage set equal to 1.0.

Figure 1D depicts a Northern blot showing that the induction of P. infestans CaMK appeared to be zoosporogenesis-specific and not just a consequence of starvation resulting from placing the spores in water. P. infestans CaMK was detected in spores during cleavage (CL), but was not detected in hyphae incubated in media lacking nitrogen (NS) or carbon (CS), or incubated in water (HS).

Figure 2 depicts a genomic Southern blot showing the detection of the CaMK gene in the genome of P. infestans. Genomic DNA was digested with BamHl (B), EcoRl (E), Hindil (H), or Pstl (P) and probed with a P. infestans CaMK probe. A single 6 kb band was detected in the Hindlll digest, suggesting that the P. infestans CaMK gene is a single copy gene in the genome of P. infestans. Note that BamHl, EcoRl, and Pstl sites all are found within the probe.

Figures 3A-3C depict the predicted genomic organization of the P. infestans CaMK gene, the deduced amino acid sequence, and a comparison of he predicted amino acid sequence of P. infestans CaMK with several other calcium-dependent protein kinases from other species.

Figure 3A depicts the structure of the P. infestans CaMK gene found on a 6 kb Hindlll fragment of a bacterial artificial chromosome (BAC) clone.

Indicated are the transcription start point (+1) ; a 16 nucleotide (nt) oocytes promoter motif (00) at-96 nt; a 50 nt 5'untranslated leader ; the start codon (ATG); the stop codon (TAA); a 45 nt 3'untranslated region; the positions of twelve conserved kinase domains (1-5,6a, 6b, 7-11), and the positions of restriction sites for EcoRl (Eco), Hindlll (Hin), and Pstl (Pst). The black boxes indicate amino acid sequences of the P. infestans CaMK gene that do not show related amino acid sequences in the calcium-dependent protein kinases from other species shown in Figure 3B.

Figure 3B shows the sequence of the predicted polypeptide encoded by the P. infestans CaMK gene. The twelve kinase domains are boxed and the ATP-binding domain is underlined (plain underlining). The underlining with the arrowheads indicates the position of the catalytic site.

Figure 3C depicts an alignment of the kinase domains of P. infestans CaMK (bottom sequence of each group; SEQ ID NO: 14) with calcium/calmodulin-dependent protein kinases from C. elegans (GenBank (TM) accession BAA82674; SEQ ID NO : 7), Dictyostelium (SEQ ID NO : 8); mouse (GenBank (TM) accession AAC48715; SEQ ID NO : 9) Aspergillus nidulans (GenBank (TM) accession Q00771; SEQ ID NO : 10); Saccharomyces cerevisiae (GenBank accession CAA40928; SEQ ID NO : 11); Fragaria (GenBank (TM) accession AAB88537; SEQ ID NO : 12); and Paramecium tetraurelia (GenBank (TM) accession AAC13356; SEQ ID NO : 13). The positions of the twelve kinase domains are indicated below the sequence. Dashes have been added to the Figure to maximize the alignment of the sequences.

Figure 4 depicts a Neighbor-joining tree based on the kinase domains. A consensus tree was developed based upon the amino acid sequence of the twelve kinase domain regions of various serine/threonine protein kinases. The numbers at the nodes indicate the percentage of their occurrence in 500 bootstrap replicates, and the scale represents 0.1 PAM

units. The kinases included in the analysis and their GenBank (w) accession numbers is as follows : CaMKs were from Aspergillus nidulans (AAD38850); C. elegans (BAA82674); Dictyostelium discoideum (A40811) ; Glomerella cingulata (AAC62515); human (NP001212); mouse (AAC48715); and S. cerevisiae (CAA40928). Calcium-dependent protein kinases (with calmodulin-like domains; CDPKs) were from cucumber (AAB49984); Eimeria tenella (CAA96439); Paramecium tetraurelia (AAC13356); tobacco (AAC25423); tomato (AAK52801); and Toxoplasma gondii (AF043629). A calcium and/or calcium/calmodulin regulated kinase (CCaMK) from tobacco (with visinin-like domain; AAD28791) was also tested. Plant calcium- independent protein kinases (CRKs) were from rice (AAK54157) and maize (BAA22410). PEP carboxylase kinases (PPCKs) were from Arabidopsis (AAK84668); sorghum (AAK81871), and tomato (AAF19403). A PPCK- related kinase (PEPRK) from Arabidopsis (T45842) was also tested. SNF1- like kinases (SNRKs) were from the algae Guillardia theta (AF165818); Mesembryanthemum crystallinum (Z26846), S. cerevisiae (M13971) ; Schizosaccharomyces pombe (Kin1 ; M36060); and soybean (Glycine max ; AF128443). Other serine/threonine kinases tested, which are not directly regulated by calcium or calmodulin, include the DNA damage-induced Dun1 kinase of S. cerevisiae (S43941); human phosphorylase kinase (catalytic subunit; KIHUCT) ; and two kinases in the metazoan AGC group (the mitogen and stress-activated MSK1 kinase of Homo sapiens, T13149, and the Gallus gallus ribosomal protein S6 kinase, M28488).

Figure 5 depicts the effect of actinomycin D on P. infestans CaMK mRNA. Sporangia were isolated from culture places by rubbing in water with or without 10 ug/ml actinomycin D, and incubated for 0 or 60 minutes at 10°C before RNA was extracted. Northern blots were hybridized with a probe from the P. infestans CaMK gene, and then stripped and hybridized with a probe for EF-1. The ratio of P. infestans CaMK to EF-1 signals was determined by phosphorimager analysis.

Brief Description Of The Sequence Listing SEQID NO : 1 is a nucleic acid sequence of a novel kinase induced during sporangial cleavage in P. infestans.

SEQID NO : 2 is the predicted amino acid sequence derived from the nucleic acid sequence of SEQ ID NO : 1.

SEQ ID NOs : 3-6 are the nucleotide sequences for various PCR primers used to confirm an appropriate gene disruption event in P. infestans.

Primer S1 comprises SEQID NO : 2, a 20 base pair sequence from the 5'end of the Geneticin resistance gene. Primer S2 comprises SEQ) D N0 : 3, a 20 base pair sequence from the 3'end of the Geneticin resistance gene.

Primers S1 and S2 can be used to detect the appropriate targeting of the P. infestans gene, which can then be confirmed using primers G2 and G3 (SEQ ID NOs : 4 and 5, respectively).

SEQ ID NOs : 7-13 are the amino acid sequences of the kinase domains of the polypeptides listed below. SEQ ID NO: Organism GenBank ('M) Accession No. 7 C. elegans BAA82674 8 Dictyostelium discoideum n. a. 9 Mouse AAC48715 10 Aspergillus nidulans Q00771 11 Saccharomyces cerevisiae CAA40928 12 Fragaria x ananassa AAB88537 13 Paramecium tetraurelia AAC13356 14 P. infestans n. a.

Detailed Description of the Invention To gain a better understanding of the regulation of germination and zoospore development in P. infestans, inhibitors of cellular processes were tested for their effects on direct and indirect germination. One or more stages of zoospore development were inhibited by compounds affecting calcium pathways or protein kinases. This led to a search for protein kinase

genes that were differentially expressed during zoosporogenesis, in recognition that protein phosphorylation plays major roles in regulating cellular functions in other species (Braun & Schulman 1995). A kinase- encoding gene was identified that became transcriptionally activated within minutes of placing sporangia in cool water to induce zoospore release. In contrast, no transcripts were detected in directly germinating sporangia, sporangia not induced to release zoospores, or hyphae. The product of the gene resembled Ca2+/calmodulin-regulated protein kinases but appeared to lack the C-terminal regulatory or protein-association domain typically found in such proteins. The protein kinase gene was also used to address the reliability of previous studies that assessed the effect of actinomycin D on germination.

1. Definitions For clarity, certain terms used in the specification are defined and presented as follows : The phrases"associated with"and"operatively linked"are used interchangeably and each refer to two 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 situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.

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 associated with, a nucleic acid sequence that codes for a mRNA or which is expressed as a protein, such that the regulatory nucleic acid sequence is able to regulate 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.

The term"co-factor"refers to a natural reactant, such as an organic molecule or a metal ion, that is normally required in an enzyme-catalyzed reaction. Representative co-factors include nicotinamide adenine dinucleotide (NAD); nicotinamide adenine dinucleotide phosphate (NADP); riboflavin, including flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN); folate ; molybdopterin ; thiamin; biotin; lipoic acid; pantothenic acid; coenzyme A; S-adenosylmethionine ; pyridoxal phosphate; ubiquinone; and menaquinone. In one embodiment, a co-factor can be regenerated and reused.

The phrase"coding sequence"refers to a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. In one embodiment, a coding sequence is transcribed into an RNA, which is then translated in an organism to produce a protein.

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.

The phrase"enzyme activity"refers to the ability of an enzyme to catalyze the conversion of a substrate into a product. A substrate for the enzyme comprises the natural substrate of the enzyme, but can also comprise analogues of the natural substrate, which can also be converted, by the enzyme into a product or into an analogue of a product. The activity of the enzyme can be measured, for example, by determining the amount of product in the reaction after a certain period of time, or by determining the amount of substrate remaining in the reaction mixture after a certain period of time. The activity of the enzyme 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 (e. g. adenosine triphosphate (ATP),

phosphoenolpyruvate, acetyl phosphate, or phosphocreatine) remaining in the reaction mixture after a certain period of time or by determining the amount of a used donor of free energy or energy-rich molecule (e. g. adenosine diphosphate (ADP), pyruvate, acetate, or creatine) in the reaction mixture after a certain period of time.

The term"essential", used in reference to a fungal nucleotide sequence, refers to a nucleotide sequence encoding a protein such as, for example, a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the fungus.

The phrase"expression cassette"as used herein refers to a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest which is operatively linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but can also code for a functional RNA of interest : for example, 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 ; i. e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must be 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 or organ or stage of development.

The term"gene"is used broadly to refer to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to 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 (e. g. a DNA sequence) or 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 are expressed to yield exogenous polypeptides.

The term"homologous", as used herein to describe a nucleic acid (e. g. DNA) sequence, refers to a nucleic acid (e. g. DNA) sequence naturally associated with a host cell into which it is introduced.

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 (e. g., total cellular) DNA or RNA. The phrase"bind (s) substantially"refers to complementary hybridization between a probe nucleic acid 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"as used herein 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 terms"fungicide"and"fungicidal compound"are used interchangeably and each 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/fungi.

The term"interaction"as used herein 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 (antagonists) or enhancing (agonists).

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"as used herein refers to plants that are genetically identical, except that they can differ by the presence or absence of a heterologous DNA sequence.

The term"isolated"as used herein in the context of an isolated DNA molecule or an isolated enzyme refers to 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.

The phrase"mature protein"as used herein refers to a protein from which the transit peptide, signal peptide, and/or propeptide portions have been removed.

The phrase"minimal promoter"as used herein refers to the smallest piece 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, the minimal promoter functions to permit transcription.

The phrase"modified enzyme activity"as used herein refers to enzyme activity 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.

The term"native"refers to a gene that is present in the genome of an untransformed plant cell.

The term"naturally occurring"is used to describe 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 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.

The term"pre-protein"as used herein 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. In one embodiment, a purified protein is in a homogeneous state although it 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.

The phrase"regulatory elements"as used herein refers 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.

The phrase"significant increase"as used herein refers to an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique, 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 yet another embodiment an increase by about 10-fold or greater.

The phrase"significantly less"as used herein refers to an amount of a product of an enzymatic reaction that 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 phrases"specific binding"and"immunological cross-reactivity"as used herein each refers 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)<BR> immunoreactive 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 biologics.

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 can require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to the protein with the 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 & 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 in one embodiment at least twice background signal or noise, and in another embodiment 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 5gC 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 to no other sequences.

The term"subsequence"as used herein 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.

The term"substrate"as used herein refers to a molecule that an enzyme naturally recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function, or is a modified version of the molecule, which is also recognized by the enzyme and is converted by the enzyme to a product in an enzymatic reaction similar to the naturally-occurring reaction.

The term"transformation"as used herein refers to a process for introducing heterologous DNA into a plant cell, plant tissue, or plant.

Transformed plant cells, plant tissue, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. Representative techniques for transforming dicotyledonous and monocotyledonous plants are known in the art. See e. g. U. S. Patent No. 6,091, 004, the contents of which are herein fully incorporated by reference.

The phrase"a plant, or parts thereof", as used herein shall mean an entire plant ; and shall mean the individual parts thereof, including but not limited to seeds, leaves, stems, and roots, as well as plant tissue cultures.

Transgenic plants of the present invention are understood to encompass not only the end product of a transformation method, but also transgenic progeny thereof.

The terms"transformed","transgenic", and"recombinant"as used herein each 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 or

the nucleic acid molecule 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.

The term"viability"as used herein 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.

Representative plants for the methods disclosed herein include but are not limited to rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, tobacco, tomato, sorghum and sugarcane.

I. A. Nucleic acids The nucleic acid molecules provided by the present invention include an isolated nucleic acid molecule of SEQ ID NO : 1 ; sequences substantially identical to SEQ ID NO : 1 ; conservative variants thereof; subsequences and elongated sequences thereof; complementary DNA molecules ; and corresponding RNA molecules. The present invention also encompasses genes, cDNAs, chimeric genes, and vectors comprising disclosed nucleic acid sequences.

The term"nucleic acid molecule"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 properties as the reference natural nucleic acid. Unless otherwise indicated,

a particular nucleotide sequence also implicitly encompasses conservatively modified variants thereof (e. g., degenerate codon substitutions), complementary sequences, subsequences, elongated sequences, as well as the sequence explicitly indicated. The terms"nucleic acid molecule"or <BR> <BR> "nucleotide sequence"can also be used in place of"gene", "cDNA", or "mRNA". Nucleic acids can be derived from any source, including any organism.

The term"isolated", as used in the context of a nucleic acid molecule, indicates that the nucleic acid molecule exists apart from its native environment and is not a product of nature. An isolated DNA molecule can exist in a purified form or can exist in a non-native environment such as a transgenic host cell.

The term"purified", when applied to a nucleic acid, denotes that the nucleic acid is essentially free of other cellular components with which it is associated in the natural state. Preferably, a purified nucleic acid molecule is a homogeneous dry or aqueous solution. The term"purified"denotes that a nucleic acid gives rise to essentially one band in an electrophoretic gel.

Particularly, it means that the nucleic acid is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.

The term"substantially identical", in the context of two nucleotide sequences, refers to two or more sequences or subsequences that have at least 60%, preferably about 70%, more preferably about 80%, more preferably about 90-95%, and most preferably about 99% nucleotide identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms (described herein below under the heading"Nucleotide and Amino Acid Sequence Comparisons"or by visual inspection. Preferably, the substantial identity exists in nucleotide sequences of at least 50 residues, more preferably in nucleotide sequence of at least about 100 residues, more preferably in nucleotide sequences of at least about 150 residues, and most preferably in

nucleotide sequences comprising complete coding sequences. In one aspect, polymorphic sequences can be substantially identical sequences.

The term"polymorphic"refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. An allelic difference can be as small as one base pair.

Another indication that two nucleotide sequences are substantially identical is that the two molecules specifically or substantially hybridize to each other under stringent conditions. In the context of nucleic acid hybridization, two nucleic acid sequences being compared can be designated a"probe"and a"target". A"probe"is a reference nucleic acid molecule, and a"target"is a test nucleic acid molecule, often found within a heterogeneous population of nucleic acid molecules. A"target sequence"is synonymous with a"test sequence".

A representative nucleotide sequence employed for hybridization studies or assays includes probe sequences that are complementary to or mimic at least an about 14 to 40 nucleotide sequence of a nucleic acid molecule of the present invention. In one embodiment, probes comprise 14 to 20 nucleotides, or even longer where desired, such as 30,40, 50,60, 100, 200,300, or 500 nucleotides or up to the full length of that set forth as SEQ ID NO : 1. Such fragments can be readily prepared by, for example, directly synthesizing the fragment by chemical synthesis, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production.

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 nucleic acid mixture (e. g., total cellular DNA or RNA).

The phrase"hybridizing substantially to"refers to complementary hybridization between a probe nucleic acid molecule and a target nucleic acid molecule and embraces minor mismatches that can be accommodated

by reducing the stringency of the hybridization media to achieve the desired hybridization.

"Stringent hybridization conditions"and"stringent hybridization wash conditions"in the context of nucleic acid hybridization experiments such as Southern and Northern blot analysis are both sequence-and environment- dependent. 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 specifically to its target subsequence, but to no 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 Southern or Northern Blot analysis of complementary nucleic acids having more than about 100 complementary residues is overnight hybridization in 50% formamide with 1 mg of heparin at 42°C. An example of highly stringent wash conditions is 15 minutes in 0. 1x SSC, SM NaCI at 65°C. An example of stringent wash conditions is 15 minutes in 0.2X SSC buffer at 65°C (See Sambrook & Russell 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 example of medium stringency wash conditions for a duplex of more than about 100 nucleotides, is 15 minutes in 1X SSC at 45°C. An example of low stringency wash for a duplex of more than about 100 nucleotides, is 15 minutes in 4-6X SSC at 40°C. For short probes (e. g. , about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1M Na+ ion, typically about 0.01 to 1M Na+ ion concentration (or other salts) at pH 7.0-8. 3, and the temperature is typically 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 2-fold (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.

The following are examples of hybridization and wash conditions that can be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a probe nucleotide sequence preferably hybridizes to a target nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5M NaP04, 1mM EDTA at 50°C followed by washing in 2X SSC, 0. 1% SDS at 50°C ; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5M NaP04, 1 mM EDTA at 50°C followed by washing in 1X SSC, 0. 1% SDS at 50°C ; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0. 5M NaP04, 1 mM EDTA at 50°C followed by washing in 0.5X SSC, 0. 1% SDS at 50°C ; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5M NaP04, 1mM EDTA at 50°C followed by washing in 0. 1X SSC, 0. 1% SDS at 50°C ; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5M NaP04, 1mM EDTA at 50°C followed by washing in 0. 1 X SSC, 0. 1% SDS at 65°C.

A further indication that two nucleic acid sequences are substantially identical is that proteins encoded by the nucleic acids are substantially identical, share an overall three-dimensional structure, are biologically functional equivalents, or are immunologically cross-reactive. These terms are defined further under the heading"Polypeptides"herein below. Nucleic acid molecules that do not hybridize to each other under stringent conditions are still substantially identical if the corresponding proteins are substantially identical. This can occur, for example, when two nucleotide sequences are significantly degenerate as permitted by the genetic code.

The term"conservatively substituted variants"refers to nucleic acid sequences having degenerate codon substitutions wherein 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 a/., 1994).

The term"subsequence"refers to a sequence of nucleic acids that comprises a part of a longer nucleic acid sequence. An exemplary subsequence is a probe, described herein above, or a primer. The term "primers used herein refers to a contiguous sequence comprising in one embodiment about 8 or more deoxyribonucleotides or ribonucleotides, in another embodiment 10-20 nucleotides, and in still another embodiment 20- 30 nucleotides of a selected nucleic acid molecule. The primers of the invention encompass oligonucleotides of sufficient length and appropriate sequence so as to provide initiation of polymerization on a nucleic acid molecule of the present invention.

The term"elongated sequence"refers to an addition of nucleotides (or other analogous molecules) incorporated into the nucleic acid. For example, <BR> <BR> a polymerase (e. g. , a DNA polymerase) can add sequences at the 3' terminus of the nucleic acid molecule. In addition, the nucleotide sequence can be combined with other DNA sequences, such as promoters, promoter regions, enhancers, polyadenylation signals, intronic sequences, additional restriction enzyme sites, multiple cloning sites, and other coding segments.

The term"complementary sequences", as used herein, indicates two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between base pairs. As used herein, the term"complementary sequences" means nucleotide sequences which are substantially complementary, as can be assessed by the same nucleotide comparison set forth above, or is defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein. A particular example of a complementary nucleic acid segment is an antisense oligonucleotide.

The term"gene"refers broadly to any segment of DNA associated with a biological function. A gene encompasses sequences including but not limited to a coding sequence, a promoter region, a cis-regulatory sequence, a non-expressed DNA segment that is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof. A gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation of an existing sequence.

The term"gene expression"generally refers to the cellular processes by which a biologically active polypeptide is produced from a DNA sequence.

The present invention also encompasses chimeric genes comprising the disclosed sequences. The term"chimeric gene", as used herein, refers to a promoter region operatively linked to a coding sequence, a nucleotide sequence producing an antisense RNA molecule, a RNA molecule having tertiary structure, such as a hairpin structure, or a double-stranded RNA molecule.

The term"operatively linked", as used herein, refers to a promoter region that is connected to a nucleotide sequence in such a way that the transcription of that nucleotide sequence is controlled and regulated by that promoter region. Techniques for operatively linking a promoter region to a nucleotide sequence are known in the art.

The terms"heterologous gene","heterologous DNA sequence", "heterologous nucleotide sequence", "exogenous nucleic acid molecule", or "exogenous DNA segment", as used herein, each refer to a sequence that originates from a source foreign to an intended 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, for example by mutagenesis or by isolation from native cis-regulatory sequences. The terms also include non-naturally occurring

multiple copies of a naturally occurring nucleotide 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 wherein the element is not ordinarily found.

The term"transcription factor"generally refers to a protein that modulates gene expression by interaction with the cis-regulatory element and cellular components for transcription, including RNA Polymerase, Transcription Associated Factors (TAFs), chromatin-remodeling proteins, and any other relevant protein that impacts gene transcription.

The present invention further includes vectors comprising the disclosed nuclear sequences, including plasmids, cosmids, and viral vectors.

The term"vector", as used herein refers to a DNA molecule having sequences that enable its replication in a compatible host cell. A vector also includes nucleotide sequences to permit ligation of nucleotide sequences within the vector, wherein such nucleotide sequences are also replicated in a compatible host cell. A vector can also mediate recombinant production of a polypeptide, as described further herein below. A preferred host cell is a bacterial cell, an insect cell, or a plant cell.

Nucleic acids of the present invention can be cloned, synthesized, recombinantly altered, mutagenized, or combinations thereof. Standard recombinant DNA and molecular cloning techniques used to isolate nucleic acids are known in the art. Exemplary, non-limiting methods are described by Sambrook & Russell 2001; by Silhavy et al., 1984; by Ausubel et al., 1992; and by Glover 1985. Site-specific mutagenesis to create base pair changes, deletions, or small insertions are also known in the art as exemplified by publications. See e. g., Adelman et al., 1983; Sambrook & Russell 2001.

Sequences detected by methods of the invention can be detected, subcloned, sequenced, and further evaluated by any measure known in the art using any method usually applied to the detection of a specific DNA sequence including but not limited to dideoxy sequencing, PCR, oligomer

restriction (Saiki et al., 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner et al., 1983), and oligonucleotide ligation assays (OLAs ; Landegren etal., 1988a). SeealsoLandegren etal., 1988b.

I. B. Polypeptides The polypeptides provided by the present invention include the isolated polypeptide set forth as SEQ ID NO : 2; polypeptides substantially identical to SEQ ID NO : 2; polypeptide fragments (preferably biologically functional fragments), fusion proteins comprising the disclosed amino acid sequences, biologically functional analogs, and polypeptides that cross-react with an antibody that specifically recognizes a disclosed polypeptide.

The term"isolated", as used in the context of a polypeptide, indicates that the polypeptide exists apart from its native environment and is not a product of nature. An isolated polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell.

The term"purified", when applied to a polypeptide, denotes that the polypeptide is essentially free of other cellular components with which it is associated in the natural state. Preferably, a polypeptide is a homogeneous solid or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. The term"purified"denotes that a polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the polypeptide is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.

The term"substantially identical"in the context of two or more polypeptide sequences is measured as polypeptide sequences having about in one embodiment 35%, in another embodiment 45%, in another embodiment 45-55%, and in still another embodiment 55-65% of identical or functionally equivalent amino acids. In another embodiment, two or more

"substantially identical"polypeptide sequences will have about 70%, in another embodiment about 80%, in another embodiment about 90%, in another embodiment about 95%, and in still another embodiment about 99% identical or functionally equivalent amino acids. Percent"identity"and methods for determining identity are defined herein below under the heading "Nucleotide and Amino Acid Sequence Comparisons".

Substantially identical polypeptides also encompass two or more polypeptides sharing a conserved three-dimensional structure.

Computational methods can be used to compare structural representations, and structural models can be generated and easily tuned to identify similarities around important active sites or ligand binding sites. See Henikoff et a/., 2000; Huang et a/., 2000; Saqi et al., 1999; Barton 1998.

The term"functionally equivalent"in the context of amino acid sequences is known in the art and is based on the relative similarity of the amino acid side-chain substituents. See Henikoff & Henikoff 2000. Relevant factors for consideration include side-chain hydrophobicity, hydrophilicity, charge, and size. For example, arginine, lysine, and histidine are all positively charged residues; that alanine, glycine, and serine are all of similar size; and that phenylalanine, tryptophan, and tyrosine all have a generally similar shape. By this analysis, described further herein below, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine; are defined herein as biologically functional equivalents.

In making biologically functional equivalent amino acid substitutions, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+ 4.5) ; valine (+ 4.2) ; leucine (+ 3.8) ; phenylalanine (+ 2.8) ; cysteine (+ 2.5) ; methionine (+ 1.9) ; alanine (+ 1.8) ; glycine (-0.4) ; threonine (-0.7) ; serine (-0.8) ; tryptophan (- 0.9) ; tyrosine (-1.3) ; proline (-1.6) ; histidine (-3.2) ; glutamate (-3.5) ;

glutamin (-3.5) ; aspartate (-3.5) ; asparagine (-3.5) ; lysine (-3.9) ; and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al. (1982) J Mol Biol 157 : 105). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within 2 of the original value is preferred, those that are within 1 of the original value are particularly preferred, and those within 0. 5 of the original value are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U. S. Patent No.

4,554, 101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, e. g., with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein.

As detailed in U. S. Patent No. 4,554, 101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+ 3.0) ; lysine (+ 3.0) ; aspartate (+ 3. 01) ; glutamate (+ 3. 01) ; serine (+ 0.3) ; asparagine (+ 0.2) ; glutamin (+ 0.2) ; glycine (0); threonine (-0.4) ; proline (-0. 51) ; alanine (-0.5) ; histidine (-0.5) ; cysteine (-1.0) ; methionine (-1.3) ; valine (-1.5) ; leucine (-1.8) ; isoleucine (-1.8) ; tyrosine (-2.3) ; phenylalanine (-2.5) ; tryptophan (- 3.4).

In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within 2 of the original value is preferred, those that are within 1 of the original value are particularly preferred, and those within 0. 5 of the original value are even more particularly preferred.

The present invention also encompasses polypeptide fragments or functional portions of a polypeptide. Such functional portion need not comprise all or substantially all of the amino acid sequence of a native gene product. The term"functional"includes any biological activity or feature, including immunogenicity.

The present invention also includes longer sequences of a disclosed polypeptide, or portion thereof. For example, one or more amino acids can be added to the N-terminus or C-terminus of a polypeptide. Fusion proteins comprising the polypeptide sequences are also provided within the scope of the present invention. Methods of preparing such proteins are known in the art.

The present invention also encompasses functional analogs of a polypeptide. Functional analogs share at least one biological function with a polypeptide. An exemplary function is immunogenicity. In the context of amino acid sequence, biologically functional analogs, as used herein, are peptides in which certain, but not most or all, of the amino acids can be substituted. Functional analogs can be created at the level of the corresponding nucleic acid molecule, altering such sequence to encode desired amino acid changes. In one embodiment, changes can be introduced to improve a biological function of the polypeptide, e. g., to improve the antigenicity of the polypeptide. In another embodiment, a polypeptide sequence is varied so as to assess the activity of a mutant polypeptide.

The present invention also encompasses recombinant production of the disclosed polypeptides. Briefly, a nucleic acid sequence encoding a polypeptide, or portion thereof, is cloned into an expression cassette, the cassette is introduced into a host organism, where it is recombinantly produced.

The term"expression cassette"as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively

linked to the nucleotide sequence of interest which is operatively linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest can be chimeric. 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 of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. Exemplary promoters include Simian virus 40 early promoter, a long terminal repeat promoter from retrovirus, an action promoter, a heat shock promoter, and a metallothionein promoter. In the case of a multicellular organism, the promoter and promoter region can direct expression to a particular tissue or organ or stage of development.

Suitable expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: viruses such as vaccinia virus or adenovirus, baculovirus vectors, yeast vectors, bacteriophage vectors (e. g., lambda phage), plasmid and cosmid DNA vectors, and transposon-mediated transformation vectors.

The term"host cell", as used herein, refers to a cell into which a heterologous nucleic acid molecule has been introduced. Transformed cells, tissues, or organisms are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.

A host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. For example, different host cells have characteristic and specific mechanisms for the translational and post-transactional processing and modification (e. g., glycosylation, phosphorylation of proteins). Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.

Expression in a bacterial system can be used to produce a non-glycosylated

core protein product. Expression in yeast will produce a glycosylated product. Expression in insect cells can be used to ensure"native" glycosylation of a heterologous protein.

Expression constructs are transfected into a host cell by any standard method, including electroporation, calcium phosphate precipitation, diethyl <BR> <BR> aminoethyl (DEAE) -Dextran transfection, liposome-mediated transfection, transposon-mediated transformation and infection using a retrovirus. The encoding nucleotide sequence carried in the expression construct can be stably integrated into the genome of the host or it can be present as an extrachromosomal molecule.

Isolated polypeptides and recombinantly produced polypeptides can be purified and characterized using a variety of standard techniques that are known to the skilled artisan. See e. g., Ausubel et al., 1992; Bodanszky et al., 1976; Zimmer et al., 1993.

I. C. Nucleotide and Amino Acid Sequence Comparisons The terms"identical"or percent"identity"in the context of two or more nucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms disclosed herein or by visual inspection.

The term"substantially identical"in regards to a nucleotide or polypeptide sequence means that a particular sequence varies from the sequence of a naturally occurring sequence by one or more deletions, substitutions, or additions, the net effect of which is to retain at least some of biological activity of the natural gene, gene product, or sequence. Such sequences include"mutant"sequences, or sequences wherein the biological activity is altered to some degree but retains at least some of the original biological activity. The term"naturally occurring", as used herein, is used to describe a composition 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, which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.

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 entered into a computer program, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are selected. The sequence comparison algorithm then calculates the percent sequence identity for the designated test sequence (s) relative to the reference sequence, based on the selected program parameters.

Optimal alignment of sequences for comparison can be conducted, e. g., 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 Wisconsin Genetics Software Package, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection.

See generally, Ausubel et al., 1992.

A preferred algorithm 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. nim. 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.

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=11, an expectation E=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 less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

I. D. Antibodies Also provided is an antibody that specifically a polypeptide of the present invention. The term"antibody"indicates an immunoglobulin protein, or functional portion thereof, including a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, Fab fragments, and a

Fab expression library."Functional portion"refers to the part of the protein that binds a molecule of interest. In a preferred embodiment, an antibody of the invention is a monoclonal antibody. Techniques for preparing and characterizing antibodies are known in the art. See e. g., Harlow & Lane 1988. A monoclonal antibody of the present invention can be readily prepared through use of well-known techniques such as the hybridoma techniques exemplified in U. S. Patent No 4,196, 265 and the phage- displayed techniques disclosed in U. S. Patent No. 5,260, 203.

The phrase"specifically (or selectively) binds to an antibody", or "specifically (or selectively) immunoreactive with", when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biological materials. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not show significant binding to other proteins present in the sample. Specific binding to an antibody under such conditions can require an antibody that is selected based on 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 unrelated proteins.

The use of a molecular cloning approach to generate antibodies, particularly monoclonal antibodies, and more particularly single chain monoclonal antibodies, are also provided. The production of single chain antibodies has been described in the art. See e. g., U. S. Patent No.

5,260, 203. For this approach, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning on tissue that expresses the polypeptide. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by heavy (H) and light (L)

chain combinations in a single chain, which further increases the chance of finding appropriate antibodies. Thus, an antibody of the present invention, or a"derivative"of an antibody of the present invention, pertains to a single polypeptide chain binding molecule which has binding specificity and affinity substantially identical to the binding specificity and affinity of the light and heavy chain aggregate variable region of an antibody described herein.

The term"immunochemical reaction", as used herein, refers to any of a variety of immunoassay formats used to detect antibodies specifically bound to a particular protein, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, enzyme linked immunosorbent assay (ELISA), "sandwich"immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e. g., using colloidal gold, enzyme or radioisotope labels), Western blot analysis, precipitation reactions, agglutination assays (e. g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. See Harlow & Lane 1988 for a description of immunoassay formats and conditions.

In another aspect, the present invention provides a method of producing an antibody immunoreactive with a disclosed polypeptide, the method comprising recombinantly or synthetically producing the polypeptide, or portion thereof, to be used as an antigen. The polypeptide is formulated so that it is can be used as an effective immunogen. An animal is immunized with the formulated polypeptide to generate an immune response in the animal. The immune response is characterized by the production of antibodies that can be collected from the blood serum of the animal.

Optionally, cells producing an antibody can be fused with myeloma cells, whereby a monoclonal antibody can be selected. Representative embodiments of the method use a polypeptide set forth as SEQ ID NO : 2.

The present invention also encompasses antibodies and cell lines that produce monoclonal antibodies as described herein.

The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the polypeptide sequences of the invention, e. g., for cloning of nucleic acids, immunopurification of polypeptides, imaging polypeptides in a biological sample, and measuring levels thereof in appropriate biological samples.

LE Methods for Detecting a Nucleic Acid In another aspect of the invention, a method is provided for detecting a nucleic acid molecule that encodes a disclosed polypeptide. Such methods can be used to detect gene variants and related gene sequences.

The disclosed methods facilitate genotyping, cloning, gene mapping, and gene expression studies.

The nucleic acids of the present invention can be used to clone genes and genomic DNA comprising the disclosed sequences. Alternatively, the nucleic acids of the present invention can be used to clone genes and genomic DNA of related sequences. Using the nucleic acid sequences disclosed herein, such methods are known to one skilled in the art. See, for example, Sambrook & Russell 2001. Representative nucleic acids used for this method comprise a sequences set forth as SEQ ID NO : 1.

In one embodiment, genetic assays based on nucleic acid molecules of the present invention can be used to screen for genetic variants by a number of PCR-based techniques, including single-strand conformation polymorphism (SSCP) analysis (Orita et al., 1989), SSCP/heteroduplex analysis, enzyme mismatch cleavage, direct sequence analysis of amplified exons (Kestila et al., 1998; Yuan et al., 1999), allele-specific hybridization (Stoneking et al., 1991), and restriction analysis of amplified genomic DNA containing the specific mutation. Automated methods can also be applied to large-scale characterization of single nucleotide polymorphisms (Brookes 1999; Wang et al., 1998). Preferred detection methods are non- electrophoretic, including, for example, the TAQMANTM allelic discrimination

assay, PCR-OLA, molecular beacons, padlock probes, and well fluorescence. See Landegren et al., 1998.

I. F. Methods for Detecting a Polypeptide In another aspect of the invention, a method is provided for detecting a level of a disclosed polypeptide using an antibody that specifically recognizes the polypeptide, or portion thereof. In a preferred embodiment, biological samples from an experimental subject and a control subject are obtained, and the polypeptide is detected in each sample by immunochemical reaction with the antibody. More preferably, the antibody recognizes an amino acid of SEQ ID NO : 2; and is prepared according to a method of the present invention for producing such an antibody.

In one embodiment, an antibody is used to screen a biological sample for the presence of a disclosed polypeptide. A biological sample to be screened can be a biological fluid such as extracellular or intracellular fluid, or a cell or tissue extract or homogenate. A biological sample can also be an isolated cell (e. g., in culture) or a collection of cells such as in a tissue sample or histology sample. A tissue sample can be suspended in a liquid medium or fixed onto a solid support such as a microscope slide. In accordance with a screening assay method, a biological sample is exposed to an antibody immunoreactive with a polypeptide whose presence is being assayed, and the formation of antibody-polypeptide complexes is detected.

Techniques for detecting such antibody-antigen conjugates or complexes are known in the art and include but are not limited to centrifugation, affinity chromatography and the like, and binding of a labeled secondary antibody to the antibody-candidate complex.

The disclosed methods for detecting a polypeptide can be useful to determine altered levels of gene expression that are associated with particular conditions.

II. Recombinant Production of 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 of the SEQ ID NO : 1 presented below or nucleotide sequences substantially similar to SEQ ID NO : 1 presented below, or polypeptides encoded thereby, 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, such as a mammal, a fly, or a yeast. In another embodiment, the nucleotide sequences of the invention are derived from a fungus. The nucleic acid molecules are also produced using available synthetic methods known in the art.

The choice of the specific regulatory sequences such as promoter, signal sequence, 5'and 3'untranslated sequence, and enhancer 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 linking 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 & Summers 1988. Additional<BR> suitable expression vectors are baculovirus expression vectors, e. g. , those derived from the genome of Autographica califomica 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 califomica baculovirus DNA (Pharmingen, San Diego, California, United States of America). The resulting virus is used to infect HIGH FIVET" Tricoplusia ni cells (Invitrogen Corporation, La Jolla, California, United States of America).

Recombinantly produced proteins can be 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. Ausubel et al., 1995.

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, whose target 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. 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 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+ cheated on iminodiacetic acid agarose. The protein is then labeled with a fluorescent tag such as carboxytetramethylrhodamine or BODIPYe (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 & Yip, 1993). When coupled to a time-of-flight (TOF) mass spectrometer, SELDI provides a mean to rapidly analyze molecules retained on a chip. It can be applied to ligand-protein interaction analysis by covalently binding the target protein on the chip and analyzed the small molecules that bind to this protein by mass spectroscopy (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.

Biacoree (Uppsala, Sweden) 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 microlitre 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 (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.

Another important use for the P. infestans nucleic acids and polypeptides of the present invention is using a two-hybrid system to identify interacting proteins. Such interacting proteins may represent substrates of the kinase, regulatory subunits, or other proteins. The term"two-hybrid system"is meant to define a screening method to identify protein-protein interactions, using a known gene as a"bait"or target to screen a library of expressed genes and their corresponding encoded products for specific interactions with the"bait". This can be performed in bacteria or, more commonly, in the yeast Saccharomyces cerevisiae. Kits are available from several commercial sources, including the MATCHMAKER kit from BD Biosciences Clontech (Palo Alto, California, United States of America).

Methods for library construction and use of visual marker genes for yeast two-hybrid screens are well known and can be found in Hannon & Bartel, 1995.

This method, as done in the yeast S. cerevisiae, exploits the fact that a functional transcription factor can be separated into two components. One component is a DNA-binding domain and the other is an activation domain.

When held together non-covalently (as on separate proteins), these two domains can still bind DNA and activate transcription. The system is constructed as follows : a DNA-binding domain is localized 5'to a reporter gene, for example luciferase or ß-galactosidase, and transformed into a yeast strain. The nucleic acid sequence for the DNA-binding domain of the transcription factor is ligated to the gene (or partial gene sequence) being used as bait, in this case the P. infestans gene of interest. Commonly used DNA-binding domains include those from lexA protein from E. coli, and the Gal4 protein in yeast. Expression of this DNA-binding domain-bait fusion is driven, for example by the yeast adh1 of gall promoter. A"library"of gene- fusions is also produced, using the activation domain of the transcriptional factor fused to genes (or gene fragments) from an expression library of interest (referred to as the activation domain hybrid). This library is typically constructed from clones of cDNA fused to the activation domain, which commonly include portions of the B42 (bacterial) and Gal4 (yeast) proteins.

Expression of the activation domain hybrids can also be performed, for example, using the yeast adh1 or gall promoter. To perform the two-hybrid screen, plasmids encoding the DNA-binding domain hybrid and a library of activation domain-cDNA hybrids are introduced into a yeast strain already containing the reporter. Transformed yeast in which the activation domain hybrid specifically binds to the DNA-binding domain hybrid express the reporter, since it binds to the promoter of the reporter gene and stimulates transcription. Positives clones are further characterized by recovering the cDNA sequence, analyzing that sequence to discern the potential structure of the interacting protein, and performing further tests of the biological relevance of the interaction between the two P. infestans proteins.

Another use for the P. infestans nucleic acids and polypeptides of the present invention is to identify interacting proteins using in vitro affinity purification methods. Such interacting proteins can represent substrates of the kinase, regulatory subunits, or other proteins.

Polypeptides such as the polypeptides of the present invention can be coupled to a solid support. The solid support can be packed, for example, in an affinity chromatography column or bound to a bead matrix. These can be used to purify peptide tagged proteins from complex mixtures such as cell extracts. Once the tagged test protein is purified from the other components of the cell lysate, and then eluted from the affinity column or solid support, it can be analyzed by various analytical techniques. For example the purified protein, or peptide produced from the protein by protease digestion, can be analyzed by the use of matrix assisted laser desorption ionization time-of- flight (MALDI-TOF) mass spectroscopy; by comparison with predicted peptide databases for P. infestans, this can identify the gene encoding the kinase-binding protein and thus its entire sequence. Alternatively, the eluted protein can be sequenced by Edman degradation or other analysis techniques. By screening genomics databases, the resulting data can be used to identify the gene encoding the kinase-binding protein and thus its entire sequence. Alternatively, the sequence of the eluted protein can be used to design a degenerate oligonucleotide probe that can be hybridized to library of P. infestans DNA; this can be used to identify a clone containing the gene corresponding to a kinase-interacting protein.

A related affinity method for purifying interacting proteins involves generating an antibody to the kinase. Methods of making and assaying for antibody binding specificity and affinity are well known in the art; when presented as an immunogen to rabbits or other animals, foreign proteins such as the kinase will elicit the production of polyclonal antibodies. These can be purified and coupled to a solid matrix using either individual particles or particles assembled into a chromatography column. Cell extracts can then be applied to the particles for the purpose of isolating complexes of the protein kinase and interacting proteins. These complexes can be eluted from the column and analyzed as described above by MALDI-TOF, Edman degradation, or other methods.

IV. Production of Peptides Phage particles displaying diverse peptide libraries permits 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.

V. 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 is preferably 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.

VI. 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.

Vil. Funqal 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, e. g. SEQ ID NO : 1, 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 & Russell 2001; Silhavy et al., 1984; Reiter et al., 1992; Ausubel et al., 1994; and Schultz et al., 1998.

Example 1 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 homology to the target gene locus the target gene locus being identified as described in Wendland et al., 2000 Gene 242: 381-391, and 20 nucleotides 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 : 3) 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 : 4). The polymerase chain reaction (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 ug of the desired Geneticin resistance module is obtained by linearizing a vector containing the Geneticin resistance gene positioned behind an appropriate fungal promoter (for example, the Saccharomyces cerevisiae TEF1 promoter) and subjecting the linearized template to approximately 35 rounds of a PCR reaction consisting of the following steps: Step 1: Denaturation at 96°C for 30 seconds; Step 2: Primer annealing at 50°C for 30 seconds; Step 3: Elongation reaction at 72°C for 2.5 minutes.

Following the 35th round of this protocol, a final elongation period of 5 minutes at 72°C is carried out.

Transformation of the PCR product resulting from amplification with the S1/S2 primer pair is done by electroporation as follows : 1) Inoculate 100 ml of AFM media (1% casein peptone, 2% glucose, 1% yeast extract, 0. 1% myo-inositol) with an Ashbya spore suspension of approximately 107 spores.

2) Incubate at 30°C for a maximum of 18 hours at a shaker speed of 200 rpm.

3) Collect the resultant fungal mycelia by filtration and wash once with sterile water.

4) Resuspend 1 gram of mycelia (wet weight) in 40 ml of 50 mM potassium phosphate buffer, pH 7.5 containing 25mM dithiothreitol (DTT) and incubate at 30°C for 30 minutes with gentle shaking.

5) Collect the mycelia by filtration and wash once with 50 mi of cold STM buffer (275 mM sucrose, 10 mM Tris-HCI, pH 7.5, 2 mM MgCl2).

6) Resuspend the mycelia to a dense mixture in STM buffer.

7) Mix approximately 150 lli of the mycelial mixture with 10 ug of PCR product (in a maximum volume of 50 tl) in an Eppendorf tube and transfer the mixture to an electroporation cuvette with a 4 mm gap distance.

8) Apply an electric field pulse of 1.5 kilovolts (kV), 100 ohms, 25 microfarads (, uF), which will result in a pulse length of approximately 2.3 milliseconds. Add 1 mi of AFM media to the cuvette and spread equal amounts onto 3 pre-dried AFM agar plates.

9) Incubate plates for a minimum of 4 hours at 30°C.

10) Overlay the plates with 8 ml of a 0.5% agarose containing Geneticin (G418) at a final concentration of 200 gg/ml.

11) Incubate at 30°C for approximately 3 days to allow sufficient growth of Geneticin resistant transformants.

Verification of the desired transformation event resulting in homologous integration of the Geneticin resistance module in the target of interest is achieved by PCR using verification primers designated G1 (positioned upstream of the S1 region) and G4 (positioned downstream of the S2 region) and template DNA purified from putative Phytophthora transformants. Additional verification primers designated G2 (5'- GTTTAGTCTGACCATCTCATCTG-3'; SEQ ID NO : 5) and G3 (5'- TCGCAGACCGATACCAGGATC-3'; SEQ ID NO : 6) are derived from the open reading frame of the selectable Geneticin resistance gene such that the detection of a G1/G2 PCR product and or a G3/G4 PCR product of a predictable size serves to verify the desired gene disruption event. Also, verification of the desired gene disruption can be determined by standard DNA hybridization experiments.

Determination of whether a gene is essential to 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 nuclei as well. Thus, if an essential gene is disrupted in the transformed nucleus, the essential gene product can, in many instances, still be supplied by the non-transformed nuclei within the same cell. Such primary transformants usually exhibit normal growth and sporulation, and spores are collected from primary transformants 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 2 Expression of the Polypeptides of the Invention in Heteroloaous Expression Systems The coding region of the polypeptides of the invention are subcloned into previously described expression vectors, and transformed into Escherichia coli using the manufacturer's conditions. Specific examples include plasmids such as pBLUESCRIPI'° 11 (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 Corp. , La Jolla, 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 3 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 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 (Pharmacia Biotech, Piscataway, New Jersey, United States of America) 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 Bio-Rad Gene Pulser and the manufacturer's conditions (Bio-Rad 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 restreaking. 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 4 In Vitro Recombination of the Nucleotide Sequences 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 pBLUESCRIPTe 11 vector (Stratagene, La Jolla, California, United States of America). 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 5 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 6 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 Ll 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 (i. e. 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.

Overview of Examples 7-11 The protein kinase inhibitor K252a, the calcium channel blocker verapamil, and the calmodulin antagonist trifluoroperazine had little impact on the direct germination of sporangia of Phytophthora infestans, but major effects on indirect germination. Verapamil and trifluoroperazine strongly inhibited zoospore release and encystment, but only modestly impaired cyst germination. K252a perturbed zoospore release, caused zoospores to lyse before and during encystment, and blocked cyst germination. This led to the identification of a gene encoding a member of the Ca+2/camodulin-regulated family of protein kinases that was specifically expressed during indirect germination. Its transcription began within minutes of placing sporangia in cool water, conditions that induce sporangial cytoplasm to cleave and release zoospores, but before cleavage or zoospore release was evident.

The transcript was also detected in zoospores and germinated cysts, but not in other tissues including hyphae or directly germinating sporangia. The structure of the predicted protein was novel as its C-terminal region, which binds calmodulin in related proteins, was unusually short. Adding actinomycin D to sporangia at concentrations blocking direct germination was insufficient to prevent transcription of the kinase gene during cleavage, raising questions about the role of de novo transcription during

zoosporogenesis developed in previous studies of the transcriptional inhibitor.

To gain a better understanding of the regulation of germination and zoospore development in P. infestans, inhibitors of cellular processes were tested for their effects on direct and indirect germination. One or more stages of zoospore development were inhibited by compounds affecting calcium pathways or protein kinases. This led to a search for protein kinase genes that were differentially expressed during zoosporogenesis, in recognition that protein phosphorylation plays major roles in regulating cellular functions in other species (Braun & Schulman 1995). A kinase- encoding gene was identified that became transcriptionally activated within minutes of placing sporangia in cool water to induce zoospore release. In contrast, no transcripts were detected in directly germinating sporangia, sporangia not induced to release zoospores, or hyphae. The predicted product of the gene resembled Ca2+/calmodulin-regulated protein kinases but appeared to lack the C-terminal regulatory or protein-association domain typically found in such proteins. The protein kinase gene was also used to address the reliability of previous studies that assessed the effect of actinomycin D on germination.

Example 7 Effects Of Inhibitors On Differentiation Compounds antagonizing protein kinases (K252a), calcium channels (verapamil), calmodulin (trifluoroperazine), and transcription (actinomycin D) were tested for their effects on direct germination and the stages of zoospore development in P. infestans. The effects of kinase inhibitors on these pathways have not been previously reported, but the other compounds have been tested against a subset of the developmental stages in Phytophthora or Pythium (Donaldson & Deacon 1993; Deacon & Donaldson 1993; Warburton & Deacon 1998; Von Broembsen & Deacon 1996; Penington et a/., 1989; Donaldson & Deacon 1992). The inhibitors were tested by adding them to sporangia 5 minutes before chilling spore suspensions to induce

zoosporogenesis; to sporangia 5 minutes before placing them in rye media to allow direct germination; to swimming zoospores 5 minutes before inducing encystment by vortexing in 0.5 mM Call2 ; and to zoospore cysts 10 minutes after vortexing to study their effects on cyst germination.

Actinomycin D blocked cyst germination and direct germination, but had little effect on zoospore release (including cleavage) or encystment. It follows that de novo transcription is not required for cleavage or encystment, as previously noted (Clark et al., 1978; Penington et al., 1986). However, this assumes that actinomycin D enters sporangia with enough rapidity to inhibit transcription. As described below, this might not be the case.

Inhibitors of calcium pathways (verapamil and trifluoroperazine) strongly inhibited zoospore release. This occurred in a dose-dependent manner for both compounds, however for trifluoroperazine differential effects on cleavage as opposed to zoospore release were noted. At 2. 5, uM, no development was observed in 25% sporangia, 3% appeared to release zoospores normally, while 72% of sporangia released a single "megazoospore. "This appeared to be an uncleaved mass of cytoplasm bearing multiple flagella (>10) that was expelled from the sporangium and swam for 5 to 15 minutes before lysing or encysting. At 12. 5, uM trifluoroperazine and higher, no form of development occurred. Verapamil and trifluoroperazine also inhibited zoospore encystment, however with distinct effects. Both altered the directionality of swimming, as previously reported (Donaldson & Deacon 1993). Verapamil prevented the encystment of most zoospores, leaving the rest in a motile state. When trifluoroperazine was added to zoospores little effect was noted, but most zoospores immediately lysed when they were vortexed to induce encystment. Both compounds had minor but dose-dependent effects on cyst germination, which were less than reported for P. parasitica (Warburton & Deacon 1998).

This was likely due to the inclusion of 0.5 mM Cas) 2 in the encystment solution, a treatment that may attenuate the effects of the inhibitors

(Warburton & Deacon 1998). W7, another calmodulin inhibitor, had effects that parallele those of trifluoroperazine.

Verapamil and trifluoroperazine had no obvious effects on the germination of zoospore cysts. The direct germination of sporangia was also not affected. This was based on the fraction of germinated spores and the length of germ tubes measured after 3 and 16 hours. As observed for the calcium pathway inhibitors, the protein kinase inhibitor K252a strongly affected the zoospore pathway but had little effect on direct germination.

Zoospore release was moderately sensitive to K252a, but strikingly most zoospores lysed a few minutes after emergence. K252a appeared to inhibit encystment, however this was challenging to quantitate since many zoospores appeared to lyse just prior to or coincident with the encystment stimulus ; after vortexing, zoospores either appeared lysed or encysted but never motile. The germination of cysts was inhibited in a dose-dependent manner by K252a; cysts either germinated or remained in a dormant but unlysed form. The concentration of K252a required to observe these phenomena resembled that needed to detect cellular effects in other systems (Grosskopf etal., 1990).

Example 8 Identification Of Protein Kinase Induced During Cleavage Based on the inhibitor studies, a program to identify protein kinases potentially regulating zoospore development was implemented through a process resembling Digital Differential Display (Scheurle et a/., 2000).

Expressed sequence tags (ESTs) from a proprietary database (Lam 2001) were sorted based on the source of mRNA used to construct the corresponding cDNA libraries. These included sporangia, sporangia cleaving into zoospores, germinating zoospore cysts, hyphae, directly germinating sporangia, and other tissues. The ESTs were first clustered within each library, and then contigs (EST clusters) from zoospore-related libraries were assembled against non-zoospore contigs. This identified contigs constructed mainly from zoospore-related ESTs. Those potentially

encoding protein kinases were then identified by comparison to sequences in GenBank (TM) using BLAST. Next, Fisher's Exact Test was used to determine whether the differential distribution of the underlying ESTs in zoospore versus non-zoospore libraries was statistically significant. Using a nonstringent cut-off of P>0.8, three protein kinase-like genes possibly up- regulated during zoospore development were identified. This compares to 220 protein kinase contigs present in the original EST libraries.

One gene candidate proved to be specific to zoospore development based on RNA blot analysis. Hybridization was detected against mRNA from zoospores, but not from nonsporulating hyphae, hyphae decorated with sporangia, ungerminated sporangia, or directly germinating sporangia (Fig.

1A). The mRNA detected was 1.3 kb, and BLAST analysis of the existing partial-length cDNA sequence indicated similarity to Ca2+/calmodulin- regulated protein kinases (CaMKs). Subsequent studies indicated that the putative kinase gene started to be transcribed very early during zoospore development. This involved flooding culture plates with cool water, rubbing off the spores, incubating the spores at 10°C, and then extracting RNA for analysis. Thirty minutes after water was added, the abundance of the transcript was already at 61% of its maximum level relative to an EF-1 control (Fig. 1B). This was before cytoplasmic changes associated with cleavage were evident by light microscopy and before any zoospores were released (Fig. 1 B).

Several data suggested that transcription initiated within minutes of adding water to sporangia. However, the timing was difficult to precisely assess since variation was observed between experiments, and a true"zero- timepoint"is difficult to obtain since five minutes are normally required to harvest sporangia from culture plates. In some preparations of freshly harvested sporangia, no transcript was detected while in others it was already at one-third of its maximum level. Fig. 1 B shows the most common result, where the transcript is-1% of its maximum. Such discrepancies likely reflect variation in the swiftness at which sporangia were harvested, or

differences between batches of sporangia as some release zoospores faster than others. It is notable that no transcript was ever detected in blots of RNA, even after long exposures, from sporulating cultures (containing unseparated hyphae and sporangia, harvested without water).

The abundance of the kinase mRNA peaked during cleavage and zoospore release, and fell afterwards (Fig. 1 C). In cysts germinated in water for eight hours, for example, the abundance of the transcript had dropped 40% from its peak. This could understate the decline in mRNA abundance after cyst germination, however, as only 55% of cysts germinated in the experiment shown, a fraction typical for P. infestans The induction of the gene appeared to be a zoosporogenesis-specific response, and not simply the consequence of starvation resulting from placing spores in water. The latter scenario needed to be considered since oomycete sporangia are metabolically active. However, the kinase transcript was not present in hyphae incubated in media lacking nitrogen or carbon, or in water (Fig. 1 D). Also, the gene was not induced in sporangia held at 24°C for one hour in water.

Example 9 Gene Structure A single copy of the gene seemed to be present within the genome based on studies of independent BAC clones and blot analysis of genomic DNA. On each of several BACs analyzed, the gene resided on a 6 kb Hindlil fragment (Fig. 2A). When that fragment was hybridized against Hindlil- digested P. infestans DNA, a single 6 kb band was detected (Fig. 3). Other digests were also consistent with a single copy of the gene (Fig. 3; note that BamHl, EcoRl, and Psfl cleave within the probe). Similar results were obtained in analyses of three diverse isolates of P. infestans.

The gene appeared to be expressed as an intron-lacking transcript bearing 5'and 3'untranslated regions of 50 and 45 nt, respectively. This was determined by isolating and sequencing several full-length cDNAs, which were compared to the genomic clone. The sequence of the gene and

flanking regions are deposited in GENBANK (M) under accession number (to be provided). The sequence CCAGATTCCACCATTT (SEQ ID NO : 15) was noted 96 nt upstream of the transcription start site, which resembles a motif found upstream of most oomycete genes (GCTCATTYYNCAATTT ; SEQ ID <BR> <BR> NO : 16) although it is farther upstream than average (Pieterse et al., 1994) ).

An A-T rich region resembling a consensus polyadenylation site was detected 20 nt upstream of the polyA tail.

Example 10 Protein structure The gene was predicted to encode a protein of 398 amino acids, which resembled Ca2+ or calmodulin-regulated protein kinases from other species (Fig. 3). The strongest match against sequences in GENBANK was against a predicted calcium-dependent protein kinase (CDPK) from Arabidopsis thaliana, (Accession number S46283, BLAST E=10-41), followed by a known CDPK from the protozoan Toxoplasma gondii (AF043629, E=10- 37). The P. infestans protein includes each of the twelve kinase subdomains diagnostic of serine/threonine kinases, including the ATP-binding and catalytic motifs. Twenty-one amino acids previously shown to be invariant or nearly invariant in other protein kinases are each conserved in the predicted P. infestans protein (Hanks & Hunter, 1995). Twelve phosphorylation sites were predicted by PROSITE.

The relationship between the P. infestans gene product and Ca2+/calmodulin-regulated protein kinases (CaMKs) was strengthened by performing alignments between the kinase subdomains of representative proteins. The eukaryotic serine/threonine kinase subfamily can be classified into seven major groups (AGC, CaMK, CK1, CMGC, GCyc, PTK, STE) plus several other protein kinase groups or OPKs (Hanks and Hunter, 1995).

Amino acid alignments between the P. infestans protein and three representatives of each major group, plus ten OPKs, indicated strongest affinity with the CaMK kinases.

However, strong affinity to any particular group of Ca2+ or calmodulin- regulated protein kinases was not revealed by alignment studies (Fig. 4).

This analysis included representative animal and fungal CaMKs, plant and protozoan calcium-dependent calmodulin-like domain kinases (CDPKs), Ca2+ and/or Ca2+/calmodulin-regulated visinin-like kinases (CCaMKs), plus proteins not directly regulated by calcium or calmodulin but resembling such kinases including the calcium-dependent kinase-related kinases (CRKs), phosphorylase kinase, SNF1-related kinases (SNRKs), PEP carboxylase kinases (PPCKs), the DUN1 checkpoint kinase and PPCK-related kinases (PEPCKs). Best affinity was observed against the CaMK, CCaMK, and protozoan CDPK groups.

While the data suggested an evolutionary relationship between the P. infestans protein and Ca2+ or calmodulin-regulated kinases, the P. infestans protein was novel in structure. The region N-terminal to the kinase subdomains was 91 amino acids in length, which is larger than usually observed (Harmon et al., 2001). In addition, in the P. infestans protein the region C-terminal to the kinase subdomains was only 28 amino acids compared to 41 to >200 amino acids in other proteins (Braun & Schulman 1995; Harmon et a/., 2001). Such C-terminal regions generally include autoinhibitory domains, a calmodulin and/or calmodulin binding domains, and association domains for other proteins (Braun & Schulman 1995; Harmon et al., 2001). Known motifs for such functions (CaM domains, EF-1 hands, polyglutamine tracts) were not detected in the P. infestans protein, although these are known to be poorly conserved.

Example 11 Effect Of Actinomycin D On Transcription Of The Kinase Gene The failure of actinomycin D to block cleavage and encystment has been used to suggest that these steps do not require de novo transcription (Penington et al., 1989; Clark et al., 1978). To test whether this inhibitor effectively blocks transcription in sporangia, the kinase gene was probed against RNA from sporangia incubated in water at 10°C for 60 minutes in the

presence and absence of 10, ug/ml actinomycin D. By comparison, direct germination and hyphal growth are totally blocked by 1, ug/ml or less of this compound. Sporangia were harvested from culture plates flooded with 10 //g/mi actinomycin D to ensure that they contacted the inhibitor as quickly as possible.

Nevertheless, the kinase gene was still induced to 60% of normal levels (Fig. 5). Since studies on directly germinating sporangia and hyphae indicate that P. infestans is inherently sensitive to actinomycin D, this may mean that passage of the inhibitor into sporangia occurred too slowly to block the transcription of genes induced early in the pathway.

Discussion of Examples 7-11 This study presents the first report of a gene induced during zoosporogenesis in an oomycete, which appears to encode a novel serine/threonine protein kinase. In eukaryotes, such proteins regulate diverse cellular functions by modifying transcription factors, metabolic enzymes, ion channels, cytoskeletal components, and other targets (Braun & Schulman 1995). The P. infestans gene is induced within minutes of exposing sporangia to cool water, which initiates cleavage and zoospore release.

Consequently, the gene is a marker for the earliest biochemical and molecular changes associated with development. The kinase is also a candidate for a key regulator of one or more steps of the zoospore pathway, although identifying the stage at which its acts remains to be determined and is subject to interpretations of the effect of actinomycin D on sporangia. The inhibitor data presented also indicate that a protein kinase, plus calcium pathways, are essential for zoospore development although it premature to suggest that the identified kinase is the critical target of K252a.

A model to explain how the cleavage pathway is triggered can be proposed based on the fact that sporangia must be exposed to both water and cool temperatures. The primary effect of water is likely not hydration of the sporangium, since these are not desiccated like typical spores of true

fungi. Instead, water may release a germination inhibitor. Chilling may act by reducing membrane fluidity, which probably alters the activity of membrane-associated proteins such as phospholipase C or G proteins.

Similar changes are invoked to explain chilling responses in plants (Bigot & Boucaud 2000; Suh et al., 2001). Within seconds, chilling could result in the synthesis of inositol triphosphate (IP3) and the release of calcium from IP3 receptor-gated channels in the endoplasmic reticulum. This could initiate changes in the cytoskeleton, possibly due to activating presynthesized Ca2+lcalmodulin-regulated protein kinases. Transcription factors could similarly be enabled, resulting in the expression of genes such that encoding the 398 amino acid protein kinase.

That cleavage and encystment are insensitive to transcription inhibitors such as actinomycin D has been interpreted to indicate that these steps do not required de novo RNA synthesis, excluding the involvement of the protein kinase. However, the observation that 10, ug/ml actinomycin D only partially inhibits transcription of the kinase gene forces a re-evaluation of this interpretation.

Further analysis of the P. infestans kinase is required to determine its substrates and mode of regulation. In other species, some kinases are dedicated to a single substrate while others are multifunctional, and some are regulated solely by transcription while others are controlled through the binding of regulatory subunits or calmodulin, or phosphorylation. The P. infestans protein has the strongest sequence similarity to Ca2+ and calmodulin-regulated kinases. However, compared to such kinases, the C- terminal domain of the P. infestans protein appears too small to have conventional binding sites for calmodulin or other proteins. In this regard, it resembles the phosphoenolpyruvate carboxylase kinases (PPCKs) of plants and the Dictyostelium myosin light chain kinase (Hartwell et al., 1999; Tan & Spudich 1990). These proteins are also members of the Ca2+/calmodulin- regulated group and have short C-terminal domains, and are not regulated by Ca2+ or calmodulin. Plant PPCKs are controlled only at the level of

transcription, while the Dictyostelium kinase is regulated by transcription and phosphorylation.

Experimental procedures for Examples 7-11 Growth and manipulation of P. infestans. Isolates used in this study were 88069 (A1, The Netherlands) and 1306 (A1, United States). These were generally grown at 18°C on rye A agar (Caten & Jinks 1968), or clarified rye broth prepared by centrifuging rye A media for 10 min at 5 x kg.

Nitrogen and carbon-starved cultures utilized a defined media (Xu 1982) modified by reducing NH4SO4 levels to 0.5 mM, or omitting glucose and reducing fumarate levels to 20 mM, respectively. This reduced growth rates by 70% relative to the complete defined media.

Tissues used to study the effects of inhibitors, or as sources of RNA, were prepared as follows. Nonsporulating hyphae were obtained from 5 day rye broth cultures inoculated with 104/ml sporangia. Sporulating hyphae were scraped off polycarbonate membranes laid upon rye agar that had been inoculated 8-10 days earlier. Sporangia were isolated by flooding 8-12 day rye agar cultures with water, rubbing the sporangia free with a glass rod, and then separating sporangia from hyphal fragments by passage through 50um nylon mesh. To directly germinate sporangia, they were placed in clarified rye broth at 104/mi. Indirect germination was induced by cooling a sporangial suspension (105/ml) to about 10°C by placing it on ice for about 20 minutes, followed by incubation in a 10°C chamber for 30 minutes (for "cleaved"sporangia) or about 90 additional minutes (for zoospores).

Zoospores were purified from sporangia by passage through 15, um nylon mesh. Germinated zoospore cysts were isolated by adding 1 mM CaCts to zoospores, vortexing for 30 seconds to induce encystment, and then incubating the cysts in water at 10°C for 6 hours.

Sequence analysis. An expressed sequence tag (EST) database was generated in collaboration with the Syngenta Agricultural Biotechnology Research Institute (SABRI, North Carolina, United States of America). This was established using libraries prepared individually from mRNA extracted

from the tissues described above, plus other samples including some prepared at SABRI. cDNAs were cloned into pSPORT1 (Invitrogen Corporation, Carlsbad, California, United States of America) prior to 5'sequencing by SABRI.

Starting from a FASTA flat file of all sequences, the ESTs were clustered using Seqman (DNASTAR, Wisconsin, United States of America) prior to searching for zoospore-relevant kinases. The membership of library-specific clusters was then compared for ESTs preferentially present during developmental stages. ESTs appearing differentially abundant at P>. 90 based on Fisher's Exact Test were then tested in RNA blots.

Alignments of protein sequences were performed using a version of ClustalW as implemented in Alignment (Vector NTI Suite, Informax), with graphic outputs generated with the aid of BOXSHADE. Output from Alignment was also used to generate phylograms using Phylip for Macintosh, after trimming sequences to contain only the 12 kinase subdomains. Bootstrap replicates (500) were generated using SEQBOOT, distances were determined using the PAM option of PROTDIST, neighbor- joining trees were developed using NEIGHBOR, and a consensus tree was developed using CONSENSE.

RNA and DNA blot analysis. P. infestans RNA and DNA were extracted as described (Judelson 1993). RNA blots were prepared by electrophoresing 5, ug of glyoxylated total RNA in 1% agarose followed by transfer to nylon membranes (Rao 1994). Band sizes were determined using a 0.28-6. 58 kb RNA ladder (Promega Corporation, Madison, Wisconsin, United States of America) stained with SybrGreen II (Molecular Probes, Oregon, United States of America). To control for variation in sample loading, a probe for the P. infestans elongation factor-1 gene (kindly provided by F. Govers) was used. DNA blots were prepared using 0.8% agarose gels in 1x TBE-buffer (89 mM Tris, 89 mM H3BO3, 2 mM Na2EDTA) that were blotted to nylon membranes.

Hybridizations were performed using probes prepared by random hexamer labeling as described (Judelson et a/., 1995). Signals were detected using a Personal FX Phosphorimager (Biorad, Hercules, California, United States of America). Quantitation was performed using Quantity One software (BioRad) for Macintosh.

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

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It will be understood that various details of the invention can be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation--the invention being defined by the claims.