EP1244347A2 - Peptide synthetase gene cps1 - Google Patents

Peptide synthetase gene cps1

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Publication number
EP1244347A2
EP1244347A2 EP00986231A EP00986231A EP1244347A2 EP 1244347 A2 EP1244347 A2 EP 1244347A2 EP 00986231 A EP00986231 A EP 00986231A EP 00986231 A EP00986231 A EP 00986231A EP 1244347 A2 EP1244347 A2 EP 1244347A2
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Prior art keywords
nucleic acid
acid molecule
seq
polypeptide
cpsl
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German (de)
French (fr)
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Olen C. Yoder
Barbara C. Turgeon
Shun-Wen Lu
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Cornell Research Foundation Inc
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Cornell Research Foundation Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • the present invention relates to nucleic acid molecules encoding peptide synthetase homologs of Cochliobolus heterostrophus, Pyrenophora teres, Fusarium graminearium, and Alter naria solani and uses thereof.
  • Cochliobolus there are approximately 30 species included in the genus Cochliobolus, nearly all of which are pathogens of wild grasses or cereals (Yoder et al, "Cochliobolus spp. And Their Host-Specific Toxins, in Carroll, eds., The Mycota Vol. 5: Plant Relationships, Part A, Berlin:Springer-Verlag, pp. 145-166 (1997)).
  • Cochliobolus heterostrophus represents the most widely distributed species in the genus and can be found in many tropical and subtropical areas in the world. As a natural pathogen of corn, C.
  • heterostrophus causes a disease frequently called leaf spot of maize in the old literature (Drechsler, "Leafspot of Maize Caused by Ophiobolus Heterostrophus n. sp., The Ascigerous Stage of a Helminthosporium Exhibiting Bipolar Germination," J. Agr.
  • Southern Corn Leaf Blight Hooker, "Cytoplasmic Susceptibility in Plant Disease,” Ann. Rev. Phytopathol., 12:167-179 (1974)
  • Southern Corn Leaf Blight was only known as an endemic disease and was not considered to be major economic importance in the United States. But in 1970, it suddenly broke into a severe epidemic that destroyed 15% of the U.S. corn crop and caused losses estimated at more than $1 billion. This serious damage made Southern Corn Leaf Blight one of the most widely known crop diseases in the U.S.
  • race O C. heterostrophus
  • Helminthosporium maydis (Cochliobolus heterostrophus)," Phytopathology, 65:273-276 (1975); Yoder, "Evaluation of the Role oi Helminthosporium maydis, Race T Toxin in Southern Corn Leaf Blight, in Tomiyama, eds., Biochemistry and Cytology of Plant Parasite Interaction, New York, New York:Elsevier, pp. 16-24 (1976); Yoder, "Toxins in Pathogenesis,” Ann. Rev. Phytopathol.. 18:103-129 (1980)).
  • T-cytoplasm stands for Texas male sterile cytoplasm, a unique cytoplasm with a trait for maternally inherited male sterility, characterized by the failure to produce pollen (Levings, "The Texas Cytoplasm of Maize: Cytoplasmic Male Sterility and Disease Susceptibility," Science. 250:942-947 (1990)).
  • T- cytoplasm corn was widely used for hybrid seed production and breeding to avoid hand or mechanical emasculation in the 1950s and the 1960s. It was the coexistence of large acreages of intensively planted T-cytoplasm corn and the sudden appearance of race T of C. heterostrophus that resulted in the epidemic of the disease in 1970. This discovery first opened the door to understanding pathogenesis by C. heterostrophus.
  • T-toxin production and high virulence on T-cytoplasm corn are controlled by a single genetic locus defined as Toxl (Leach et al., "Dominance at the Toxl Locus Controlling T-Toxin Production by Cochliobolus heterostrophus," Physiol. Plant Pafhol., 21 :327-333 (1982)).
  • T-toxin is required by C heterostrophus for its high virulence on T-cytoplasm corn. This hypothesis was first tested by the generation of induced T-toxin deficient mutants using different mutagenesis procedures. All mutants with a tight Tox" phenotype cause disease symptoms that are indistinguishable from those caused by race O when tested on both T and N-cytoplasm corn, suggesting that T-toxin is indeed a virulence factor (Yang et al., 1992; Lu et al., "Tagged Mutations at the Toxl Locus of
  • cutinase Oeser et al., "Pathogenesis by Cochliobolus heterostrophus Transformants Expressing a Gene Encoding Cutinase from Nectria haematococca,” Mol. Plant-Microbe Int.. 7:282-288 (1994)
  • polygalacturonase and xylanase Lyngholm et al., "Mutants oi Cochliobolus heterostrophus Deficient in Extracellular Enzymes," Fungal Genet.
  • heterostrophus is known to produce a nonhost specific toxin called ophiobolin (or cochliobolin), a C 5 sesterterpenoid compound, which is toxic to many organisms, including plants, bacteria, fungi and nematodes, there is no evidence that ophiobolins are involved in pathogenesis by C. heterostrophus or other phytopathogenic fungi. No other pathogenesis-related toxins have been isolated from C. heterostrophus so far, but studies on closely related Cochliobolus species and other phytopathogenic fungi suggest that pathogenesis by this group of fungi also involves peptide toxins.
  • peptide phytotoxins victorin, HC-toxin, AM-toxin, and enniatins
  • pathogenicity or virulence factors are all small cyclic peptides (4-6 residues), containing unusual amino acids or hydroxy acids, and they can be either host specific or non-host specific in terms of plant toxicity.
  • a number of peptide phytotoxins are believed to be synthesized nonribosomally.
  • peptide synthetases catalyzing the biosynthetic process (Laland et al., "The Protein Thiotemplate Mechanism of Synthesis for the Peptide Antibiotics Produced by Bacillus Brevis,” Essays in Biochemistry, 7:31- 57 (1973); Lipmann, "Bacterial Production of Antibiotic Polypeptides by Thiol- Linked Synthesis on Protein Templates,” Adv. Microbiol. Physiol., 21 :277-266 (1980)).
  • Peptide synthetases can catalyze biosynthesis of a variety of peptides. In terms of bioactivity, they can be antibiotics, enzyme inhibitors, plant or animal toxins and immunosuppressants (Stachelhaus et al., "Modular Structure of Peptide Synthetases Revealed by Dissection of the Multifunctional Enzyme GrsA.," Journal of Biological Chemistry. 270(11):6163-6169 (1995)). In terms of chemical structure, they can be either linear (i.e. ACV, the penicillin precursor and gramicidin) or cyclic (most are). The latter can be further classified into three subgroups: 1) The "standard" cyclic peptides (i.e.
  • gramicidin S tyrocidine, HC- toxin and cyclosporin
  • cyclic lactones i.e. destruxin
  • cyclic depsipeptides i.e. beauvericin and enniatin
  • a minimal amino acid-activating module must contain six core sequences, whose functions (except for core 1 ) have been proposed based on mutational analysis of several peptide synthetases. Core sequences 1-5 are grouped into an amino acid adenylation domain and core 6 is a thioester formation domain ( Figure 1 A). All bacterial peptide synthetase genes contain "type I modules” - the minimal amino acid activating modules which were previously called “type I domains"
  • Two fungal genes, acvA and HTS1 also have this modular structure.
  • two fungal genes, esynl and simA contain type II modules, in which an insertion (about 400 amino acids) is found between cores 5 and 6 of a normal type I module. This region contains a motif (VLE/DXGXGXG (SEQ. ID. No.
  • safB contains two type I amino acid activating modules.
  • One module has all six highly conserved core sequences, but another, believed to activate alanine (the first amino acid in the linear tetrapeptide precursor of saframycin Mxl), lacks core 5 and has a weakly conserved core 1 (Pospiech et al., "Two Multifunctional Peptide Synthetases and an O-methyltransferase Are Involved in the Biosynthesis of the DNA-Binding Antibiotic and Antitumour Agent Saframycin Mxl from Myxococcus xanthus " Microbiology, 142(4):741-746 (1996)) (Figure 1A). This suggests that some of the motifs in the amino acid adenylation domain are dispensable or not critical for domain function. It also raises the possibility that other variations might be found in yet unknown peptide synthetase genes.
  • T-toxin is a host-specific vimlence factor in Southern Com Leaf Blight (Yoder et al., "Molecular-Genetic Evaluation of Fungal Molecules for Roles in Pathogenesis in Plants," J. Genet.. 75(3):425- 440 (1996); Yoder et al., Cochliobolus spp. and Their Host-Specific Toxins, Carroll eds., The Mycota Vol.
  • C. heterostrophus needs additional factors, presumably general factors for pathogenesis to corn plants, since race O, which does not produce T-toxin, can be an effective com pathogen. Attempts to identify additional general factors required by C. heterostrophus for pathogenesis have been unsuccessful. Cloning and characterizing additional C. heterostrophus genes that control biosynthesis of novel fungal molecules involved in critical pathogenic processes may be important because it offers potential targets for the design of products that might interfere with the com plant infection process. The present invention is directed to achieving these objectives.
  • the present invention relates to an isolated nucleic acid molecule encoding CPSl peptide synthetase homologs.
  • the DNA molecule comprises a nucleotide sequence which hybridizes to a DNA molecule having a sequence as set forth in at least one of SEQ. ID. No. 2, SEQ ID No. 41, SEQ ID No. 43, or SEQ ID No. 45.
  • Another aspect of the present invention relates to a method for identifying inhibitors of a CPSl protein or polypeptide which involves providing the CPSl protein or polypeptide, contacting the protein with potential inhibitor compounds, determining peptide synthetase activity, and selecting compounds which decrease the peptide synthetase activity.
  • Still another aspect of the present invention relates to a method of imparting disease resistance to a plant by overexpressing a CPSl protein or polypeptide in the plant cell.
  • Figure 1 provides the structure of amino-acid activating modules identified in peptide synthetase genes (adapted from Stachelhaus and Marahiel, 1995; Pospiech 1995; Marahiel, 1997).
  • Figure 1A shows the domain arrangements in two types of modules. Structural variations in the first module (safBl) of the gene safB are also indicated below type I.
  • Figure IB shows the correlation between module types and the nature of residues in two fungal peptides. Open box: type I module; filled box: type II module. Each peptide sequence is given below.
  • Figure 2 depicts the recovery of DNA flanking the REMI vector insertion site (arrows) in mutant R.C4.2696. Circled numbers indicate restriction enzyme sites used for recovery of each plasmid.
  • p214B7 contains 4.2 kb flanking DNA (3.4 left; 0.7 right);
  • p214Ml contains 0.1 kb left flank that overlaps with p214B7 and 1.1 kb right flank that overlaps with p214S 1 , which contains 3.2 kb flanking DNA on the left only.
  • Figures 3A-B illustrate the extension of the tagged sequence by targeted integration and plasmid rescue.
  • Figure 3A shows a general scheme illustrating chromosome walking strategy (only one direction is indicated).
  • "X” indicates a restriction enzyme site used for recovery of vector (indicated by "T") with flanking genomic DNA ("L” for left flank; “R” for right flank; R' for a fragment from "R” that is subcloned into a subsequent transformation vector).
  • Each integration site is indicated by a vertical arrowhead pointed at the selectable marker on the vector.
  • Overlapped flanking DNA is represented by bars drawn in the same pattern.
  • Figure 3B shows two strategies used for construction of a targeted integration vector.
  • a genomic DNA fragment (indicated by the letter “a") is subcloned into a vector directly and the subsequent integration occurs by a single crossover ( “a” is duplicated).
  • “a” is subcloned into a vector via multiple steps (see Figure 6 for an example) that allows linearization with restriction enzyme "Y” and the subsequent integration occurs by a double crossover resulting in a simple insertion.
  • Figure 4 shows how the targeted integration vector p214SNP was constructed.
  • the sites for restriction enzyme digestion are indicated by arrows.
  • p214SNP was originally designed for a double crossover integration after digested with 5stXI and BcR, but it was found later that the E. coli strain (DH5 ) used for amplification of the plasmid is Dam + which blocks the BcR site (TGA TCA to TG m TCA).
  • the plasmid was linearized with .SstXI only and used for transformation.
  • Figure 5 depicts the recovery of genomic DNA flanking the targeted integration vector p214SNP in transformant #118.
  • the vector integrated into the target site by a single crossover that resulted in duplicated Hindlll-Sac fragments (indicated by letter b and b ).
  • the second genomic DNA fragment carried by the vector is also duplicated in the genome (N ⁇ rl-Hwdlll, indicated by letter a and a').
  • Genomic D ⁇ A was digested with BgRl (which does not cut the vector) or BcR (which cuts once in fragment a' on the vector) as indicated by numbers in a circle.
  • the two recovered plasmids carry sequences that extend the right flank only by 170 and 800 bp respectively beyond the genomic D ⁇ A on P 214S1.
  • FIG 6 shows how the targeted integration vector pi 18BSP was constructed.
  • pi 18BSP was linearized with BgRl and transformed into a wild type C. heterostrophus strain.
  • Figure 7 depicts the recovery of genomic DNA flanking the targeted integration vector pi 18BSP in transformant #9. The vector integrated into the target site by a double crossover. Genomic DNA was digested with Pstl and relegated.
  • the recovered plasmid p9P2 includes the entire pUC18 sequence on pi 18BSP and 4.6 kb of genomic DNA that contains all of ORF1 (CPSl), including the stop codon (TAG) and 3.0 kb of genomic region 3' of the stop codon.
  • Figure 8 shows how the targeted integration vector pi 18BCS was constructed.
  • the sites for restriction enzyme digestion are indicated by arrows.
  • Figure 9 depicts the recovery of genomic DNA flanking the targeted integration vector pi 18BSP in transformant #12.
  • the vector integrated into the target site by a single crossover that resulted in a duplicated Sspl-Sspl fragment in the transformant genome (indicated by the letter "a").
  • Genomic DNA was digested with Hindlll (arrows) and ligated.
  • the recovered plasmid pl2H6 contains the entire pi 18BSP sequence and a 2.1 kb genomic DNA (Sacl-Hindill) on the left region that overlaps with the sequence carried on p9P2 (see Figure 7).
  • Figures 1 OA-B are photographs which show that the REMI mutant R.C4.2696 grows like wild type in culture ( Figure 10A) and produces normal appressoria ( Figure 10B).
  • Figure 10A plates containing complete medium (CM) were inoculated with a conidia-bearing mycelium plug and incubated at 22°C under warm white light (F40/350BL) (Sylvania Inc., Danvers, MA). The photograph in Figure 10A was taken 6 days after inoculation. Left to right: Mutant R.C4.2696; wild type.
  • Figure 10B conidia of mutant (left) or wild type (right) were placed in a drop of water and incubated at 32°C for 6 hrs. No significant difference in percentage of appressorium-forming conidia (arrows) was detected.
  • FIGS 11 A-B are photographs which show that the REMI mutant R.C4.2696 produces wild type levels of T-toxin ( Figure 1 IB) but has reduced vimlence on T-cytoplasm com ( Figure 11 A).
  • Figure 11 A two week old T- cytoplasm com plants were inoculated with conidial suspensions of (left to right) mutant R.C42696, wild type race T, and a Tox+ mutant progeny from a cross between R.C4.2696 and a race O tester, and symptoms (represented by three diseased leaves for each strain) were recorded 5 days after inoculation. Leaves infected by wild type race T collapsed (arrow) but those infected by mutants remained alive.
  • Figure 12 is a photograph which displays that the REMI mutant
  • R.C4.2696 produces lesions much smaller than wild type on N-cytoplasm com.
  • Two week old N-cytoplasm com plants were inoculated with conidial suspensions at the same concentration and symptoms were recorded 7 days after inoculation.
  • the mutant (right) produces the same number of lesions as wild type (left) but the size of lesions was dramatically reduced compared to wild type (arrows).
  • Figures 13A-B show that the REMI mutant R.C4.2696 has a 60% reduction of vimlence compared to wild type.
  • lengths of 100 typical lesions from com leaves inoculated with wild type race O and a mutant progeny R45 (Tox ⁇ , hygB R ) carrying the R.C4.2696 mutation were measured 7 days after inoculation and values plotted.
  • Figure 13B shows the statistical analysis that 86% of the mutant lesions are less than 4 mm in length (average size 3.5 mm), 60% reduced compared to that of wild type (8.5 mm).
  • Figures 14A-B illustrate the genetic analysis showing that a tagged, single site mutation is responsible for the mutant phenotype.
  • Figure 14A is a photograph of a plant assay where N-cytoplasm com was inoculated with parents and progeny indicated in Figure 14B (a complete tetrad from the second cross is shown here; random spore assay for the two crosses gave the same results).
  • Figure 14B is a table which shows progeny segregation data.
  • progeny segregated 1 : 1 for parental type only and all hygromycin B resistant progeny showed the same small lesion phenotype (represented by progeny 1-4 in Figure 14 A) as the mutant parent (parent 1 in Figure 14A); all hygromycin B sensitive progeny show the large lesion phenotype (represented by progeny 5-6 in Figure 14A) of the wild type parent (parent 2 in Figure 14A).
  • Figure 15 is a restriction map of the cloned sequences surrounding the tagged site.
  • a 11.3 kb genomic region (thick line) was cloned and completely sequenced.
  • the original REMI insertion point in the mutant R.C4.2696 is indicated by a vertical arrow.
  • the asterisks indicate two targeted integration sites in the wild type genome.
  • Two open reading frames (in opposite directions), ORF1 (CPSl, 5.4 kb) and ORF2 (TES1, 1.1 kb) are indicated by open boxes below the map (the positions of putative introns are indicated by vertical bars).
  • Locations of seven overlapping plasmid clones used for sequencing are indicated by thin lines on the top of the map (filled triangles represent the vector sequence in each clone). Sequencing strategy is indicated by arrows above each clone line.
  • Figure 16 graphically depicts the G+C content of the cloned sequences surrounding the tagged site.
  • the 11.3 kb of genomic DNA sequence ( Figure 15) was examined as 200 bp fragments and G+C content of each fragment was calculated and plotted. Regions A and C have normal G+C content; regions B and D show high G+C content. The overall percentage of G+C in each region is indicated by underlined numbers.
  • the positions of ORFl (CPSl) and ORF2 (TES1) are indicated by arrows and the percentage of G+C of each ORF is given below (in parentheses).
  • Figure 17 displays the nucleotide sequence oiCPSl. 5,725 base pairs from the 11.3 kb sequenced region ( Figure 15) are shown. The deduced amino acid sequence of CPSl protein is given below the DNA sequence. The position of start codon ATG (bold and underlined) is designated +1 and the open reading frame stops at position 5381 (TAG, in bold and underlined). Five putative "CAAT" boxes (bold and indicated by asterisks) are found at the positions -36, - 58, -67, -172 and -309.
  • Three putative introns are located at positions 2070-2114, 3542-3592 and 4197-4249. conserveed core sequences are shaded.
  • the GXSXG motif is boxed.
  • a putative polyadenylation signal is located at position 5604 (bold and overlined).
  • Figure 18 displays the nucleotide sequence oi TESl. 1,901 base pairs 5' oiCPSl are shown. The deduced amino acid sequence of the TES1 protein is given below the DNA sequence.
  • the CT motif (bold and indicated by asterisks) is found at position -46.
  • a "AT"-rich region (bold and italicized) is found at position -12.
  • the position of ATG start codon (bold and underlined) is designated +1 and the open reading frame (transcribed in the opposite direction from CPSl) stops at position 1153 (TGA, in bold and underlined).
  • One putative intron (in lowercase, border sequences are bold; branch sites are underlined) is located at position 518-566.
  • a putative polyadenylation signal is located at position 1345 (bold and overlined).
  • the putative active site (VHS) is shaded.
  • Figures 19A-C are schematic representations which show the characterization of modular structure oiCPSl.
  • Peptide synthetase and thioesterase are indicated by open boxes; shaded boxes inside indicate functional domains and modules; vertical bars in the shaded boxes indicate highly conserved core sequences.
  • Figure 19A illustrates the general structure of bacterial and fungal peptide synthetases (adapted from Marahiel 1997, which is herein incorporated by reference). A peptide synthetase gene cluster is shown on the top.
  • amino acid activating module cyclosporine synthetase has 11
  • some peptide synthetases have thioesterase domains (TE), which can be either integrated into modules or encoded by a separate gene.
  • TE thioesterase domains
  • Each synthetase can have type I, type II or both modules.
  • a type I (minimal) module is enlarged to show organization of core sequences and domains.
  • Some peptide synthetases also have condensation or epimerization domains.
  • Figure 19B illustrates the organization of saframycin Mxl synthetase containing 4 amino acid activating modules (Pospiech et.
  • TES 1 is a thioesterase encoded by a separate gene (TES1).
  • Figures 20A-F are comparative alignments of core amino acid sequences in CPSl A and CPS1B with those of other peptide synthetases.
  • Figures 20A-E are comparative amino acid sequence alignments of amino-acid-activating domains (cores 1-5);
  • Figure 20F is a comparative amino acid sequence alignment of the thioester formation domain (core 6).
  • the first column shows the names of peptide synthetases; the second indicates the position of the first residue aligned in the original amino acid sequence of each protein; the last column on the right indicates the number of amino acids between two cores ( Figures 20A-E, in parentheses) or the distance between two adjacent amino-acid- activating modules ( Figure 20F, in parentheses).
  • SafBl the first module in saframycin Mxl synthetase B of Myxococcus xanthus (Genbank accession U24657); GrsA: gramicidin S synthetase A oi Bacillus brevis (SWISS PROT accession P14687); HTS1 A and HTS1B: the first two modules in HC-toxin synthetase of Cochliobolus carbonum (Q01886); EsynA and EsynB: two modules in enniatin synthetase oi Fusarium scirpi (EMBL accession Z 18755); ACVA and ACVB: the first two modules in ACV synthetase of Aspergillus nidulans (SWISS PROT PI 9787); CsynA and CsynB: the first two modules in cyclosporine synthetase of Tolypocladium nivenm
  • Figure 21 is a comparative alignment of amino acid sequences of active sites of thioesterase domains (TE) in CPSl with those of other peptide synthetases.
  • ACV ACV synthetase (Swiss-PROT accession P19787); GrsB: gramicidin S synthetase B (PI 4688); GrsT: the thioesterase encoded by grsT (P 14686) in gramicidin S synthetase gene cluster; SrfA: surfactin synthetase A-3 (Q08787); TycC: tyrocidine synthetase C (Genbank accession AF004853); TycF: the thioesterase encoded by tycF (AF004853) in the tyrocidine synthetase gene cluster.
  • GXSXG The highly conserved residues (GXSXG) are indicated by asterisks.
  • the number on the left of each amino acid sequence indicates the original position of the first residue; the number on the right (in parentheses) indicates the distance between the last residue shown to the end of each protein.
  • Figure 22 is a comparative alignment of the amino acid sequence of the TES1 protein (CH-TES1) with that of other type II thioesterases.
  • HS-TEII Homo sapiens thioesterase II (EMBL accession X86032)
  • EC-TESB E. coli acyl- coA thioesterase II (Genbank accession M63308)
  • MT-TESB Mycobactrium tuberculosis homolog to E. coli.
  • acyl-coA thioesterase II (EMBL Z95387).
  • the identical residues in all four proteins are in bold.
  • the putative active site VHS motif is indicated by asterisks.
  • the numbers on the right column indicate the original position of the last residue of the line in each protein sequence.
  • the entire protein sequence of each TES was aligned using the Jotun Hein Method. Amino acids corresponding to the positions 142-171, 236-265 and 356-367 in CH- TES1 have no significant similarity among the four proteins.
  • Figures 23A-B are photographs displaying a plant assay ( Figure 23A) and a gel blot ( Figure 23B). Targeted gene disruption suggests that CPSl is involved in fungal pathogenesis.
  • Figure 23 A N-cytoplasm corn was inoculated with (left to right): The REMI mutant R.C4.2696; wild type race T; wild type race O and five disruptants obtained using the linearized p214B7 as vector. All disruptants give the small lesions similar to the original mutant.
  • Figure 23B total genomic DNA was digested with BgRl and probed with both 5' and 3' end flanking DNA fragments carried on p214B7 separately (strain order is the same as above).
  • a single band (4.2 kb) is present in both wild type race T and race O but replaced by a 9.3 kb band (increased by the size of the vector, 5.1 kb) in all strains that showed the mutant phenotype in Figure 23 A.
  • Figure 24 displays a gel blot analysis showing targeted integration of the chromosome walking vector p214SNP into the wild type genome.
  • Lane 1 wild type race O.
  • Lanes 2-8 seven transformants obtained by transforming wild type race O (strain C5) with p214SNP.
  • Genomic DNAs were digested with BgRl and probed with 3.2 kb flanking genomic DNA fragment cloned on p214Sl which gives three bands in wild type (4.2, 2.1 and 0.5 kb, indicated by arrows on the left).
  • the targeted site is in the 2.1 kb fragment which is missing in all disruptants (indicated by an arrow on the right). Other two bands are intact as predicted.
  • disruptants showed the same mutant phenotype in the plant assay as shown in Figure 23.
  • Five disruptants (lanes 2, 3, 5, 7 and 8) resulted from a single crossover integration. Integration in two other disruptants (lanes 4 and 6) has not been determined.
  • One of the disruptants (#118, lane 2) was used to recover the plasmid pi 18B14 and pi 18BC4.
  • Figure 25 displays a gel blot analysis showing targeted integration of the chromosome walking vector pi 18BSP into the wild type genome.
  • Lane 1 wild type race O
  • Lane 2 wild type race T
  • Lanes 3, 4 and 5 three transformants obtained by transforming wild type race O (strain C5) with pi 18BSP.
  • Genomic DNAs were digested with Pst I (which cuts pUCATPH) and probed with the 3.2 kb flanking genomic DNA fragment cloned on p214Sl which gives two bands in wild type (6.6 and 2.5 kb, indicated by arrows on the left).
  • the targeted site is in the 6.6 kb region which is missing in all three disruptants (indicated by arrows on the right).
  • the 2.5 kb band is intact as predicted.
  • the third band (part of vector plus 4.6 kb genomic DNA) does not hybridize to the probe. All disruptants showed the same mutant phenotype in the plant assay as shown in Figure 23. Two disruptants (lanes 3 and 4) resulted from a double crossover integration. Integration in the third disruptant (lane 5) has not been determined. One of the disruptants (#9, lane 3) was used to recover the plasmid p9P2.
  • Figure 26 displays a gel blot showing the detection of CPSl homologs in C. victoriae and C. carbonum.
  • Genomic DNAs were digested with BgRl and probed with the 3.4 kb CPSl fragment cloned on p214B7 ( Figure 2) which includes most of the 4.2 kb BgRl fragment oiCPSl ( Figure 15).
  • Lanes 1 and 2 C. heterostrophus race T (C4) and race O (C5), both of which hybridized to ' the 4.2 kb fragment.
  • Lanes 3 C. victoriae (Hvw).
  • Lanes 4, 5 and 6 C. carbonum race 1 (26R13), race 2 (YugY) and race 3 (BZ1209). Note that both C. victoriae and C. carbonum (three races) hybridized to a 5.0 kb fragment and the hybridization signals of all three species showed about the same intensity.
  • Figure 27 is a high-performance liquid chromatography (HPLC) profile of culture extracts from wild type C. victoriae (HvW, top left) and three transformants (Tx7, Tx2 and Tx9). The major peak for victorin C is indicated by arrowheads. No significant differences in victorin production were detected between the wild type and transformants. Three other transformants (Tx4, Tx5 and Tx8) gave the same results.
  • HPLC high-performance liquid chromatography
  • Figure 28A-B are photographs which display a plant assay ( Figure 28 A) and a gel blot ( Figure 28B), showing targeted disruption of the CPSl homolog in C. victoriae.
  • Figure 28 A shows C. victoriae transformants (Tx) with reduced or wild type pathogenicity to susceptible oats. Oat seeds were inoculated with conidial suspensions of (left to right) wild type, Tx7, Tx2 and Tx9 (the last pot on the right is the uninoculated control).
  • Two transformants (Tx7 and Tx2) showed dramatically reduced pathogenicity as indicated by the substantial growth of the oat plants. These two transformants resulted from a homologous integration (lanes 2 and 3 in Figure 28B).
  • Tx9 which killed all oat plants as wild type, resulted from a ectopic integration (lane 4 in Figure 28B). All three transformants produced wild type level of victorin as determined by HPLC analysis ( Figure 27).
  • Figure 28B shows disruption oiCPSl homolog in the wild type genome. Genomic DNAs were digested with BgRl and probed with the 3.2 kb CPSl fragment (Kpnl-Sacl) cloned on p214Sl ( Figure 2) which hybridized to two fragments (4.2 and 2.2 kb, see Figure 15) in C. heterostrophus (lane 1), but to three fragments (5.0, 1.8 and 0.2 kb) in the C. victoriae (lane 2).
  • Tx7 (lane 3) and Tx2 (lane 4), one or two of the wild type fragments was replaced by a larger fragment (8.0 kb for Tx7 and 9.4 kb for Tx2) containing the transforming vector.
  • the actual size increase by the vector integration can not be predicted because the presence of polymorphic bands in C. victoriae genome and the presence of duplicated CPSl fragments on the transforming vector ( Figure 5).
  • Tx9 which caused wild type symptoms shown in Figure 28A, all three wild type bands (indicated by arrows) are intact, confirming an ectopic integration.
  • Figure 29 shows the REMI vector pUCATPH.
  • This vector was constmcted by insertion of a 2.4 kb SaR fragment containing the selectable marker cassette (the largest arrow) from pDH25 (Cullen et al., 1987) into the SaR site of polylinker site of pUC18 (between lad and lacZ). Only six-base-pair restriction enzyme sites are shown on the map. Italicized sites are unique; three of them, H dlll, Kpnl, and Sad (bold) have been used for REMI transformation. Non- cutting enzymes are listed below the map (enzymes that recognize six-base-pair sites are underlined), amp, Ampicillin resistance gene; hygB, hygromycin B resistance gene; PtrpC, A. nidulans trpC promoter; TtrpC, A. nidulans trpC terminator; ori, Escherichia coli origin of replication.
  • Figures 30A-30C are photographs of DNA gel blots showing DNA-DNA hybridization of ChCPSl to other fungal genera and species.
  • the gel was loaded with Cochliobolus species (lanes 1-17) as follows: C. heterostrophs race T, race O; C. carbonum race 1, race 2; C. victoriae isolates FI3, HvW; C. bicolor, C. dactyloctenii, C. chloridis, C. homomorphus, C. intermedius, C. melinidis, C. melinidis, C. peregianensis, C. perotidis, C. ravenelii and C. sativus.
  • Figure 30B is a photograph of a DNA gel blot from a gel loaded with other Ascomycete genera (lanes 1-14) as follows: C. carbonum racel (control), Setosphaeria rostrata, Stemphyllium spp., Pyrenophora tritici repentis, Bipolaris sacchari, Alternaria spp., A. solani, Nectria haematococca, Fusarium oxysporum, Glomerella spp. Magnaporthe grisea, F. moniliforme, F. moniliforme (repeat) and A. solani (repeat).
  • Figure 30C is is a photograph of a DNA gel blot from a gel comparing Candida albicans to C. heterostrophus and closely related species (lanes 1-7): C. heterostrophs race T, Bipolaris sacchari, Setosphaeria rostrata, Stemphyllium spp., Pyrenophora tritici repentis, Alternaria spp. and Candida ⁇ /&/ ⁇ ws(arrowhead).
  • Genomic DNAs were digested with Hind ⁇ ll (A, lanes 1-17; B, lanes 1-11; C, lanes 1-7), Xhol (B, lanes 12 and 14) or BgRl (B, lane 13) and probed with the 3.2 kb fragment oiCPSl from p214Sl (Fig. 2) at high stringency. Weak signals in lanes 3 and 17 (panel A) are due to insufficient DNA loading (confirmed by a repeat experiment).
  • Figure 31 A is a structural comparison of the ioufyCPSl homologs to ChCPSl .
  • ORFs are indicated by the open boxes; shaded boxes inside indicate functional domains; vertical bars indicate conserved motif sequences found in nonribosomal peptide synthetases (NRPS) as defined by Stachelhaus and Marahiel (Stachelhaus and Marahiel, 1995; Marahiel, 1997) (dashed bars indicate weak conservation).
  • the black bulbs indicate the position of putative introns.
  • Cores 1-5 adenylation; core 6: thioaltion; TE: thioesterase. The distance between core sequences is not drawn in exact scale.
  • the name of proteins is on the left of the ORF boxes and the number of amino acids on the right.
  • the unidentified regions oiAsCPSl and PtCPSl are indicated by dash-lined boxes.
  • the similarity to ChCPSl(i ⁇ the overlapping region only, see text for details) is given in the parentheses under the protein names in the order: nucleotide identity/ amino acid identity/ amino acid similarity.
  • the positions of the ChCPSl amino acid 1040 is indicated by the open arrow; the positions 511 and 1269 (to the first and the last amino acids of AsCPSl and PtCPSl) are indicated by filled triangles.
  • Figure 3 IB is an amino acid alignment of the four CPSl homologs to ChCPSl . 530 amino acids aligned to the amino acids 511-1040 of ChCPS 1 (shown in A) are shown.
  • Figure 32 is the nucleotide sequence oiFgCPSl. 6,003 base pairs cloned using the plasmid rescue procedure are shown.
  • the amino acid sequence of FgCPSl protein is given below the DNA sequence.
  • the position of the start codon ATG (bold and underlined) is designated +1 and the open reading frame stops at position 5123 (TGA, bold and underlined).
  • a "CT” motif (italicized and underlined) and two putative "CAAT” boxes (bold with asterisks) are found at positions -30, -204 and -302.
  • a putative intron in lowercase with 5' and 3' splice sequences in bold; branch sites underlined is located at positions 4245-4290.
  • Figure 33 shows the nucleotide sequence oi AsCPSl. 2,369 base pairs amplified by PCR are shown. The amino acid sequence of AsCPSl protein is given below the DNA sequence. The sequence is not complete. Two putative introns (in lowercase with 5' and 3' splice sequences in bold; branch sites underlined) are located at positions 540-584 and 2012-2059. Conserved core sequences are shaded and the putative cyclization domain motif "DXXXXDXXS" (positions 694-720 ) is underlined. The PCR primer binding sites at the 5' and 3' end are underlined.
  • Figure 34 shows the nucleotide sequence oi PtCPSl. 2,320 base pairs amplified by PCR are shown. The amino acid sequence of PtCPSl protein is given below the DNA sequence. The sequence is not complete. A putative intron (in lowercase with 5' and 3' splice sequences in bold; branch sites underlined) is located at positions 540-583. conserveed core sequences are shaded and the putative cyclization domain motif "DXXXXDXXS" (positions 693-719) is underlined. The PCR primer binding sites at the 5' and 3' end are underlined.
  • Figure 35 A is a photograph of a DNA gel blot showing that the 2.2 kb wild type band (arrowhead) is disrupted in homologous transformants
  • Genomic DNAs were digested with Clal and probed with pFgC8 which carries a 1.0 kb FgCPSl fragment.
  • Figure 35B is a photograph of plants from a vimlence assay showing F. graminearum transformants (Tx) with reduced or wild type vimlence to wheat.
  • Wheat heads were inoculated with conidial suspensions (10 4 /ml) of (left to right) wild type, TxFgC8-4, -10, -HI, and -11 or with water only. Photograph was taken 7 days after inoculation. Note that most spikelets of wheat heads inoculated with homologous transformants looked "healthy” in contrast to those inoculated with ectopic transformants that were completely “bleached” (indistinguishable from wild type).
  • the present invention generally relates to an isolated DNA molecule from a plant pathogen encoding a CPS 1 peptide synthetase.
  • the DNA molecule has a nucleotide sequence which hybridizes to a DNA molecule having a sequence corresponding to SEQ. ID. No. 2 as follows: TGCCTGCGCC TGTGCTTGTG CCTGTGGAAT GTCGCGGCCC GCTGCTGCAT AGCCTATCTG 60
  • CAGCAGAACC CACCCCAACC ATCCAGTGAG GGCTCTCGCT CCCGCACCGC ATCCTTTGCT 420
  • CAACAGGATC CAATGGCTAC AAGAAACTTC AAGAAAAACA CAGAACCCAA CTTCGCCTCC 2040
  • GGACTGTACG AAGATCGCAT CAGACAGCGT GTTGAATGGG TAGAAAATGG TCAGCTTGAA 2820 GCCGAGCATC GATACTTTTT TGTGCAGCAC CTGGTCACAA GCATTATGAA GGCCGTGCCA 2880
  • the plant pathogen is Cochliobolus heterostrophus.
  • the plant pathogen is Cochliobolus carbonum, C. victoriae, C. sativus, C. specifer, C. homomorphus, C. dactyloctenii, Setosphaeria turcica, S. rostrata, or Bioplaris sacchari.
  • the peptide synthetase of SEQ. ID. No. 2 has a deduced amino acid sequence corresponding to SEQ. ID. No. 3 as follows:
  • an isolated nucleic acid molecule encoding a CPS 1 peptide synthetase homolog and which hybridizes to a nucleic acid molecule having a sequence corresponding to SEQ ID No. 41 as follows :
  • CTACTACATA TCATTGCTGA CTTCAAGGTC AAGGCTATCC TCGTCAATGC TGGCGTAGAC 1440 CACCTGATGA AGGTCAAGCA AGTATCGCAG CACATCAAAC AGTCAGCAGT CATTCTCAAG 1500
  • GCTATTGCAC GAGGTGCTGG CAAGAACATG GCTCTGCACG AGCTCAAGAA CCTCATGATC 1980 GCGACTGACG GTCGGCCGCG CGTAGACGTC TGTAAGTGTT GCGATCCTGT ATAAGCATCT 2040
  • the CPSl nucleic acid molecule which hybridizes to a nucleic acid molecule having a sequence as set forth in SEQ ID No. 41 is from the plant pathogen Altenaria solani.
  • the CPSl gene is from a plant pathogen such as Alternaia alternatherae, A. alternata, A. amaranthi, A. araliae, A. brassicae, A. brassicicola, A. camelliae, A. cassiae, A. cheiranthi, A. cinerariae, A. gossypii, A. helianthi, A. helianthinficiens, A. mali, or A. raphani.
  • the peptide synthetase product of SEQ ID NO:41 has a deduced amino acid sequence as follows (SEQ ID NO:42):
  • an isolated nucleic acid molecule encoding a CPS 1 peptide synthetase homolog and which hybridizes to a nucleic acid molecule having a sequence corresponding to SEQ ID No. 43 as follows :
  • GTTCTCTCGC TGGAGGATCG ACCACTGCTC ACCGCGACTC CCTCTTCTTC TCCCCCTCCC 600 ATCTCGAACC TGAAACCCGG ACAGGTACTA TGATGTCGGG CGACTATGCA TTCAGACCCG 660
  • AGGACTACCA ACGCTTGAAC CACATTCTTA CTACAACGCA GGCCCATCTA GCGCTGACCA 1380 CCGATAACAA CCTCAAAGCC TTTCAACGAG ACATTACTAC ACAAAAGTTG ACATGGCCAA 1440
  • TCGATCCCCG TCAGGGCATT GGCATGATTC TGAGCGTGCT ACTGACCGTC TACGGCGGCC 1800 ACACCACTGT TTGGTTCGAC AACAAAGCTG TTGATGTTCC TGGACTGTAC GCCCACCTCC 1860
  • GAGAAGAGCG CATGGGATGC CCATTAAAGC TTGAACTTGG GGAGGATACA GAGTCTGACG 2220
  • CAGCACTCGC AATACGAACA CCCACCACAA TCAGCCGGTT CTCAAGCACC AGCCCAGCTG 5460 AACCTTTCTC ACCAGCCCGA TCAAGGATTC GATATGGACT TTTCACGATA TAGTTCAGCA 5520
  • the CPSl nucleic acid molecule which hybridizes to a nucleic acid molecule having a sequence as set forth in SEQ ID No. 43 is from the plant pathogen Fusarium graminearium.
  • the CPSl gene is from a plant pathogen such as Fusarium avenaceum, F. carpineum, F. chlamydosporum, F. coccophilum, F. culmorum, F. episphaeria, F. equiseti, F. flocciferum, F. moniliforme, F. oxysporum, F. redolens, F. sambucinum, F. solani, F. subglutinans, F. trichothecioides, F. udum, or F. ventricosum.
  • the peptide synthetase product of SEQ ID NO:43 has a deduced amino acid sequence as follows (SEQ ID No. 44):
  • RVGSFGYPIP DATLSWDPE TGLLASPHSV GEIWVDSPSL SGGFWAQPKN
  • VKSFYGSKDA FDAERFDGRA LDGDPNIQYI RTGDLGFLHN VSRPIGPNGA QVDMQVLFVL 1380
  • PVIVNAILNE HQIIVDIVAF VNKGDFPRSR LGEKQRGKIL GGWVSRKLRT LAQFSIRDMD 1500 AESTAGDMMD PSRASMVSVR SGGGAAPGSS SLRNVEPAPQ ILEEEHDQMT PRHEYEAAPT 1560
  • an isolated nucleic acid molecule encoding a CPSl peptide synthetase homolog and which hybridizes to a nucleic acid molecule having a sequence corresponding to SEQ ID No. 45 as follows :
  • TTCTTCGTGC AGCACCTCGT CACCAGTATC ATGAAGGCTG TTCCCAAGAT CTACGACTGG 540 TAAGTCTTCT CATGTTTTAG ATGAGCGTTC TAACACTATG CAGCTCATCT TTCGACTCGT 600 ACGTCAATGG CGAATACCTG CCTATCATCC TCATCGAGAC ACAGGCTGCA TCGACAGCCC 660
  • CAGCGCCAAA CACATTACCA CGAGTTGTTA AGAATGGTCG ACGAGAAATT GGCAACATGC 840 TCTGTCGAAG AGAATTTGAT AATGGCTCAT TACCTTGTGT CCACGTCAAG TTTGGTGTTG 900
  • the CPSl nucleic acid molecule which hybridizes to a nucleic acid molecule having a sequence as set forth in SEQ ID No. 45 is from the plant pathogen Pyrenophora teres.
  • the CPSl gene is from a plant pathogen such as Pyrenophora avenae, P. bromi, P. leuceienes, P. phaeocomes, P. schroeteri, P. trichostoma, or P. tritici-repentis.
  • the peptide synthetase product of SEQ ID No:45 has a deduced amino acid sequence as follows (SEQ ID No. 46):
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid and polymers thereof in either a single or double stranded form.
  • nucleic acid also encompasses nucleic acids containing known analogs of naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof such as degenerate codon substitutions and complementary sequences.
  • nucleotide sequence refers to a polymer of DNA or RNA which may be single or double stranded and may contain synthetic, non-natural, or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • the terms “nucleic acid” , “nucleic acid molecule”, “nucleic acid fragment”, or nucleic acid sequence or segment”, may also be used interchangeably with the terms “gene”, “cDNA”, “DNA” and "RNA”.
  • DNA molecules of the present invention include DNA molecules that have a nucleic acid sequence which is more than 70% identical to the nucleotide sequence of SEQ. ID. Nos. 2, 41, 43, or 45. Nucleotide sequence similarity may be determined by the BLAST program with the default parameters (Altschul et al., "Basic Local Alignment Search Tool," J. Mol. Biol.. 215:403-410 (1990), which is hereby inco ⁇ orated by reference).
  • Preferred sequences include those DNA molecules which will hybridize to a nucleic acid molecule having the sequence of SEQ. ID No. 2, 41, 43, 45 or their compliments.
  • stringent conditions are selected to be about 50°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • the T m is dependent upon the solution conditions and the base composition of the probe, and may be calculated using the following equation:
  • Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase.
  • wash conditions are typically performed at or below stringency.
  • suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are asas set forth above. More or less stringent conditions may also be selected.
  • level of stringency For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook, J., E.F. Fritsch, et al. 1989 "Molecular Cloning: a Laboratory Manual, 2 nd Edition, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, at 11.45.
  • An example of low stringency conditions is 4-6X SSC/0.1 -0.5% w/v SDS at 37°-45° C for 2-3 hours.
  • alternative conditions of stringency may be employed such as medium stringent conditions. Examples of medium stringent conditions include 1-4X SSC/0.25% w/v SDS at > 45° C for 2-3 hours.
  • An example of high stringency conditions includes 0.1-1X SSC/0.1% w/v SDS at 60 C for 1-3 hours.
  • the skilled artisan is aware of various parameters which may be altered during hybridization and washing and which will either maintain or change the stringency conditions.
  • another stringent hibridization condition is hybridization at 4X SSC at 65° C, followed by a washing in 0. IX SSC at 65° C for about one hour.
  • an exemplary stringent hybridization condition is in 50% formamide, 4XSSC, at 42° C.
  • Still another example of stringent conditions include hybridization at 62° C in 6X SSC, .05X BLOTTO, and washing at 2X SSC, 0.1% SDS at 62° C.
  • proteins or polypeptides of the present invention include polypeptides that have an amino acid sequence having at least 75 % similarity to the amino acid sequence of at least one of SEQ. ID. No. 3, SEQ ID No. 42, SEQ ID No. 44, or SEQ ID No. 46.
  • the protein or polypeptide will have at least 90% similarity with at least one of SEQ. ID No. 3, SEQ ID No. 42, SEQ ID No. 44, or SEQ ID No. 46.
  • Protein sequence similarity may be determined by the BLAST program with the default parameters (Altschul et al., "Basic Local Alignment Search Tool," J. Mol. Biol..
  • the CPSl protein of SEQ. ID. No. 3 has a molecular weight of about 190-200 kDa, preferably 193.2 kDa.
  • the CPSl protein contains two structurally similar modules, both of which are similar to SafBl, the first module of saframycin synthetase B (overall 25% identity; 50% similarity) and have apparent amino-acid-activating and thiolation domains with core sequences conserved in known peptide synthetases.
  • the DNA molecule encoding a CPSl protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e., not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding- sequences.
  • U.S. Patent No. 4,237,224 to Cohen and Boyer which is hereby inco ⁇ orated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase.
  • recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eukaryotic cells grown in culture.
  • Recombinant genes may also be introduced into vimses, such as vaccinia vims.
  • Recombinant vimses can be generated by transfection of plasmids into cells infected with vims.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby inco ⁇ orated by reference), pQE, pIH821 , pGEX, pET series (see Studier et.
  • viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325,
  • host- vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used.
  • Host- vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with vims (e.g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i.e. biolistics).
  • the expression elements of these vectors vary in their strength and specificities. Depending upon the host- vector system utilized, any one of a number of suitable transcription and translation elements can be used.
  • RNA transcription and messenger RNA Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.
  • the DNA sequences of eukaryotic promoters differ from those of procaryotic promoters.
  • eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eukaryotic cells.
  • SD Shine-Dalgarno
  • This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein.
  • the SD sequences are complementary to the 3 '-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome.
  • Promoters vary in their "strength" (i.e., their ability to promote transcription). For the pu ⁇ oses of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E.
  • promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters of coliphage lambda and others, including but not limited, to / ⁇ cUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lac ⁇ JV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters of coliphage lambda and others, including but not limited, to / ⁇ cUV5, ompF, bla, Ipp, and the like, may be used to direct high levels
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced.
  • the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
  • the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside).
  • IPTG isopropylthio-beta-D-galactoside.
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
  • the DNA expression vector which contains a promoter, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno ("SD") sequence about 7- 9 bases 5' to the initiation codon ("ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed.
  • Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving inco ⁇ oration of synthetic nucleotides may be used.
  • the present invention also relates to anti-sense nucleic acid for essential cell proteins, such as replication proteins, which serve to render the host cells incapable of further cell growth and division.
  • Anti-sense regulation has been described by Rosenberg et al., "Production of Phenocopies by Kruppel Antisense RNA Injection Into Drosophila Embryos," Nature. 313:703-706 (1985); Preiss et al., "Molecular Genetics of Kruppel, A Gene Required for Segmentation of the Drosophila Embryo," Nature. 313:27-32 (1985); Melton, “Injected Anti-sense RNAs Specifically Block Messenger RNA Translation In vivo," Proc. Natl. Acad. Sci. USA.
  • Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like. In the present invention, the host cells may be from plants such as corn, oat, grass, weed, bamboo, and sugarcane.
  • One aspect of the present invention involves using an inhibitor of the CPSl protein to interfere with the plant infection process in order to impart disease resistance to plants.
  • the peptide synthetase CPSl could be inhibited by an appropriate drug, thereby causing the plants to be resistant to fungal attack.
  • the CPSl nonribosomal product could be degraded by an enzyme for which it is a suitable substrate, and, when the gene encoding this enzyme is genetically engineered into plants, the plants will become resistant to fungal attack.
  • large numbers of compounds can be screened for their activity as inhibitors of CPSl protein by a high-throughput screening assay as described in U.S. Patent No. 5,876,946 to Burbaum et al., which is hereby incorporated by reference.
  • a library of compounds is assayed for inhibition of an enzyme catalyzed reaction and the amounts of fluorescence bound to individual suspendable solid supports measured to determine the degree of inhibition. For example, the amount of fluorescence bound to a microbead in the presence of inhibitory compounds is greater than for non-inhibitory compounds.
  • the amounts of fluorescence bound to individual beads are determined by confocal microscopy.
  • inhibition can be determined of a peptide synthetase such as CPSl.
  • the substrate can be amino acids (or hydroxy acids), linked at one end to the microbead and at the other end to a fluorescent label.
  • the enzyme inhibitors can be utilized to impart fungal resistance to a variety of plants including oats, grasses, weeds, sugarcane, and corn in particular.
  • the present invention provides a method for identifying inhibitors of a CPSl protein , wherein said CPSl protein is a peptide synthetase of a plant pathogen.
  • the method comprises: providing a CPSl protein or polypeptide, contacting the protein or polypeptide with potential inhibitor compounds; determining peptide synthetase activity, and selecting compounds which decrease the peptide synthetase activity.
  • the method is especially useful in identifying inhibitors of a CPSl protein from plant pathogens of the genera Cochliobolus, Alternaia, Fusarium, and Pyrenophora such as those described hereinabove.
  • the method may be used to identify inhibitors of a CPSl protein from Alternaria solani, Fusarium graminearium, and Pyrenophora teres.
  • Another aspect of the present invention involves using one or more of the above DNA molecules encoding a CPSl protein or polypeptide or a gene encoding an enzyme that degrades the CPSl N.R.P. product to transform plants in order to impart fungal resistance to the plants.
  • This concept of pathogen-derived resistance is that host resistance to a particular parasite can effectively be engineered by introducing a gene, gene fragment, or modified gene or gene fragment of the pathogen into the host.
  • the procedure for making plants, for example, resistant to infection by one or more fungus involves isolating DNA coding for a gene such as CPSl of a fungus, operably linking the DNA within an expression vector, transforming the plant cell or plant tissue with the expression vector, and growing the transformed plant cells or plant tissue in the presence of the fungus such as e.g., Cochliobolus heterostrophus, Alternaria solani, Fusarium graminearium, or Pyrenophora teres, where the CPSl DNA is expressed as a gene product and the CPS protein disrupts the essential activity of the fungi.
  • a gene such as CPSl of a fungus
  • the present invention provides a method of imparting disease resistance to a plant by over-expressing a CPSl polypeptide in the plant, wherein the polypeptide has protein synthetase activity.
  • the plant may be any plant in which it is desired to impart disease resistance.
  • the plant may be an agrigultural crop or ornamental plant.
  • the plant may be herbacious or woody.
  • the plant may be a monocot or dicot. Examples of plants which may be used in practicing the present invention, include but are not limited to, com, oats, grasses, weeds, sugarcane, barley, wheat, rice, tomato, potato, citms, malus, rye, cotton, brassica, cabbage, and carrot.
  • plants which serve as hosts for Cochliobolus sp., Fusarium sp., Alternaria sp., and Pyrenophera sp. may be used. Reference to host plants may be conveniently made to Fungi on Plants and Plant Products in the United States, David, F. Farr et al. editors, American Phytopathological Society Press, St. Paul, Minnesota, 1989.
  • the CPSl peptide is from the genera Cochliobolus, Alternaria, Fusarium ,or Pyrenophora.
  • the CPSl gene is from Cochliobolus heterostrophus, Alternaria solani, Fusarium graminearium or Pyrenophora teres.
  • Promoters and other regulatory regions which function in plants are well known and include e.g., constitutive promoters, inducible promoters, temporally regulated and tissue specific promoters.
  • constitutive promters include e.g., actin, CAMV 35S, MAS, ubiquitin, rice cyclophilin, maize H3 histone, and actin 2.
  • tissue specific promoters include e.g., leaf specific promoters such as the RuBisCo ssu, Cab (chlorophyll a/b/binding) protein, and the AldP gene promoter from rice (Kagaya et al., 1995 Molecular and General. Genetics 248:668-614.
  • root specific promoters examples include e.g., beta tubulin (Oppenheimer et al. Gene 65:87, 1988), and SbPRPl (Suzuki et al., Plant Mol. Biol. 27:109-119, 1993.
  • beta tubulin Oppenheimer et al. Gene 65:87, 1988
  • SbPRPl Suzuki et al., Plant Mol. Biol. 27:109-119, 1993.
  • plants are transformed with a vector which replicates in a plant cell and which have a promoter which directs expression of the CPS gene product in the plant.
  • Methods of plant transformation are well known in the art.
  • a vector comprising a subject nucleic acid molecule coding for a CPS gene or fragment thereof may be introduced into a plant by leaf disk transforamtion-regeneration procedure as described by Horsh et al. (1985) Science 227:1229-1231.
  • Other methods of transformation such as protoplast culture (Horsh et al. 1984 Science, 225:496; DeBlock et al. (1984) Embo J. 2:2143; Barton et al. (1983) Cell, 52:1033) may also be used and are within the scope of this invention.
  • plants may be transformed Agrobacterium-de ⁇ ved vectors such as those described in Klett et al. (1987) Annu. Rev. Plant Physiol, 38:467.
  • Other well known methods are available to insert the subject CPS genes into plant cells.
  • Such alternative methods include biolistic approaches (Klein et al. 1987, Nature:311:10), electroporation, microinjection (Potrykus and Spangenberg eds., Gene Transfer to Plants, Springer Verlag, Berlin , 1995), chemically-induced DNA uptake, the use of vimses or pollen as vectors, liposome mediated transformation, transformation using wounded or enzyme-degraded immature embryos, or wounded or enzyme degraged embryonic callus.
  • transformation refers to the transfer of an exogenous nucleic acid molecule into a host cell.
  • the nucleic acid molecule may be stably or transiently introduced into the host cell and may be maintained non- integrated for example, as a plasmid, or alternatively, may be integrated into the host genome.
  • the resulting transformed plant cell(s) may then be used to regenerate a transformed plant via standard methods.
  • Tox + (Tox + ; MAT-2; hygB R ) is a C4-derived mutant generated using the REMI mutagenesis procedure (Lu et al., "Tagged Mutations at the Toxl Locus of Cochliobolus heterostrophus Using Restriction Enzyme-Mediated Integration," Proc. Natl. Acad. Sci. USA. 91 :12649-12653 (1994), which is hereby inco ⁇ orated by reference).
  • Strains 1301R33 (To ⁇ -, MAT-2; hygB R ), 1301R45 (Tox ⁇ ; MAT-1; hygB R ), 1301R26 (Tox + ; MAT-2; hygB R ) are progeny of the cross C5 X R.C4.2696.
  • Culture media including CM (complete medium), CMX (complete medium with xylose instead of glucose), CMNS (CM with salts omitted), and MM (minimal medium) have been described, as have mating procedures (Leach et al., 1982; Turgeon et al., "Transformation of the Fungal Maize Pathogen
  • CMX stable transformants were transferred to CMX containing the same drug but at a higher concentration (120 ug/ml) to compensate for reduced drug activity due to the inhibition by the salts in the medium.
  • Single conidia were picked up under a dissecting microscope and grown on CMNS hygromycin B plates; stable colonies were then transferred to individual CMX/ hygromycin B plates. All purified transformants were stored at -70°C in CM liquid medium containing 25% of glycerol in 96-well microtiter dishes. Bioassays. Fungal strains were grown on CMX plates (100 X
  • T-toxin-sensitive E. coli (DH5a) cells were evenly spreaded on LB medium containing ampicillin (100 ug/ml) and the plates were allowed to air dry for 30 min in a laminar hood. Agar plugs bearing fungal mycelia were inoculated (upside down) onto the E. coli cell lawn and the plates were incubated at 32°C. Wild type race T and race O were used as controls for each assay plate. T-toxin-producing strains of the fungus will inhibit growth of the E. coli cells and produce halos.
  • Tox ⁇ mutants can be distinguished from wild type by failure to produce a halo (tight) or by production of halos smaller (leaky) or larger than wild type (ove ⁇ roducing). All Tox ⁇ mutants were transferred to Fries medium (Pringle et al., "The Isolation of the Toxin oi Helminthosporium victoriae,” Phytopathology, 47:369-371 ( 1957), which is hereby inco ⁇ orated by reference), which optimizes toxin production, and retested. T-cytoplasm corn plants (inbred W64A) are used to verify the Tox ⁇ mutants identified from the E. coli assay using the procedure described below.
  • N-cytoplasm com plants (inbred W64A) grown in the green house (5-6 plants in one 4" X 6" pot) were inoculated with 5 ml conidial suspensions (10 5 conidia/ml) using a pressurized Preval Spray Gun Power Unit thin layer chromatography sprayer (Alltech Associates, Deerfield, IL), incubated in the mist chamber for 24 hours (23°C) and then taken to the growth chamber (23°C, 80% humidity, 14 hours of light).
  • the mutant phenotypes were determined by occurrence of apparent variations in disease symptom development, mainly by lesion size comparison. Mutants producing lesions smaller than wild type were retested and lengths of typical lesions from each mutant were compared with wild type 7 days after inoculation and measurements were taken for statistical evaluation.
  • Genomic and plasmid DNA preparation, restriction enzyme digestions, gel electrophoresis and gel blot analysis were done using standard protocols (Sambrook, et al., Molecular
  • RP reverse primer
  • FP forward primer
  • Primers designed to genomic DNA sequences are numbered in order. For stock tube and the notebook, Primers 1-17 have a leading number “214"; 18-20 with “118”; 21-29 with “9P2” and 30-31 with “9P5".
  • M13RMT(a M13R mutant version; there is a mutation in the polylinker of pUC18) and M13F-40 are provided by Cornell DNA Sequenceing Facility.
  • T ⁇ C primer site is in the pUCATPH TrpC promoter region 38 bp from SaR site with sequencing direction from SaR to Kpnl. b.
  • each primer corresponds to the assembled sequence (CPSl + TES1, total 11.3 kb found in Lu's folder in the computer Yoder labl) c.
  • Each primer sequence is given in the 5' to 3' direction d.
  • Original sequences that were used for primer design can be found in the CPSl sequence notebook or in Shunwen Lu's folder (CPSl sequence) in the computer Yoder labl under the same names as listed.
  • Genomic DNA of mutant R.C4.2696 was digested with BgRl, Mscl (no sites in pUCATPH) or Sacl (which cuts the vector once) and purified by phenol extraction and ethanol precipitation, then dissolved in TE (pH 8.0). Ligation was performed in 50 ul reaction mixture, containing 1 x T4 DNA ligase buffer with 10 mM ATP, 60 units T4 DNA ligase (New England Biolabs, Beverly, MA) and 3 ug of 5g/II-digested genomic DNA, at 14°C overnight.
  • p214B7 was amplified and plasmid DNA purified by equilibrium centrifugation in CsCl-ethidium bromide gradients (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 n Ed., Cold Spring Harbor, New York:Cold Spring Harbor Laboratory Press (1989), which is hereby inco ⁇ orated by reference).
  • 30 ug of plasmid DNA (linearized with BgRl for double crossover integration as illustrated in Figure 3) were used to transform wild type and the transformants were purified by isolation of single conidia, assayed for pathogenicity and characterized by gel blot analysis.
  • p214Sl was digested with N ⁇ rl and religated to create p214SlN, which was then digested with H dIII and ligated into the HmdIII site of pUCATPH to create p214SNP for transformation of race O (C5).
  • One transformant (Txl 18) resulting from homologous integration was used for plasmid rescue as described above.
  • Two new plasmids pi 18B14 and pi 18BC4 were recovered, both of which carry sequence at the 3' end but only 172 and 680 bp more than p214Sl, respectively ( Figure 5).
  • pi 18B14 was digested with Sacl and ligated into the Sacl site of pUCATPH to create pi 18BSP ( Figure 6).
  • This vector was transformed into wild type and one plasmid, p9P2 was recovered (from transformant Tx9), which extends 4.4 kb into the region 3' of pi 18BC4 and contains the 3' end oiCPSl ( Figure 7).
  • a third experiment was done in an attempt to recover a 15 kb Xhol fragment at the 3' end of that tagged gene, pi 18BCS ( Figure 8) was constructed by subcloning a 0.8 kb Sspl fragment into the same site of pUCATPHN.
  • mutant R.C4.2696 grows just like wild type with no variations in growth rate, color and mo ⁇ hological features ( Figure 10A). It produces normal conidia that germinate and form infection structures like wild type when induced on artificial surfaces ( Figure 10B) and shows normal mating ability when crossed to wild type testers. No pleiotropic phenotypes associated with the mutation have been detected so far.
  • the mutant differs from wild type in the ability to cause disease on com plants.
  • the mutant When tested on T-cytoplasm corn, the mutant produces race T type symptoms but the disease develops more slowly than with wild type although it produces wild type levels of T-toxin as detected in a microbial assay ( Figure 1 1), suggesting that the reduced vimlence is not related to a deficiency in the ability to produce T-toxin. This is clearer on N-cytoplasm corn where the mutant produces lesions significantly smaller than those produced by wild type ( Figure 12).
  • mutant phenotype When the mutant was crossed to a wild type race O tester, the small lesion phenotype and ability to produce T-toxin segregated independently, indicating that mutant phenotype is not associated with the reduced fitness trait tightly linked with the Toxl locus (Klittich et al., "Reduced Fitness Associated With Toxl of Cochliobolus heterostrophus," Phytopathology, 76:1294-1298 (1986), which is hereby inco ⁇ orated by reference).
  • the statistical evaluation of lesion size in the race O genetic background indicates that the mutation causes 60% reduction in the fungal vimlence to com plants (Figure 13).
  • the mutant phenotype is caused by a tagged, single site mutation.
  • progeny segregated 1 1 for parental types only and all hygromycin B-resistant progeny produced lesions similar to the mutant parent; all hygromycin B-sensitive progeny produced wild type lesions ( Figure 14), indicating that a tagged mutation is responsible for the reduced pathogenicity of the mutant.
  • EXAMPLE 3 Cloning and sequencing of DNA flanking the REMI vector insertion site.
  • ORFl The G+C content of ORFl is 51.5%, which is similar to most Cochliobolus genes (Turgeon et al., "Cloning and Analysis of the Mating Type Genes from Cochliobolus heterostrophus " Mol. Gen. Gene., 238(l-2):270-284 (1993); VanWert et al., "Structure of the Cochliobolus-heterostrophus
  • ORFl is flanked by two regions of G+C rich DNA. The first (1.4 kb, 60.7% G+C) is found between ORFl and ORF2; the second (1.2 kb, 60.3% G+C) is found 1.8 kb downstream of the stop codon of ORFl ( Figure 16).
  • ORF2 starts about 1.6 kb upstream of the start codon oi CPSl and is transcribed in the opposite direction ( Figure 15).
  • No "TATA" box-like element and CAAT box are found; instead, an AT-rich sequence "AAAACTAT” (SEQ. ID. No. 40) is located 11 bp upstream of the start codon ATG and a CT motif is found in the -30 region, which is characteristic of a number of fungal genes that lack a CAAT box in their promoter region (Gurr et al., "The Structure and Organization of Nuclear Genes of Filamentous Fungi," in Kinghorn, ed., Gene Stmcture in Eukaryotic Microbes, Vol.
  • ORF2 encodes a protein with high similarity to Homo sapiens thioesterase II (hTE, Liu et al., "Binding of HIV-1 Nef to a Novel Thioesterase Enzyme Correlates with Nef-Mediated CD4 Down-Regulation,” J. Biol. Chem.. 272(21 :13779-13785 (1997), which is hereby inco ⁇ orated by reference) and E. coli thioesterase II encoded by the tesB gene (Naggert et al., "Cloning, Sequencing and Characterization oi Escherichia-coli Thioesterase II," J. Biol. Chem., 266(17)11044-11050 (1991), which is hereby inco ⁇ orated by reference).
  • the nucleotide sequence of ORF2 (designated TES1) is given in Figure 18.
  • EXAMPLE 5 Modular structure of CPSl.
  • Predicted CPSl protein (1743 amino acids, M r 193235) contains two structurally similar modules, both of which are similar to SafBl, the first module of saframycin synthetase B (overall 25% identity; 50% similarity) and have apparent amino-acid-activating and thiolation domains but lack methyltransferase activity, thus appearing to be typical type I modules ( Figure 19).
  • the number of amino acids in each module is different: the first module (CPSl A) consists of 574 amino acids (from the first residue of core 1 to the last residue of core 6), which is larger than most type I modules; the second module (CPS IB) has 530 amino acids, which is average.
  • the distance between the two modules is 193 amino acids, much shorter than most peptide synthetases (500-600 aa), but this distance is not highly conserved, i.e., an opposite variation is found in HC-toxin synthetase and cyclosporine synthetase, both of which have about 1 ,000 aa between the first and second amino-acid-activating module ( Figure 20F).
  • Tolypocladium niveum is Encoded by a Giant 45.8-Kilobase Open Reading Frame," Current Genetics. 26(2): 120-125 (1994), which is hereby inco ⁇ orated by reference) and tyrocidine synthetase A (Mootz et al., "The Tyrocidine Biosynthesis Operon of Acillus brevis: Complete Nucleotide Sequence and Biochemical Characterization of Functional Internal Adenylation Domains," J.
  • CPSl In addition to amino- acid-activating and thiolation domains, CPSl also has an integrated thioesterase domain (TE) in the carboxy-terminal end of CPS IB ( Figure 19).
  • TE thioesterase domain
  • a signature sequence GXSXG which is highly conserved in animal fatty acid thioesterase type II enzymes and several peptide synthetases, is found in this domain ( Figure 21).
  • EXAMPLE 6 Sequence homology analysis of TES1 protein.
  • the predicted TES1 protein consists of 367 amino acids (M r 41013). Amino acid alignment of TES1 to hTE, TESB and Mycobacterium tuberculosis TESB homolog (Philipp et al., "An Integrated Map of the Genome of the Tubercle bacillus, Mycobacterium tuberculosis H37Rv, and Comparison with Mycobacterium leprae " Proc. Natl. Acad. Sci. USA. 93(7):3132-3137 (1996), which is hereby inco ⁇ orated by reference) showed that these proteins have an overall 40% identity and 60% similarity.
  • VHS motif (putative active site) is found in the C-terminal region of TES1 at a conserved position ( Figure 22). All these thioesterases have no sequence similarity with the previously identified animal type I or type II thioesterases known to be involved in the chain termination of fatty acid synthesis (Naggert et al., "Cloning, Sequencing and Characterization oi Escherichia-coli Thioesterase II," J. Biol.
  • C. Cochliobolus.
  • S. Setosphaeria.
  • B. Bioplaris.
  • the name of isolates (or lab strains) of each species are given in parentheses and those known to produce host-specific toxins are underlined. * Provided by Tsukiboshi Takao (Japan) and the isolate could be either BZ1209 or BZ1703.
  • Genotype susceptible to the host-specific toxin-producing isolate is given in parentheses.
  • Genomic DNAs from a previously prepared gel blot filter, Rose et al., "A Decarboxylase Required for Polyketide Toxin Production and High Virulence by
  • Genomic DNAs for probing were prepared according to Yoder ("Cochliobolus heterostrophus, Cause of Southern Corn Leaf Blight,” in Sidhu, ed., Genetics of Plant Pathogenic Fungi, Vol. 6, San Diego,
  • heterostrophus cloned on p214B7 (3.4 kb left flank) and p214Sl (3.2 kb right flank) ( Figure 2) were prepared by restriction enzyme digestion of the plasmid DNAs followed by purification using the QIAquick Gel Extraction Kit (QIAGEN Inc., Chatsworth, CA).
  • the plasmid pi 18B14, which carries the 2.2 kb Bgl II fragment of CPSl interrupted by the hygB cassette (see Figure 5) was linearized with BgRl and introduced into HvW genome. Transformants were purified by isolation of single conidia and genomic DNAs were digested with BgRl and probed with the CPSl 3.1 kb fragment.
  • Bioassays Pathogenicity was determined by an oat plant assay. Fungal strains were grown on individual oat meal agar medium plates (60 X 15 mm) containing hygromycin B (60 ug/ml) for 10 days at 24°C under lights. Conidia were scraped from the plates and suspended in 6 ml of sterilized distilled water. One ml of conidial suspension of each strain was mixed with 60 seeds of susceptible or resistant oats. Inoculated seeds were planted in 4" X 6" pots and seedlings were allowed to grow for two weeks. Seed germination rate and symptom development were recorded at different stages (4, 6, 8 and 24 days after inoculation). Detection of victorin production using HPLC analysis was done by Alice Churchill in Dr. Vladimir Macko's lab at Boyce Thompson Institute for Plant Research.
  • CPSl homologs appear to be polymo ⁇ hic among different species, i.e., all species gave one or two unique bands when BgRl or Hindlll digested genomic DNAs were probed (except for C. victoriae, which showed the same hybridization pattern as C. carbonum) (Table 4 and Figure 26).
  • EcoRI digested genomic DNAs of the same species did not show polymo ⁇ hisms; all species hybridized to a large fragment ( ⁇ 23 kb, Table 4), indicating the absence of an EcoRI site in all CPSl homologs as in the C. heterostrophus gene.
  • C. heterostrophus gene In C.
  • heterostrophus a > 12 kb of genomic region which includes CPSl (5.4 kb), TES1 (1.1 kb) and sequence downstream of the 3' end of CPSl has no EcoRI sites.
  • CPSl homologs appear to be highly conserved among different isolates of the same species.
  • C. heterostrophus race T and race O hybridized to the same 4.2 kb BgRl fragment (or 5.2 and 3.2 kb Hindlll fragments); all three C. carbonum races hybridized to the same 5.0 kb BgRl fragment (or 6.6 kb Hr ⁇ dlll fragment) (Table 4, Figure 26) and B. sacchari isolates 764-1 and 1249-10 hybridized to the same H dIII fragments (5.4 and 2.5 kb) (Table 4).
  • EXAMPLE 10 Targeted disruption of CPSl homolog in C. victoriae. Twenty transformants were obtained from transformation of the victorin-producing isolate HvW with Z?g/II-linearized plasmid pi 18B14 (Figure 5). Six transformants were purified and assayed for both victorin production and pathogenicity to susceptible oat plants. All transformants produced wild type levels of victorin as determined by HPLC analysis ( Figure 27), but four of them (Tx7, Tx2, Tx5 and Tx8) showed dramatically reduced vimlence in the plant assay.
  • the seed germination rate on the eighth day after inoculation is only 13- 25% for wild type and two transformants (Tx9 and Tx4), but 45-63%) for the other four transformants.
  • Tx9 or Tx4 were killed but most (29-63%) from the seeds inoculated with Tx2, Tx7, Tx5 or Tx8 still survived (Table 5, Figure 28A).
  • Gel blot analysis confirmed that transformants showing the reduced vimlence phenotype resulted from homologous integration of the transforming vector that dismpted the wild type CPSl homolog in C.
  • Control- 1 uninoculated susceptible oat seeds.
  • Control-2 and Control-3 resistant and susceptible oat seeds inoculated with wild type C. victoriae (isolate HvW), respectively.
  • Six transformants were tested on both resistant and susceptible seeds, but only data for the later are shown (all transformants gave the same results as Control-2 when tested on resistant seeds). Repeat experiments gave similar results (data not shown).
  • d Recorded on day 24 after inoculation. The percentage of survivors is based on the number of plants recorded on days 8 and 24.
  • CPSl Cochliobolus heterostrophus gene
  • CPSl encodes a peptide synthetase that appears to be a general factor for fungal vimlence to their hosts.
  • CPSl has been found to be highly conserved among at least 9 fungal species belonging to 3 genera including the genus Cochliobolus and closely related genera Bioplaris and Setosphaeria; it has been demonstrated to be required for pathogenesis of three different plant pathogens i.e., C. heterostrophus race O , race T to com and C.
  • Fungal genomic DNAs were prepared according to a previously described procedure (Yoder, 1988. "Cochliobolus heterostrophus, cause of Southern Com Leaf Blight”. Genetics of Plant Pathogenic Fungi. G. S. Sidhu. San Diego, Academic Press. 6: 93-112). Plasmid DNA preparations, restriction enzyme digestions and preparation of DNA gel blots were performed following standard protocols (Sambrook, J., E. F. Fritsch, et al. 1989 "Molecular Cloning: A Laboratory Manual, 2nd Edition".
  • ChCPSl fragment (corresponding to ChCPSl amino acids 173-1208) was obtained by restriction enzyme digestion of a plasmid clone p214Sl (Lu, 1998, Ph.D thesis), (see Figure 2), followed by purification using the QIAquick Gel Extraction Kit (QIAGEN Inc., Chatsworth, CA). The purified ChCPSl fragment was labeled with ⁇ -[ 32 P]dCTP (Turgeon, B. G., H. Bohlmann, et al, 1993. "Cloning and analysis of the mating type genes from Cochliobolus heterostrophus " Mol. Gen. Genet. 238: 270-284.).
  • DNA-DNA hybridization was carried out at 62° C in 6 X SSC, 0.05 X BLOTTO (Sambrook et al., 1989). Filters were washed in 2 X SSC, 0.1% SDS at 62° C for 60 minutes.
  • Cochliobolus species including the known plant pathogens C. carbonum, C. victoriae, C. miyabeanus, C. sativus and C. spicifer, and five genera closely related to Cochliobolus, i.e., Pyrenophora, Setosphaeria, Bipolaris, Stemphyllium and Alternaria showed hybridization intensities comparable to that of C. heterostrophus itself (Fig. 30A).
  • DNAs of species from nine distantly related genera including several of economic importance (e. g., Magnaporthe grisea, Fusarium graminearum, Gaeumannomyces graminis) or of medical importance (e. g., Candida albicans) hybridized weakly to CPSl (FigS. 30B, 30C) whereas no signal was detected in DNA of the basidiomycete Ustilago maydis.
  • CPSl homolog genes were cloned and characterized. Three of them were cloned from phytopathogenic fungi, including the wheat head scab fungus Fusarium graminearum (FgCPSl, 6003 bp, SEQ. No. 43), the potato early blight fungus Alternaria solani, (AsCPSl, 2369bp, SEQ. No. 41) and the barley net blotch fungus Pyrenophora teres (PtCPSl, 2306 bp, SEQ. No. 45).
  • phytopathogenic fungi including the wheat head scab fungus Fusarium graminearum (FgCPSl, 6003 bp, SEQ. No. 43), the potato early blight fungus Alternaria solani, (AsCPSl, 2369bp, SEQ. No. 41) and the barley net blotch fungus Pyrenophora teres (PtCPS
  • FgCPSl was cloned as a full length gene using both PCR amplification and the plasmid rescue procedure that was preceded by targeted gene dismption in the genome.
  • AcCPSl and PtCPSl homologs were partially cloned by direct PCR amplification.
  • PCR Polymerase Chain Reaction
  • ChCPSl degenerate primers designed to conserved regions of C. heterostrophus CPSl
  • Two sets of degenerate primers were designed to amino acids at or close to conserved core sequences of C. heterostrophus CPSl (ChCPSl).
  • the first pair of primers 5'TGYTTYATHGCNGGN GTNGTNGCNGTNCC3 ' (CHFP6, corresponding to positions 493-521 oi ChCPSl) and 5 ⁇ TGYTGNGGNGGNCCNCCNGGRTT3' (CHRP4, 2197-2220 of ChCPSl), was used to amplify CPSl from Fusarium graminearum.
  • the second pair of primers 5'-AARAARAARGGNCCNACNGAG-3' (FP4CB, corresponding to positions 1531-1550 oi ChCPSl) and 5'SRYTGNA CCCADATYTCNCC3' (RP2DB, corresponding to positions 3883-3902 of ChCPSl), was used to amplify CPSl from A. solani and Pyrenophora teres. PCR was carried out in a Perkin Elmer Cetus 9600-thermocycler with fungal (Fusarium graminearium, Alternaria solani, and Pyreophora teres) genomic DNA as a template.
  • Reaction mixtures contained about 500 ng of genomic DNA in 100 ul of reaction buffer [ 1 x Ex Taq buffer, 0.2 mM dNTPs, 0.2 uM of each primer and 0.05 U/ml Takara Ex Taq (Pan Vera Co ⁇ oration)].
  • An initial denaturing step of 95 °C for 3 min. was followed by 30 cycles of 94 °C for 1 min, 47 °C (for G. zeae) or 55 °C (for A. solni and Pyrenophora teres) for 3 min, and 72°C for 3 min. Reactions were cooled to 4 °C after a final extension of 72 °C for 10 min.
  • PCR products were examined (10 ul of each reaction) by agarose-gel (0.75%)) electrophoresis.
  • Sequencing of each plasmid clone was initiated with vector-specific primers or primers designed to previously determined sequences. Sequences were analyzed using MapDraw and MegAlign (DNASTAR) and nucleotide or protein database searches were performed with the BLAST program (Altschul et al., 1990, 1997).
  • the FgCPSl open reading frame (5125 bp, SEQ. No. 43) has 50% nucleotide identity to ChCPSl (SEQ ID No.2) in about 4.4 kb of overlap (Fig. 2).
  • No "TATA" box-like element was found in the 5' untranslated region, but other promoter sequences including two putative "CAAT” boxes and a "CT” motif were located upstream of the start codon (ATG) in FgCPSl (Fig. 32). Only one putative intron was found 1508 bp upstream the stop codon (TGA) in contrast to three in ChCPSl (Figs. 31 A, 3 IB and 32).
  • a putative polyadenylation signal "AATAA” was located 62 bp downstream of the stop codon (Fig. 32).
  • the predicted FgCPSl protein (1692 amino acids, M ⁇ 187983 Da, SEQ ID No. 44) has 68% identity, 73% similarity to ChCPSl (SEQ ID No. 3) in a about 1 ,500 amino acid overlap (Figs. 31 A and 3 IB) that contains two structurally similar modules highly similar to those of ChCPS 1.
  • FgCPS 1 has no significant similarity to ChCPSl at the C-terminus, which is relatively shorter and lacks the thioeterase domain as seen in ChCPSl (Figs. 31 A and 3 IB).
  • the annotated FgCPSl sequence is given in Figure 32.
  • AsCPSl (2369 bp, SEQ. No. 41) has 76% nucleotide identity to ChCPSl (SEQ ID No.2) in the entire cloned region which contains two conserved introns (Figs. 31 A and B).
  • the translated AsCPSl protein (partial) includes 758 amino acids corresponding to amino acids 511-1269 in ChCPSl and has up to 93% identity, 95% similarity to ChCPSl (Fig. 2).
  • the annotated AsCPSl sequence is given in Figure 33.
  • PtCPSl (2306 bp, SEQ. No. 45) has 78% nucleotide identity to ChCPSl (SEQ IDNo.2) in the entire cloned region which contains only one intron (Fig. 2).
  • the translated PtCPSl protein (partial) includes 758 amino acids corresponding to amino acids 511-1269 in ChCPSl and has 93% identity, 96% similarity to ChCPSl (Fig. 2).
  • the annotated PtCPSl seqence is given in Figure 34.
  • EXAMPLE 12 Targeted disruption of CPSl homolog in F. graminearum.
  • a 2.2 kb Xbal fragment from pUCATPH (Lu, et al, 1994) containing the bacterial hygmycin resistance gene (hygB) driven by the Aspergillus nidulans trpC promoter was inserted into the Xbal site of a PCR clone pFgC8, which carries the 1.0 kb internal fragment oi FgCPSl, to create pFgC8- hygB.
  • This constmct was transformed (in a circular form or linearized with Hindlll or BgRl) into an isolate (GZ3639) of wild type F. graminearum.
  • F. graminearum strains were grown on PDA (or PDA plus hygromycin B for transformants) plates (100 X 15 mm) for 7-15 days at 24 C under black lights (Sylvania Inc., Danvers, MA) for maximum conidiation.
  • a susceptible spring wheat cultivar, Norm Hard Red (kindly provided by G. Bergstrom, Cornell university) was used.
  • Two months old wheat plants grown in the greenhouse at anthesis (10 plants in one 4" X 6" pot) were sprayed with 10 ml conidial suspensions (10 ⁇ or 10 ⁇ conidia/ml) using a pressurized Preval Spray Gun Power Unit thin layer chromatography sprayer (Alltech Associates,

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Abstract

The present invention relates to genes cloned from the plant pathogens Cochliobolus heterostrophus, Alternaria solani, Fusarium graminearium, and Pyrenophora teres, that encode a CPS1 peptide synthetase required for fungal pathogenesis. The nucleic acid molecules in a vector, a host cell, or a plant is also disclosed. The invention further provides a protein or polypeptide encoded by the CPS1 genes. Other aspects of the invention relate to a method of imparting disease resistance to a plant by overexpressing a protein of the present invention in a plant and a method for identifying inhibitors of a CPS1 protein in a sample.

Description

PEPTIDE SYNTHETASE GENE CPSl
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation in part application of U.S. Application Serial No. 09/448,215, filed November 23, 1999.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
The present invention was made with support from the United States Government under Grant No. 96-35303-3198 from the USDA/NRI. The United States Government may have certain rights in the invention.
FIELD OF THE INVENTION The present invention relates to nucleic acid molecules encoding peptide synthetase homologs of Cochliobolus heterostrophus, Pyrenophora teres, Fusarium graminearium, and Alter naria solani and uses thereof.
BACKGROUND OF THE INVENTION
There are approximately 30 species included in the genus Cochliobolus, nearly all of which are pathogens of wild grasses or cereals (Yoder et al, "Cochliobolus spp. And Their Host-Specific Toxins, in Carroll, eds., The Mycota Vol. 5: Plant Relationships, Part A, Berlin:Springer-Verlag, pp. 145-166 (1997)). Cochliobolus heterostrophus represents the most widely distributed species in the genus and can be found in many tropical and subtropical areas in the world. As a natural pathogen of corn, C. heterostrophus causes a disease frequently called leaf spot of maize in the old literature (Drechsler, "Leafspot of Maize Caused by Ophiobolus Heterostrophus n. sp., The Ascigerous Stage of a Helminthosporium Exhibiting Bipolar Germination," J. Agr. Res., 31 :701-726 (1925); Drechsler, "Phytopathological and Taxonomic Aspects of Ophiobolus, Pyrenophora, Helminthosporium, and a New Genus, Cochliobolus," Phytopathol., 24:953-983 (1934); Yu, "Studies on Helminthosporium Leaf Spot of Maize," 3:273-318 (1933); Orillo, "Leafspot of Maize Caused by Helminthosporium maydis," 36:327-395 (1952). In the United States, C. heterostrophus is usually found in the warmer southern states, thus, the disease is commonly known as Southern Corn Leaf Blight (Hooker, "Cytoplasmic Susceptibility in Plant Disease," Ann. Rev. Phytopathol., 12:167-179 (1974)). For many years, Southern Corn Leaf Blight was only known as an endemic disease and was not considered to be major economic importance in the United States. But in 1970, it suddenly broke into a severe epidemic that destroyed 15% of the U.S. corn crop and caused losses estimated at more than $1 billion. This serious damage made Southern Corn Leaf Blight one of the most widely known crop diseases in the U.S.
Prior to the outbreak of the disease, only one race of C. heterostrophus (race O) was known in the field. In late 1969 when the disease became an epidemic, a new race of the fungus was identified from infected corn leaves collected in severely diseased areas. It was soon designated as race T because of its high virulence on T-cytoplasm corn and the ability to produce a phytotoxin called T-toxin, which specifically affects T-corn. In contrast, race O does not produce T-toxin and is mildly virulent on both T-cytoplasm and N- cytoplasm (normal cytoplasm) corn (Hooker et al., "Physiological Races of Helminthosporium maydis and Disease Resistance," Plant Pis. Reptr.. 54: 1109- 1110 (1970); Scheifele, "Cytoplasmically Inherited Susceptibility to Diseases as Related to Cytoplasmically Controlled Pollen Sterility in Maize," 25:110-138 (1970); Smith et al., "Physiologic Races oi Helminthosporium maydis," 54:819- 822 (1970); Yoder et al., "Segregation of Pathogenicity Types and Host-Specific Toxin Production in Progenies of Crosses Between Races T and O of
Helminthosporium maydis (Cochliobolus heterostrophus)," Phytopathology, 65:273-276 (1975); Yoder, "Evaluation of the Role oi Helminthosporium maydis, Race T Toxin in Southern Corn Leaf Blight, in Tomiyama, eds., Biochemistry and Cytology of Plant Parasite Interaction, New York, New York:Elsevier, pp. 16-24 (1976); Yoder, "Toxins in Pathogenesis," Ann. Rev. Phytopathol.. 18:103-129 (1980)). T-cytoplasm stands for Texas male sterile cytoplasm, a unique cytoplasm with a trait for maternally inherited male sterility, characterized by the failure to produce pollen (Levings, "The Texas Cytoplasm of Maize: Cytoplasmic Male Sterility and Disease Susceptibility," Science. 250:942-947 (1990)). T- cytoplasm corn was widely used for hybrid seed production and breeding to avoid hand or mechanical emasculation in the 1950s and the 1960s. It was the coexistence of large acreages of intensively planted T-cytoplasm corn and the sudden appearance of race T of C. heterostrophus that resulted in the epidemic of the disease in 1970. This discovery first opened the door to understanding pathogenesis by C. heterostrophus.
Early genetic analysis suggested that both T-toxin production and high virulence on T-cytoplasm corn are controlled by a single genetic locus defined as Toxl (Leach et al., "Dominance at the Toxl Locus Controlling T-Toxin Production by Cochliobolus heterostrophus," Physiol. Plant Pafhol., 21 :327-333 (1982)). This was demonstrated by crosses between race T and race O in which only parental phenotypes segregated in a 1 :1 ratio (Tox+:Tox~); all T-toxin producing progeny are highly virulent on T-cytoplasm corn while all T-toxin nonproducing progeny are weakly virulent (Yoder et al., "Segregation of Pathogenicity Types and Host-Specific Toxin Production in Progenies of Crosses Between Races T and O oi Helminthosporium maydis (Cochliobolus heterostrophus), Phytopathology. 65:273-275 (1975); Leach et al., "Dominance at the Toxl Locus Controlling T-Toxin Production by Cochliobolus heterostrophus," Physiol. Plant Pathol.. 21 :327-333 (1982)). Further investigation by comparison of electrophoretic karyotypes and chromosome-specific DNA hybridizations indicated that Toxl is tightly linked to a reciprocal translocation breakpoint and is associated with as much as a megabase of DNA (mostly highly repeated and A+T- rich) that is missing in race O (Bronson, "Ascospore Abortion in Crosses of Cochliobolus heterostrophus Heterozygous for the Virulence Locus Toxl," Genome. 30:12-18 (1988); Tzeng et al., "A Restriction Fragment Length Polymorphism Map and Electrophoretic Karyotype of the Fungal Maize Pathogen Cochliobolus heterostrophus," Genetics. 130(l):81-96 (1992); Chang et al., "A Reciprocal Translocation and Possible Insertion(s) Tightly Associated with Host- Specific Virulence in Cochliobolus heterostrophus," Genome. 39(3):549-557
(1996)). Surprisingly, recent analyses of several Tox~ mutants revealed that Toxl is not a single locus but rather two loci, each on a different translocated chromosome (Yoder et al., "Molecular Determinants of the Plant/Fungus Interaction," in Kohmoto, eds., Host-Specific Toxin: Biosynthesis, Receptor and Molecular Biology, Tortori, Japan:Faculty of Agriculture, Tottori Univ., pp. 23-32 (1994); Turgeon et al., "Function and Chromosomal Location of the Cochliobolus heterostrophus Toxl Locus," Can. J. Bot.. 73 (suppl. 1 Sect. E-H):S1071-S1076 (1995)). These two Toxl loci have been designated Toxl A and ToxlB (Yoder et al., "Cochliobolus spp. and Their Host-Specific Toxins," in Carroll, eds., The Mycota Vol. 5: Plant Relationships, Part A, Berlin: Springer-Verlag, pp. 145-166 (1997)). Two genes PKS1 and DEC1 have been cloned from the two loci respectively; both are required for biosynthesis of T-toxin and are found only in race T isolates of C. heterostrophus (Yang, "The Molecular Genetics of T-Toxin Biosynthesis by Cochliobolus heterostrophus," Ph.D. Thesis, Cornell University (1995); Yang et al., "A Polyketide Synthase is Required for Fungal Virulence and Production of the Polyketide T-Toxin," Plant Cell. 8(11):2139-2150 (1996); Rose et al., "A Decarboxylase Required for Poloyketide Toxin Production and High Virulence by Cochliobolus heterostrophus," 8th Int. Symp. Mol. Plant-Microbe Int, Knoxville, p. J-49 (1996)).
Genetic analysis also suggested that T-toxin is required by C heterostrophus for its high virulence on T-cytoplasm corn. This hypothesis was first tested by the generation of induced T-toxin deficient mutants using different mutagenesis procedures. All mutants with a tight Tox" phenotype cause disease symptoms that are indistinguishable from those caused by race O when tested on both T and N-cytoplasm corn, suggesting that T-toxin is indeed a virulence factor (Yang et al., 1992; Lu et al., "Tagged Mutations at the Toxl Locus of
Cochliobolus heterostrophus Using Restriction Enzyme-Mediated Integration," Proc. Natl. Acad. Sci. USA. 91 :12649-12653 (1994 ; Rose et al.. "A Decarboxylase Required for Polyketide Toxin Production and High Virulence by Cochliobolus heterostrophus," 8th Int. Symp. Mol. Plant-Microbe Int., Knoxville, p. J-49 (1996)). This conclusion was firmly supported by the site-specific disruption of the PKS1 or DEC1 in the wild type race T genome; disruptants lost the ability to produce T-toxin and caused race O type symptoms on both T-com and N-com (Yang et al., "A Polyketide Synthase is Required for Fungal Virulence and Production of the Polyketide T-Toxin," Plant Cell. 8(11):2139-2150 (1996); Rose et al., "A Decarboxylase Required for Polyketide Toxin Production and High Virulence by Cochliobolus heterostrophus," 8th Int. Symp. Mol. Plant- Microbe Int.. Knoxville, p. J-49 (1996)). These experiments have given a very clear resolution for the role of T-toxin in pathogenesis. They also implied that pathogenesis by C. heterostrophus must involve additional pathogenicity factors because race O which does not produce T-toxin and race T-derived Tox~ mutants are effective pathogens on corn.
A number of fungal molecules have been identified as general pathogenicity or virulence factors in several plant pathogenic fungi (Yoder et al., "Molecular-Genetic Evaluation of Fungal Molecules for Roles in Pathogenesis in Plants," J. Genet.. 75(3):425-440 (1996)). These include potential penetration factors such as melanin (Guillen et al., "Linkage Among Melanin Biosynthetic Mutations in Cochliobolus heterostrophus," Fungal Genet. Newsl.. 41 :41 -42 (1994)), cutinase (Oeser et al., "Pathogenesis by Cochliobolus heterostrophus Transformants Expressing a Gene Encoding Cutinase from Nectria haematococca," Mol. Plant-Microbe Int.. 7:282-288 (1994)) and polygalacturonase and xylanase (Lyngholm et al., "Mutants oi Cochliobolus heterostrophus Deficient in Extracellular Enzymes," Fungal Genet. Newsl., 42:46- 47 (1995)) or possible mechanisms involved in colonization such as phytotoxin detoxification (Schafer et al., "One Enzyme Makes a Fungal Pathogen, But Not a Saprophyte, Virulent on a New Host Plant," Science. 246:247-249 (1989)) or components of signal transduction pathways (Horwitz et al., "A G Protein Alpha Subunit Gene From the Com Pathogen Cochliobolus heterostrophus is Involved in Two Complex Developmental Pathways: Mating and Appressorium Formation (unpublished) (1997)). Although C. heterostrophus is known to produce a nonhost specific toxin called ophiobolin (or cochliobolin), a C 5 sesterterpenoid compound, which is toxic to many organisms, including plants, bacteria, fungi and nematodes, there is no evidence that ophiobolins are involved in pathogenesis by C. heterostrophus or other phytopathogenic fungi. No other pathogenesis-related toxins have been isolated from C. heterostrophus so far, but studies on closely related Cochliobolus species and other phytopathogenic fungi suggest that pathogenesis by this group of fungi also involves peptide toxins.
Four peptide phytotoxins (victorin, HC-toxin, AM-toxin, and enniatins) have been characterized as pathogenicity or virulence factors. They are all small cyclic peptides (4-6 residues), containing unusual amino acids or hydroxy acids, and they can be either host specific or non-host specific in terms of plant toxicity. A number of peptide phytotoxins are believed to be synthesized nonribosomally. Early in the 1960s, several biochemists working on the bacterial peptide antibiotics gramicidin and tyrocidine found that these polypeptides can be synthesized in RNAase-treated particle-free extracts oi Bacillus brevis that are known to produce the same antibiotics; adding protein-synthesis inhibitors to the extracts does not affect this process. This indicated the existence of a peptide biosynthetic system in which ribosomes and mRNAs are not needed. Further studies revealed that in this system, peptides are synthesized on a protein-template and this template itself is a multifunctional enzyme or a complex of several such enzymes, collectively called peptide synthetases, catalyzing the biosynthetic process (Laland et al., "The Protein Thiotemplate Mechanism of Synthesis for the Peptide Antibiotics Produced by Bacillus Brevis," Essays in Biochemistry, 7:31- 57 (1973); Lipmann, "Bacterial Production of Antibiotic Polypeptides by Thiol- Linked Synthesis on Protein Templates," Adv. Microbiol. Physiol., 21 :277-266 (1980)).
Peptide synthetases can catalyze biosynthesis of a variety of peptides. In terms of bioactivity, they can be antibiotics, enzyme inhibitors, plant or animal toxins and immunosuppressants (Stachelhaus et al., "Modular Structure of Peptide Synthetases Revealed by Dissection of the Multifunctional Enzyme GrsA.," Journal of Biological Chemistry. 270(11):6163-6169 (1995)). In terms of chemical structure, they can be either linear (i.e. ACV, the penicillin precursor and gramicidin) or cyclic (most are). The latter can be further classified into three subgroups: 1) The "standard" cyclic peptides (i.e. gramicidin S, tyrocidine, HC- toxin and cyclosporin); 2) cyclic lactones (i.e. destruxin); 3) cyclic depsipeptides (i.e. beauvericin and enniatin). There have been over 300 different carboxy compounds that can be activated by peptide synthetases.
Although the first peptide synthetase, Gramicidin S synthetase, was purified and used for the cell-free synthesis of the peptide early in the 1960s (Tomino et al., "Cell-Free Synthesis of Gramicidin S," Biochem.. 6:2552-2560 (1967)), the first bacterial peptide synthetase gene, tycA, which encodes the tyrocidine synthetase 1 in B. brevis was not cloned until almost twenty years later (Marahiel et al., "Cloning of the Tyrocidine Synthetase 1 Gene from Bacillus- brevis and Its Expression in Escherichia-coli, Mol. Gen. Genet., 201 (2): 1986 (1985)). Since then, more than twenty peptide synthetase genes have been reported for both bacteria and filamentous fungi, but only fourteen have complete nucleotide sequences published. All are larger than 3.3 kb and range between 3.3- 19.5 kb for bacterial genes and 9.4-45.8 kb for fungal ones. Interestingly, all fungal peptide synthetase genes reported lack introns, even the cyclosporin A synthetase gene simA, which has a 45.8 kb of open reading frame (the largest genomic ORF so far recorded). Although biosynthesis of bacterial peptides differs from that of fungal ones in terms of the number of multifunctional enzymes involved, the genes encoding these enzymes are similar to each other in both function and structure. Comparison of nucleotide sequences reveals one or more highly conserved regions at certain positions in each peptide synthetase gene. These regions formerly called "amino acid activating domains"
(Stachelhaus et al., "Modular Structure of Peptide Synthetases Revealed by Dissection of the Multifunctional Enzyme GrsA," Journal of Biological Chemistry. 270(11):6163-6169 (1995)), now called "amino acid activating modules" (Marahiel, "Protein Templates for the Biosynthesis of Peptide Antibiotics," Chem. Biol., 4(8):561-567 (1997)) consist of a set of domains (formerly called "modules") believed to have specific functions such as recognization, activation and thioesterification of individual constituent amino or hydroxy acids, and in some cases methylation and racemation for modification of certain residues before incorporation into the peptide chain (Stachelhaus et al., "Modular Structure of Peptide Synthetases Revealed by Dissection of the
Multifunctional Enzyme GrsA," Journal of Biological Chemistry, 270(11):6163- 6169 (1995)). The most convincing evidence supporting this assignment is that in most cases, the number of conserved functional units in each gene or gene cluster is equal to the number of amino acids in the respective peptide. This one-for-one match is very clear between three of four fungal peptides and their biosynthetic genes. The total number of modules in three of four bacterial gene clusters also matches the number of amino acids in the respective peptides. Sequence alignment of amino acid-activating modules reveals strictly conserved sequence motifs that contain active residues for module functions. These motifs are called "core sequences" (Marahiel, "Multidomain Enzymes Involved in Peptide Synthesis," FEBS Lett.. 307(l):40-43 (1992)). A minimal amino acid-activating module must contain six core sequences, whose functions (except for core 1 ) have been proposed based on mutational analysis of several peptide synthetases. Core sequences 1-5 are grouped into an amino acid adenylation domain and core 6 is a thioester formation domain (Figure 1 A). All bacterial peptide synthetase genes contain "type I modules" - the minimal amino acid activating modules which were previously called "type I domains"
(Stachelhaus et al., "Modular Structure of Peptide Synthetases Revealed by Dissection of the Multifunctional Enzyme GrsA," Journal of Biological Chemistry. 270(1 1):6163-6169 (1995)). Two fungal genes, acvA and HTS1 also have this modular structure. In addition to the type I module, two fungal genes, esynl and simA, contain type II modules, in which an insertion (about 400 amino acids) is found between cores 5 and 6 of a normal type I module. This region contains a motif (VLE/DXGXGXG (SEQ. ID. No. 1)), highly conserved in S- adenosyl-methionine (SAM)-dependent methyltransferases, hence, it is referred to as a N-methylation domain (Figure 1A). Additional evidence for methyltransferase activity of this module is that the number and position of type II modules in esynl and simA exactly match that of N-methylated amino acids in ennatin and cyclosporin sequences (Figure IB).
Although the modular structure described above is highly conserved among most peptide synthetase genes, some variations have been found in the latest cloned peptide synthetase gene safB, which is the first gene in the saframycin Mxl synthetase gene cluster (Pospiech et al., "A New Myxococcus xanthus Gene Cluster for the Biosynthesis of the Antibiotic Saframycin Mxl Encoding a Peptide Synthetase," Microbiology. 141(8):1793-1803 (1995)). safB contains two type I amino acid activating modules. One module has all six highly conserved core sequences, but another, believed to activate alanine (the first amino acid in the linear tetrapeptide precursor of saframycin Mxl), lacks core 5 and has a weakly conserved core 1 (Pospiech et al., "Two Multifunctional Peptide Synthetases and an O-methyltransferase Are Involved in the Biosynthesis of the DNA-Binding Antibiotic and Antitumour Agent Saframycin Mxl from Myxococcus xanthus " Microbiology, 142(4):741-746 (1996)) (Figure 1A). This suggests that some of the motifs in the amino acid adenylation domain are dispensable or not critical for domain function. It also raises the possibility that other variations might be found in yet unknown peptide synthetase genes.
Although C. heterostrophus has been a model eukaryotic plant pathogen since the 1970s, most molecular genetic analyses conducted in this . system have focused on production of the polyketide T-toxin by race T isolates of the fungus. Solid evidence now indicates that T-toxin is a host-specific vimlence factor in Southern Com Leaf Blight (Yoder et al., "Molecular-Genetic Evaluation of Fungal Molecules for Roles in Pathogenesis in Plants," J. Genet.. 75(3):425- 440 (1996); Yoder et al., Cochliobolus spp. and Their Host-Specific Toxins, Carroll eds., The Mycota Vol. 5: Plant Relationships, Part A. Berlin: Springer- Verlag, pp. 145-166 (1997)). It is clear, however, that C. heterostrophus needs additional factors, presumably general factors for pathogenesis to corn plants, since race O, which does not produce T-toxin, can be an effective com pathogen. Attempts to identify additional general factors required by C. heterostrophus for pathogenesis have been unsuccessful. Cloning and characterizing additional C. heterostrophus genes that control biosynthesis of novel fungal molecules involved in critical pathogenic processes may be important because it offers potential targets for the design of products that might interfere with the com plant infection process. The present invention is directed to achieving these objectives.
SUMMARY OF THE INVENTION The present invention relates to an isolated nucleic acid molecule encoding CPSl peptide synthetase homologs. The DNA molecule comprises a nucleotide sequence which hybridizes to a DNA molecule having a sequence as set forth in at least one of SEQ. ID. No. 2, SEQ ID No. 41, SEQ ID No. 43, or SEQ ID No. 45.
Another aspect of the present invention relates to a method for identifying inhibitors of a CPSl protein or polypeptide which involves providing the CPSl protein or polypeptide, contacting the protein with potential inhibitor compounds, determining peptide synthetase activity, and selecting compounds which decrease the peptide synthetase activity.
Still another aspect of the present invention relates to a method of imparting disease resistance to a plant by overexpressing a CPSl protein or polypeptide in the plant cell.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides the structure of amino-acid activating modules identified in peptide synthetase genes (adapted from Stachelhaus and Marahiel, 1995; Pospiech 1995; Marahiel, 1997). Figure 1A shows the domain arrangements in two types of modules. Structural variations in the first module (safBl) of the gene safB are also indicated below type I. Figure IB shows the correlation between module types and the nature of residues in two fungal peptides. Open box: type I module; filled box: type II module. Each peptide sequence is given below.
Figure 2 depicts the recovery of DNA flanking the REMI vector insertion site (arrows) in mutant R.C4.2696. Circled numbers indicate restriction enzyme sites used for recovery of each plasmid. p214B7 contains 4.2 kb flanking DNA (3.4 left; 0.7 right); p214Ml contains 0.1 kb left flank that overlaps with p214B7 and 1.1 kb right flank that overlaps with p214S 1 , which contains 3.2 kb flanking DNA on the left only.
Figures 3A-B illustrate the extension of the tagged sequence by targeted integration and plasmid rescue. Figure 3A shows a general scheme illustrating chromosome walking strategy (only one direction is indicated). "X" indicates a restriction enzyme site used for recovery of vector (indicated by "T") with flanking genomic DNA ("L" for left flank; "R" for right flank; R' for a fragment from "R" that is subcloned into a subsequent transformation vector). Each integration site is indicated by a vertical arrowhead pointed at the selectable marker on the vector. Overlapped flanking DNA is represented by bars drawn in the same pattern. Figure 3B shows two strategies used for construction of a targeted integration vector. Left: A genomic DNA fragment (indicated by the letter "a") is subcloned into a vector directly and the subsequent integration occurs by a single crossover ( "a" is duplicated). Right: "a" is subcloned into a vector via multiple steps (see Figure 6 for an example) that allows linearization with restriction enzyme "Y" and the subsequent integration occurs by a double crossover resulting in a simple insertion.
Figure 4 shows how the targeted integration vector p214SNP was constructed. The sites for restriction enzyme digestion are indicated by arrows. p214SNP was originally designed for a double crossover integration after digested with 5stXI and BcR, but it was found later that the E. coli strain (DH5 ) used for amplification of the plasmid is Dam+ which blocks the BcR site (TGA TCA to TGmTCA). As an alternative, the plasmid was linearized with .SstXI only and used for transformation.
Figure 5 depicts the recovery of genomic DNA flanking the targeted integration vector p214SNP in transformant #118. The vector integrated into the target site by a single crossover that resulted in duplicated Hindlll-Sac fragments (indicated by letter b and b ). The second genomic DNA fragment carried by the vector is also duplicated in the genome (Nαrl-Hwdlll, indicated by letter a and a'). Genomic DΝA was digested with BgRl (which does not cut the vector) or BcR (which cuts once in fragment a' on the vector) as indicated by numbers in a circle. The two recovered plasmids carry sequences that extend the right flank only by 170 and 800 bp respectively beyond the genomic DΝA on P214S1.
Figure 6 shows how the targeted integration vector pi 18BSP was constructed. The sites for restriction enzyme digestion on the vectors are indicated by arrows, pi 18B14 contains two duplicated Narl-Hindll -Sac fragments (numbered region, a + b = a' + b') but only one is present in pi 18BSP. pi 18BSP was linearized with BgRl and transformed into a wild type C. heterostrophus strain. Figure 7 depicts the recovery of genomic DNA flanking the targeted integration vector pi 18BSP in transformant #9. The vector integrated into the target site by a double crossover. Genomic DNA was digested with Pstl and relegated. The recovered plasmid p9P2 includes the entire pUC18 sequence on pi 18BSP and 4.6 kb of genomic DNA that contains all of ORF1 (CPSl), including the stop codon (TAG) and 3.0 kb of genomic region 3' of the stop codon.
Figure 8 shows how the targeted integration vector pi 18BCS was constructed. The sites for restriction enzyme digestion are indicated by arrows.
Figure 9 depicts the recovery of genomic DNA flanking the targeted integration vector pi 18BSP in transformant #12. The vector integrated into the target site by a single crossover that resulted in a duplicated Sspl-Sspl fragment in the transformant genome (indicated by the letter "a"). Genomic DNA was digested with Hindlll (arrows) and ligated. The recovered plasmid pl2H6 contains the entire pi 18BSP sequence and a 2.1 kb genomic DNA (Sacl-Hindill) on the left region that overlaps with the sequence carried on p9P2 (see Figure 7).
Figures 1 OA-B are photographs which show that the REMI mutant R.C4.2696 grows like wild type in culture (Figure 10A) and produces normal appressoria (Figure 10B). In Figure 10A, plates containing complete medium (CM) were inoculated with a conidia-bearing mycelium plug and incubated at 22°C under warm white light (F40/350BL) (Sylvania Inc., Danvers, MA). The photograph in Figure 10A was taken 6 days after inoculation. Left to right: Mutant R.C4.2696; wild type. In Figure 10B, conidia of mutant (left) or wild type (right) were placed in a drop of water and incubated at 32°C for 6 hrs. No significant difference in percentage of appressorium-forming conidia (arrows) was detected.
Figures 11 A-B are photographs which show that the REMI mutant R.C4.2696 produces wild type levels of T-toxin (Figure 1 IB) but has reduced vimlence on T-cytoplasm com (Figure 11 A). In Figure 11 A, two week old T- cytoplasm com plants were inoculated with conidial suspensions of (left to right) mutant R.C42696, wild type race T, and a Tox+ mutant progeny from a cross between R.C4.2696 and a race O tester, and symptoms (represented by three diseased leaves for each strain) were recorded 5 days after inoculation. Leaves infected by wild type race T collapsed (arrow) but those infected by mutants remained alive. In Figure 1 IB, a plate containing T-toxin-sensitive E. coli cells was inoculated with agar blocks bearing mycelia of three strains (in the same order as in Figure 11 A) and incubated at 32°C overnight (the inoculum at bottom is race O control). No significant differences in T-toxin production (indicated by halos) among the three strains were detected.
Figure 12 is a photograph which displays that the REMI mutant
R.C4.2696 produces lesions much smaller than wild type on N-cytoplasm com. Two week old N-cytoplasm com plants were inoculated with conidial suspensions at the same concentration and symptoms were recorded 7 days after inoculation. The mutant (right) produces the same number of lesions as wild type (left) but the size of lesions was dramatically reduced compared to wild type (arrows).
Figures 13A-B show that the REMI mutant R.C4.2696 has a 60% reduction of vimlence compared to wild type. In Figure 13 A, lengths of 100 typical lesions from com leaves inoculated with wild type race O and a mutant progeny R45 (Tox~, hygBR) carrying the R.C4.2696 mutation were measured 7 days after inoculation and values plotted. Figure 13B shows the statistical analysis that 86% of the mutant lesions are less than 4 mm in length (average size 3.5 mm), 60% reduced compared to that of wild type (8.5 mm).
Figures 14A-B illustrate the genetic analysis showing that a tagged, single site mutation is responsible for the mutant phenotype. Figure 14A is a photograph of a plant assay where N-cytoplasm com was inoculated with parents and progeny indicated in Figure 14B (a complete tetrad from the second cross is shown here; random spore assay for the two crosses gave the same results). Figure 14B is a table which shows progeny segregation data. In both crosses, progeny segregated 1 : 1 for parental type only and all hygromycin B resistant progeny showed the same small lesion phenotype (represented by progeny 1-4 in Figure 14 A) as the mutant parent (parent 1 in Figure 14A); all hygromycin B sensitive progeny show the large lesion phenotype (represented by progeny 5-6 in Figure 14A) of the wild type parent (parent 2 in Figure 14A).
Figure 15 is a restriction map of the cloned sequences surrounding the tagged site. A 11.3 kb genomic region (thick line) was cloned and completely sequenced. The original REMI insertion point in the mutant R.C4.2696 is indicated by a vertical arrow. The asterisks indicate two targeted integration sites in the wild type genome. Two open reading frames (in opposite directions), ORF1 (CPSl, 5.4 kb) and ORF2 (TES1, 1.1 kb) are indicated by open boxes below the map (the positions of putative introns are indicated by vertical bars). Locations of seven overlapping plasmid clones used for sequencing are indicated by thin lines on the top of the map (filled triangles represent the vector sequence in each clone). Sequencing strategy is indicated by arrows above each clone line.
Figure 16 graphically depicts the G+C content of the cloned sequences surrounding the tagged site. The 11.3 kb of genomic DNA sequence (Figure 15) was examined as 200 bp fragments and G+C content of each fragment was calculated and plotted. Regions A and C have normal G+C content; regions B and D show high G+C content. The overall percentage of G+C in each region is indicated by underlined numbers. The positions of ORFl (CPSl) and ORF2 (TES1) are indicated by arrows and the percentage of G+C of each ORF is given below (in parentheses).
Figure 17 displays the nucleotide sequence oiCPSl. 5,725 base pairs from the 11.3 kb sequenced region (Figure 15) are shown. The deduced amino acid sequence of CPSl protein is given below the DNA sequence. The position of start codon ATG (bold and underlined) is designated +1 and the open reading frame stops at position 5381 (TAG, in bold and underlined). Five putative "CAAT" boxes (bold and indicated by asterisks) are found at the positions -36, - 58, -67, -172 and -309. Three putative introns (in lowercase with 5' and 3' splice sequences in bold; branch sites are underlined) are located at positions 2070-2114, 3542-3592 and 4197-4249. Conserved core sequences are shaded. The GXSXG motif is boxed. A putative polyadenylation signal is located at position 5604 (bold and overlined). Figure 18 displays the nucleotide sequence oi TESl. 1,901 base pairs 5' oiCPSl are shown. The deduced amino acid sequence of the TES1 protein is given below the DNA sequence. The CT motif (bold and indicated by asterisks) is found at position -46. A "AT"-rich region (bold and italicized) is found at position -12. The position of ATG start codon (bold and underlined) is designated +1 and the open reading frame (transcribed in the opposite direction from CPSl) stops at position 1153 (TGA, in bold and underlined). One putative intron (in lowercase, border sequences are bold; branch sites are underlined) is located at position 518-566. A putative polyadenylation signal is located at position 1345 (bold and overlined). The putative active site (VHS) is shaded.
Figures 19A-C are schematic representations which show the characterization of modular structure oiCPSl. Peptide synthetase and thioesterase are indicated by open boxes; shaded boxes inside indicate functional domains and modules; vertical bars in the shaded boxes indicate highly conserved core sequences. Figure 19A illustrates the general structure of bacterial and fungal peptide synthetases (adapted from Marahiel 1997, which is herein incorporated by reference). A peptide synthetase gene cluster is shown on the top. There can be one or more amino acid activating module (cyclosporine synthetase has 11) in each protein; some peptide synthetases have thioesterase domains (TE), which can be either integrated into modules or encoded by a separate gene. Each synthetase can have type I, type II or both modules. A type I (minimal) module is enlarged to show organization of core sequences and domains. Some peptide synthetases also have condensation or epimerization domains. Figure 19B illustrates the organization of saframycin Mxl synthetase containing 4 amino acid activating modules (Pospiech et. al., "Two Multifunctional Peptide Synthetases and an O-mefhyltransferase are Involved in the Biosynthesis of the DNA-Binding Antibiotic and Antitumour Agent Saframycin Mxl from Myxococcus xanthus," Microbiology. 142(4): 741-746 (1996)). SafBl from the first module is enlarged. Core sequences 1 and 5 in safB 1 are weakly conserved (indicated by dashed vertical bars). The remaining domains are typical of type I as shown in Figure 19A. SafC is a putative O-methyltransferase. Figure 19C illustrates the organization oiCPSl. Sequence analysis revealed two amino acid activating modules (CPSl A and CPS1B), both of which have high similarity to safBl except that core 2 is weakly conserved. A thioesterase domain is found at the C-terminal region of CPSl B. Three vertical arrows indicate the positions of targeted gene disruptions in the wild type genome that yielded the mutant phenotype. TES 1 is a thioesterase encoded by a separate gene (TES1).
Figures 20A-F are comparative alignments of core amino acid sequences in CPSl A and CPS1B with those of other peptide synthetases. Figures 20A-E are comparative amino acid sequence alignments of amino-acid-activating domains (cores 1-5); Figure 20F is a comparative amino acid sequence alignment of the thioester formation domain (core 6). In each subfigure, the first column shows the names of peptide synthetases; the second indicates the position of the first residue aligned in the original amino acid sequence of each protein; the last column on the right indicates the number of amino acids between two cores (Figures 20A-E, in parentheses) or the distance between two adjacent amino-acid- activating modules (Figure 20F, in parentheses). There is an extra column on the right in Figure 20F, showing the total number (underlined) of residues in each amino-acid-activating module in which the aligned core sequence is located. The consensus of each core sequence is on the top, which includes identical or similar residues found in all peptide synthetases or with only a few exceptions (active site is also indicated by asterisks). SafBl : the first module in saframycin Mxl synthetase B of Myxococcus xanthus (Genbank accession U24657); GrsA: gramicidin S synthetase A oi Bacillus brevis (SWISS PROT accession P14687); HTS1 A and HTS1B: the first two modules in HC-toxin synthetase of Cochliobolus carbonum (Q01886); EsynA and EsynB: two modules in enniatin synthetase oi Fusarium scirpi (EMBL accession Z 18755); ACVA and ACVB: the first two modules in ACV synthetase of Aspergillus nidulans (SWISS PROT PI 9787); CsynA and CsynB: the first two modules in cyclosporine synthetase of Tolypocladium nivenm (EMBL Z28383).
Figure 21 is a comparative alignment of amino acid sequences of active sites of thioesterase domains (TE) in CPSl with those of other peptide synthetases. ACV: ACV synthetase (Swiss-PROT accession P19787); GrsB: gramicidin S synthetase B (PI 4688); GrsT: the thioesterase encoded by grsT (P 14686) in gramicidin S synthetase gene cluster; SrfA: surfactin synthetase A-3 (Q08787); TycC: tyrocidine synthetase C (Genbank accession AF004853); TycF: the thioesterase encoded by tycF (AF004853) in the tyrocidine synthetase gene cluster. The highly conserved residues (GXSXG) are indicated by asterisks. The number on the left of each amino acid sequence indicates the original position of the first residue; the number on the right (in parentheses) indicates the distance between the last residue shown to the end of each protein.
Figure 22 is a comparative alignment of the amino acid sequence of the TES1 protein (CH-TES1) with that of other type II thioesterases. HS-TEII: Homo sapiens thioesterase II (EMBL accession X86032); EC-TESB: E. coli acyl- coA thioesterase II (Genbank accession M63308); MT-TESB: Mycobactrium tuberculosis homolog to E. coli. acyl-coA thioesterase II (EMBL Z95387). The identical residues in all four proteins are in bold. The putative active site VHS motif is indicated by asterisks. The numbers on the right column indicate the original position of the last residue of the line in each protein sequence. The entire protein sequence of each TES was aligned using the Jotun Hein Method. Amino acids corresponding to the positions 142-171, 236-265 and 356-367 in CH- TES1 have no significant similarity among the four proteins.
Figures 23A-B are photographs displaying a plant assay (Figure 23A) and a gel blot (Figure 23B). Targeted gene disruption suggests that CPSl is involved in fungal pathogenesis. In Figure 23 A, N-cytoplasm corn was inoculated with (left to right): The REMI mutant R.C4.2696; wild type race T; wild type race O and five disruptants obtained using the linearized p214B7 as vector. All disruptants give the small lesions similar to the original mutant. In Figure 23B, total genomic DNA was digested with BgRl and probed with both 5' and 3' end flanking DNA fragments carried on p214B7 separately (strain order is the same as above). A single band (4.2 kb) is present in both wild type race T and race O but replaced by a 9.3 kb band (increased by the size of the vector, 5.1 kb) in all strains that showed the mutant phenotype in Figure 23 A.
Figure 24 displays a gel blot analysis showing targeted integration of the chromosome walking vector p214SNP into the wild type genome. Lane 1 : wild type race O. Lanes 2-8: seven transformants obtained by transforming wild type race O (strain C5) with p214SNP. Genomic DNAs were digested with BgRl and probed with 3.2 kb flanking genomic DNA fragment cloned on p214Sl which gives three bands in wild type (4.2, 2.1 and 0.5 kb, indicated by arrows on the left). The targeted site is in the 2.1 kb fragment which is missing in all disruptants (indicated by an arrow on the right). Other two bands are intact as predicted. All disruptants showed the same mutant phenotype in the plant assay as shown in Figure 23. Five disruptants (lanes 2, 3, 5, 7 and 8) resulted from a single crossover integration. Integration in two other disruptants (lanes 4 and 6) has not been determined. One of the disruptants (#118, lane 2) was used to recover the plasmid pi 18B14 and pi 18BC4.
Figure 25 displays a gel blot analysis showing targeted integration of the chromosome walking vector pi 18BSP into the wild type genome. Lane 1 : wild type race O; Lane 2: wild type race T; Lanes 3, 4 and 5: three transformants obtained by transforming wild type race O (strain C5) with pi 18BSP. Genomic DNAs were digested with Pst I (which cuts pUCATPH) and probed with the 3.2 kb flanking genomic DNA fragment cloned on p214Sl which gives two bands in wild type (6.6 and 2.5 kb, indicated by arrows on the left). The targeted site is in the 6.6 kb region which is missing in all three disruptants (indicated by arrows on the right). The 2.5 kb band is intact as predicted. The third band (part of vector plus 4.6 kb genomic DNA) does not hybridize to the probe. All disruptants showed the same mutant phenotype in the plant assay as shown in Figure 23. Two disruptants (lanes 3 and 4) resulted from a double crossover integration. Integration in the third disruptant (lane 5) has not been determined. One of the disruptants (#9, lane 3) was used to recover the plasmid p9P2.
Figure 26 displays a gel blot showing the detection of CPSl homologs in C. victoriae and C. carbonum. Genomic DNAs were digested with BgRl and probed with the 3.4 kb CPSl fragment cloned on p214B7 (Figure 2) which includes most of the 4.2 kb BgRl fragment oiCPSl (Figure 15). Lanes 1 and 2: C. heterostrophus race T (C4) and race O (C5), both of which hybridized to ' the 4.2 kb fragment. Lanes 3: C. victoriae (Hvw). Lanes 4, 5 and 6: C. carbonum race 1 (26R13), race 2 (YugY) and race 3 (BZ1209). Note that both C. victoriae and C. carbonum (three races) hybridized to a 5.0 kb fragment and the hybridization signals of all three species showed about the same intensity.
Figure 27 is a high-performance liquid chromatography (HPLC) profile of culture extracts from wild type C. victoriae (HvW, top left) and three transformants (Tx7, Tx2 and Tx9). The major peak for victorin C is indicated by arrowheads. No significant differences in victorin production were detected between the wild type and transformants. Three other transformants (Tx4, Tx5 and Tx8) gave the same results.
Figure 28A-B are photographs which display a plant assay (Figure 28 A) and a gel blot (Figure 28B), showing targeted disruption of the CPSl homolog in C. victoriae. Figure 28 A shows C. victoriae transformants (Tx) with reduced or wild type pathogenicity to susceptible oats. Oat seeds were inoculated with conidial suspensions of (left to right) wild type, Tx7, Tx2 and Tx9 (the last pot on the right is the uninoculated control). Two transformants (Tx7 and Tx2) showed dramatically reduced pathogenicity as indicated by the substantial growth of the oat plants. These two transformants resulted from a homologous integration (lanes 2 and 3 in Figure 28B). Tx9, which killed all oat plants as wild type, resulted from a ectopic integration (lane 4 in Figure 28B). All three transformants produced wild type level of victorin as determined by HPLC analysis (Figure 27). Figure 28B shows disruption oiCPSl homolog in the wild type genome. Genomic DNAs were digested with BgRl and probed with the 3.2 kb CPSl fragment (Kpnl-Sacl) cloned on p214Sl (Figure 2) which hybridized to two fragments (4.2 and 2.2 kb, see Figure 15) in C. heterostrophus (lane 1), but to three fragments (5.0, 1.8 and 0.2 kb) in the C. victoriae (lane 2). In Tx7 (lane 3) and Tx2 (lane 4), one or two of the wild type fragments was replaced by a larger fragment (8.0 kb for Tx7 and 9.4 kb for Tx2) containing the transforming vector. The actual size increase by the vector integration can not be predicted because the presence of polymorphic bands in C. victoriae genome and the presence of duplicated CPSl fragments on the transforming vector (Figure 5). In Tx9 (lane 5), which caused wild type symptoms shown in Figure 28A, all three wild type bands (indicated by arrows) are intact, confirming an ectopic integration. Figure 29 shows the REMI vector pUCATPH. This vector was constmcted by insertion of a 2.4 kb SaR fragment containing the selectable marker cassette (the largest arrow) from pDH25 (Cullen et al., 1987) into the SaR site of polylinker site of pUC18 (between lad and lacZ). Only six-base-pair restriction enzyme sites are shown on the map. Italicized sites are unique; three of them, H dlll, Kpnl, and Sad (bold) have been used for REMI transformation. Non- cutting enzymes are listed below the map (enzymes that recognize six-base-pair sites are underlined), amp, Ampicillin resistance gene; hygB, hygromycin B resistance gene; PtrpC, A. nidulans trpC promoter; TtrpC, A. nidulans trpC terminator; ori, Escherichia coli origin of replication.
Figures 30A-30C are photographs of DNA gel blots showing DNA-DNA hybridization of ChCPSl to other fungal genera and species. In Figure 30A, the gel was loaded with Cochliobolus species (lanes 1-17) as follows: C. heterostrophs race T, race O; C. carbonum race 1, race 2; C. victoriae isolates FI3, HvW; C. bicolor, C. dactyloctenii, C. chloridis, C. homomorphus, C. intermedius, C. melinidis, C. melinidis, C. peregianensis, C. perotidis, C. ravenelii and C. sativus. Figure 30B is a photograph of a DNA gel blot from a gel loaded with other Ascomycete genera (lanes 1-14) as follows: C. carbonum racel (control), Setosphaeria rostrata, Stemphyllium spp., Pyrenophora tritici repentis, Bipolaris sacchari, Alternaria spp., A. solani, Nectria haematococca, Fusarium oxysporum, Glomerella spp. Magnaporthe grisea, F. moniliforme, F. moniliforme (repeat) and A. solani (repeat). Figure 30C is is a photograph of a DNA gel blot from a gel comparing Candida albicans to C. heterostrophus and closely related species (lanes 1-7): C. heterostrophs race T, Bipolaris sacchari, Setosphaeria rostrata, Stemphyllium spp., Pyrenophora tritici repentis, Alternaria spp. and Candida α/&/αws(arrowhead). Genomic DNAs were digested with Hindϊll (A, lanes 1-17; B, lanes 1-11; C, lanes 1-7), Xhol (B, lanes 12 and 14) or BgRl (B, lane 13) and probed with the 3.2 kb fragment oiCPSl from p214Sl (Fig. 2) at high stringency. Weak signals in lanes 3 and 17 (panel A) are due to insufficient DNA loading (confirmed by a repeat experiment).
Figure 31 A is a structural comparison of the ioufyCPSl homologs to ChCPSl . ORFs are indicated by the open boxes; shaded boxes inside indicate functional domains; vertical bars indicate conserved motif sequences found in nonribosomal peptide synthetases (NRPS) as defined by Stachelhaus and Marahiel (Stachelhaus and Marahiel, 1995; Marahiel, 1997) (dashed bars indicate weak conservation). The black bulbs indicate the position of putative introns. Cores 1-5: adenylation; core 6: thioaltion; TE: thioesterase. The distance between core sequences is not drawn in exact scale. The name of proteins is on the left of the ORF boxes and the number of amino acids on the right. The unidentified regions oiAsCPSl and PtCPSl are indicated by dash-lined boxes. The similarity to ChCPSl(iτι the overlapping region only, see text for details) is given in the parentheses under the protein names in the order: nucleotide identity/ amino acid identity/ amino acid similarity. The positions of the ChCPSl amino acid 1040 is indicated by the open arrow; the positions 511 and 1269 (to the first and the last amino acids of AsCPSl and PtCPSl) are indicated by filled triangles. Figure 3 IB is an amino acid alignment of the four CPSl homologs to ChCPSl . 530 amino acids aligned to the amino acids 511-1040 of ChCPS 1 (shown in A) are shown. The identical residues are in uppercase and the similar residues in lowercase. Consensus of sequences similar to the typical NRPS signature motifs is underlined. The putative cyclization domain motif "DXXXXD/ EXXS/ A" is underlined.
Figure 32 is the nucleotide sequence oiFgCPSl. 6,003 base pairs cloned using the plasmid rescue procedure are shown. The amino acid sequence of FgCPSl protein is given below the DNA sequence. The position of the start codon ATG (bold and underlined) is designated +1 and the open reading frame stops at position 5123 (TGA, bold and underlined). A "CT" motif (italicized and underlined) and two putative "CAAT" boxes (bold with asterisks) are found at positions -30, -204 and -302. A putative intron (in lowercase with 5' and 3' splice sequences in bold; branch sites underlined) is located at positions 4245-4290. Conserved core sequences are shaded and the putative cyclization domain motif "DXXXXEXXA" (position 2323-2346) is underlined. A putative polyadenylation signal "AATAA" at position 5188 is bold and overlined.
Figure 33 shows the nucleotide sequence oi AsCPSl. 2,369 base pairs amplified by PCR are shown. The amino acid sequence of AsCPSl protein is given below the DNA sequence. The sequence is not complete. Two putative introns (in lowercase with 5' and 3' splice sequences in bold; branch sites underlined) are located at positions 540-584 and 2012-2059. Conserved core sequences are shaded and the putative cyclization domain motif "DXXXXDXXS" (positions 694-720 ) is underlined. The PCR primer binding sites at the 5' and 3' end are underlined.
Figure 34 shows the nucleotide sequence oi PtCPSl. 2,320 base pairs amplified by PCR are shown. The amino acid sequence of PtCPSl protein is given below the DNA sequence. The sequence is not complete. A putative intron (in lowercase with 5' and 3' splice sequences in bold; branch sites underlined) is located at positions 540-583. Conserved core sequences are shaded and the putative cyclization domain motif "DXXXXDXXS" (positions 693-719) is underlined. The PCR primer binding sites at the 5' and 3' end are underlined.
Figure 35 A is a photograph of a DNA gel blot showing that the 2.2 kb wild type band (arrowhead) is disrupted in homologous transformants
TxFgC8-4, -10, -11 and -5 but is intact in the ectopic transformant TxFgC8-Hl, - H2 and -Bl [generated using Hindlll (H) or £g/7/(B)-digested pFgC8-hygB]. Genomic DNAs were digested with Clal and probed with pFgC8 which carries a 1.0 kb FgCPSl fragment. Figure 35B is a photograph of plants from a vimlence assay showing F. graminearum transformants (Tx) with reduced or wild type vimlence to wheat. Wheat heads were inoculated with conidial suspensions (104/ml) of (left to right) wild type, TxFgC8-4, -10, -HI, and -11 or with water only. Photograph was taken 7 days after inoculation. Note that most spikelets of wheat heads inoculated with homologous transformants looked "healthy" in contrast to those inoculated with ectopic transformants that were completely "bleached" (indistinguishable from wild type).
DETAILED DESCRIPTION OF THE INVENTION
The present invention generally relates to an isolated DNA molecule from a plant pathogen encoding a CPS 1 peptide synthetase. In one embodiment, the DNA molecule has a nucleotide sequence which hybridizes to a DNA molecule having a sequence corresponding to SEQ. ID. No. 2 as follows: TGCCTGCGCC TGTGCTTGTG CCTGTGGAAT GTCGCGGCCC GCTGCTGCAT AGCCTATCTG 60
TACATACAAC ACCATCCCAT CCCGCTTCAC CTGCCTTGCC TCCCTCCTCG TGCCACACAT 120
CCGCCGCCCA CAACACCATG GCTGCGACCA ACCCCGAGCT GCAGGCCAAA CTGCAGGAGC 180
TGGACCACGA GCTCGAGGAG GGCGATATTA CACAAAAAGG GTCCGTACTG CTGCACCACC 240 ACCGCCATCC GCCTCTCTGC GTGCGCTAAT CAGTCGCATA GCTATGAAAA ACGTCGCACC 300
GTGCTGCTGT CGCAGTATCT AGGGCCTGAC TTTGCTGCCC AGTTGCAGGC CGACCTGAAC 360
CAGCAGAACC CACCCCAACC ATCCAGTGAG GGCTCTCGCT CCCGCACCGC ATCCTTTGCT 420
ATTCCGTCCG GTCCGAGTCC ATCACNGCGA CCACAACCCC CACATATCCA GCTCCCCCGC 480
CCCGACTCAT ACCATGACGC TTCCGCACAG GGCCAATTGG GCGCACCCAT GCCATATGCG 540 AACGCCTCCG CCGCTGCCTC GGGGGGCTCG CAGTACATGG CATACCCGCC CAGCCAAGTC 600
GGCCGTTTTC AAGAGAAGCA GCTGGGCCTG CGTACAAATT CGCTCCAGCG CAATTCCTCA 660
CAGCTGTCGC AAGGAAGCGA GACGTTCATT CCACGGCCTC AAACGCCTGA ATACAACCAC 720
TCGCGCGAGC CCACCATGAT GGGCAACTAC GCCTTCAATC CAGACAATCA GCAAAGTTAT 780
GATGGCCAAT TTGGCTCTCC GGGAGAGGCC AGTCGAAGGA GCACCATGCT CGAGGTAAAC 840 CAGGGTTATT TTTCCGACTT CACAGGCCAG CAGATGCAAG ACAATCGCGA CTCGTATGGG 900
GGACCCAACC GCTACTCGTC GGGAGATGCC TTTTCTCCTA CCGCCGCGAT TCCACCTCCC 960
ATGATGAACC CCAACGATCT CCCCTTGGGC GCTGCTGAAA CCATGATGCC GCTAGAGCCC 1020
CGCGATCTGC CTTTTGACGT TTACGACCCT CACAACCCCA ATGTCAAAAT GTCAAAGTTT 1080
GACAACATTG GCGCTGTCTT GCGTCACCGA AGTCGCACAC AGCCAAGGAC GACTGCCTTC 1140 TGGGTCCTTG ACGCAAAAGG CAAAGAGACG GCGTCCATCA CCTGGGAAAA GGTGGCTAGT 1200
CGCGCGGAAA AGGTGGCCAA AGTGATTCGG GACAAGAGCA ACCTCTATCG AGGCGACCGT 1260
GTGGCATTAG TGTACAGGGA TACAGAAATC ATTGATTTTG TCGTGGCGTT GATGGGCTGC 1320
TTCATTGCGG GCGTTGTAGC GGTACCCATC AATAGCGTCG ACGACTACCA GAAACTCATT 1380
CTTCTCCTAA CGACAACTCA AGCTCATCTC GCATTGACCA CAGACAACAA TCTCAAGGCC 1440 TTTCATCGTG ACATTAGTCA GAACCGTCTG AAATGGCCGA GTGGGGTAGA GTGGTGGAAG 1500
ACGAACGAGT TTGGCAGCCA CCACCCCAAG AAACATGACG ATACTCCAGC TTTGCAAGTA 1560
CCAGAGGTTG CCTATATTGA GTTCTCGCGT GCACCTACTG GTGACCTTCG CGGTGTGGTG 1620
CTTAGTCACC GGACTATTAT GCACCAAATG GCCTGCATCA GTGCCATGAT TAGCACGATA 1680 CCCACCAACG CTCAGAGCCA AGACACGTTC AGCACTAGCC TACGGGATGC AGAGGGAAAG 1740
TTCGTTGCTC CAGCACCGTC CAGAAACCCC ACAGAAGTGA TCCTCACGTA CCTCGACCCG 1800
CGCGAAAGCG CTGGTCTCAT TCTCAGTGTC TTGTTTGCAG TTTATGGAGG CCACACCACC 1860
GTATGGCTCG AGACAGCGAC CATGGAAACC CCGGGTCTAT ATGCACATCT CATCACCAAA 1920 TACAAGTCCA ACATACTGCT AGCGGATTAC CCAGGCCTCA AGCGCGCTGC ATACAACTAC 1980
CAACAGGATC CAATGGCTAC AAGAAACTTC AAGAAAAACA CAGAACCCAA CTTCGCCTCC 2040
GTGAAGATCT GTCTGATTGA CACGCTTACC GTCGACTGTG AATTTCACGA AATTCTCGGA 2100
GATCGATATT TCAGGCCACT GCGAAACCCT AGAGCGCGAG AACTGATCGC GCCAATGCTC 2160
TGCTTGCCAG AACATGGTGG AATGATAATA TCTGTACGCG ACTGGCTAGG TGGAGAGGAG 2220 CGCATGGGCT GCCCGCTAAG CATAGCAGTA GAAGAGTCAG ATAATGATGA AGATGATACA 2280
GAGGATAAGT ATGCAGCGGC AAATGGCTAC TCCAGTCTTA TTGGTGGTGG CACTACAAAG 2340
AACAAAAAGG AGAAGAAGAA GAAAGGCCCG ACAGAGCTTA CAGAAATCTT GCTGGACAAG 2400
GAAGCTCTGA AGATGAACGA AGTCATTGTT CTGGCCATTG GAGAAGAAGC AAGCAAGCGG 2460
GCAAACGAGC CCGGCACCAT GCGAGTCGGT GCCTTTGGAT ACCCCATACC GGATGCGACA 2520 CTAGCTATTG TAGACCCTGA GACAAGTCTT CTATGTTCAC CATACTCGAT AGGCGAGATC 2580
TGGGTAGATT CGCCTTCACT CTCTGGTGGC TTCTGGCAGC TGCAGAAGCA TACAGAGACC 2640
ATTTTCCATG CTCGACCATA CCGTTTCGTT GANGGTAGCC CTACGCCACA GTTGCTTGAA 2700
CTCGAGTTTC TGCGTACTGG ACTCCTCGGC TTTGTTGTAG ANGGAAAAAT ATTTGTCCTT 2760
GGACTGTACG AAGATCGCAT CAGACAGCGT GTTGAATGGG TAGAAAATGG TCAGCTTGAA 2820 GCCGAGCATC GATACTTTTT TGTGCAGCAC CTGGTCACAA GCATTATGAA GGCCGTGCCA 2880
AAAATTTACG ACTGGTAAGT GAGCTGCCAA CAGAGCAAGG ACTGTCTAAC GTGTCATAGC 2940
TCGTCGTTTG ATTCTTATGT AAATGGTGAA TACCTGCCAA TCATTCTCAT CGAGACGCAG 3000
GCCGCATCGA CTGCGCCCAC AAACCCAGGT GGACCACCAC AACAATTGGA TATACCATTT 3060
TTGGATTCAC TATCTGAGAG GTGCATGGAG GTCCTTTACC AAGAGCATCA TTTACGGGTA 3120 TACTGCGTGA TGATTACAGC ACCTAATACA CTTCCACGAG TCATCAAGAA CGGACGGCGA 3180
GAAATTGGCA ATATGCTGTG TANGAGANAG TTTGACAATG GCTCTCTGCC CTGTGTNCAC 3240
GTNAAGTTTG GCATTGAGCG ATCAGTGCAG AACATTGCGC TCGGTGACGA TCCCGCTGGC 3300
GGCATGTGGT CATTTGAGGC ATCAATGGCA CGTCAGCAAT TCTTGATGCT CCAAGACAAG 3360 CAATACTCTG GTGTCGATCA TCGCGAAGTC GTCATTGACG ACAGGACATC GACTCCACTC 3420
AATCAGTTCT CGAATATCCA CGACCTGATG CAATGGCGTG TATCTCGGCA GGCCGAGGAA 3480
CTTGCTTACT GCACTGTCGA CGGTCGAGGA AAAGAGGGCA AAGGCGTCAA TTGGAAGAAG 3540
TTTGATCAAA AGGTTGCGGG CGTAGCAATG TACCTCAAGA ACAAGGTCAA GGTCCAGGCC 3600 GGCGATCATC TCCTTCTGAT GTACACGCAT TCAGAAGAAT TTGTTTATGC TGTTCATGCA 3660
TGTTTTGTGC TTGGAGCTGT TTGCATACCA ATGGCGCCAA TTGATCAGAA CCGGTTGAAT 3720
GAGGATGCGC CGGCCTTGCT GCATATCCTT GCAGATTTCA AGGTCAAAGC CATTCTTGTC 3780
AACGCTGACG TTGACCATCT GATGAAGATC AAGCAAGTAT CGCAGCACAT CAAACAATCG 3840
GCCGCTATCC TCAAGATCAG TGTGCCAAAC ACATACAGCA CAACAAAGCC GCCAAAGCAA 3900 TCCAGTGGCT GCCGCGACCT CAAGCTTACA ATTCGACCGG CATGGATTCA GGCGGGTTTC 3960
CCAGTGCTAG TCTGGACATA CTGGACGCCC GATCAACGTC GTATCGCAGT TCAGCTGGGC 4020
CATAGCCAAA TCATGGCACT GTGCAAGGTC CAAAAAGAAA CATGCCAAAT GACAAGTACA 4080
CGACCAGTCC TTGGTTGTGT CCGGAGCACG ATAGGACTTG GTTTCCTTCA CACTTGTCTC 4140
ATGGGAATCT TCCTTGCCGC ACCCACATAC CTGGTGTCAC CTGTTGACTT TGCACAAAAC 4200 CCTAATATTC TGTTCCAAAC GCTTTCGCGG TACAAGATCA AGGATGCATA TGCAACGAGT 4260
CAAATGTTGG ACCACGCCAT CGCACGCGGA GCTGGTAAGA GTATGGCTCT GCACGAGCTG 4320
AAGAATCTCA TGATTGCGAC TGATGGAAGA CCACGCGTTG ATGTTTGTAA GTGAACATTT 4380
GTATGAGAGG ACTTTCATGA TTGCTAACTC AATGCAGACC AAAGAGTGCG TGTGCACTTT 4440
GCGCCAGCCA ACTTAGACCC AACCGCAATC AACACTGTCT ACTCACATGT ATTGAACCCA 4500 ATGGTAGCAT CACGATCATA CATGTGTATT GAGCCAGTCG AGCTCCATCT CGATGTGCAT 4560
GCTCTGCGAC GCGGCCTCGT CATGCCCGTT GACCCTGACA CAGAGCCCAA CGCTTTGCTC 4620
GTCCAAGACT CGGGCATGGT GCCAGTGAGC ACGCAAATAT CCATTGTCAA CCCAGAGACC 4680
AACCAACTGT GCTTGAACGG CGAGTACGGC GAGATCTGGG TGCAGTCCGA GGCGAATGCT 4740
TATAGCTTCT ACATGTCGAA AGAGCGCTTG GATGCAGAAC GCTTCAATGG GAGGACGATT 4800 GACGGAGACC CAAATGTGCG ATATGTTCGT ACAGGCGATT TAGGATTTTT GCACAGCGTG 4860
ACACGGCCCA TTGGACCCAA CGGTGCACCT GTTGATATGC AGGTGCTTTT CGTGCTTGGA 4920
AGCATAGGTG ACACTTTTGA AGTCAACGGA CTGAACCATT TCTCTATGGA CATTGAGCAG 4980
TCTGTTGAAC GTTGTCACCG GAATATTGTC CCTGGAGGCT GGTACGTTTC TTCGATTCGC 5040 TGTTATTTAG TAAATACTTA CTAACACTCT ACAGTGCTGT TTTCCAGGCA GGTGGGCTTG 5100
TTGTTGTCGT TGTGGAAATC TTCCGACGCA ACTTCCTCGC AAGCATGGTG CCTGTGATTG 5160
TCAATGCAAT TTTGAACGAG CATCAGCTGG TCATTGACAT TGTCTCGTTT GTGCAAAAGG 5220
GCGACTTCCA CCGGTCTCGT CTGGGCGAGA AGCAACGCGG AAAGATTCTT GCAGGATGGG 5280 TCACACGGAA GATGCGCACA ATAGCCCAGT ACAGTATACG GGATCCTAAT GGACAGGATT 5340
CCCAGATGAT CACGGAAGAG CCTGGTCCAC GGGCTAGATG ACTGGAAGTA TGCTTGGGCG 5400
AATGGGCGGC CCAGCCAGTA TCAAGGCCGG GTCGACAAGA GCACCGAGTC TAATGGGCAT 5460
GACAGCGACT ATGAATAATC TATCCCTTAC ACAGCAGCAA CAGCAGCAAT ACCAACAGCC 5520
GGGTATGTAT GCTCAACAGC AAGGCATGCA CCCCCAGCAA CAACACCAAT TTAGCATGTC 5580 CAACACGCCA CCACAAGGTC CACCCCAAGG CGTAGAACTA CATGATCCTA GCGACCGCAC 5640
ACCAACAGAC AACCGGCACT CTTTCCTTGC CGACCCGCGT ATGCAGAACC AGGGCCAAAT 5700
GAACGAGACG GGCGCCTACG AACCCATGAA CTATCAAAAC GCGTATCATC CGCATCAACA 5760
ACAATACGAA TCTGAAGACG GGGGGAGCAG ACTCAGCGGC CCCGTGCCAG ACGTGCTGCG 5820
GCCGGGTCCT TCATCCGGGT CCATAGAGCA GCACGACCAA GCTAACAACG ACAACAATAT 5880 GTGGAATAAT CGCGAGTACT ATGGTAACAG CCCATCGTAT GCAGGCGGAT ACACGCAAGA 5940
TGGCAATATC CACGAGCAGC AACAACACGA TGAGTACACG AGTAATGCGT CATATGGCGG 6000
AAATCAAGGA GCAGGCGGAG GCAGCGGCGG CGGTGGCGGT CTCCGAGTTG CAAATCGTGA 6060
CAGCTCCGAC AGCGAGGGTG CAGATGACGA CGCTTGGAGA CGTGATGCCC TTGCTCAGAT 6120
CAATTTTGCG GGCGGCGCTG CTGCTGCCTC CGCTGGAGCA CCTGCTGCTG GTGCTTCTTC 6180 TTCGCAGCCG GGCCATGCGC AGTAGACGGG ATATGCGTGA GTTTTTTTTT AAATTTCGTA 6240
CATAGAGACC GTTGTATACG CAGGTTTCAA ATTAGAAGAG CGAATATGCA TATCAGCTGT 6300
TGTTCAATGT TCTAGTTTGG GAAGGTTAAC CCCCCCCCCT TCCCCTTCCA AGACTTTTCA 6360
CTTGTTTGTG TGTGATTTAA ATCTGGAGAT TTCAAATCTA CATCTCGCTA TACATAGGTG 6420
TTGTTTGATA ACGTAGGGGG CAGAAGGGTA TCTCGTGATA TTAGACTGGG AGTTGCATGA 6480 ATCAAGGTGT TGAGCAAAAA AAGAGAGAGC GGTGAAGGGC GGGGGGGATA GGTGGTGTGC 6540
ACGTGGCTG 6549
In a preferred embodiment, the plant pathogen is Cochliobolus heterostrophus. In another preferred embodiment, the plant pathogen is Cochliobolus carbonum, C. victoriae, C. sativus, C. specifer, C. homomorphus, C. dactyloctenii, Setosphaeria turcica, S. rostrata, or Bioplaris sacchari.
The peptide synthetase of SEQ. ID. No. 2 has a deduced amino acid sequence corresponding to SEQ. ID. No. 3 as follows:
Met Leu Glu Val Asn Gin Gly Tyr Phe Ser Asp Phe Thr Gly Gin 15
Gin Met Gin Asp Asn Arg Asp Ser Tyr Gly Gly Pro Asn Arg Tyr 30
Ser Ser Gly Asp Ala Phe Ser Pro Thr Ala Ala lie Pro Pro Pro 45
Met Met Asn Pro Asn Asp Leu Pro Leu Gly Ala Ala Glu Thr Met 60
Met Pro Leu Glu Pro Arg Asp Leu Pro Phe Asp Val Tyr Asp Pro 75 His Asn Pro Asn Val Lys Met Ser Lys Phe Asp Asn lie Gly Ala 90
Val Leu Arg His Arg Ser Arg Thr Gin Pro Arg Thr Thr Ala Phe 105
Trp Val Leu Asp Ala Lys Gly Lys Glu Thr Ala Ser lie Thr Trp 120
Glu Lys Val Ala Ser Arg Ala Glu Lys Val Ala Lys Val lie Arg 135
Asp Lys Ser Asn Leu Tyr Arg Gly Asp Arg Val Ala Leu Val Tyr 150 Arg Asp Thr Glu lie lie Asp Phe Val Val Ala Leu Met Gly Cys 165
Phe lie Ala Gly Val Val Ala Val Pro lie Asn Ser Val Asp Asp 180
Tyr Gin Lys Leu lie Leu Leu Leu Thr Thr Thr Gin Ala His Leu 195
Ala Leu Thr Thr Asp Asn Asn Leu Lys Ala Phe His Arg Asp He 210
Ser Gin Asn Arg Leu Lys Trp Pro Ser Gly Val Glu Trp Trp Lys 225 Thr Asn Glu Phe Gly Ser His His Pro Lys Lys His Asp Asp Thr 240
Pro Ala Leu Gin Val Pro Glu Val Ala Tyr He Glu Phe Ser Arg 255
Ala Pro Thr Gly Asp Leu Arg Gly Val Val Leu Ser His Arg Thr 270
He Met His Gin Met Ala Cys He Ser Ala Met He Ser Thr He 285
Pro Thr Asn Ala Gin Ser Gin Asp Thr Phe Ser Thr Ser Leu Arg 300 Asp Ala Glu Gly Lys Phe Val Ala Pro Ala Pro Ser Arg Asn Pro 315
Thr Glu Val He Leu Thr Tyr Leu Asp Pro Arg Glu Ser Ala Gly 330
Leu He Leu Ser Val Leu Phe Ala Val Tyr Gly Gly His Thr Thr 345
Val Trp Leu Glu Thr Ala Thr Met Glu Thr Pro Gly Leu Tyr Ala 360
His Leu He Thr Lys Tyr Lys Ser Asn He Leu Leu Ala Asp Tyr 375 Pro Gly Leu Lys Arg Ala Ala Tyr Asn Tyr Gin Gin Asp Pro Met 390
Ala Thr Arg Asn Phe Lys Lys Asn Thr Glu Pro Asn Phe Ala Ser 405
Val Lys He Cys Leu He Asp Thr Leu Thr Val Asp Cys Glu Phe 420
His Glu He Leu Gly Asp Arg Tyr Phe Arg Pro Leu Arg Asn Pro 435 Arg Ala Arg Glu Leu He Ala Pro Met Leu Cys Leu Pro Glu His 450
Gly Gly Met He He Ser Val Arg Asp Trp Leu Gly Gly Glu Glu 465
Arg Met Gly Cys Pro Leu Ser He Ala Val Glu Glu Ser Asp Asn 480
Asp Glu Asp Asp Thr Glu Asp Lys Tyr Ala Ala Ala Asn Gly Tyr 495
Ser Ser Leu He Gly Gly Gly Thr Thr Lys Asn Lys Lys Glu Lys 510 Lys Lys Lys Gly Pro Thr Glu Leu Thr Glu He Leu Leu Asp Lys 525
Glu Ala Leu Lys Met Asn Glu Val He Val Leu Ala He Gly Glu 540
Glu Ala Ser Lys Arg Ala Asn Glu Pro Gly Thr Met Arg Val Gly 555
Ala Phe Gly Tyr Pro He Pro Asp Ala Thr Leu Ala He Val Asp 570
Pro Glu Thr Ser Leu Leu Cys Ser Pro Tyr Ser He Gly Glu He 585 Trp Val Asp Ser Pro Ser Leu Ser Gly Gly Phe Trp Gin Leu Gin 600
Lys His Thr Glu Thr He Phe His Ala Arg Pro Tyr Arg Phe Val 615
Xaa Gly Ser Pro Thr Pro Gin Leu Leu Glu Leu Glu Phe Leu Arg 630
Thr Gly Leu Leu Gly Phe Val Val Glu Gly Lys He Phe Val Leu 645
Gly Leu Tyr Glu Asp Arg He Arg Gin Arg Val Glu Trp Val Glu 660 Asn Gly Gin Leu Glu Ala Glu His Arg Tyr Phe Phe Val Gin His 675
Leu Val Thr Ser He Met Lys Ala Val Pro Lys He Tyr Asp Cys 690
Ser Ser Phe Asp Ser Tyr Val Asn Gly Glu Tyr Leu Pro He He 705
Leu He Glu Thr Gin Ala Ala Ser Thr Ala Pro Thr Asn Pro Gly 720
Gly Pro Pro Gin Gin Leu Asp He Pro Phe Leu Asp Ser Leu Ser 735 Glu Arg Cys Met Glu Val Leu Tyr Gin Glu His His Leu Arg Val 750
Tyr Cys Val Met He Thr Ala Pro Asn Thr Leu Pro Arg Val He 765
Lys Asn Gly Arg Arg Glu He Gly Asn Met Leu Cys Arg Arg Glu 780
Phe Asp Asn Gly Ser Leu Pro Cys Val His Val Lys Phe Gly He 795
Glu Arg Ser Val Gin Asn He Ala Leu Gly Asp Asp Pro Ala Gly 810 Gly Met Trp Ser Phe Glu Ala Ser Met Ala Arg Gin Gin Phe Leu 825 Met Leu Gin Asp Lys Gin Tyr Ser Gly Val Asp His Arg Glu Val 840
Val He Asp Asp Arg Thr Ser Thr Pro Leu Asn Gin Phe Ser Asn 855
He His Asp Leu Met Gin Trp Arg Val Ser Arg Gin Ala Glu Glu 870
Leu Ala Tyr Cys Thr Val Asp Gly Arg Gly Lys Glu Gly Lys Gly 885 Val Asn Trp Lys Lys Phe Asp Gin Lys Val Ala Gly Val Ala Met 900
Tyr Leu Lys Asn Lys Val Lys Val Gin Ala Gly Asp His Leu Leu 915
Leu Met Tyr Thr His Ser Glu Glu Phe Val Tyr Ala Val His Ala 930
Cys Phe Val Leu Gly Ala Val Cys He Pro Met Ala Pro He Asp 945
Gin Asn Arg Leu Asn Glu Asp Ala Pro Ala Leu Leu His He Leu 960 Ala Asp Phe Lys Val Lys Ala He Leu Val Asn Ala Asp Val Asp 975
His Leu Met Lys He Lys Gin Val Ser Gin His He Lys Gin Ser 990
Ala Ala He Leu Lys He Ser Val Pro Asn Thr Tyr Ser Thr Thr 1005
Lys Pro Pro Lys Gin Ser Ser Gly Cys Arg Asp Leu Lys Leu Thr 1020
He Arg Pro Ala Trp He Gin Ala Gly Phe Pro Val Leu Val Trp 1035 Thr Tyr Trp Thr Pro Asp Gin Arg Arg He Ala Val Gin Leu Gly 1050
His Ser Gin He Met Ala Leu Cys Lys Val Gin Lys Glu Thr Cys 1065
Gin Met Thr Ser Thr Arg Pro Val Leu Gly Cys Val Arg Ser Thr 1080
He Gly Leu Gly Phe Leu His Thr Cys Leu Met Gly He Phe Leu 1095
Ala Ala Pro Thr Tyr Leu Val Ser Pro Val Asp Phe Ala Gin Asn 1110 Pro Asn He Leu Phe Gin Thr Leu Ser Arg Tyr Lys He Lys Asp 1125
Ala Tyr Ala Thr Ser Gin Met Leu Asp His Ala He Ala Arg Gly 1140
Ala Gly Lys Ser Met Ala Leu His Glu Leu Lys Asn Leu Met He 1155
Ala Thr Asp Gly Arg Pro Arg Val Asp Val Tyr Gin Arg Val Arg 1170
Val His Phe Ala Pro Ala Asn Leu Asp Pro Thr Ala He Asn Thr 1185 Val Tyr Ser His Val Leu Asn Pro Met Val Ala Ser Arg Ser Tyr 1200
Met Cys He Glu Pro Val Glu Leu His Leu Asp Val His Ala Leu 1215
Arg Arg Gly Leu Val Met Pro Val Asp Pro Asp Thr Glu Pro Asn 1230
Ala Leu Leu Val Gin Asp Ser Gly Met Val Pro Val Ser Thr Gin 1245
He Ser He Val Asn Pro Glu Thr Asn Gin Leu Cys Leu Asn Gly 1260 Glu Tyr Gly Glu He Trp Val Gin Ser Glu Ala Asn Ala Tyr Ser 1275 Phe Tyr Met Ser Lys Glu Arg Leu Asp Ala Glu Arg Phe Asn Gly 1290
Arg Thr He Asp Gly Asp Pro Asn Val Arg Tyr Val Arg Thr Gly 1305
Asp Leu Gly Phe Leu His Ser Val Thr Arg Pro He Gly Pro Asn 1320
Gly Ala Pro Val Asp Met Gin Val Leu Phe Val Leu Gly Ser He 1335 Gly Asp Thr Phe Glu Val Asn Gly Leu Asn His Phe Ser Met Asp 1350
He Glu Gin Ser Val Glu Arg Cys His Arg Asn He Val Pro Gly 1365
Gly Cys Ala Val Phe Gin Ala Gly Gly Leu Val Val Val Val Val 1380
Glu He Phe Arg Arg Asn Phe Leu Ala Ser Met Val Pro Val He 1395
Val Asn Ala He Leu Asn Glu His Gin Leu Val He Asp He Val 1410 Ser Phe Val Gin Lys Gly Asp Phe His Arg Ser Arg Leu Gly Glu 1425
Lys Gin Arg Gly Lys He Leu Ala Gly Trp Val Thr Arg Lys Met 1440
Arg Thr He Ala Gin Tyr Ser He Arg Asp Pro Asn Gly Gin Asp 1455
Ser Gin Met He Thr Glu Glu Pro Gly Pro Arg Ala Ser Met Thr 1470
Gly Ser Met Leu Gly Arg Met Gly Gly Pro Ala Ser He Lys Ala 1485 Gly Ser Thr Arg Ala Pro Ser Leu Met Gly Met Thr Ala Thr Met 1500
Asn Asn Leu Ser Leu Thr Gin Gin Gin Gin Gin Gin Tyr Gin Gin 1515
Pro Gly Met Tyr Ala Gin Gin Gin Gly Met His Pro Gin Gin Gin 1530
His Gin Phe Ser Met Ser Asn Thr Pro Pro Gin Gly Pro Pro Gin 1545
Gly Val Glu Leu His Asp Pro Ser Asp Arg Thr Pro Thr Asp Asn 1560 Arg His Ser Phe Leu Ala Asp Pro Arg Met Gin Asn Gin Gly Gin 1575
Met Asn Glu Thr Gly Ala Tyr Glu Pro Met Asn Tyr Gin Asn Ala 1590
Tyr His Pro His Gin Gin Gin Tyr Glu Ser Glu Asp Gly Gly Ser 1605
Arg Leu Ser Gly Pro Val Pro Asp Val Leu Arg Pro Gly Pro Ser 1620
Ser Gly Ser He Glu Gin His Asp Gin Ala Asn Asn Asp Asn Asn 1635 Met Trp Asn Asn Arg Glu Tyr Tyr Gly Asn Ser Pro Ser Tyr Ala 1650
Gly Gly Tyr Thr Gin Asp Gly Asn He His Glu Gin Gin Gin His 1665
Asp Glu Tyr Thr Ser Asn Ala Ser Tyr Gly Gly Asn Gin Gly Ala 1680
Gly Gly Gly Ser Gly Gly Gly Gly Gly Leu Arg Val Ala Asn Arg 1695
Asp Ser Ser Asp Ser Glu Gly Ala Asp Asp Asp Ala Trp Arg Arg 1710 Asp Ala Leu Ala Gin He Asn Phe Ala Gly Gly Ala Ala Ala Ala 1725 Ser Ala Gly Ala Pro Ala Ala Gly Ala Ser Ser Ser Gin Pro Gly 1740
His Ala Gin 1743
In another embodiment of the invention, there is provided an isolated nucleic acid molecule encoding a CPS 1 peptide synthetase homolog and which hybridizes to a nucleic acid molecule having a sequence corresponding to SEQ ID No. 41 as follows :
AAGAAGAAAG GGCCGACCGA GTTGACCGAA ATATTGCTAG ATAAGGAAGC ACTGAAGCTG 60
AACGAAGTTG TTGTTTTGGC CATTGGAGAG GAAGTGAGCA AGCGTGTCAA CGAACCCGGC 120
ACTATGAGAG TCGGTGCTTT TGGCTACCCG ATACCAGATG CGACGCTGGC CGTCGTCGAT 180
CCGGAAACTA ATCTTTTGTG TTCACCCTAT TCCATAGGAG AGATCTGGGT AGACTCGCCA 240 TCATTGTCCG GAGGGTTTTG GCAGCTGCAG AAGCACACTG AGACTATTTT CCACGCTCGG 300
CCATATCGTT TCGTAGAGGG CAGCCCAACC CCGCAACTAC TCGAACTGGA GTTTCTACGC 360
ACTGGACTGC TCGGATGCGT GGTAGAAGGC AAAATCTTCG TATTAGGCCT GTACGAGGAC 420
CGGATTAGGC AGCGCGTTGA ATGGGTAGAG CACGGTCAGC TAGAAGCCGA ACATAGGTAT 480
TTCTTCGTGC AGCATCTTGT CACCAGCATT ATGAAAGCTG TTCCAAAGAT TTACGACTGG 540 TAAGTGCTAT CGAATCTCTG GGTAATCAAC CTAACATTGC GCAGCTCGTC TTTCGATTCC 600
TATGTCAACG GCGAATACTT ACCAATCATC CTTATCGAGA CACAGGCCGC ATCAACTGCT 660
CCCACAAATC CAGGCGGGCC ACCACAACAA CTTGACATTC CTTTCCTAGA CTCTCTTTCT 720
GAGCGATGTA TGGAGGTACT GTATCAAGAA CACCACCTTC GGGTGTATTG TGTGATGATC 780
ACTGCACCGA ACACACTCCC GCGAGTCATC AAGAACGGTC GACGAGAAAT TGGAAACATG 840 CTTTGCCGGA GAGAATTTGA CAATGGCTCG CTACCCTGCG TTCACGTCAA GTTTGGCGTC 900
GAGAGGTCGG TCCAGAATAT TGCGCTAGGT GATGACCCTG CTGGCGGCAT GTGGTCTTAC 960
GAGGCGTCGA TGGCACGCCA GCAGTTCCTG ATGCTTCAAG ATAAGCAGTA CTCTGGAGTA 1020
GATCACAGAG AAGTCGTTAT TGACGACAGA ACGTCGACGC CGCTCAACCA GTTCTCCAAC 1080
ATTCATGACC TTATGCAATG GCGCGTACAA CGACAAGCTG AAGAGCTCGC CTACTGCACG 1140 GTAGATGGTC GAGGTAAAGA GGGCAAAGGC GTCAACTGGA AGAAGTTCGA CCAGAAGGTC 1200
GCAGGTGTCG CCATGTACCT GAAGAACAAG GTCAAGGGTC AGACTGGTGA CCACCTGCTC 1260
TTGATGTACA CCCACTCGGA AGACTTTGTC TATGCCGTAC ACGCGTGTTT CGTCCTTGGA 1320
GCTGTGTGTA TACCCATGGC ACCAATCGAC CAGAACAGGC TAAATGAAGA CGCGCCCGCA 1380
CTACTACATA TCATTGCTGA CTTCAAGGTC AAGGCTATCC TCGTCAATGC TGGCGTAGAC 1440 CACCTGATGA AGGTCAAGCA AGTATCGCAG CACATCAAAC AGTCAGCAGT CATTCTCAAG 1500
ATCAACGTAC CGAATACCTA TAACACCACA AAACCACCTA AGCAGTCTAG TGGTTGCCGC 1560 GATCTTAAGC TCACAATACG ACCTGCTTGG ATACAATCTG GTTTCCCTGT TCTAGTATGG 1620
ACATACTGGA CACCTGACCA GAGACGCATA GCTGTGCAAT TAGGTCATAG CCAAATCATG 1680 GCGCTATGCA AAGTTCAGAA AGAAACGTGC CAGATGACGA GCACACGGCC CGTCCTTGGA 1740
TGTGTTCGTA GCACGATCGG TCTTGGCTTC ATACACACCT GTGTTATGGG TATCTTCCTC 1800
GCAGCGCCAA CTTACCTTGT GTCACCTGTC GATTTTGCGC AAAACCCGAA CATCCTCTTC 1860
CAGACCATGT CGAGATACAA GATCAAGGAC GCGTATGCGA CCAGCCAAAT GCTGGACCAC 1920
GCTATTGCAC GAGGTGCTGG CAAGAACATG GCTCTGCACG AGCTCAAGAA CCTCATGATC 1980 GCGACTGACG GTCGGCCGCG CGTAGACGTC TGTAAGTGTT GCGATCCTGT ATAAGCATCT 2040
GAAATCTAAT TCTTGATAGA CCAGCGTGTG CGAGTACACT TCTCGCCAGC AAGTTTGGAC 2100
CGAACGGCAA TCAATACTGT TTACTCACAC GTACTGAATC CTATGGTCGC ATCGCGGTCA 2160
TACATGTGCA TCGAACCCAT AGAACTACAT CTCGATGTCG GTGCCCTTCG AAGAGGTCTC 2220
ATCATGCCTG TCGACCCAGA CACGGAACCT GGTGCTCTCT TAGTCCAGGA CTCGGGTATG 2280 GTACCAGTTA GTACACAAAT TTCAATCGTG AATCCAGAGA CAAACCAGCT TTGCCTAGTC 2340
GGCGAGTATG GCGAAATCTG GGTCCAACC 2370
Preferably, the CPSl nucleic acid molecule which hybridizes to a nucleic acid molecule having a sequence as set forth in SEQ ID No. 41 is from the plant pathogen Altenaria solani. In another preferred embodiment, the CPSl gene is from a plant pathogen such as Alternaia alternatherae, A. alternata, A. amaranthi, A. araliae, A. brassicae, A. brassicicola, A. camelliae, A. cassiae, A. cheiranthi, A. cinerariae, A. gossypii, A. helianthi, A. helianthinficiens, A. mali, or A. raphani.
The peptide synthetase product of SEQ ID NO:41 has a deduced amino acid sequence as follows (SEQ ID NO:42):
KKKGPTELTE I LDKEALKL NEVWLAIGE EVSKRVNEPG TMRVGAFGYP IPDATLAWD 60 PETNLLCSPY SIGEIWVDSP SLSGGF QLQ KHTETIFHAR PYRFVEGSPT PQLLELEFLR 120
TGLLGCWEG KIFV GLYED RIRQRVEWVE HGQLEAEHRY FFVQHLVTSI MKAVPKIYDC 180
SSFDSYVNGE YLPIILIETQ AASTAPTNPG GPPQQLDIPF LDSLSERCME VLYQEHHLRV 240
YCVMITAPNT LPRVIKNGRR EIGNMLCRRE FDNGSLPCVH VKFGVERSVQ NIALGDDPAG 300
GMWSYEASMA RQQFLMLQDK QYSGVDHREV VIDDRTSTPL NQFSNIHD M Q RVQRQAEE 360 LAYCTVDGRG KEGKGVNWKK FDQKVAGVAM YLKNKVKGQT GDHLLLMYTH SEDFVYAVHA 420
CFVLGAVCIP MAPIDQNRLN EDAPA LHII ADFKVKAI V NAGVDHLMKV KQVSQHIKQS 480 AVI KINVPN TYNTTKPPKQ SSGCRDLKLT IRPAWIQSGF PVLVWTYWTP DQRRIAVQLG 540
HSQIMALCKV QKETCQMTST RPVLGCVRST IGLGFIHTCV MGIFLAAPTY LVSPVDFAQN 600 PNILFQTMSR YKIKDAYATS QMLDHAIARG AGKNMALHEL KNLMIATDGR PRVDVYQRVR 660
VHFSPASLDR TAINTVYSHV LNPMVASRSY MCIEPIELHL DVGALRRGLI MPVDPDTEPG 720
ALLVQDSGMV PVSTQISIVN PETNQLCLVG EYGEIWVQ 760
In another embodiment of the invention, there is provided an isolated nucleic acid molecule encoding a CPS 1 peptide synthetase homolog and which hybridizes to a nucleic acid molecule having a sequence corresponding to SEQ ID No. 43 as follows :
CTCGAGGTTA GTAAAAGATC CCCGTTTGTT CCACAAATCT CCATCTCCCT CTCAATGCCT 60
TTCTTGGCGC CTCAACCCGC TATTTTGAAG ACAGTTTGTT GTTGTCGCAT GCGACCAAAA 120
ATCATCCTCT CAAGTTTTCA TCGCTGACCT GTTTCTTGGC GTAGGAAGGA GATATCACAC 180
AGAAAGGGTA AGCTGCTTTG CGTCCAGAGT ACTTACAATT GCTTCTCAAT TACTTACGCG 240
CCGGCAGCTA CCAAAAGCGA CGAACTCAAC TTTTCTCCCA ATTCCTCGGT GCACCTCCAC 300 CTCAGATTGC TGCTCTCGCC GAGCCTCAGT CTGGCCTACG CATACACTCG CCCGATGACT 360
CCGACCACCC TTCAGGCGAT GGCCATCGCG CTACCGCCTA TGCCGCTCTC GGTAGCAGCA 420
GCGGTCCAAT CCCAGATTCA CCAGACTCAC CTATGTACCG ACCGCACTCT GGTTATGCTC 480
CTTCAGAATC ACCAAGACCT TCTCCAGCAC AACCTCCACC TTCCCTGCTG CGCCCGGGGG 540
GTTCTCTCGC TGGAGGATCG ACCACTGCTC ACCGCGACTC CCTCTTCTTC TCCCCCTCCC 600 ATCTCGAACC TGAAACCCGG ACAGGTACTA TGATGTCGGG CGACTATGCA TTCAGACCCG 660
AGCAGCAAGG CACATATGGC GAATCCCAGC ATCAACAGCA CCAGTTCCAG CAACAGCAAC 720
AGCCACAGCA GCAACAGCAG TACGATGGGC AGCAGTATGA TGGACGAACT ACAACGCTTC 780
TCGATTCGCA AGGATACTTT TCGGATTTTG CGGGACAGCA GCACTATGAT CAGACTCAAA 840
CCGTTGAGTA TGTGGGACCT CAGCAGCGGT ATTCTTCCAG CGATGCATTC TCTCCAACCG 900 CCGCAATGGC ACCTCCAATG CTTACAACCA ACGACCTCCC ACCGCCGGAA GCGCTTGAGT 960
ACCAGCTGCC CCTTGACCCT CGCGAGGTAC CATTCGCTAT TCAAGATCCC CATGATGATT 1020
CTACGCCAAT GTCAAAGTTC GATAACATCG CAGCTGTACT CAGACATAGA GGCCGAACGA 1080
TTGCTAAGAA GCCGGCATAC TGGGTGTTGG ATAGTAAGGG CAAGGAGATT GCATCGATTA 1140
CGTGGGATAA GCTGGCATCT AGAGCCGAAA AGGTTGCGCA AGTCATCCGC GACAAAAGCT 1200 CTCTGTACCG GGGTGATCGG GTTGCTCTCA TCTACCGCGA TTCAGAGGTT ATTGATTTCG 1260
CCATTGCCTT GCTGGGATGC TTCATTGCTG GAGTTGTTGC CGTTCCCATC AATGATCTGC 1320
AGGACTACCA ACGCTTGAAC CACATTCTTA CTACAACGCA GGCCCATCTA GCGCTGACCA 1380 CCGATAACAA CCTCAAAGCC TTTCAACGAG ACATTACTAC ACAAAAGTTG ACATGGCCAA 1440
AGGGTGTCGA ATGGTGGAAG ACAAACGAGT TTGGCAGTTA TCACCCCAAG AAGAAGGAGG 1500 ATGTCCCGGC TTTGGTTGTT CCCGATCTGG CATATATCGA GTTTTCGCGG GCCCCAACTG 1560
GAGACTTGAG AGGTGTTGTT CTGAGCCACC GAACCATTAT GCACCAAATG GCTTGTCTTA 1620
GTGCGATTAT TTCTACTATC CCGGGTAATG GACCTGGCGA CACTTTCAAC CCGTCTCTTC 1680
GCGACAAGAA TGGTCGACTT ATTGGTGGCG GCGCAAGCAG CGAAATTTTG GTGTCGTACC 1740
TCGATCCCCG TCAGGGCATT GGCATGATTC TGAGCGTGCT ACTGACCGTC TACGGCGGCC 1800 ACACCACTGT TTGGTTCGAC AACAAAGCTG TTGATGTTCC TGGACTGTAC GCCCACCTCC 1860
TTACCAAGTA CAAATCGACC ATCATGATTG CCGACTACCC AGGATTGAAG CGAGCCGCCT 1920
ACAACTACCA GCAAGAGCCA ATGGTGACCC GAAATTTTAA GAAGGGAATG GAGCCAAACT 1980
TTCAAATGAT CAAGCTTTGC TTGATTGACA CCTTGACTGT AGACAGCGGG TCCCACGAAG 2040
TTTTGGCTGA CCGATGGCTA CGACCGTTGA GAAACCCTCG TGCCCGTGAG GTTGTCGCAC 2100 CTATGCTTTG TCTACCTGAA CACGGAGGCA TGGTGATTAG TGTGCGTGAC TGGCTAGGAG 2160
GAGAAGAGCG CATGGGATGC CCATTAAAGC TTGAACTTGG GGAGGATACA GAGTCTGACG 2220
AAGAGAAAGA GGAAACAGAG AAGCCAGCAG TTTCCAATGG CTTTGGTAGT CTCTTGTCAG 2280
GTGGTGGCAC AGCAACAACC GAAGAGAGGG CAAAGAATGA GCTTGGCGAA GTCCTTTTGG 2340
ATCGTGAGGC TCTAAAGACC AACGAAGTTG TGGTGGTGGC CATAGGTAAC GATGCCCGTA 2400 AAAGGGTGAC GGATGACCCA GGCTTGGTAC GGGTCGGTTC TTTTGGATAC CCCATACCCG 2460
ATGCCACACT CTCCGTCGTC GATCCAGAAA CGGGTTTACT GGCGTCACCA CATTCCGTGG 2520
GTGAAATCTG GGTCGACTCC CCTTCTCTTT CAGGTGGTTT CTGGGCGCAG CCAAAGAATA 2580
CTGAGCTGAT TTTCCATGCT CGTCCTTACA AGTTTGACCC AGGTGATCCT ACACCGCAGC 2640
CCGTCGAGCC CGAATTCCTG CGAACAGGCT TGCTGGGCAC CGTCATCGAG GGTAAAATCT 2700 TTGTTCTGGG CCTTTACGAA GACCGAATTC GACAAAAGGT TGAGTGGGTT GAGCATGGAC 2760
ACGAACTAGC AGAGTACCGC TACTTCTTTG TTCAGCACAT CGTTGTGAGC ATTGTCAAGA 2820
ACGTTCCAAA GATATACGAT TGTTCAGCCT TTGACGTCTT TGTCAATGAC GAACACCTGC 2880
CAGTCGTGGT GCTGGAGTCA GCAGCTGCGT CAACGGCACC ATTGACATCT GGAGGACCTC 2940
CTCGACAACC GGATACAGCT CTGCTAGAGT CATTGGCTGA GCGCTGCATG GAGGTTCTCA 3000 TGTCAGAGCA TCATCTGAGA CTGTACTGCG TTATGATCAC AGCACCCGAC ACTTTGCCTC 3060
GAGTTGTTAA GAACGGACGA CGCGAAATTG GTAACATGCT TTGCCGTCGG GAGTTTGATC 3120
TCGGCAACCT TCCATGTGTG CACGTCAAGT TTGGCGTGGA GCATGCAGTA CTTAACCTCC 3180
CTATTGGTGT AGACCCTATA GGTGGTATCT GGTCACCGTT GGCGTCCGAT TCTCGTGCCG 3240
AATTCTTATT GCCAGCTGAC AAGCAATACT CTGGTGTCGA CAGGCGCGAA GTCGTTATCG 3300 ATGACCGTAC TTCAACGCCC CTAAACAATT TCTCTTGCAT TTCGGATCTT ATCCAATGGC 3360 GCGTGGCCCG TCAACCAGAA GAGCTAGCGT ACTGCACAAT CGATGGCAAA AGCCGAGAAG 3420
GTAAGGGTGT AACATGGAAG AAATTCGACA CCAAGGTCGC TTCCGTTGCC ATGTACCTGA 3480 AGAACAAGGT CAAGGTGAGG CCGGGAGACC ACATCATCCT CATGTACACA CATTCAGAGG 3540
AGTTTGTCTT TGCCATCCAT GCCTGCATTT CCTTGGGCGC AATTGTCATT CCCATCGCAC 3600
CCCTCGACCA GAACCGATTG AACGAAGATG TCCCAGCTTT CCTGCATATT GTATCTGATT 3660
ACAACGTCAA GGCTGTGCTG GTCAACGCTG AGGTCGATCA TCTAATCAAG GTAAAGCCTG 3720
TGGCTAGCCA TATCAAACAG TCAGCCCAGG TTCTCAAGAT CACGAGCCCT GCCATCTACA 3780 ACACAACTAA GCCGCCAAAG CAAAGTAGTG GATTGAGGGA TTTGAGATTC ACCATTGACC 3840
CTGCCTGGAT TCGGCCTGGC TACCCCGTCA TTGTTTGGAC TTATTGGACC CCCGATCAAC 3900
GACGAATTTC AGTTCAGCTT GGACATGACA CCATTATGGG CATGTGCAAG GTTCAAAAGG 3960
AAACTTGCCA AATGACAAGT TCAAGACCTG TGCTTGGATG TGTACGAAGC ACGACTGGCC 4020
TAGGCTTTAT TCATACGGCT CTGATGGGAA TTTATATCGG AACACCAACC TACCTCCTAT 4080 CACCTGTCGA GTTTGCAGCC AACCCCATGT CTCTATTCGT CACCTTGTCG AGATACAAGA 4140
TTAAGGATAC TTATGCGACA CCACAGATGC TTGATCATGC CATGAACTCC ATGCAGGCCA 4200
AGGGCTTTAC ACTTCATGAA CTTAAGAACA TGATGATCAC TGCCGAGAGC CGACCAAGAG 4260
TTGATGTTTT CCAAAAGGTC AGACTTCACT TTGCTGGGGC TGGGCTCGAT AGAACTGCTA 4320
TTAACACGGT CTATTCGCAT GTCCTCAACC CCATGGTAGC GTCGCGATCT TATATGTGCA 4380 TCGAGCCTAT TGAGCTTTGG TTGGACACGC AAGCGCTTCG ACGTGGTCTG GTTATTCCTG 4440
TGGACCCTGA ATCAGATCCT CTGGCCCTAC TGGTACAGGA CAGCGGTATG GTTCCAGTTT 4500
CAACCCAAAT AGCCATCATC AACCCTGAAA GCAGAATACA CTGCCTCGAT GGTGAGTATG 4560
GTGAAATTTG GGTCGACTCT GAAGCCTGCG TCAAGTCATT CTATGGCTCC AAAGACGCTT 4620
TTGACGCTGA GCGCTTTGAT GGCCGAGCTC TTGACGGCGA TCCCAACATT CAGTATATCC 4680 GTACCGGAGA CTTGGGTTTC CTTCATAATG TTAGTCGACC TATTGGCCCT AATGGTGCCC 4740
AGGTGGACAT GCAAGTGTTG TTTGTTCTCG GCAACATTGG CGAGACTTTT GAGATCAACG 4800
GATTGAGCCA TTTCCCAATG GATATTGAGA ACTCGGTGGA AAAATGCCAC AGAAACATTG 4860
TGGCGAATGG CTGGTAAGTA TAAAATCTCT ATTTGAAGCG AATATGCTAA CAAAGTCAGT 4920
GCGGTGTTCC AAGCTGGTGG CTTGGTGGTT GTTCTGGTTG AAGTCAACCG CAAGCCATAC 4980 CTGGCATCGA TTGTTCCCGT CATTGTCAAC GCTATCCTCA ATGAACACCA AATCATTGTA 5040
GATATCGTCG CATTCGTCAA CAAGGGAGAC TTCCCACGGT CTCGTCTAGG AGAGAAGCAG 5100
CGTGGCAAGA TTCTTGGTGG CTGGGTTAGT AGAAAGCTGA GGACTCTTGC CCAGTTCTCG 5160
ATTCGCGATA TGGACGCCGA ATCCACAGCT GGTGATATGA TGGATCCTTC TAGAGCATCA 5220
ATGGTCAGCG TACGAAGCGG AGGCGGTGCT GCTCCCGGAT CTTCTAGTTT GAGGAATGTC 5280 GAACCTGCGC CTCAAATCTT GGAGGAGGAA CATGACCAGA TGACTCCTCG TCACGAATAC 5340 GAAGCAGCCC CTACCATGAT TTCTGAACTT CCCGACGGCC AAGAGACACC GACAGGGTTT 5400
CAGCACTCGC AATACGAACA CCCACCACAA TCAGCCGGTT CTCAAGCACC AGCCCAGCTG 5460 AACCTTTCTC ACCAGCCCGA TCAAGGATTC GATATGGACT TTTCACGATA TAGTTCAGCA 5520
GAGCCCGATC ACGGCCCTGT CCACAGACGT CCAGTCCCAG GCCAAGCCCA ACAACCCGAG 5580
CCTATGCAAG GGTACGGTCA AGCGCCGCCC CAGATCCGGC TACCAGGTGT TGATGGACGA 5640
GAGGAGGGAG GGTTCTGGTC ACAGCAGGAA AAGAACGAGA AGAGTGAAGA AGACTGGACA 5 00
ACTGATGCCA TGATGCATAT GAATCTGGCA GGTGATATGA AACCGCCACG ATGATAATAC 5760 ACAACATAAG AGCGAAGTGA CGAAGCGGAG TCGGAGTTGG GAAGCATTTA GAAACGAATA 5820
ACAAACAATT GGACTTGTCG GTCTGATGGC CTATTTACTT CATTCATAGA TGAGGATTGG 5880
ATAGTGAATA TGTGATTGGA TAAAGCCTGG GTTTGTGAGT TTGTGAATGC AGTGGGTGCT 5940
TGCTATAAGC TGTTTTATTG AGGTCTTTGG AGGAGTGTCT AACAAAGATG CAAAGTTACT 6000
AGT 6003
Preferably, the CPSl nucleic acid molecule which hybridizes to a nucleic acid molecule having a sequence as set forth in SEQ ID No. 43 is from the plant pathogen Fusarium graminearium. In another preferred embodiment, the CPSl gene is from a plant pathogen such as Fusarium avenaceum, F. carpineum, F. chlamydosporum, F. coccophilum, F. culmorum, F. episphaeria, F. equiseti, F. flocciferum, F. moniliforme, F. oxysporum, F. redolens, F. sambucinum, F. solani, F. subglutinans, F. trichothecioides, F. udum, or F. ventricosum.
The peptide synthetase product of SEQ ID NO:43 has a deduced amino acid sequence as follows (SEQ ID No. 44):
MMSGDYAFRP EQQGTYGESQ HQQHQFQQQQ QPQQQQQYDG QQYDGRTTTL LDSQGYFSDF 60
AGQQHYDQTQ TVEYVGPQQR YSSSDAFSPT AAMAPPMLTT NDLPPPEALE YQLPLDPREV 120
PFAIQDPHDD STPMSKFDNI AAVLRHRGRT IAKKPAYWVL DSKGKEIASI TWDKLASRAE 180 KVAQVIRDKS SLYRGDRVAL IYRDSEVIDF AIALLGCFIA GWAVPINDL QDYQRLNHIL 240
TTTQAHLALT TDNNLKAFQR DITTQKLTWP KGVEWWKTNE FGSYHPKKKE DVPALWPDL 300
AYIEFSRAPT GDLRGWLSH RTIMHQMACL SAIISTIPGN GPGDTFNPSL RDKNGRLIGG 360
GASSEILVSY LDPRQGIGMI LSVLLTVYGG HTTVWFDNKA VDVPGLYAHL LTKYKSTIMI 420
ADYPGLKRAA YNYQQEPMVT RNFKKGMEPN FQMIKLCLID TLTVDSGSHE VLADRWLRPL 480 RNPRAREWA PMLCLPEHGG MVISVRDWLG GEERMGCPLK LELGEDTESD EEKEETEKPA 540
VSNGFGSLLS GGGTATTEER AKNELGEVLL DREALKTNEV VWAIGNDAR KRVTDDPGLV 600
RVGSFGYPIP DATLSWDPE TGLLASPHSV GEIWVDSPSL SGGFWAQPKN TELIFHARPY 660 KFDPGDPTPQ PVEPEFLRTG LLGTVIEGKI FVLGLYEDRI RQKVEWVEHG HELAEYRYFF 720
VQHIWSIVK NVPKIYDCSA FDVFVNDEHL PWVLESAAA STAPLTSGGP PRQPDTALLE 780
SLAERCMEVL MSEHHLRLYC VMITAPDTLP RWKNGRREI GNMLCRREFD LGNLPCVHVK 840
FGVEHAVLNL PIGVDPIGGI WSPLASDSRA EFLLPADKQY SGVDRREWI DDRTSTPLNN 900 FSCISDLIQW RVARQPEELA YCTIDGKSRE GKGVTWKKFD TKVASVAMYL KNKVKVRPGD 960
HIILMYTHSE EFVFAIHACI SLGAIVIPIA PLDQNRLNED VPAFLHIVSD YNVKAVLVNA 1020
EVDHLIKVKP VASHIKQSAQ VLKITSPAIY NTTKPPKQSS GLRDLRFTID PAWIRPGYPV 1080
IVWTYWTPDQ RRISVQLGHD TIMGMCKVQK ETCQMTSSRP VLGCVRSTTG LGFIHTALMG 1140
IYIGTPTYLL SPVEFAANPM SLFVTLSRYK IKDTYATPQM LDHAMNSMQA KGFTLHELKN 1200 MMITAESRPR VDVFQKVRLH FAGAGLDRTA INTVYSHVLN PMVASRSYMC IEPIELWLDT 1260
QALRRGLVIP VDPESDPLAL LVQDSGMVPV STQIAIINPE SRIHCLDGEY GEIWVDSEAC 1320
VKSFYGSKDA FDAERFDGRA LDGDPNIQYI RTGDLGFLHN VSRPIGPNGA QVDMQVLFVL 1380
GNIGETFEIN GLSHFPMDIE NSVEKCHRNI VANGCAVFQA GGLVWLVEV NRKPYLASIV 1440
PVIVNAILNE HQIIVDIVAF VNKGDFPRSR LGEKQRGKIL GGWVSRKLRT LAQFSIRDMD 1500 AESTAGDMMD PSRASMVSVR SGGGAAPGSS SLRNVEPAPQ ILEEEHDQMT PRHEYEAAPT 1560
MISELPDGQE TPTGFQHSQY EHPPQSAGSQ APAQLNLSHQ PDQGFDMDFS RYSSAEPDHG 1620
PVHRRPVPGQ AQQPEPMQGY GQAPPQIRLP GVDGREEGGF WSQQEKNEKS EEDWTTDAMM 1680
HMNLAGDMKP PR 1692
In another embodiment of the invention, there is provided an isolated nucleic acid molecule encoding a CPSl peptide synthetase homolog and which hybridizes to a nucleic acid molecule having a sequence corresponding to SEQ ID No. 45 as follows :
AAAAAGAAGG GGCCTACGGA GTTGACCGAG ATATTGCTAG ATAAGGAAGC GCTCAAGATG 60
AACGATGTTG TGGTCCTTGC AATAGGAGAA GAGGCCAGTA AACGTGCGAA TGAGCCTGGC 120
ACAATGCGAG TTGGCGCTTT TGGATACCCA ATACCAGATG CGACGCTAGC CGTCGTAGAT 180
CCAGAGACGA ATCTCTTGTG TTCACCCTAC TCGATAGGAG AGATTTGGGT AGACTCACCT 240 TCATTGTCTG GTGGTTTCTG GCAATTGCAG AAGCACACTG AAACTATATT TCACGCCCGC 300
CCATACCGCT TTGTGGAGGG CAGTCCTACC CCGCAGTTGC TTGAGCTTGA GTTTCTCCGG 360
ACAGGCTTAC TCGGATTCGT CGTAGAGGGC AAGGTCTTTA TCCTTGGTCT CTATGAAGAT 420
CGCATCAGGC AGCGCGTTGA ATGGGTAGAA CATGGTCAGC TGGAAGCTGA ACACAGATAC 480
TTCTTCGTGC AGCACCTCGT CACCAGTATC ATGAAGGCTG TTCCCAAGAT CTACGACTGG 540 TAAGTCTTCT CATGTTTTAG ATGAGCGTTC TAACACTATG CAGCTCATCT TTCGACTCGT 600 ACGTCAATGG CGAATACCTG CCTATCATCC TCATCGAGAC ACAGGCTGCA TCGACAGCCC 660
CTACGAACCC TGGTGGACCG CCACAGCAAC TCGACATCCC CTTCCTAGAC TCACTGTCTG 720
AGCGATGCAT GGAAGTGTTG TATCAAGAAC ACCATCTGCG AGTATACTGC GTCATGATCA 780
CAGCGCCAAA CACATTACCA CGAGTTGTTA AGAATGGTCG ACGAGAAATT GGCAACATGC 840 TCTGTCGAAG AGAATTTGAT AATGGCTCAT TACCTTGTGT CCACGTCAAG TTTGGTGTTG 900
AGAGGTCAGT TCTCAACATC GCGTTGGGTG ATGACCCCTC CGGAGGCATG TGGTCATATG 960
AAGCCTCGAT GGCGCGTCAG CAGTTCTTGA TGCTCCAAGA CAAGCAGTAT TCTGGAGTAG 1020
ATCACCGCGA AGTCGTCATG GATGACAGAA CATCGACACC TCTCAACCAA TTCTCCAACA 1080
TTCACGACCT CATGCAATGG CGCGTATCAC GGCAGGCTGA AGAGCTCGCA TATTGCACAG 1140 TCGACGGTCG AGGCAAAGAA GGCAAGGGCG TCAACTGGAA GAAGTTCGAC CAGAAAGTTG 1200
CGGGTGTCGC AATGTACCTG AAGAACAAGG TCAAAGTGCA AACCGGCGAT CATCTGCTTC 1260
TGATGTATAC GCACTCGGAA GACTTTGTAT ATGCGGTACA TGCATGCTTT GTGCTTGGCG 1320
CTGTATGCAT ACCAATGGCA CCAATCGACC AGAACCGATT GAATGAGGAT GCACCTGCAT 1380
TGCTGCACAT CCTTGCAGAC TTCAAGGTCA AGGCCATCCT CGTCAATGCC GATGTGGATC 1440 ATCTCATGAA GGTCAAGCAA GTATCGCAGC ACATCAAACA ATCAGCAGCC ATCTTCAAGA 1500
TCAACGTGCC GCACACTTAC AACACAACCA AGCCACCTAA GCAGTCGAGT GGTTGTCGGG 1560
ATCTCAAGCT CACAATACGG CCTGCCTGGG TACAGCCTGG TTTCCCAGTT CTTGTATGGA 1620
CATACTGGAC TCCAGATCAA CGCCGTATAG CCGTACAACT AGGTCATAGC CAAATCATGG 1680
CACTAGGCAA GGTCCAGAAG GAGACTTGTC AAATGACAAG TACAAGGCCA GTCCTAGGAT 1740 GTGTACGGAG TACCATCGGA CTTGGCTTCA TTCATACCTG CATCATGGGC ATCTTCCTTG 1800
CCGCACCCAC TTACCTCGTG TCGCCTGTCG ACTTTGCACA AAATCCAAAC ATACTCTTCC 1860
AGACGTTATC AAGATACAAG ATCAAGAATG CGTACGCAAC CAGTCAAATG TTGGATCACG 1920
CTATTGCCCG TGGGGCTGGA AAGAACATGG CCCTGCACGA ACTCAAGAAT CTCATGATTG 1980
CGACTGATGG TAGGCCGCGT GTTGATGTTT ACCAGAGAGT GCGCGTACAC TTTTCACCAG 2040 CAAGCTTGGA CCGGACAGCG ATTAACACAG TCTACTCTCA CGTGCTCAAC CCAATGGTAG 2100
CATCGCGATC ATACATGTGC ATCGAGCCAA TAGAACTGCA TCTCGACGTC AACGCTCTTC 2160
GAAGAGGTCT GATCATGCCC GTCGACCCAG ATACCGAGCC TGGCGCTCTA ATGGTCCAGG 2220
ACTCTGGTAT GGTGCCAGTC TCCACACAAA TAGCAATTGT GAACCCAGAG ACAAACCAGC 2280
TTTGCTTGGT TGGCGAATAT GGCGAAATCT GGGTTCAATC 2320
Preferably, the CPSl nucleic acid molecule which hybridizes to a nucleic acid molecule having a sequence as set forth in SEQ ID No. 45 is from the plant pathogen Pyrenophora teres. In another preferred embodiment, the CPSl gene is from a plant pathogen such as Pyrenophora avenae, P. bromi, P. leuceienes, P. phaeocomes, P. schroeteri, P. trichostoma, or P. tritici-repentis.
The peptide synthetase product of SEQ ID No:45 has a deduced amino acid sequence as follows (SEQ ID No. 46):
KKKGPTELTE ILLDKEALKM ND WLAIGE EASKRANEPG TMRVGAFGYP IPDATLAWD 60 PETNLLCSPY SIGEIWVDSP SLSGGFWQLQ KHTETIFHAR PYRFVEGSPT PQLLELEFLR 120 TGLLGFWEG KVFILGLYED RIRQRVEWVE HGQLEAEHRY FFVQHLVTSI MKAVPKIYDC 180
SSFDSYVNGE YLPIILIETQ AASTAPTNPG GPPQQLDIPF LDSLSERCME VLYQEHHLRV 240
YCVMITAPNT LPRWKNGRR EIGNMLCRRE FDNGSLPCVH VKFGVERSVL NIALGDDPSG 300 GMWSYEASMA RQQFLMLQDK QYSGVDHREV VMDDRTSTPL NQFSNIHDLM QWRVSRQAEE 360
LAYCTVDGRG KEGKGVNWKK FDQKVAGVAM YLKNKVKVQT GDHLLLMYTH SEDFVYAVHA 420
CFVLGAVCIP MAPIDQNRLN EDAPALLHIL ADFKVKAILV NADVDHLMKV KQVSQHIKQS 480
AAIFKINVPH TYNTTKPPKQ SSGCRDLKLT IRPAWVQPGF PVLVWTYWTP DQRRIAVQLG 540
HSQIMALGKV QKETCQMTST RPVLGCVRST IGLGFIHTCI MGIFLAAPTY LVSPVDFAQN 600 PNILFQTLSR YKIKNAYATS QMLDHAIARG AGKNMALHEL KNLMIATDGR PRVDVYQRVR 660
VHFSPASLDR TAINTVYSHV LNPMVASRSY MCIEPIELHL DVNALRRGLI MPVDPDTEPG 720
ALMVQDSGMV PVSTQIAIVN PETNQLCLVG EYGEIWVQ 758
As used herein, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid and polymers thereof in either a single or double stranded form. As used herein, "nucleic acid" also encompasses nucleic acids containing known analogs of naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof such as degenerate codon substitutions and complementary sequences. As used herein, the term "nucleotide sequence" refers to a polymer of DNA or RNA which may be single or double stranded and may contain synthetic, non-natural, or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms "nucleic acid" , "nucleic acid molecule", "nucleic acid fragment", or nucleic acid sequence or segment", may also be used interchangeably with the terms "gene", "cDNA", "DNA" and "RNA".
Other DNA molecules of the present invention include DNA molecules that have a nucleic acid sequence which is more than 70% identical to the nucleotide sequence of SEQ. ID. Nos. 2, 41, 43, or 45. Nucleotide sequence similarity may be determined by the BLAST program with the default parameters (Altschul et al., "Basic Local Alignment Search Tool," J. Mol. Biol.. 215:403-410 (1990), which is hereby incoφorated by reference).
Preferred sequences include those DNA molecules which will hybridize to a nucleic acid molecule having the sequence of SEQ. ID No. 2, 41, 43, 45 or their compliments. Generally, stringent conditions are selected to be about 50°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition of the probe, and may be calculated using the following equation:
Tm = 79.8°C + (18.5 x Log[Na+])
+ (58.4°C x %[G+C]) - (820 / #bp in duplex)
(0.5 x % formamide) More preferred stringent conditions are when the temperature is 20°C below Tm, and the most preferred stringent conditions are when the temperature is 10°C below Tm. Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase.
Wash conditions are typically performed at or below stringency. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are asas set forth above. More or less stringent conditions may also be selected.
For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook, J., E.F. Fritsch, et al. 1989 "Molecular Cloning: a Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, at 11.45. An example of low stringency conditions is 4-6X SSC/0.1 -0.5% w/v SDS at 37°-45° C for 2-3 hours. Depending on the source and concentration of the nucleic acid involved in the hybridization, alternative conditions of stringency may be employed such as medium stringent conditions. Examples of medium stringent conditions include 1-4X SSC/0.25% w/v SDS at > 45° C for 2-3 hours. An example of high stringency conditions includes 0.1-1X SSC/0.1% w/v SDS at 60 C for 1-3 hours. The skilled artisan is aware of various parameters which may be altered during hybridization and washing and which will either maintain or change the stringency conditions. For example, another stringent hibridization condition is hybridization at 4X SSC at 65° C, followed by a washing in 0. IX SSC at 65° C for about one hour. Alternatively, an exemplary stringent hybridization condition is in 50% formamide, 4XSSC, at 42° C. Still another example of stringent conditions include hybridization at 62° C in 6X SSC, .05X BLOTTO, and washing at 2X SSC, 0.1% SDS at 62° C.
Other proteins or polypeptides of the present invention include polypeptides that have an amino acid sequence having at least 75 % similarity to the amino acid sequence of at least one of SEQ. ID. No. 3, SEQ ID No. 42, SEQ ID No. 44, or SEQ ID No. 46. In a preferred embodiement of the invention, the protein or polypeptide will have at least 90% similarity with at least one of SEQ. ID No. 3, SEQ ID No. 42, SEQ ID No. 44, or SEQ ID No. 46. Protein sequence similarity may be determined by the BLAST program with the default parameters (Altschul et al., "Basic Local Alignment Search Tool," J. Mol. Biol.. 215:403-410 (1990), which is hereby incorporated by reference). The CPSl protein of SEQ. ID. No. 3 has a molecular weight of about 190-200 kDa, preferably 193.2 kDa. The CPSl protein contains two structurally similar modules, both of which are similar to SafBl, the first module of saframycin synthetase B (overall 25% identity; 50% similarity) and have apparent amino-acid-activating and thiolation domains with core sequences conserved in known peptide synthetases.
The DNA molecule encoding a CPSl protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e., not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding- sequences. U.S. Patent No. 4,237,224 to Cohen and Boyer, which is hereby incoφorated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eukaryotic cells grown in culture. Recombinant genes may also be introduced into vimses, such as vaccinia vims. Recombinant vimses can be generated by transfection of plasmids into cells infected with vims.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incoφorated by reference), pQE, pIH821 , pGEX, pET series (see Studier et. al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology, vol. 185 (1990), which is hereby incoφorated by reference), and any derivatives thereof. Suitable vectors are continually being developed and identified. Recombinant molecules can be introduced into cells via transformation, transduction, conjugation, mobilization, or electroporation using methods well known in the art. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Springs
Laboratory, Cold Springs Harbor, New York (1982), which is hereby incoφorated by reference.
A variety of host- vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host- vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with vims (e.g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i.e. biolistics). The expression elements of these vectors vary in their strength and specificities. Depending upon the host- vector system utilized, any one of a number of suitable transcription and translation elements can be used.
Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA ("mRNA") translation). Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of procaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eukaryotic cells. Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eukaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno ("SD") sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3 '-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incoφorated by reference.
Promoters vary in their "strength" (i.e., their ability to promote transcription). For the puφoses of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to /αcUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lac\JV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene. Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno ("SD") sequence about 7- 9 bases 5' to the initiation codon ("ATG") to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incoφoration of synthetic nucleotides may be used.
The present invention also relates to anti-sense nucleic acid for essential cell proteins, such as replication proteins, which serve to render the host cells incapable of further cell growth and division. Anti-sense regulation has been described by Rosenberg et al., "Production of Phenocopies by Kruppel Antisense RNA Injection Into Drosophila Embryos," Nature. 313:703-706 (1985); Preiss et al., "Molecular Genetics of Kruppel, A Gene Required for Segmentation of the Drosophila Embryo," Nature. 313:27-32 (1985); Melton, "Injected Anti-sense RNAs Specifically Block Messenger RNA Translation In vivo," Proc. Natl. Acad. Sci. USA. 82:144-148 (1985); Izant et al., "Constitutive and Conditional Suppression of Exogenous and Endogenous Genes by Anti-sense RNA," Science, 229:345-352 (1985); Kim et al., "Stable Reduction of Thymidine Kinase Activity in Cells Expressing High Levels of Anti-sense RNA," Cell, 42:129-138 (1985); Pestka et al., "Anti-mRNA: Specific Inhibition of Translation of Single mRNA Molecules," Proc. Natl. Acad. Sci. USA. 81 :7525-7528 (1984); Coleman et al., "The Use of RNAs Complementary to Specific mRNAs to Regulate the Expression of Individual Bacterial Genes," Cell. 37:429-436 (1984); and McGarry et al., "Inhibition of Heat Shock Protein Synthesis by Heat-Inducible Antisense RNA," Proc. Natl. Acad. Sci. USA. 83:399-403 (1986), which are hereby incoφorated by reference.
Once the isolated DNA molecules encoding the CPSl protein or polypeptide, as described above, has been cloned into an expression system, they are ready to be incoφorated into a host cell. Such incoφoration can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like. In the present invention, the host cells may be from plants such as corn, oat, grass, weed, bamboo, and sugarcane.
One aspect of the present invention involves using an inhibitor of the CPSl protein to interfere with the plant infection process in order to impart disease resistance to plants. In one mechanism, the peptide synthetase CPSl could be inhibited by an appropriate drug, thereby causing the plants to be resistant to fungal attack. Alternatively, the CPSl nonribosomal product could be degraded by an enzyme for which it is a suitable substrate, and, when the gene encoding this enzyme is genetically engineered into plants, the plants will become resistant to fungal attack.
In this aspect of the present invention, large numbers of compounds can be screened for their activity as inhibitors of CPSl protein by a high-throughput screening assay as described in U.S. Patent No. 5,876,946 to Burbaum et al., which is hereby incorporated by reference. Generally, a library of compounds is assayed for inhibition of an enzyme catalyzed reaction and the amounts of fluorescence bound to individual suspendable solid supports measured to determine the degree of inhibition. For example, the amount of fluorescence bound to a microbead in the presence of inhibitory compounds is greater than for non-inhibitory compounds. The amounts of fluorescence bound to individual beads are determined by confocal microscopy. Using this type of assay, inhibition can be determined of a peptide synthetase such as CPSl. For CPSl, the substrate can be amino acids (or hydroxy acids), linked at one end to the microbead and at the other end to a fluorescent label. The enzyme inhibitors can be utilized to impart fungal resistance to a variety of plants including oats, grasses, weeds, sugarcane, and corn in particular.
Thus, the present invention provides a method for identifying inhibitors of a CPSl protein , wherein said CPSl protein is a peptide synthetase of a plant pathogen. The method comprises: providing a CPSl protein or polypeptide, contacting the protein or polypeptide with potential inhibitor compounds; determining peptide synthetase activity, and selecting compounds which decrease the peptide synthetase activity. The method is especially useful in identifying inhibitors of a CPSl protein from plant pathogens of the genera Cochliobolus, Alternaia, Fusarium, and Pyrenophora such as those described hereinabove. Preferably, the method may be used to identify inhibitors of a CPSl protein from Alternaria solani, Fusarium graminearium, and Pyrenophora teres.
Another aspect of the present invention involves using one or more of the above DNA molecules encoding a CPSl protein or polypeptide or a gene encoding an enzyme that degrades the CPSl N.R.P. product to transform plants in order to impart fungal resistance to the plants. This concept of pathogen-derived resistance, according to U.S. Patent No. 5,840,481 to Johnston and Sanford, which is hereby incoφorated by reference, is that host resistance to a particular parasite can effectively be engineered by introducing a gene, gene fragment, or modified gene or gene fragment of the pathogen into the host. This approach is based on the fact that in any parasite-host interaction, there are certain parasite-encoded cellular functions (activities) that are essential to the parasite but not to the host and that when one of the essential functions of the parasite such as survival or reproduction is disrupted, the parasitic process will be stopped. "Disruption" refers to any change that diminishes the survival, reproduction, or infectivity of the parasite. Such essential functions, which are under the control of the parasite's genes, can be dismpted by the presence of a corresponding gene product in the host which is (1) dysfunctional, (2) in excess, or (3) appears in the wrong context or at the wrong developmental stage in the parasite's life cycle. If such faulty signals are designed specifically for parasitic cell functions, they will have little effect on the host. Therefore, the procedure for making plants, for example, resistant to infection by one or more fungus involves isolating DNA coding for a gene such as CPSl of a fungus, operably linking the DNA within an expression vector, transforming the plant cell or plant tissue with the expression vector, and growing the transformed plant cells or plant tissue in the presence of the fungus such as e.g., Cochliobolus heterostrophus, Alternaria solani, Fusarium graminearium, or Pyrenophora teres, where the CPSl DNA is expressed as a gene product and the CPS protein disrupts the essential activity of the fungi.
Thus, the present invention provides a method of imparting disease resistance to a plant by over-expressing a CPSl polypeptide in the plant, wherein the polypeptide has protein synthetase activity. In praticing this aspect of the invention, the plant may be any plant in which it is desired to impart disease resistance. Thus, the plant may be an agrigultural crop or ornamental plant. The plant may be herbacious or woody. The plant may be a monocot or dicot. Examples of plants which may be used in practicing the present invention, include but are not limited to, com, oats, grasses, weeds, sugarcane, barley, wheat, rice, tomato, potato, citms, malus, rye, cotton, brassica, cabbage, and carrot. Many other plants may also be used in the practice of the present invention. As a guideline, plants which serve as hosts for Cochliobolus sp., Fusarium sp., Alternaria sp., and Pyrenophera sp., may be used. Reference to host plants may be conveniently made to Fungi on Plants and Plant Products in the United States, David, F. Farr et al. editors, American Phytopathological Society Press, St. Paul, Minnesota, 1989. In another preferred embodiment, the CPSl peptide is from the genera Cochliobolus, Alternaria, Fusarium ,or Pyrenophora. In an even more preferred embodiment, the CPSl gene is from Cochliobolus heterostrophus, Alternaria solani, Fusarium graminearium or Pyrenophora teres.
Promoters and other regulatory regions which function in plants are well known and include e.g., constitutive promoters, inducible promoters, temporally regulated and tissue specific promoters. Examples of constitutive promters include e.g., actin, CAMV 35S, MAS, ubiquitin, rice cyclophilin, maize H3 histone, and actin 2. Examples of tissue specific promoters include e.g., leaf specific promoters such as the RuBisCo ssu, Cab (chlorophyll a/b/binding) protein, and the AldP gene promoter from rice (Kagaya et al., 1995 Molecular and General. Genetics 248:668-614. Examples of root specific promoters include e.g., beta tubulin (Oppenheimer et al. Gene 65:87, 1988), and SbPRPl (Suzuki et al., Plant Mol. Biol. 27:109-119, 1993. Thus, using well known methods and widely available regulatory sequences, the skilled artisan is direct expression of a subject CPS nucleic acid molecule in a plant.
To provide regulated expression of a CPS gene of the present invention, plants are transformed with a vector which replicates in a plant cell and which have a promoter which directs expression of the CPS gene product in the plant. Methods of plant transformation are well known in the art. A vector comprising a subject nucleic acid molecule coding for a CPS gene or fragment thereof may be introduced into a plant by leaf disk transforamtion-regeneration procedure as described by Horsh et al. (1985) Science 227:1229-1231. Other methods of transformation such as protoplast culture (Horsh et al. 1984 Science, 225:496; DeBlock et al. (1984) Embo J. 2:2143; Barton et al. (1983) Cell, 52:1033) may also be used and are within the scope of this invention.
In transforming dicot plant species, plants may be transformed Agrobacterium-deήved vectors such as those described in Klett et al. (1987) Annu. Rev. Plant Physiol, 38:467. Other well known methods are available to insert the subject CPS genes into plant cells. Such alternative methods include biolistic approaches (Klein et al. 1987, Nature:311:10), electroporation, microinjection (Potrykus and Spangenberg eds., Gene Transfer to Plants, Springer Verlag, Berlin , 1995), chemically-induced DNA uptake, the use of vimses or pollen as vectors, liposome mediated transformation, transformation using wounded or enzyme-degraded immature embryos, or wounded or enzyme degraged embryonic callus.
For transformation of monocot plant species, a number of well known methods may be used such as biolistic methods (Tang, K. W. et al. 2000, "Acta Biotechnologies 20(2): 175-183; de-Villiers, S. M. et al., 2000, South African Journal of Plant and Soil. 17(1): 50- 53) protoplast transformation, electroporation of partially permeabilized cells, and introduction of DNA using glass fibers, etc. Agrobacterium may also be used (Raineri, D. M., et al., 1990 Bio/Technology Vol. 8 :33-38).
As used herein, "transformation" refers to the transfer of an exogenous nucleic acid molecule into a host cell. The nucleic acid molecule may be stably or transiently introduced into the host cell and may be maintained non- integrated for example, as a plasmid, or alternatively, may be integrated into the host genome. The resulting transformed plant cell(s) may then be used to regenerate a transformed plant via standard methods.
The invention is further illustrated by the following examples which are not intended in any way to limit the scope of the invention.
EXAMPLES
EXAMPLE 1 - Materials and Methods for Examples 2- 7
Strains, Media, Crosses and Transformation. C4 (Toxl+; MAT-
2) and C5 (Toxl~; MAT-1) are member of near-isogenic C. heterostrophus strains (Leach et al., 1982, which is hereby incoφorated by reference). R.C4.2696
(Tox+; MAT-2; hygBR) is a C4-derived mutant generated using the REMI mutagenesis procedure (Lu et al., "Tagged Mutations at the Toxl Locus of Cochliobolus heterostrophus Using Restriction Enzyme-Mediated Integration," Proc. Natl. Acad. Sci. USA. 91 :12649-12653 (1994), which is hereby incoφorated by reference). Strains 1301R33 (Toχ-, MAT-2; hygBR), 1301R45 (Tox~; MAT-1; hygBR), 1301R26 (Tox+; MAT-2; hygBR) are progeny of the cross C5 X R.C4.2696. Culture media, including CM (complete medium), CMX (complete medium with xylose instead of glucose), CMNS (CM with salts omitted), and MM (minimal medium) have been described, as have mating procedures (Leach et al., 1982; Turgeon et al., "Transformation of the Fungal Maize Pathogen
Cochliobolus heterostrophus Using the Aspergillus nidulans amdS Gene," Mol. Gen. Genet., 201 :450-453 (1985), which are hereby incoφorated by reference). All strains were grown at 24°C under the warm white light or black light (F40/350BL) (Sylvania Inc., Danvers, MA). Ascospore germination was done at 32°C in the dark for 3 days. REMI transformants were purified by transferring the transformants from the original REMI plates to fresh CMNS medium containing hygromycin B (CalbiochemR) at 80 ug/ml. For conidiation, stable transformants were transferred to CMX containing the same drug but at a higher concentration (120 ug/ml) to compensate for reduced drug activity due to the inhibition by the salts in the medium. Single conidia were picked up under a dissecting microscope and grown on CMNS hygromycin B plates; stable colonies were then transferred to individual CMX/ hygromycin B plates. All purified transformants were stored at -70°C in CM liquid medium containing 25% of glycerol in 96-well microtiter dishes. Bioassays. Fungal strains were grown on CMX plates (100 X
15mm) for 7-10 days at 24°C under the light for maximum conidiation. To verifiy normal T-toxin production by a race T isolate, 1.0 ml of T-toxin-sensitive E. coli (DH5a) cells were evenly spreaded on LB medium containing ampicillin (100 ug/ml) and the plates were allowed to air dry for 30 min in a laminar hood. Agar plugs bearing fungal mycelia were inoculated (upside down) onto the E. coli cell lawn and the plates were incubated at 32°C. Wild type race T and race O were used as controls for each assay plate. T-toxin-producing strains of the fungus will inhibit growth of the E. coli cells and produce halos. Tox~ mutants can be distinguished from wild type by failure to produce a halo (tight) or by production of halos smaller (leaky) or larger than wild type (oveφroducing). All Tox~ mutants were transferred to Fries medium (Pringle et al., "The Isolation of the Toxin oi Helminthosporium victoriae," Phytopathology, 47:369-371 ( 1957), which is hereby incoφorated by reference), which optimizes toxin production, and retested. T-cytoplasm corn plants (inbred W64A) are used to verify the Tox~ mutants identified from the E. coli assay using the procedure described below. Mutants defective in T-toxin production fail to produce typical race T symptoms on T-com. Pathogenicity phenotype on N-cytoplasm com and virulence of Tox+ strains to T-cytoplasm corn were determined by a plant assay where about 3,000 transformants generated using the REMI mutagenesis procedure (Lu et al., "Tagged Mutations at the Toxl Locus of Cochliobolus heterostrophus Using Restriction Enzyme-Mediated Integration," Proc. Natl. Acad. Sci. USA, 91 : 12649-12653 (1994), which is hereby incoφorated by reference) were screened for mutants defective in ability to cause disease on corn plants. Two week old N-cytoplasm com plants (inbred W64A) grown in the green house (5-6 plants in one 4" X 6" pot) were inoculated with 5 ml conidial suspensions (105 conidia/ml) using a pressurized Preval Spray Gun Power Unit thin layer chromatography sprayer (Alltech Associates, Deerfield, IL), incubated in the mist chamber for 24 hours (23°C) and then taken to the growth chamber (23°C, 80% humidity, 14 hours of light). The mutant phenotypes were determined by occurrence of apparent variations in disease symptom development, mainly by lesion size comparison. Mutants producing lesions smaller than wild type were retested and lengths of typical lesions from each mutant were compared with wild type 7 days after inoculation and measurements were taken for statistical evaluation.
DNA manipulations and sequencing. Genomic and plasmid DNA preparation, restriction enzyme digestions, gel electrophoresis and gel blot analysis were done using standard protocols (Sambrook, et al., Molecular
Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor, New YoricCold Spring Harbor Laboratory Press (1989), which is hereby incoφorated by reference). DNA was sequenced at the Cornell DNA Sequencing Facility using TaqCycle automated sequencing with DyeDeoxy terminators (Applied Biosystems, Foster City, CA). pUCATPH was used for subcloning (Table 1). Primers used for sequencing (Table 2) were designed using Primer Select (DNASTAR Inc., LaserGene system) and synthesized by the Cornell Oligonucleotide Synthesis Facility. Sequencing of each plasmid clone was initiated with vector-specific primers or primers designed to previously determined sequences. Sequences obtained were analyzed using the same system and nucleotide or protein database searches were performed with the BLAST program (Altschul et al., "Basic Local Alignment Search Tool," J. Mol. Biol., 215:403-419 (1990), which is hereby incoφorated by reference).
Table 1. Transformation vectors and clones used
Plasmid Length Characteristics Reference
(kb)a pUCATPH 5.1 See Figure 29. pUCATPH 4.6 Cloning vector, same as pUCATPH but Figure 8
N lacking a 420 bp Narl fragment containing the Hwdlll site p214B7 9,2 A clone containing pUCATPH recovered from Figure 2 the tagged site in mutant R.C4.2696 by religation of Z?g/II-digested genomic DNA p214Ml 63 As above but with Mscl-digested genomic Figure 2
DNA p214Sl 93 As above but with Sαcl-digested genomic Figure 2
DNA p214SlN 12 Nαrl fragment derived from 214S1 Figure 4 containing a 0.8 kb Narl-Sacl fragment of genomic DΝA ligated to pUC 18 p214SNP M Vector for targeted integration constructed Figure 4 by ligating HmdIII-digested pUCATPH into the Hindlll site of p214SlΝ pl l8BSP 13 Vector for targeted integration constmcted Figure 6 by ligation of a 2.2 kb Sacl fragment of pi 18B14 into the Sacl site of pUCATPH pl l8BCS 14 Vector for targeted integration constmcted Figure 9 by ligation of a 0.8 kb Sspl fragment of pi 18BC4 into the Sspl site of pUCATPHN pl l8B14 10.4 A clone recovered from the p214SNP Figure 5 integration site in transformant #118 by ligation of a 5g/II-digested genomic DNA fragment containing the entire vector pl l8BC4 6 _ A clone recovered from same site as above Figure 5 but by ligation of a βc/I-digested genomic
DNA fragment containing part of vector
(214SNP) sequence p9P2 13 A clone recovered from the p 118BSP Figure 7 integration site in transformant #9 by ligation of a Pstl-digested genomic DNA fragment containing pUC 18 pl2H6 _2 A clone recovered from the pi 18BCS Figure 9 integration site in transformant #12 by ligation of a Hwdlll-digested genomic DNA fragment containing the entire vector. a. An underlined kb number indicates that the plasmid carries genomic DNA sequences (see related figures for details). Table 2. Primers used for sequencing recovered genomic DNA flanking the REMI insertion site at the R.C4 2696 mutation.
Name" Position15 Sequence0 Plasmidd Origine
M13RMT GCGGATAACAATTTCACACAGGA A pUC18 SEQ. ID. No. 4
1. RPlb 775 AGGCCCAGCTGCTTCTCTTG A 214B7TrpC SEQ. ID. No. 5
2. RP2 604 ACTCGGACCGGACGGAATAACAA A 214B7RPlb SEQ. ID. No. 6
3. RP3 119 CGGAAGGAGTGCGAACAA A 214B7RP2 SEQ. ID. No. 7
4. RP4 -232 GCTGCTTGCATCTGGTCTTG A 214B7RP3 SEQ. ID. No. 8
5. RP5 -812 AGACCCAGCTGTTGCCCATTG A 214B7RP4 SEQ. ID. No. 9
6. RP5b -1215 CGGAGACGCAAAGCCTGAGA A 214B7RP4 SEQ. ID. No. 10
7. RP6 -1392 TGCCAGCTGCGTCCAAGAAG A 214B7RP5 SEQ. ID. No. 1 1
8. RP7 -1839 GCTAGCATGGCCCTCACAC A 214B7RP6 SEQ. ID. No. 12
TrpC TGTGTTGACCTCCACTAGCTC A PUCATPH SEQ. ID. No. 13
9. FP1 1885 CTACGGGATGCAGAGGGAAAGT A 214B7TrpC SEQ. ID. No. 14
10. FPlb 1828 GCCATGATTAGCACGATACCC B 214B7TrpC SEQ. ID. No. 15
1 1. FP2 2028 CGCGCTGCATACAACTACCAA B 214MlFPlb SEQ. ID. No. 16
12. FP3 2490 TGGTGGCACTACAAAGAACA C 214M1FP2 SEQ. ID. No. 17
13. FP4 2949 CAGCGTGTTGAATGGGTAGAA C 214S1FP3 SEQ. ID. No. 18
14. FP4B 2745 CTGGGTAGATTCGCCTTCAC C 214S1 FP4 SEQ. ID. No. 19
15. FP5 3421 GAGCGATCAGTGCAGAACATT C 214S1 FP4 SEQ. ID. No. 20
16. FP6 3948 CGCTGACGTTTGACCATCTGA C 214S1FP5 SEQ. ID. No. 21
17. FP7 4411 GCATATGCAACGAGTCAAA C, D 214S1FP6 SEQ. ID. No. 22
18. FP8 5035 ACGGTGCACCTGTTGATA D 1 18B14FP7 SEQ. ID. No. 23
19. FP9 5457 ATGCGCACAATAGCCCAGTA 1 18BC4FP8 SEQ. ID. No. 24
20. RP48 2865 TTCAAGCAACTGTGGCGTAGG D 214S1FP6 SEQ. ID. No. 25
21. FP10 5790 GATCCTAGCGACCGCACACCAAC F 9P2FP9 SEQ. ID. No. 26
22. FP1 1 6327 CCTGCTGCTGGTGCTTCT F 9P2FP10 SEQ. ID. No. 27 23. FPl lb 621 1 GAGTTGCAAATCGTGACAGC 9P2FP10
SEQ. ID. No. 28
24. FP12 6457 TATCAGCTGTTGTTCAATGTTCTA 9P2FP1 1
SEQ. ID. No. 29
25. FP13 6854 TGTTATCCCATTGCCATTG 9P2FP12
SEQ. ID. No. 30
26. FP14 7400 AAGGACGGAGATTGGTGGAG 9P2FP13
SEQ. ID. No. 31
27. FP15 7771 GGAGATGGCGGTGACGA 9P2FP14
SEQ. ID. No. 32
28. FP16 8145 GCATGGCTTGTGGAGGAC 9P2FP15
SEQ. ID. No. 33
29. FP17 8492 AGATTGTGGCTAGTATGGAGGTAA 9P2FP16
SEQ. ID. No. 34
M 13F40 GTTTTCCCAGTCACGAC pUC18
SEQ. ID. No. 35
30. RP1 8953 TACTACTAGCATACCAGCATACCT 9P5M13F4
SEQ. ID. No. 36
31. RP2 8559 TCAACCTCGGAATACCAAGTC 9P5RP1
SEQ. ID. No. 37
a. "RP" indicates reverse primer; "FP" indicates forward primer. Primers designed to genomic DNA sequences are numbered in order. For stock tube and the notebook, Primers 1-17 have a leading number "214"; 18-20 with "118"; 21-29 with "9P2" and 30-31 with "9P5". M13RMT(a M13R mutant version; there is a mutation in the polylinker of pUC18) and M13F-40 are provided by Cornell DNA Sequenceing Facility. TφC primer site is in the pUCATPH TrpC promoter region 38 bp from SaR site with sequencing direction from SaR to Kpnl. b. The position of the first base of each primer corresponds to the assembled sequence (CPSl + TES1, total 11.3 kb found in Lu's folder in the computer Yoder labl) c. Each primer sequence is given in the 5' to 3' direction d. Plasmids used as templates for each sequencing reaction. A = p214B7; B = p214Ml; C = p214Sl; D = pl l8B14; E = pl l8BC4; F = p9P2. G = p9P5 (=9P2) e. Original sequences that were used for primer design can be found in the CPSl sequence notebook or in Shunwen Lu's folder (CPSl sequence) in the computer Yoder labl under the same names as listed.
Recovery of tagged DNA from the REMI insertion site and targeted gene disruption. Genomic DNA of mutant R.C4.2696 was digested with BgRl, Mscl (no sites in pUCATPH) or Sacl (which cuts the vector once) and purified by phenol extraction and ethanol precipitation, then dissolved in TE (pH 8.0). Ligation was performed in 50 ul reaction mixture, containing 1 x T4 DNA ligase buffer with 10 mM ATP, 60 units T4 DNA ligase (New England Biolabs, Beverly, MA) and 3 ug of 5g/II-digested genomic DNA, at 14°C overnight. 10 ul of ligation mixture was used to transform 200 ul of competent DH5a cells, prepared using the calcium chloride treatment (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, New York:Cold Spring Harbor Laboratory Press (1989), which is hereby incoφorated by reference), to ampicillin resistance. Ampicillin resistant clones were analyzed by digestion of plasmid DNA with several diagnostic restriction enzymes and clones containing the REMI vector plus flanking genomic DNA were sequenced using the vector-specific primers (M13R or TrpC). Three plasmids, p214B7, p214Ml and p214Sl (Figure 2) were recovered and used for sequencing. For targeted gene disruption in wild type, p214B7 was amplified and plasmid DNA purified by equilibrium centrifugation in CsCl-ethidium bromide gradients (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2n Ed., Cold Spring Harbor, New York:Cold Spring Harbor Laboratory Press (1989), which is hereby incoφorated by reference). 30 ug of plasmid DNA (linearized with BgRl for double crossover integration as illustrated in Figure 3) were used to transform wild type and the transformants were purified by isolation of single conidia, assayed for pathogenicity and characterized by gel blot analysis.
Sequence extension by targeted integration and plasmid rescue. Two overlapping cosmid clones were isolated by probing a genomic DNA library of C4 constmcted on a cosmid vector, but both extended into the left region only of p214B7. To extend to the right, a chromosome walking strategy was employed (Figure 3). Three targeted gene disruption experiments (each followed by plasmid rescue) were done successively. In the first experiment, a vector was constructed as follows (see Figure 4 for details): p214Sl was digested with Nαrl and religated to create p214SlN, which was then digested with H dIII and ligated into the HmdIII site of pUCATPH to create p214SNP for transformation of race O (C5). One transformant (Txl 18) resulting from homologous integration (confirmed by gel blot analysis) was used for plasmid rescue as described above. Two new plasmids pi 18B14 and pi 18BC4 were recovered, both of which carry sequence at the 3' end but only 172 and 680 bp more than p214Sl, respectively (Figure 5). To continue the walk, pi 18B14 was digested with Sacl and ligated into the Sacl site of pUCATPH to create pi 18BSP (Figure 6). This vector was transformed into wild type and one plasmid, p9P2 was recovered (from transformant Tx9), which extends 4.4 kb into the region 3' of pi 18BC4 and contains the 3' end oiCPSl (Figure 7). A third experiment was done in an attempt to recover a 15 kb Xhol fragment at the 3' end of that tagged gene, pi 18BCS (Figure 8) was constructed by subcloning a 0.8 kb Sspl fragment into the same site of pUCATPHN. Plasmid rescue using Xhol digested-genomic DNA of a transformant (TX12) failed to recover the 15 kb Xhol fragment, but pl2H6 was recovered using Hwdlll-digested genomic DNA of the same transformant; the genomic DNA matched that already cloned on p9P2 (Figure 9).
EXAMPLE 2 - Characterization of the REMI mutant. 1) In all culture conditions used in the lab, mutant R.C4.2696 grows just like wild type with no variations in growth rate, color and moφhological features (Figure 10A). It produces normal conidia that germinate and form infection structures like wild type when induced on artificial surfaces (Figure 10B) and shows normal mating ability when crossed to wild type testers. No pleiotropic phenotypes associated with the mutation have been detected so far.
2) The mutant differs from wild type in the ability to cause disease on com plants. When tested on T-cytoplasm corn, the mutant produces race T type symptoms but the disease develops more slowly than with wild type although it produces wild type levels of T-toxin as detected in a microbial assay (Figure 1 1), suggesting that the reduced vimlence is not related to a deficiency in the ability to produce T-toxin. This is clearer on N-cytoplasm corn where the mutant produces lesions significantly smaller than those produced by wild type (Figure 12). When the mutant was crossed to a wild type race O tester, the small lesion phenotype and ability to produce T-toxin segregated independently, indicating that mutant phenotype is not associated with the reduced fitness trait tightly linked with the Toxl locus (Klittich et al., "Reduced Fitness Associated With Toxl of Cochliobolus heterostrophus," Phytopathology, 76:1294-1298 (1986), which is hereby incoφorated by reference). The statistical evaluation of lesion size in the race O genetic background indicates that the mutation causes 60% reduction in the fungal vimlence to com plants (Figure 13).
3) The mutant phenotype is caused by a tagged, single site mutation. In crosses between the mutant and wild type testers, progeny segregated 1 : 1 for parental types only and all hygromycin B-resistant progeny produced lesions similar to the mutant parent; all hygromycin B-sensitive progeny produced wild type lesions (Figure 14), indicating that a tagged mutation is responsible for the reduced pathogenicity of the mutant.
EXAMPLE 3 - Cloning and sequencing of DNA flanking the REMI vector insertion site.
A total of 11.3 kb of genomic DNA surrounding the insertion site was cloned and completely sequenced (Figure 15). The sequence was derived from seven plasmid clones. The first three (p214B7, p214Ml and p214Sl) were recovered from the tagged site in mutant R.C4.2696 and cover about 60 % (6.6 kb) of the entire region. The rest (pi 18B14, pi 18BC4, p9P2 and pl2H6) were recovered from transformants generated using the chromosome walking strategy. DNA to the left of the insertion site (3.4 kb) was cloned on p214B7; DNA on the right (7.9 kb) was cloned on different overlapping plasmids. p9P2 carries the largest amount (4.6 kb) including genomic DNA on pl2H6 (Figure 15).
EXAMPLE 4 - Identification of CPSl and TES1 at the sequenced region.
Analysis of the combined sequences revealed two open reading frames (ORFs). 0RF1(5.4 kb) starts 576 bp upstream of the REMI vector insertion site and ends with an in- frame stop codon (TAG) 3029 bp from the end of the sequenced region in the right flank (Figure 15). No "TATA" box-like element is found in the expected position, but five putative "CAAT" boxes are located upstream of the start codon (ATG), three of them are in the range found in most filamentous fungal promoters (60-200 bp) (Gurr et al., 1987, which is hereby incoφorated by reference). Sequence around ATG of ORFl (CACCATGCT (SEQ. ID. No. 38)) is similar to the fungal consensus (CACCATGGC (SEQ. ID. No. 39)). Although there are several ATGs found upstream, they are less likely to be used as a start codon because the surrounding sequences lack similarity to the consensus. Three putative introns are identified by their conserved 5' and 3' border sequences and potential branch sites (Table 3). Splicing these introns eliminated stop codons which would otherwise interrupt the 5.4 kb open reading frame. Three introns have similar size (45-53 bp respectively) which is in the range of intron size determined from most fungal genes. A putative polyadenylation signal (ATAA) is found 223 bp downstream of the translation termination site. The G+C content of ORFl is 51.5%, which is similar to most Cochliobolus genes (Turgeon et al., "Cloning and Analysis of the Mating Type Genes from Cochliobolus heterostrophus " Mol. Gen. Gene., 238(l-2):270-284 (1993); VanWert et al., "Structure of the Cochliobolus-heterostrophus
Glyceraldehyde-3 -Phosphate Dehydrogenase Gene," Curr. Genet., 22(l):29-35 (1992); Yang et al., "A Polyketide Synthase is Required for Fungal Vimlence and Production of the Polyketide T-Toxin," Plant Cell. 8(11):2139-2150 (1996); Rose et al., "A Decarboxylase Required for Polyketide Toxin Production and High Virulence by Cochliobolus heterostrophus," 8th Int. Symp. Mol. Plant-Microbe Int., Knoxville, p. J-49 (1996), which are hereby incoφorated by reference). Interestingly, ORFl is flanked by two regions of G+C rich DNA. The first (1.4 kb, 60.7% G+C) is found between ORFl and ORF2; the second (1.2 kb, 60.3% G+C) is found 1.8 kb downstream of the stop codon of ORFl (Figure 16). Database searches using the translated protein sequence of ORF 1 revealed high similarity to SafB, one of the multifunctional enzymes catalyzing the biosynthesis of the cyclic peptide antibiotic saframycin Mxl produced by the bacterium Myxococcus xanthus (Pospiech et al., "Two Multifunctional Peptide Synthetases and an O-methyltransferase are Involved in the Biosynthesis of the DNA-Binding Antibiotic and Antitumour Agent Saframycin Mxl from Myxococcus xanthus," Microbiology, 142(4):741-746 (1996), which is hereby incoφorated by reference). The entire nucleotide sequence of ORFl (designated CPSl ) is given in Figure 17.
Table 3. Characteristics of putative introns in CPSl and TES1
Gene Intron Size(bp) Location 5 'Border 3 'Border Branch Site
CPSl I 45 3060-3105 GTAAGT TAG GTCTAAC
II 51 4532-4582 GTAAGT CAG TGCTAAC
III 53 5187-5239 GTACGT CAG TACTAAC
TES1 I 49 528-566 GTAAGT TAG CCTTAAG
Consensus GTAA/cGT T/CAG YNCTAAC* * Y = Pyrimidine (T or C); N = purine or pyrimidine.
ORF2 starts about 1.6 kb upstream of the start codon oi CPSl and is transcribed in the opposite direction (Figure 15). No "TATA" box-like element and CAAT box are found; instead, an AT-rich sequence "AAAACTAT" (SEQ. ID. No. 40) is located 11 bp upstream of the start codon ATG and a CT motif is found in the -30 region, which is characteristic of a number of fungal genes that lack a CAAT box in their promoter region (Gurr et al., "The Structure and Organization of Nuclear Genes of Filamentous Fungi," in Kinghorn, ed., Gene Stmcture in Eukaryotic Microbes, Vol. 22, published by the Society for General Microbiology, Oxford, EnglandTRL Press, pp. 93-140 (1987), which is hereby incoφorated by reference). The sequence around ATG matches perfectly fungal gene consensus. A putative intron (50 bp) is found in the middle of ORF2 with conserved 5' and 3' border sequences and a potential branch site (Table 3). A putative polyadenylation signal (AAATA) is found 189 bp downstream of the translation stop codon TGA. The G+C content of ORF2 is 55.5%, which is slightly higher than the normal range because the 5' end of ORF2 is located in the region of G+C rich DNA upstream of ORFl (Figure 16). Database search revealed that ORF2 encodes a protein with high similarity to Homo sapiens thioesterase II (hTE, Liu et al., "Binding of HIV-1 Nef to a Novel Thioesterase Enzyme Correlates with Nef-Mediated CD4 Down-Regulation," J. Biol. Chem.. 272(21 :13779-13785 (1997), which is hereby incoφorated by reference) and E. coli thioesterase II encoded by the tesB gene (Naggert et al., "Cloning, Sequencing and Characterization oi Escherichia-coli Thioesterase II," J. Biol. Chem., 266(17)11044-11050 (1991), which is hereby incoφorated by reference). The nucleotide sequence of ORF2 (designated TES1) is given in Figure 18.
EXAMPLE 5 - Modular structure of CPSl. Predicted CPSl protein (1743 amino acids, Mr 193235) contains two structurally similar modules, both of which are similar to SafBl, the first module of saframycin synthetase B (overall 25% identity; 50% similarity) and have apparent amino-acid-activating and thiolation domains but lack methyltransferase activity, thus appearing to be typical type I modules (Figure 19). The number of amino acids in each module is different: the first module (CPSl A) consists of 574 amino acids (from the first residue of core 1 to the last residue of core 6), which is larger than most type I modules; the second module (CPS IB) has 530 amino acids, which is average. The distance between the two modules is 193 amino acids, much shorter than most peptide synthetases (500-600 aa), but this distance is not highly conserved, i.e., an opposite variation is found in HC-toxin synthetase and cyclosporine synthetase, both of which have about 1 ,000 aa between the first and second amino-acid-activating module (Figure 20F). Amino acid alignment of the two modules of CPSl to SafBl indicated that these modules are highly similar to each other in both overall amino acid composition and conserved motif sequences as defined by Stachelhaus and Marahiel (Stachelhaus et al., "Modular Structure of Peptide Synthetases Revealed by Dissection of the Multifunctional Enzyme GrsA," J. Biol. Chem..
270(1 1):6163-6169 (1995); Marahiel, "Protein Templates for the Biosynthesis of Peptide Antibiotics," Chem. Biol.. 4(8): 561-567 (1997), which are hereby incoφorated by reference). When aligned to other bacterial or fungal peptide synthetases, CPSl only showed local similarity to cyclosporine synthetase (Weber et al., "The Peptide Synthetase Catalyzing Cyclosporine Production in
Tolypocladium niveum is Encoded by a Giant 45.8-Kilobase Open Reading Frame," Current Genetics. 26(2): 120-125 (1994), which is hereby incoφorated by reference) and tyrocidine synthetase A (Mootz et al., "The Tyrocidine Biosynthesis Operon of Acillus brevis: Complete Nucleotide Sequence and Biochemical Characterization of Functional Internal Adenylation Domains," J.
Bacteriol., 179(21):6843-6850 (1997), which is hereby incoφorated by reference), but when the amino acids in motif regions were aligned, a overall conservation was observed. Both CPSl A and CPS IB have all five core sequences in the amino-acid-activating domain (Figure 20A-E). Cores 3 and 4 are well conserved except for the replacement of an aspartic acid residue of core 4 by a leucine in
CPSl A. Cores 1, 2 and 5 show weak conservation, but similar variations are also seen in SafB 1. A thiolation domain is found in both modules, which contains a highly conserved motif (core 6, Figure 20F). The serine residue in this motif has been shown to be the active site for 4'-phosphopantetheine attachment (Schlumbohm et al, "An Active Serine is Involved in Covalent Substrate Amino Acid Binding at Each Reaction Center of Gramicidin S Synthetase," J. Biol. Chem., 266(34):23135-23141 (1991); Stein et al., "Detection of 4'- Phosphopantetheine at the Thioester Binding Site for L-Valine of Gramicidins Synthetase 2," FEBS Lett.. 340(1 -2):39-44 (1994), which are hereby incorporated by reference). The distances between the six core sequences in the two modules are also largely conserved. Two exceptions are found in the first module, which has 312 aa between cores 2 and 3, larger than normal (150-200); 61 between cores 5 and 6, only half of that of most peptide synthetases. SafBl also shows distance variations at these two interval regions (Figure 20B and E). In addition to amino- acid-activating and thiolation domains, CPSl also has an integrated thioesterase domain (TE) in the carboxy-terminal end of CPS IB (Figure 19). A signature sequence GXSXG, which is highly conserved in animal fatty acid thioesterase type II enzymes and several peptide synthetases, is found in this domain (Figure 21).
EXAMPLE 6 — Sequence homology analysis of TES1 protein.
The predicted TES1 protein consists of 367 amino acids (Mr 41013). Amino acid alignment of TES1 to hTE, TESB and Mycobacterium tuberculosis TESB homolog (Philipp et al., "An Integrated Map of the Genome of the Tubercle bacillus, Mycobacterium tuberculosis H37Rv, and Comparison with Mycobacterium leprae " Proc. Natl. Acad. Sci. USA. 93(7):3132-3137 (1996), which is hereby incoφorated by reference) showed that these proteins have an overall 40% identity and 60% similarity. A highly conserved VHS motif (putative active site) is found in the C-terminal region of TES1 at a conserved position (Figure 22). All these thioesterases have no sequence similarity with the previously identified animal type I or type II thioesterases known to be involved in the chain termination of fatty acid synthesis (Naggert et al., "Cloning, Sequencing and Characterization oi Escherichia-coli Thioesterase II," J. Biol.
Chem., 266(17): 11044-11050 (1991), which is hereby incoφorated by reference). Interestingly, TES 1 has more homology to hTE than to two bacterial genes, suggesting that both proteins belong to a new family of eucaryotic thioesterases. EXAMPLE 7 - Targeted disruption of CPSl.
Disruption of either CPSl A or CPS IB restored the original mutant phenotype. Ten transformants from each of four individual disruption experiments using different constructs, including the plasmid recovered from the REMI insertion site in the mutant (p214B7) and three vectors for chromosome walking (p214SNP, pi 18BSP and pi 18BCS) were purified and assayed on N- cytoplasm com. All transformants showed the same small lesion phenotype as that of the original REMI mutant. Gel blot analysis confirmed that all transformants showing the mutant phenotype resulted from homologous integration of the transforming vector that dismpted the wild type CPSl (Figures 24-26). No transformants showing the wild type phenotype were obtained, presumably because of the large genomic DNA fragments (over 800 bp in all disruption experiments) on the transforming vector that resulted from high efficiency of homologous recombination and the low chance to recover transformants with ectopic integration.
EXAMPLE 8 - Methods and Materials for Examples 9-10
Strains, growth conditions and transformation. Strains of Cochliobolus species and relatives used for genomic DNA hybridization are listed in Table 4. The strain HvW, a victorin-producing isolate of C. victoriae was recovered from storage and grown on CMX medium (Turgeon et al., "Transformation of the Fungal Maize Pathogen Cochliobolus heterostrophus Using the Aspergillus nidulans amdS Gene," Mol. Gen. Genet., 201 :450-453 (1985), which is hereby incoφorated by reference) for conidiation or on oat meal agar medium (Churchill et al., "Victorin-Deficient REMI Mutants oi Cochliobolus victoriae Demonstrate a Requirement for Victorin in Pathogenesis," Fungal Genet. Newsl., 42A:41 (1995), which is hereby incoφorated by reference) for victorin detection at 24°C under warm white lights (Sylvania Inc., Danvers, MA). Transformation was done using the C. heterostrophus procedure (Turgeon et al., "Cloning and Analysis of the Mating Type Genes from Cochliobolus heterostrophus " Mol. Gen. Gene.. 238(l-2):270-284 (1993), which is hereby incorporated by reference). Table 4. Detection of CPSl homologs in Cochliobolus spp and relatives
Hybridization
Strain2 Host" EcoRI Hindlll BgRl digest0 digestd digest6
C. heterostrophus Com race T (C4) (Turf-13) + 5.2 3.2 4.2 race 0 (C5) + 5.2 3.2 4.2
C. carbonum Corn1 race l (26R13 (hmlhml) + 6.6 5.0 race 2 (YugY) N 6.6 5.0 race 3 (BZ1703)* N 6.6 5.0
C. victoriae (HvW) d s (Vb) + N 5.0
C. sativus (A20) Grasses2 + 3.0 N
C. specifer (D5-7) Grasses2 + N N
C. homomorphus Unknown N 5.8 N (ATCC 13409)
C. dactyloctenii Unknown N 5.9 N (7938-9)
5. twrc cα (NK2) Sorghum and + N N maize
5. rostrata (32191) Weeds and + 2.8 N bamboo4
B. sacchari Sugarcane (764-1) + 5.4 2.5 N (1249-10) N 5.4 2.5 N
a. C. — Cochliobolus. S. = Setosphaeria. B. = Bioplaris. The name of isolates (or lab strains) of each species are given in parentheses and those known to produce host-specific toxins are underlined. * Provided by Tsukiboshi Takao (Japan) and the isolate could be either BZ1209 or BZ1703. b. Genotype susceptible to the host-specific toxin-producing isolate is given in parentheses. References for hosts of those species not mentioned in the previous chapters are as follows: 1 : Welz et al., "Phenotypic Variation and Parasitic Fitness of Races of Cochliobolus-carbonum on Co in North Carolina," Phytopathology. 83(6)593-601 (1993); Leonard et al., "Genetic Diversity in Field Populations of Cochliobolus-carbonum on Com in North Carolina USA," Phytopathology, 80(11):1154-1159 (1990) (for races 2 and 3 only). 2: Domsch et al., "Compendium of Soil Fungi, Vol. 1 ," New York, New York: Academic Press, pp. 216-222 (1980). 3: David et al., "Fungi on Plants and Plant Products," St. , Paul, Minnesota:APS Press, p. 635 (1989); Thakur et al., "Characterization of a New Race of Exserohilum-turcicum Virulent on Com With Resistance Gene HTN," Plant Pis., 73(2):151-155 (1989). 4: Rao et al., "New Fungal Diseases on Some Weeds," Indian Bot. Rep., 6(1):38 (1987); Bhat et al., "Unrecorded Pathogen on Bamboo Causing Blight in India," Curr. SCI. (BANGALORE).
58(20): 1148-1149 (1989). 5: Yoder, "Toxins in Pathogenesis." Ann. Rev.
Phytopathol.. 18:103-129 (1980). c. Genomic DNAs (from a previously prepared gel blot filter, Rose et al., "A Decarboxylase Required for Polyketide Toxin Production and High Virulence by
Cochliobolus heterostrophus," 81 Int. Symp. Mol. Plant-Microbe Int., Knoxville, p. J-49 (1996) were probed with the 3.4 kb CPSl fragment cloned on p214B7
(Figure 2). "+" indicates a strong hybridization signal. All species hybridized to a large fragment (~23 kb). d. Genomic DNAs selected from a lab collection were probed with the CPSl 3.2 kb fragment cloned on p214Sl (Figure 2). The size of fragments that hybridized to the probe is given in kb. The intensities of hybridization signals were similar to each other. N = not done. e. Genomic DNAs were probed with the same CPSl fragment as in c. Gel blot is shown in Figure 26.
DNA manipulations and targeted disruption of the CPSl homolog in C. victoriae. Genomic DNAs for probing were prepared according to Yoder ("Cochliobolus heterostrophus, Cause of Southern Corn Leaf Blight," in Sidhu, ed., Genetics of Plant Pathogenic Fungi, Vol. 6, San Diego,
California:Academic Press, pp. 93-112 (1988), which is hereby incoφorated by reference), or selected from a lab DNA collection (stored at 4°C). A gel blot filter bearing known genomic DNAs was also probed. Plasmid DNA preparation, restriction enzyme digestions, gel electrophoresis, gel blot analysis were done using standard protocols (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, New YoricCold Spring Harbor Laboratory Press (1989), which is hereby incoφorated by reference). For probing, CPSl fragments of C. heterostrophus cloned on p214B7 (3.4 kb left flank) and p214Sl (3.2 kb right flank) (Figure 2) were prepared by restriction enzyme digestion of the plasmid DNAs followed by purification using the QIAquick Gel Extraction Kit (QIAGEN Inc., Chatsworth, CA). The plasmid pi 18B14, which carries the 2.2 kb Bgl II fragment of CPSl interrupted by the hygB cassette (see Figure 5) was linearized with BgRl and introduced into HvW genome. Transformants were purified by isolation of single conidia and genomic DNAs were digested with BgRl and probed with the CPSl 3.1 kb fragment.
Bioassays. Pathogenicity was determined by an oat plant assay. Fungal strains were grown on individual oat meal agar medium plates (60 X 15 mm) containing hygromycin B (60 ug/ml) for 10 days at 24°C under lights. Conidia were scraped from the plates and suspended in 6 ml of sterilized distilled water. One ml of conidial suspension of each strain was mixed with 60 seeds of susceptible or resistant oats. Inoculated seeds were planted in 4" X 6" pots and seedlings were allowed to grow for two weeks. Seed germination rate and symptom development were recorded at different stages (4, 6, 8 and 24 days after inoculation). Detection of victorin production using HPLC analysis was done by Alice Churchill in Dr. Vladimir Macko's lab at Boyce Thompson Institute for Plant Research.
EXAMPLE 9 - Detection of CPSl homologs.
Genomic DNAs of 12 isolates (or lab strains) of 9 fungal species hybridized to CPSl (Table 4). All 6 Cochliobolus species, including 4 known plant pathogens (C. carbonum, C. victoriae, C. sativus and C. spicifer) and 2 species with unknown hosts (C. homomorphus and C. dactylocteniϊ) gave hybridization signals of the same intensity as that of C. heterostrophus CPSl fragments (Figure 26, only C. carbonum and C. victoriae are shown). Two phytopathogenic Setosphaeria species and Bioplaris sacchari, a sugarcane pathogen gave a similar hybridization intensity.
CPSl homologs appear to be polymoφhic among different species, i.e., all species gave one or two unique bands when BgRl or Hindlll digested genomic DNAs were probed (except for C. victoriae, which showed the same hybridization pattern as C. carbonum) (Table 4 and Figure 26). Interestingly, EcoRI digested genomic DNAs of the same species did not show polymoφhisms; all species hybridized to a large fragment (~23 kb, Table 4), indicating the absence of an EcoRI site in all CPSl homologs as in the C. heterostrophus gene. In C. heterostrophus, a > 12 kb of genomic region which includes CPSl (5.4 kb), TES1 (1.1 kb) and sequence downstream of the 3' end of CPSl has no EcoRI sites. In contrast to species-dependent polymoφhisms, CPSl homologs appear to be highly conserved among different isolates of the same species. Both C. heterostrophus race T and race O hybridized to the same 4.2 kb BgRl fragment (or 5.2 and 3.2 kb Hindlll fragments); all three C. carbonum races hybridized to the same 5.0 kb BgRl fragment (or 6.6 kb Hrødlll fragment) (Table 4, Figure 26) and B. sacchari isolates 764-1 and 1249-10 hybridized to the same H dIII fragments (5.4 and 2.5 kb) (Table 4).
EXAMPLE 10 ~ Targeted disruption of CPSl homolog in C. victoriae. Twenty transformants were obtained from transformation of the victorin-producing isolate HvW with Z?g/II-linearized plasmid pi 18B14 (Figure 5). Six transformants were purified and assayed for both victorin production and pathogenicity to susceptible oat plants. All transformants produced wild type levels of victorin as determined by HPLC analysis (Figure 27), but four of them (Tx7, Tx2, Tx5 and Tx8) showed dramatically reduced vimlence in the plant assay. The seed germination rate on the eighth day after inoculation is only 13- 25% for wild type and two transformants (Tx9 and Tx4), but 45-63%) for the other four transformants. On day 24 after inoculation, all plants emerged from the seeds inoculated with wild type, Tx9 or Tx4 were killed but most (29-63%) from the seeds inoculated with Tx2, Tx7, Tx5 or Tx8 still survived (Table 5, Figure 28A). Gel blot analysis confirmed that transformants showing the reduced vimlence phenotype resulted from homologous integration of the transforming vector that dismpted the wild type CPSl homolog in C. victoriae genome; transformants showing the wild type phenotype resulted from ectopic integration events that left the native gene intact (Figure 28B). All transformants remained nonpathogenic to resistant oats, indicating that dismption of the CPSl homolog does not affect host specificity of the fungus.
Table 5. Disease development of oat plants inoculated with C. victoriae transformants (Tx).
No. germinatedb Germination No. survivorsd
Strain3 4 6 8 Rate (%)c 24 %
Control- 1 28 41 45 75 75 100
Control-2 40 50 50 83 50 100
Control-3 1 7 12 20 0 0
Tx2 8 26 27 45 16 59
Tx4 5 15 15 25 0 0 Tx5 2 24 28 47 8 29
Tx7 14 36 38 63 24 63
Tx8 7 29 29 47 13 47
Tx9 0 3 8 13 0 0
a. Control- 1 = uninoculated susceptible oat seeds. Control-2 and Control-3 = resistant and susceptible oat seeds inoculated with wild type C. victoriae (isolate HvW), respectively. Six transformants were tested on both resistant and susceptible seeds, but only data for the later are shown (all transformants gave the same results as Control-2 when tested on resistant seeds). Repeat experiments gave similar results (data not shown). b. Sixty oat seeds were used for each strain. Emerged oat plants were counted 4, 6 and 8 days after inoculation. c. Calculation based on the data collected on the day 8. d. Recorded on day 24 after inoculation. The percentage of survivors is based on the number of plants recorded on days 8 and 24.
EXAMPLE 11 ~ Isolation of CPSl genes from other plant pathogens As dislosed in the previous examples, the Cochliobolus heterostrophus gene CPSl encodes a peptide synthetase that appears to be a general factor for fungal vimlence to their hosts. Thus, CPSl has been found to be highly conserved among at least 9 fungal species belonging to 3 genera including the genus Cochliobolus and closely related genera Bioplaris and Setosphaeria; it has been demonstrated to be required for pathogenesis of three different plant pathogens i.e., C. heterostrophus race O , race T to com and C. victoriae to oats (Lu, 1998, "Molecular-genetic analysis of general and specific pathogenesis factors in Cochliobolus heterostrophus," Ph.D thesis, Cornell University). To further explore the role of CPSl in fungal pathogenesis and its conservation in other fungi, genomic DNAs of additional species in Cochliobolus and other closely or distantly related genera were probed with ChCPSl by DNA- DNA hybridization (Lu, S.-W., B. G. Turgeon and O. C. Yoder. 1999. "A gene cluster from the com pathogen Cochliobolus heterostrophus required for nonribosomal peptide biosynthesis and general vimlence of fungi." Fungal
Genetics Conference, March 1999, Pacific Grove, California). Genomic DNAs of 40 filed isolates (or lab strains) representing 34 fungal species belonging to 16 genera hybridized when probed with ChCPSl (Figs. 30A-30C). Fungal genomic DNAs were prepared according to a previously described procedure (Yoder, 1988. "Cochliobolus heterostrophus, cause of Southern Com Leaf Blight". Genetics of Plant Pathogenic Fungi. G. S. Sidhu. San Diego, Academic Press. 6: 93-112). Plasmid DNA preparations, restriction enzyme digestions and preparation of DNA gel blots were performed following standard protocols (Sambrook, J., E. F. Fritsch, et al. 1989 "Molecular Cloning: A Laboratory Manual, 2nd Edition". Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press). A 3.2 kb ChCPSl fragment (corresponding to ChCPSl amino acids 173-1208) was obtained by restriction enzyme digestion of a plasmid clone p214Sl (Lu, 1998, Ph.D thesis), (see Figure 2), followed by purification using the QIAquick Gel Extraction Kit (QIAGEN Inc., Chatsworth, CA). The purified ChCPSl fragment was labeled with α-[32P]dCTP (Turgeon, B. G., H. Bohlmann, et al, 1993. "Cloning and analysis of the mating type genes from Cochliobolus heterostrophus " Mol. Gen. Genet. 238: 270-284.). DNA-DNA hybridization was carried out at 62° C in 6 X SSC, 0.05 X BLOTTO (Sambrook et al., 1989). Filters were washed in 2 X SSC, 0.1% SDS at 62° C for 60 minutes.
All 16 Cochliobolus species, including the known plant pathogens C. carbonum, C. victoriae, C. miyabeanus, C. sativus and C. spicifer, and five genera closely related to Cochliobolus, i.e., Pyrenophora, Setosphaeria, Bipolaris, Stemphyllium and Alternaria showed hybridization intensities comparable to that of C. heterostrophus itself (Fig. 30A).
DNAs of species from nine distantly related genera, including several of economic importance (e. g., Magnaporthe grisea, Fusarium graminearum, Gaeumannomyces graminis) or of medical importance (e. g., Candida albicans) hybridized weakly to CPSl (FigS. 30B, 30C) whereas no signal was detected in DNA of the basidiomycete Ustilago maydis.
Three CPSl homolog genes were cloned and characterized. Three of them were cloned from phytopathogenic fungi, including the wheat head scab fungus Fusarium graminearum (FgCPSl, 6003 bp, SEQ. No. 43), the potato early blight fungus Alternaria solani, (AsCPSl, 2369bp, SEQ. No. 41) and the barley net blotch fungus Pyrenophora teres (PtCPSl, 2306 bp, SEQ. No. 45). FgCPSl was cloned as a full length gene using both PCR amplification and the plasmid rescue procedure that was preceded by targeted gene dismption in the genome. AcCPSl and PtCPSl homologs were partially cloned by direct PCR amplification.
The Polymerase Chain Reaction (PCR) was carried out using degenerate primers designed to conserved regions of C. heterostrophus CPSl (ChCPSl). Two sets of degenerate primers were designed to amino acids at or close to conserved core sequences of C. heterostrophus CPSl (ChCPSl). The first pair of primers: 5'TGYTTYATHGCNGGN GTNGTNGCNGTNCC3 ' (CHFP6, corresponding to positions 493-521 oi ChCPSl) and 5ΥTGYTGNGGNGGNCCNCCNGGRTT3' (CHRP4, 2197-2220 of ChCPSl), was used to amplify CPSl from Fusarium graminearum. The second pair of primers: 5'-AARAARAARGGNCCNACNGAG-3' (FP4CB, corresponding to positions 1531-1550 oi ChCPSl) and 5'SRYTGNA CCCADATYTCNCC3' (RP2DB, corresponding to positions 3883-3902 of ChCPSl), was used to amplify CPSl from A. solani and Pyrenophora teres. PCR was carried out in a Perkin Elmer Cetus 9600-thermocycler with fungal (Fusarium graminearium, Alternaria solani, and Pyreophora teres) genomic DNA as a template.
Reaction mixtures contained about 500 ng of genomic DNA in 100 ul of reaction buffer [ 1 x Ex Taq buffer, 0.2 mM dNTPs, 0.2 uM of each primer and 0.05 U/ml Takara Ex Taq (Pan Vera Coφoration)]. An initial denaturing step of 95 °C for 3 min. was followed by 30 cycles of 94 °C for 1 min, 47 °C (for G. zeae) or 55 °C (for A. solni and Pyrenophora teres) for 3 min, and 72°C for 3 min. Reactions were cooled to 4 °C after a final extension of 72 °C for 10 min. PCR products were examined (10 ul of each reaction) by agarose-gel (0.75%)) electrophoresis.
DNA from isolated clones of the three different fungi was sequenced at the Cornell DNA Sequencing Facility using TaqCycle automated sequencing with DyeDeoxy terminators (Applied Biosystems, Foster City, CA). Primers used for sequencing were designed using Primer Select (DNASTAR) and synthesized by the Cornell Oligonucleotide Synthesis
Facility. Sequencing of each plasmid clone was initiated with vector-specific primers or primers designed to previously determined sequences. Sequences were analyzed using MapDraw and MegAlign (DNASTAR) and nucleotide or protein database searches were performed with the BLAST program (Altschul et al., 1990, 1997).
The FgCPSl open reading frame (5125 bp, SEQ. No. 43) has 50% nucleotide identity to ChCPSl (SEQ ID No.2) in about 4.4 kb of overlap (Fig. 2). No "TATA" box-like element was found in the 5' untranslated region, but other promoter sequences including two putative "CAAT" boxes and a "CT" motif were located upstream of the start codon (ATG) in FgCPSl (Fig. 32). Only one putative intron was found 1508 bp upstream the stop codon (TGA) in contrast to three in ChCPSl (Figs. 31 A, 3 IB and 32). A putative polyadenylation signal "AATAA" was located 62 bp downstream of the stop codon (Fig. 32). The predicted FgCPSl protein (1692 amino acids, Mτ 187983 Da, SEQ ID No. 44) has 68% identity, 73% similarity to ChCPSl (SEQ ID No. 3) in a about 1 ,500 amino acid overlap (Figs. 31 A and 3 IB) that contains two structurally similar modules highly similar to those of ChCPS 1. FgCPS 1 has no significant similarity to ChCPSl at the C-terminus, which is relatively shorter and lacks the thioeterase domain as seen in ChCPSl (Figs. 31 A and 3 IB). The annotated FgCPSl sequence is given in Figure 32.
AsCPSl (2369 bp, SEQ. No. 41) has 76% nucleotide identity to ChCPSl (SEQ ID No.2) in the entire cloned region which contains two conserved introns (Figs. 31 A and B). The translated AsCPSl protein (partial) includes 758 amino acids corresponding to amino acids 511-1269 in ChCPSl and has up to 93% identity, 95% similarity to ChCPSl (Fig. 2). The annotated AsCPSl sequence is given in Figure 33.
PtCPSl (2306 bp, SEQ. No. 45) has 78% nucleotide identity to ChCPSl (SEQ IDNo.2) in the entire cloned region which contains only one intron (Fig. 2). The translated PtCPSl protein (partial) includes 758 amino acids corresponding to amino acids 511-1269 in ChCPSl and has 93% identity, 96% similarity to ChCPSl (Fig. 2). The annotated PtCPSl seqence is given in Figure 34.
EXAMPLE 12 — Targeted disruption of CPSl homolog in F. graminearum.
A 2.2 kb Xbal fragment from pUCATPH (Lu, et al, 1994) containing the bacterial hygmycin resistance gene (hygB) driven by the Aspergillus nidulans trpC promoter was inserted into the Xbal site of a PCR clone pFgC8, which carries the 1.0 kb internal fragment oi FgCPSl, to create pFgC8- hygB. This constmct was transformed (in a circular form or linearized with Hindlll or BgRl) into an isolate (GZ3639) of wild type F. graminearum. Twenty transformants were obtained by transformation of a wild type isolate (GZ3696) with circular plasmid pFgC8-hygB. DNA gel blot analysis of eight such transformants confirmed that the CPSl homolog in F. graminearum was disrupted by a single cross over recombination (Fig. 35A). Two transformants were obtained when the i?g/II-digested pFgC8-hygB was used and only one recovered when the plasmid was digested with Hindlll. Gel blot analysis indicated that all three transformants obtained using linearized plasmid integrated into the genome at a ectopic location that left the wild type FgCPSl gene intact (Fig. 35A). For virulence assays, F. graminearum strains were grown on PDA (or PDA plus hygromycin B for transformants) plates (100 X 15 mm) for 7-15 days at 24 C under black lights (Sylvania Inc., Danvers, MA) for maximum conidiation. A susceptible spring wheat cultivar, Norm Hard Red (kindly provided by G. Bergstrom, Cornell university) was used. Two months old wheat plants grown in the greenhouse at anthesis (10 plants in one 4" X 6" pot) were sprayed with 10 ml conidial suspensions (10^ or 10^ conidia/ml) using a pressurized Preval Spray Gun Power Unit thin layer chromatography sprayer (Alltech Associates,
Deerfield, IL) or "injected" into the spikelets (5-10 ul/ per spikelet) using a Benchmate pipettor (NICHIRYO, Japan). The inoculated plants were incubated in a mist chamber for 48 hours (23° C) and then transferred to a growth chamber (23° C, 14 hours of light). Mutant phenotypes were identified by the comparison of the number of infected or "bleached" spikelets on each head of wheat plants.
Symptoms were recorded 7-10 days after inoculation. All cpsl~ disruptants had reduced vimlence on wheat plants in the assays while ectopic transformants caused disease symptoms indistinguishable from those of wild type (Fig. 35B). Although the invention has been described in detail for the puφoses of illustration, it is understood that such detail is solely for that puφose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED IS:
1. An isolated nucleic acid molecule from a plant pathogen encoding a CPSl synthetase, wherein the nucleic acid molecule comprises a nucleotide sequence which hybridizes under stringent conditions to a nucleic acid molecule having a sequence as set forth in SEQ ID No. 41 or a complement thereof; or wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID No. 41.
2. The isolated nucleic acid molecule according to claim 1 wherein the plant pathogen is selected from the group consisting oi Alternaria solani, Alternaria Alternaia alternatherae, A. alternata, A. amaranthi, A. araliae, A. brassicae, A. brassicicola, A. camelliae, A. cassiae, A. cheiranthi, A. cinerariae, A. gossypii, A. helianthi, A. helianthinficiens, A. mali, and A. raphani.
3. The isolated nucleic acid molecule according to claim 2 wherein the plant pathogen is Alternaria solani.
4. An isolated nucleic acid molecule coding for a polypeptide having the amino acid sequence as0set forth in SEQ ID No. 42.
5. An isolated nucleic acid molecule comprising the nucleotide sequence as set forth in SEQ ID No. 41.
6. An isolated polypeptide encoded by the nucleic acid molecule of claim 1.
7. The polypeptide according to claim 6, wherein the polypeptide has an amino acid sequence which has at least 75 % similarity to the amino acid sequence of SEQ. ID. No. 42 as determined by a BLAST program with the default parameters.
8. The polypeptide according to claim 7 wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID No. 42.
9. An isolated nucleic acid molecule from a plant pathogen encoding a CPSl synthetase, wherein the nucleic acid molecule comprises a nucleotide sequence which hybridizes under stringent conditions to a nucleic acid molecule having a sequence as set forth in SEQ ID No. 43 or a complement thereof; or wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID No. 43.
10. The isolated nucleic acid molecule according to claim 1 wherein the plant pathogen is selected from the group consisting of Fusariumi graminearium, Fusarium avenaceum, F. carpineum, F. chlamydosporum, F.. coccophilum, F. culmorum, F. episphaeria, F. equiseti, F. flocciferum, F. moniliforme, F. oxysporum, F. redolens, F. sambucinum, F. solani, F. subglutinans, F. trichothecioides, F. udum, or F. ventricosum.
11. The isolated nucleic acid molecule according to claim 10 wherein the plant pathogen is Fusarium graminearium.
12. An isolated nucleic acid molecule coding for a polypeptide having the amino acid sequence as set forth in SEQ ID No. 44.
13. An isolated nucleic acid molecule comprising the nucleotide sequence as set forth in SEQ ID No. 43.
14. An isolated polypeptide encoded by the nucleic acid of claim 9.
15. The polypeptide according to claim 14, wherein the polypeptide has an amino acid sequence which has at least 75 % similarity to the amino acid sequence of SEQ. ID. No. 44 as determined by a BLAST program with the default parameters.
16. The polypeptide of claim 15 wherein the polypeptide comprises the amino acid sequence as set forth in SEQ ID No. 44.
17. An isolated nucleic acid molecule from a plant pathogen encoding a CPSl synthetase, wherein the nucleic acid molecule comprises a nucleotide sequence which hybridizes under stringent conditions to a nucleic acid molecule having a sequence as set forth in SEQ ID No. 45 or a complement thereof; or wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID No. 45.
18. The isolated nucleic acid molecule according to claim 1 wherein the plant pathogen is selected from the group consisting oi Pyrenophora teres, Pyrenophora avenae, P. bromi, P. leuceienes, P. phaeocomes, P. schroeteri, P. trichostoma, or P. tritici-repentis.
19. The isolated nucleic acid molecule according to claim 18 wherein the plant pathogen is Pyrenophera teres.
20. An isolated nucleic acid molecule coding for a polypeptide having the amino acid sequence as set forth in SEQ ID No. 46.
21. An isolated nucleic acid molecule comprising the nucleotide sequence as set forth in SEQ ID No. 45.
22. An isolated polypeptide encoded by the nucleic acid of claim 17.
23. The polypeptide according to claim 22, wherein the polypeptide has an amino acid sequence which has at least 75 % similarity to the amino acid sequence of SEQ. ID. No. 46 as determined by a BLAST program with the default parameters.
24. The polypeptide of claim 23 wherein the polypeptide comprises the amino acid sequence as set forth in SEQ ID No. 46.
25. A vector comprising the nucleic acid molecule of any one of claims 1-5, 9-13, or 17-21.
26. A vector according to claim 25, wherein the nucleic acid molecule is operably linked to a promoter.
27. A vector according to claim 26, wherein the nucleic acid molecule is in a sense orientation.
28. A vector according to claim 26, wherein the nucleic acid molecule is in an antisense orientation.
29. The vector according to claim 26, wherein the vector is capable of maintaining and expressing the nucleic acid molecule in bacterial cells.
30. The vector according to claim 26, wherein the vector is capable of maintaining and expressing the nucleic acid molecule in plant cells.
31. A host cell transformed with the vector according to claim 25.
32. A host cell according to claim 31 , wherein the host is a plant.
33. A host cell according to claim 31 , wherein the host is selected from the group consisting of corn, oats, grasses, weeds, bamboo, and sugarcane.
34. A host cell according to claim 31 , wherein the host is com.
35. A plant transformed with the nucleic acid molecule according of any one of claims 1-5, 9-13, or 17-21.
36. An isolated DNA molecule according to claim 1, wherein the nucleic acid molecule has a nucleotide sequence which is 70% identical to the nucleotide sequence of SEQ. ID. No. 41 as determined by a BLAST program with default parameters.
37. An isolated DNA molecule according to claim 1, wherein the nucleic acid molecule is 80% identical to the nucleotide sequence of SEQ. ID. No. 41 as determined by a BLAST program with default parameters.
38. An isolated DNA molecule according to claim 1, wherein the nucleic acid molecule is 90% identical to the nucleotide sequence of SEQ. ID. No. 41 as determined by a BLAST program with default parameters.
39. An isolated DNA molecule according to claim 1, wherein the nucleic acid molecule has a nucleotide sequence which is 70% identical to the nucleotide sequence of SEQ. ID. No. 43 as determined by a BLAST program with default parameters.
40. An isolated DNA molecule according to claim 1 , wherein the nucleic acid molecule is 80% identical to the nucleotide sequence of SEQ. ID. No. 43 as determined by a BLAST program with default parameters.
41. An isolated DNA molecule according to claim 21 , wherein the nucleic acid molecule is 90% identical to the nucleotide sequence of SEQ. ID. No. 43 as determined by a BLAST program with default parameters.
42. An isolated DNA molecule according to claim 1 , wherein the nucleic acid molecule has a nucleotide sequence which is 70% identical to the nucleotide sequence of SEQ. ID. No. 45 as determined by a BLAST program with default parameters.
43. An isolated DNA molecule according to claim 1, wherein the nucleic acid molecule is 80% identical to the nucleotide sequence of SEQ. ID. No. 45 as determined by a BLAST program with default parameters.
44. An isolated DNA molecule according to claim 21, wherein the nucleic acid molecule is 90% identical to the nucleotide sequence of SEQ. ID. No. 45 as determined by a BLAST program with default parameters.
45. A method for identifying inhibitors of a CPSl protein , wherein said CPS 1 protein is a peptide synthetase of a plant pathogen, said method comprising: providing a CPSl protein or polypeptide; contacting the protein or polypeptide with potential inhibitor compounds; determining peptide synthetase activity; and selecting compounds which decrease the peptide synthetase activity.
46. The method of Claim 45 wherein the CPS 1 protein is from
Alternaria solani.
47. The method of Claim 45 wherein the CPSl protein is from Fusarium graminearium.
48. The method of Claim 45 wherein the CPSl protein is from Pyrenophora teres.
49. A method of imparting disease resistance to a plant, said method comprising over-expressing a CPSl polypeptide in the plant, wherein the polypeptide has protein synthetase activity.
50. A method according to claim 49, wherein the plant is selected from a group consisting of com, oats, grasses, weeds, sugarcane, barley, wheat, rice, tomato, potato, citms, malus, rye, cotton, brassica, cabbage, and carrot.
51. The method of claim 49 wherein the CPS 1 polypeptide is from Alternaria solani.
52. The method of Claim 49 wherein the CPSl polypeptide is from Fusarium graminearium.
53. The method of Claim 45 wherein the CPSl polyeptide is from Pyrenophora teres.
EP00986231A 1999-11-23 2000-11-22 Peptide synthetase gene cps1 Withdrawn EP1244347A2 (en)

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CA2360011A1 (en) 1999-01-05 2000-07-13 Trustees Of Boston University Ordered gene assembly
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CN110257402B (en) * 2019-07-10 2021-11-26 吉林大学 Maize small leaf spot pathogen ChCDC10 gene and application thereof
CN110305879B (en) * 2019-07-10 2022-03-29 吉林大学 Maize small leaf spot pathogen ChCDC3 gene and application thereof
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