Summary
A spontaneous uridine-requiring auxotroph of Colletotrichum graminicola was recovered by selection for resistance to 5-fluoro-orotic acid. The auxotroph lacked orotate phosphoribosyl transferase (OPRTase) and was complemented with a clone from a cosmid library of C. graminicola DNA. A 3.1 kb HindIII-SalI fragment was subcloned from the cosmid and it could efficiently transform the auxotrophic strain to uridine prototrophy and integrate by site-specific recombination. This DNA fragment contains an open reading frame that is similar to OPRTase genes of the fungi Sordaria macrospora, Trichoderma reesei, Podospora anserina, and Saccharomyces cerevisiae. Based on the sequence similarities and the ability to restore uridine prototrophy, we conclude that the fragment contains the C. graminicola gene for OPRTase, which we have named PYR1. Our results demonstrate that cloning by complementation is feasible in C. graminicola, that the gene for OPRTase from C. graminicola can be useful as a selectable marker in transformation of the fungus, and that the OPRTase gene product is similar to OPRTase from other fungi.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Beckwith JR, Pardee AB, Austrian R, Jacob F (1962) Coordination of synthesis of the enzymes in the pyrimidine pathway of E. coli. J Mol Biol 5:618–634
Berges T, Perrot M, Barreau C (1990) Nucleotide sequence of the Trichoderma reesei ura3 (OMPdecase) and ura5 (OPRTase) genes. Nucleic Acids Res 18:7183
Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast. Mol Gen Genet 197:345–346
Botstein D, Davis RW (1982) Principles and practice of recombinant DNA research with yeast. In: Strathern JN, Jones EW, Broach JR (eds) The Molecular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 607–636
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395
Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13
Jund R, LaCroute F (1972) Regulation of orotidylic acid pyrophosphorlyase in Saccharomyces cerevisiae. J Bacteriol 109:196–202
Kozak M (1980) Evaluation of the scanning model for initiation of protein synthesis in eukaryotes. Cell 22:7–8
Kozak M (1984) Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNA. Nucleic Acids Res 12:857–872
Kronstad JW, Leong SA (1989) Isolation of two alleles of the b locus of Ustilago maydis. Proc Natl Acad Sci USA 86:978–982
Kronstad JW, Wang J, Covert SF, Holden DW, McKnight GL, Leong SA (1989) Isolation of metabolic genes and demonstration of gene disruption in the phytopathogenic fungus Ustilago maydis. Gene 79:97–1106
Kubo Y, Nakamura H, Kobayashi K, Okuno T, Furusawa I (1991) Cloning of a melanin biosynthetic gene essential for appressorial penetration of Colletotrichum lagenarium. Mol Plant Microbe Interact 4:440–445
Le Chavanton L, Leblon G (1989) The ura5 gene of the ascomycete Sordaria macrospora: molecular cloning, characterization and expression in Escherichia coli. Gene 77:39–49
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
May GS, Gambino J, Weatherbee JA, Morris NR (1985) Identification and functional analyses of b-tubulin genes by site specific integrative transformation in Aspergillus nidulans. J Cell Biol 101:712–719
Miller BL, Miller KY, Timberlake WE (1985) Direct and indirect gene replacements in Aspergillus nidulans. Mol Cell Biel 5:1714–1721
de Montigny J, Belarbi A, Hubert JC, LaCroute F (1989) Structure and expression of the URA5 gene of Saccharomyces cerevisiae. Mol Gen Genet 215:455–462
de Montigny J, Kern L, Hubert JC, LaCroute F (1990) Cloning and sequencing of URA10, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae. Curr Genet 17:105–111
Oakley BR, Rinehart JE, Mitchell BL, Oakley CE, Carmona C, Gray GL, May GS (1987) Cloning, map** and molecular analysis of the pyrG (orotidine-5′-phosphate decarboxylase) gene of Aspergillus nidulans. Gene 61:385–399
Orr-Weaver T, Szostak J, Rothstein RJ (1981) Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358
Paietta JV, Marzluf GA (1985) Gene disruption by transformation in Neurospora crassa. Mol Cell Biol 5:1554–1559
Panaccione DG, Hanau RM (1990) Characterization of two divergent β-tubulin genes from Colletotrichum graminicola. Gene 86:163–170
Panaccione DG, McKiernan M, Hanau RM (1988) Colleotrichum graminicola transformed with homologous and heterologous benomyl-resistance genes retains expected pathogenicity to corn. Mol Plant Microbe Interact 1:113–120
Panaccione DG, Vaillancourt LJ, Hanau RM (1989) Conidial dimorphism in Colletotrichum graminicola. Mycologia 81:876–883
Parsons KA, Chumley FG, Valent B (1987) Genetic transformation of the fungal pathogen responsible for rice blast. Proc Natl Acad Sci USA 84:4161–4165
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Timberlake WE, Boylan MT, Cooley MB, Mirabito PM, O'Hara EB, Willet CE (1985) Rapid identification of mutation-complementing restriction fragments from Aspergillus nidulans cosmids. Exp Mycol 9:351–355
Tuite J (1969) Plant Pathological Methods. Burgess, Minneapolis USA
Turcq B, Begueret J (1987) The ura5 gene of the filamentous fungus Podospora anserina: nucleotide sequence and expression in transformed strains. Gene 53:201–209
Vaillancourt LJ, Hanau RM (1991) A method for genetic analysis of Colletotrichum graminicola (Glomerella graminicola) from maize. Phytopathology 81:530–534
Vollmer SJ, Yanofsky C (1986) Efficient cloning of genes of Neorospora crassa. Proc Natl Acad Sci USA 83:4869–4873
Woloshuk CP, Seip ER, Payne GA, Adkins CR (1989) Genetic transformation system for the aflotoxin-producing fungus Aspergillus flavus. Appl Environ Microbiol 55:86–90
Yang Z, Panaccione DG, Hanau RM (1991) Gene expression associated with light-induced conidiation in Colletotrichum graminicola. Can J Microbiol 37:165–167
Yelton MM, Timberlake WE, van den Hondel CAMJJ (1985) A cosmid for selecting genes by complementation in Aspergillus nidulans: selection of the developmentally regulated yA locus. Proc Natl Acad Sci USA 82:834–838
Author information
Authors and Affiliations
Additional information
Communicated by C.A.M.J.J. van den Handel
Rights and permissions
About this article
Cite this article
Rasmussen, J.B., Panaccione, D.G., Fang, GC. et al. The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis. Molec. Gen. Genet. 235, 74–80 (1992). https://doi.org/10.1007/BF00286183
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00286183