Identification of genes expressed during cercosporin biosynthesisin Cercospora zeae-maydis

Gray leaf spot, caused by the fungus Cercospora zeae-maydis, is the most destructive foliar disease of maize in the United States. However, little is known about the biochemical and molecular events of pathogenesis. C. zeae-maydis produces cercosporin, a phytotoxin shown to be a virulence factor in diseases caused by other Cercospora species. To identify genes involved in cercosporin biosynthesis and ultimately determine the role of cercosporin in disease development, we constructed a cDNA subtraction library of C. zeae-maydis by suppression subtractive hybridization. Poly (A)+ RNA isolated from cultures grown in a cercosporin-suppressing medium was subtracted from poly (A)+ RNA isolated from cultures grown in a cercosporin-inducing medium, resulting in transcripts that are specific to the cercosporin-producing culture. Analyses of 768 sequences in this cDNA subtraction library revealed 197 cDNAs with high similarity to genes in the GenBank and Saccharomyces Genome Database, and these genes were grouped into nine categories based on predicted functions of the encoded proteins. Northern analysis of seven selected clones with predicted functions in fatty acid metabolism (fatty acid synthase, oleate Δ-12 desaturase, and linoleate diol synthase) and secondary metabolism [cytochrome P450 oxidoreductase, cytochrome P450 monooxygenase, dihydrogeodin (phenol) oxidase, and coproporphyrinogen oxidase] indicated that those genes were expressed in cercosporin-inducing conditions. Analysis of expression kinetics confirmed that those genes are expressed concomitantly with cercosporin accumulation. Published by Elsevier Science Ltd. [Copyright &y& Elsevier]