Isolation of Light-Enhanced cDNAs of Cercospora kikuchii

Abstract

Cercospora kikuchii is a fungal pathogen of soybeans which produces a photosensitizing phytotoxic polyketide metabolite, cercosporin. Cercosporin synthesis in culture is modulated by several environmental factors. In addition to the light requirement for toxin action, cercosporin biosynthesis is also highly light regulated. As a first step towards identifying genes involved in cercosporin regulation and biosynthesis, we have used subtractive hybridization to isolate light-enhanced cDNA clones. Six distinct cDNA clones representing genes from a wild-type C. kikuchii strain for which transcript accumulation is positively regulated by light were isolated. To assess the relationship of these light-enhanced cDNAs to cercosporin biosynthesis, we compared corresponding steady-state RNA levels in the wild type and in three mutant strains altered in toxin biosynthesis. Two of the mutant C. kikuchii strains which fail to accumulate cercosporin in response to light also fail to exhibit light-enhanced accumulation of transcripts corresponding to all six light-enhanced cDNAs. Cercosporin accumulation in the third mutant strain, S2, is regulated by medium composition as well as light. S2 fails to accumulate cercosporin in complete medium, a medium which allows significant cercosporin accumulation by the wild-type strain. When cultured in complete medium, this mutant strain also fails to show the wild-type, light-enhanced accumulation of transcripts corresponding to five of the six light-enhanced cDNAs. Kinetic analysis demonstrated that transcript accumulation for two of the six light-enhanced cDNAs strongly paralleled cercosporin accumulation in light-grown wild-type culture.