The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis

Cercosporin, a photo-activated, non-host-selective phytotoxin produced by many species of the plant pathogenic fungus Cercospora, causes peroxidation of plant cell membranes by generating reactive oxygen species and is an important virulence determinant. Here we report a new gene, CTB3 that is involved in cercosporin biosynthesis in Cercospora nicotianae. CTB3 is adjacent to a previously identified CTB1 encoding a polyketide synthase which is also required for cercosporin production. CTB3 contains a putative O-methyltransferase domain in the N-terminus and a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase domain in the C-terminus. The N-terminal amino acid sequence also is similar to that of the transcription enhancer AFLS (formerly AFLJ) involved in aflatoxin biosynthesis. Expression of CTB3 was differentially regulated by light, medium, nitrogen and carbon sources and pH. Disruption of the N- or C-terminus of CTB3 yielded mutants that failed to accumulate the CTB3 transcript and cercosporin. The Δctb3 disruptants produced a yellow pigment that is not toxic to tobacco suspension cells. Production of cercosporin in a Δctb3 null mutant was fully restored when transformed with a functional CTB3 clone or when paired with a Δctb1-null mutant (defective in polyketide synthase) by cross feeding of the biosynthetic intermediates. Pathogenicity assays using detached tobacco leaves revealed that the Δctb3 disruptants drastically reduced lesion formation.