Functional characterization of three genes encoding putative oxidoreductases required for cercosporin toxin biosynthesis in the fungus Cercospora nicotianae

Cercosporin is a non-host-selective, photoactivated polyketide toxin produced by many phytopathogenic Cercospora species, which plays a crucial role during pathogenesis on host plants. Upon illumination, cercosporin converts oxygen molecules to toxic superoxide and singlet oxygen that damage various cellular components and induce lipid peroxidation and electrolyte leakage. Three genes (CTB5, CTB6 and CTB7) encoding putative FAD/FMN- or NADPH-dependent oxidoreductases in the cercosporin toxin biosynthetic pathway of C. nicotianae were functionally analysed. Replacement of each gene via double recombination was utilized to create null mutant strains that were completely impaired in cercosporin production as a consequence of specific interruption at the CTB5, CTB6 or CTB7 locus. Expression of CTB1, CTB5, CTB6, CTB7 and CTB8 was drastically reduced or nearly abolished when CTB5, CTB6 or CTB7 was disrupted. Production of cercosporin was revived when a functional gene cassette was introduced into the respective mutants. All ctb5, ctb6 and ctb7 null mutants retained wild-type levels of resistance against toxicity of cercosporin or singlet-oxygen-generating compounds, indicating that none of the genes plays a role in self-protection.