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The photoactivated toxin, cercosporin, produced by  Cercospora  species, plays an important role in pathogenesis of this fungus to host plants. Cercosporin has almost universal toxicity to cells due to its production of reactive oxygen species including singlet oxygen. For that reason,  Cercospora  species, which are highly resistant to their own toxin, are good candidates to identify genes for resistance to cercosporin and to the reactive oxygen species it produces. In previous research, the zinc cluster transcription factor  CRG 1 ( c ercosporin  r esistance  g ene 1) was found to be crucial for  Cercospora  species’ resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between  Cercospora nicotianae  wild type (wt) and a  crg1  mutant. The focus of this work was to identify and characterize the hypothetical proteins that were identified in the  Cercospora nicotianae  subtractive library as potential resistance factors. Quantitative RT-PCR analysis of the 20 genes encoding hypothetical proteins showed that two,  24cF  and  71cR , were induced under conditions of cercosporin toxicity, suggesting a role in resistance. Transformation and expression of  24cF  and  71cR  in the cercosporin-sensitive fungus,  Neurospora crassa , showed that  71cR  provided increased resistance to cercosporin toxicity, whereas no significant increase was observed in  24cF  transformants. Gene disruption was used to generate  C .  nicotianae 71cR  mutants; these mutants did not differ from wt  C .  nicotianae  in cercosporin resistance or production. Quantitative RT-PCR analysis showed induction of other resistance genes in the  71cR  mutant that may compensate for the loss of 71cR. Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta-isomerase family.

Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance