P-169 - Gene contributing to the phenotype of the A2 mutant of Sclerotinia sclerotiorum identified: Cyc8 as a regulator of oxalic acid accumulation and pathogenicity

Sclerotinia sclerotiorum is a necrotrophic fungus, with a broad host range. Plants respond to infection with an oxidative burst. S. sclerotiorum accumulates oxalic acid, neutralising the oxidative burst, allowing unimpeded infection, but pathogenicity is lost if oxalic acid accumulation is blocked. Oxalic acid accumulates when the biosynthesis gene, Oah, is upregulated in response to environmental pH by Pac1. A mutant strain called A2 has a phenotype consistent with an oah knockout but has no disruptions within Oah or Pac1. Here we give evidence that an early stop mutation in Cyc8 is responsible for the A2 phenotype. In yeast, Cyc8 is a transcriptional co-repressor operating in conjunction with Tup1. Cyc8-Tup1 is required for the expression of Pac1. We theorise that Cyc8 is required for pathogenicity via the regulation of Pac1 and Oah. Cyc8 silenced strains were generated using a hairpin-RNA expressing vector. Partial knockdown was confirmed though RT-qPCR. Screening showed that cyc8 knockdowns had a reduced ability to acidify media, reduced growth in alkaline conditions and produced smaller lesions on detached leaves. These results suggest that Cyc8 plays a role in pH response, oxalic acid accumulation and pathogenicity. Cyc8 may provide a novel target for RNA-interference based methods of crop protection, such as host, spray or virus induced gene silencing. However, generation of a true cyc8 knockout, followed by complementation is needed to confirm the role of Cyc8.