Silencing of Kex2 significantly diminishes the virulence of Cryphonectria parasitica.

Cryphonectria parasitica is the causal agent of chestnut blight. Infection of this ascomycete with Cryphonectria hypovirus 1 (CHV1) results in reduction of virulence and sporulation of the fungus. The virus affects fungal gene expression and several of the CHV1 downregulated genes encode secreted proteins that contain consensus Kex2 processing signals. Additionally, CHV1 has been shown to colocalize in infected cells primarily with fungal trans-Golgi network vesicles containing the Kex2 protease. We report here the cloning, analysis, and possible role of the C. parasitica Kex2 gene (CpKex2). CpKex2 gene sequence analysis showed high similarity to other ascomycete kexin-like proteins. Southern blot analyses of CpKex2 showed a single copy of this gene in the fungal genome. In order to monitor the expression and evaluate the function of CpKex2, antibodies were raised against expressed protein and Kex2-silenced mutants were generated. Western blots indicate that the Kex2 protein was constitutively expressed. Growth rate of the fungus was not significantly affected in Kex2-silenced strains; however, these strains showed reduced virulence, reduced sexual and asexual sporulation, and reductions in mating and fertility. The reduced virulence was correlated with reduced Kex2 enzymatic activity and reduced relative mRNA transcript levels as measured by real time reverse-transcriptase polymerase chain reaction. These results suggest that secreted proteins processed by Kex2 are important in fungal development and virulence.