Two homologous Zn₂Cys₆ transcription factors play crucial roles in host specificity of Colletotrichum orbiculare by controlling the expression of cucurbit-specific virulence effectors

Fungal plant pathogens preferentially express a set of effector genes at specific infection stages to successfully colonize the host. However, it remains unclear how effector gene expression is regulated during host infection. This study identified a Zn₂Cys₆ transcription factor, TFV1 (Transcription Factor for Virulence 1), whose deletion weakened virulence of Colletotrichum orbiculare on its cucurbit hosts. The additional deletion of a TFV1 paralog gene, TVL1 (TFV1-like 1), resulted in a further reduction in virulence on the cucurbits. Notably, TFV1 TVL1 double mutants retained wild-type virulence on the Solanaceae host Nicotiana benthamiana. Expression of putative effector genes, including four cucurbit host-specific virulence effectors (effector protein for cucurbit infection, EPC1-4), was commonly downregulated in the TFV1 knockout mutants. Yeast one-hybrid assays suggested that TFV1 binds to the putative promoter regions of EPC2, EPC3, and EPC4, indicating the importance of TFV1 for the induced expression of key effector genes in cucurbit infection. Among the effector-like genes whose expression was affected by TVL1 deletion, a novel LysM effector gene, EPC5, was identified as being specifically required for virulence on cucurbit hosts. Our study extends the knowledge of the regulatory mechanisms governing host- and stage-specific effectors in C. orbiculare.