MTF4 is a key transcription factor downstream of MOR required for plant-derived signal dependent appressorium development in Colletotrichum orbiculare

Many plant pathogenic fungi including Colletotrichum species form a specialized infection structure, called an appressorium. Appressorium formation relies on perception of physical and biochemical signals at the plant surface. In cucumber anthracnose fungus C. orbiculare, the morphogenesis-related NDR (nuclear Dbf2-related) kinase pathway (MOR) is crucial for translating plant-derived signals for appressorium development (Kodama et al., 2017). To determine the downstream target of MOR, we performed whole genome transcript profiling of the NDR kinase Cbk1-AS strain, the MOR component pag1 mutant strain and the wild type strain during appressorium formation. In the wild type strain, several transcription factor-encoding genes were upregulated on the host plant compared with artificial substrates. Among them, we identified 10 genes as MTF (MOR-related transcription factor) 1-10 that were downregulated in Cbk1-AS and/or pag1 mutants compared with the wild type. Targeted gene deletion analysis of MTF1-10 showed that mtf4 mutant phenotypes were closely similar to pag1 mutant. Introduction of the constitutively active variant of Cbk1 had no effect on mtf4 mutant phenotypes. However, introduction of overexpression construct of MTF4 to pag1 mutant complemented pag1 defects. These results indicate that MTF4 acts downstream of MOR for appressorium morphogenesis induced by plant-derived signals. To test the response of MTF4 to plant signals, we analyzed its cellular localization by fluorescent protein tagging. The Mtf4-GFP was recognized at nuclei during appressorium formation in planta, whereas no distinct localization of Mtf4-GFP was observed in vitro. The conditional inactivation of Cbk1 and pag1 deletion decreased nuclear localization of Mtf4-GFP in planta. These results suggest that MOR-dependent localization of Mtf4 responds to plant-derived signals during appressorium development. Therefore, MTF4 is a key downstream transcription factor of MOR in C. orbiculare for appressorium development in response to plant-derived signals.