VPS9 domain-containing proteins are essential for autophagy and endocytosis in Pyricularia oryzae

Pyricularia oryzae is the causal pathogen of rice blast disease. Autophagy has been shown to play important roles in P. oryzae development and plant infection. The P. oryzae endosomal system is highly dynamic and has been shown to be associated with conidiogenesis and pathogenicity as well. To date, the crosstalk between autophagy and endocytosis has not been explored in P. oryzae. Here, we identified three P. oryzae VPS9 domain-containing proteins, PoVps9, PoMuk1 and PoVrl1. We found that PoVps9 and PoMuk1 are localized to vesicles and are each co-localized with PoVps21, a recognized marker of early endosomes. Deletion of PoVPS9 resulted in severe defects in endocytosis and autophagosome degradation and impaired the localization of PoVps21 to endosomes. Additionally, deletion of the PoMUK1 gene in the ΔPovps9 mutant background exhibited more severe defects in development, autophagy and endocytosis compared with the ΔPovps9 mutant. Pull-down assay showed that PoVps9 interacts with PoVps21, PoRab11 and PoRab1, which have been verified to participate in endocytosis. Furthermore, yeast two-hybrid and co-immunoprecipitation assays confirmed that PoVps9 directly interacts with the GDP form of PoVps21. Thus, PoVps9 is a key protein involved in autophagy and in endocytosis.