Ubiquitin-like activating enzymes BcAtg3 and BcAtg7 participate in development and pathogenesis of Botrytis cinerea

In eukaryotes, the ubiquitin-like (UBL) protein-activating enzymes play a crucial role in autophagy process, however, it is poorly characterized in filamentous fungi. Here, we investigated the functions of two UBL activating enzymes, BcAtg3 (E2) and BcAtg7 (E1) in the plant pathogenic fungus Botrytis cinerea. The physical interaction of BcAtg3 with BcAtg7 was demonstrated by yeast two-hybrid system. Subcellular localization assays showed that BcAtg3 diffused in cytoplasm, and BcAtg7 localized in cytoplasm as pre-autophagosomal structures (PAS). Target gene deletion experiments revealed that both BcATG3 and BcATG7 are essential for autophagy pathway. Notably, the single deletion mutant of BcATG3 and BcATG7 displayed similar biological phenotypes, including the defects in mycelial growth, conidiation and sclerotial formation. Infection tests showed that both BcATG3 and BcATG7 were required for full virulence of B. cinerea. All of these defective phenotypes were rescued by gene complementation. These results indicate that BcATG3 and BcATG7 are necessary for autophagy to regulate fungal development and pathogenesis in B. cinerea.