Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity.

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant-fungus contact sites to facilitate colonization. Only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1, 3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretome of wild-type and Δalg3 mutants and identified 51 proteins that require Alg3 for proper secretion. These proteins are predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological function of two genes, INV1 encoding invertase 1, a secreted invertase and AMCase encoding acid mammalian chinitase, a secreted chitinase. The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation is essential for the function and secretion of AMCase. Comparative secretome analysis of M. oryzae identified N-glycosylated secretory proteins that function in fungal virulence and cell wall integrity.