Functional Analyses Of V-ATPase C’ Subunit Moyma11and Six SNARE Proteins In Magnaporthe Oryzae

Rice blast, caused by Magnaporthe oryzae, is one of the most serious rice diseases that cause substantial cultured crop losses worldwide. Genomic sequence availability and genetic tractability of both M. oryzae and rice, combined with multiple analytical tools, make them a model plant pathosystem for fungus-plant interaction research. Membrane associated proteins contribute to the growth and development of organisms. Further research in membrane associated proteins will provide novel insights into the molecular mechanism of pathogenicity for M. oryzae. In this study, V-ATPase c’ subunit Movma11and six SNARE proteins were identified and characterized in this model phytopathogenic fungus.Many functions of vacuole depend on the activity of vacuolar ATPase to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. Subcellular colocalization and quinacrine staining suggested that the V1Vo domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments in M. oryzae. Targeted gene disruption of MoVMA11gene, encoding the putative c’subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not typical pH conditional lethality. Strikingly, aerial hyphae of MoVMA11null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated MoVMA11involving in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of ΔMovma11mutant on onion epidermal cells. Finally, MoVMA11null mutant lost pathogenicity on both intact and wounded host leaves. Our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions.Soluble N-ethylmaleimide-sensitive factor attached protein receptor (SNARE) proteins are implicated in many eukaryotic cellular processes during growth and development. In this report, we characterized six members of the SNARE repertoire in M. oryzae, which we designated MoNYV1, MoTOM, MoSNC, MoSSO2, MoTLG2and MoSYN8. Functional analyses revealed that MoNYV1and MoTOM null mutants were indistinguishable from wild-type strain in phenotypes, while MoTOM, MoSNC, MoSSO2, MoTLG2and MoSYN8were involved in mycelial growth, conidiogenesis, and appressorial morphogenesis. Compared with Guy11, ΔMotlg2mutant showed reduced sizes of conidia and appressoria, as well as invasive growth ability. Our findings also indicated a role for MoSYN8in the maintenance of cell wall integrity, and carbohydrate osmotic solutions, to a large extent, complemented the growth defect of ΔMosyn8mutant. Appressoria of ΔMosyn8mutant exhibited higher turgor pressure and decreased cell wall porosity, which was closely related to the melanin layer. In addition, discruption of MoSYN8severely impaired appressorial penetration ability and pathogenicity of M. oryzae, but the pathogenicity defect could be slightly restored when leaves were abraded or supplemented with1M sorbitol.In summary, our results demonstrate that MoSNC, MoSSO2, MoTLG2and MoSYN8play an important role in mycelial growth, conidial and appressorial development, and highlight the significance of V-ATPase function for infection-related morphogenesis and pathogenesis in M. oryzae.