The Functional Analyses Of Two Vacuolar Trafficking-related Genes MoYpt7and MoMon1in Magnaporthe Oryzae

In eukaryotic cells, the vacuole/lysosome is a complex organelle with a range of various functions, such as nutrient transport, the storage of Ca2+, ion homeostasis and degradation of macromolecules. In filamentous fungi, the inheritance and morphology of vacuole are vital for fungal growth, differentiation and symbiosis and/or pathogenesis. Magnaporthe oryzae is a filamentous ascomycete fungus that has been served as an excellent model organism for studying plant-pathogen interactions. A deep understanding of the molecular background of this fungus is beneficial for the control of the rice blast.This work is aimed to investigate the relationship between vacuole trafficking and cell development, differentiation and pathogenicity in M. oryzae. We isolated and cloned two vacuole fusion-related genes, MoYpt7and MoMonl, which is a homolog of gene Ypt7or Monl respectively in Saccharomyces cerevisiae. The functional analyses of MoYpt7and MoMonl during the infection process of rice blast were discussed.An EST (expressed sequence tags) for MoYpt7was found in the appressorium of the rice blast fungus. MoYpt7is a Rab GTPase, conserved from yeast to mammalian. The ΔMoYpt7mutant, in which the MoYpt7gene has been deleted, exhibits defective in mycelial growth and conidial production. Among the few conidia generated, conidia are malformed and defective in appressorial formation. Consequently, the ΔMoYpt7mutant fails to cause diseases on rice CO-39and barley ZJ-8. The cellular localization assay shows that the MoYpt7is mainly localized to vacuolar membranes. Furthermore, the ΔMoYpt7mutant shows lots of small and fragmented vacuoles in hypha, blocked in autophagy process, a breach in cell wall integrity and more sensitive to ions stress.Similarly, a sequence homologue was isolated by using the amino acid sequence of Monl searching against M. oryzae genome databases, assigned MoMonl. A targeted gene deletion strategy was adopted to determine the role of MoMonl in fungal development and pathogenicity at the molecular level. The ΔMoMonl mutant showed accumulation of small punctuate vacuoles in the hypha and hypersensitive to monensin, an antibiotic that can block intracellular protein transport. Secondly, the ΔMoMonl mutant exhibited reduced in hyphal development, conidiogenesis and failed to produce the appressorium. Additionally, the ΔMoMonl mutant revealed blocked in autophagy and a defect in pathogenicity.In summary, MoYpt7and MoMonl also have essential function in vacuolar assembly and growth, conidiation, appressorium formation and pathogenicity in M. oryzae.