Functional Analysis Of MoRab51 Putative Interaction Proteins MoYIP3、MoVPS9 In Magnaporthe Oryzae

Rab protein is a kind of monomer form of GTPase, composed of 200 amino acids with common structure characteristic, including the G conserved domains and highly variable the c-terminal and n-terminal, and the overlapping structure similar to the Ras proteins. Rab proteins are molecular switches in intracellular vesicular transport, playing an important role at different stages of vesicle transport. Rab5 is a member of the Rab family proteins, which mainly localizes in early endosomes, controls early endosome fusion, vesicle formation and directional transportation in receptor -mediated cell swallowed involved in intracellular transport regulation in the process of functional protein.Our previous research showed that MoRab51 have important function in regulating development and pathogenicity of Magnaporthe oryzae. This paper focuses on looking for its downstream regulating effectors during the infection process of the pathogen. Two MoRab51 hypothetical interaction protein genes MoYIP3 and Mo VPS9 were obtained through bioinformation analysis. The interaction relationships of MoRab51 and these two proteins were comfirmed by the point to point yeast two hybrid method. The gene knockout approach by homologous recombination was used to analyze the two genes function.Yeast two-hybrid experiment indicated that MoYIP3 did not interact with MoRab51, while MoVPS9 interacted with MoRab51 in its dominant negative stage. This result proves that MoVPS9 is the GEF of MoRab51.Bioinformatics approaches were used to analyze the subcellular localization of MoYIP3 and MoVPS9, and the evolutionary relationship with homologous proteins in other different species fungi, The results show that the domain is highly homologous to its corresponding domain. The MoYIP3 and MoVPS9 gene deletion mutants were obtained through homologous recombination. The MoYIP3 deletion mutants displayed fewer aerial mycelia, and thinner hyphae mat on the plates, while the pathogencity showed as normal as the wild type strain ku80. These data suggested that MoYIP3 was involved in hypha formation and conidiation of M.oryzae, and it was not necessary for vegetative growth and pathogenicity The deletion mutants of MoVPS9 resulted in the reduction of growth rate,fewer aerial mycelia, conidiophores and conidia, thinner hyphae mat on the plates. Moreover, the sensitivity of the cell walls was increased and the pathogenicity was reduced dramatically in the â–³MoVPS9 mutants.The results indicated that MoVPS9 in M.oryzae plays an important roal in its vegetative growth and pathogenicity.Real-time PCR showed that compared with wild type strain ku80, the expression level of MoRab51 was down-regulated in the MoYIP3 deletion mutants, while up-regulated in MoVPS9 deletion mutants. The results indicated that the MoYIP3 and MoVPS9 may be involved in the signal transduction pathways of MoRab51.The results are benefit for better understanding of the molecule mechanism pathogenicity in M.oryzae, and also benefit to clarify the signal network pathways of Rab protein mediated directional transportation of vesicles.