The Functional Analysis Of Putative MoVps9 In Magnaporthe Oryzae

Rab/Ypt family proteins are the most important transporters and play a key role in vesicle formation and their subsequent fusion with the vacuole in eukaryotic cells.Vps9 as vacuole sorting-related proteins associated with vacuolar protein transport mediated by the vesicle system.In Vps9 protein is a guanine nucleotide exchange factor(GEF)for the Ypt5,it can interact with Ypt5,stimulate its release of GDP,and binding of GTP,activating its biological functions.Magnaporthe oryzae can cause rice blast and is taken as a model organism for the study of interaction between plant and pathogenic fungi.Studies show that the MoYpt51/52,a yeast Ypt5 homolog are related to growth and pathogenicity in rice blast fungus.To further understand the function of MoYpt51/52,we obtained three putative Vps9 proteins by amino acid sequence alignment in rice blast genome database,named MoVps9a,MoVps9b and MoVps9c,for all three proteins contained Vps9 domains.Then we deleted all MoVps9 genes,and conducted further analysis on MoVps9a,including its biological function and interaction with MoYpt5 by yeast two-hybrid system.Analysis showed that MoVps9a protein contains VPS9 domain and a CUE domain as well.Deletion of MoVps9a reduced the fungal growth rate significantly,but only reduced conidiation at 1/32 level and slightly decreased pathogenicity when compared with its wild strain Ku80.Importantly,MoVps9a deletion mutants formed smaller but more vacuoles observed by transmission electron microscope,which suggested that MoVps9a functions are similarl to vacuolar formation/assembly as MoYpt51/52.Furthernore,we also obtained MoVps9a CUE domain deletion mutants MoYpt9a?CUE.Analysis displayed no much difference with the strain Ku80,but a slight sporulation reduction by 1/16,suggesting that the CUE domain in Vps9a has some contribution for conidial formation.Yeast two-hybrid analysis indicated that MoVps9a could interact with dominant negative form of MoYpt51DN and MoYpt52DN,respectively.Rather than the dominant active form of MoYpt51 CA and MoYpt52CA,indicating that MoVps9a possibly functions as a MoYpt51/52 GEF in the rice blast fungus,but needs to be further confirmed.In addition,we gained the knockout mutants of Mo Vps9b and Mo Vps9c.The results showed that mutants only had slight reduction in sporulation compared with the wild type,which is worth to conduct further study.