Screening Of The Potential Drug Targets Based On The Endosomal Sorting Complex Required For Transport Of Magnaporthe Oryzae

Rice blast is a worldwide disease.Rice production decreases up to 11%-30%in the world each year.Rice blast is widespread and dangerous,and currently there is not a very effective way to control rice blast.Rice blast is caused by Magnaporthe oryzae,which has a typical infection cycle.The endosomal sorting complex required for transport(ESCRT)is a protein complex which is capable of identifying and sorting ubiquitinated protein cargo.It is composed of four sub-complexes(ESCRT-0,ESCRT-?,ESCRT-?,ESCRT-?)and some auxiliary components(Vps4,Vps60,Istl,Vta1,etc.).ESCRT plays a role in recognition and sorting in the lysosomal degradation pathway of proteins,and the degradation of proteins such as activated membrane molecules,misfolded proteins and growth factors on the plasma membrane is particularly important for maintaining intracellular protein level,normal metabolism and growth and development in Magnaporthe oryzae.Studies have shown that ESCRT not only involves in autophagy,nuclear membrane remodeling,membrane deformation process,but also regulates signal transduction;also relates with the tumor,neurodegenerative diseases and other major human diseases.With the rapid development of molecular biology,especially the rapid development of genomics,proteomics and bioinformatics,screening of drug targets has become a key means of developing new drugs.The protein as a drug target must be related to disease,and can be combined with small molecules in a certain affinity and chemical properties.In this study,the potential drug targets were screened and validated from the ERCRT family of M.oryzae by gene knockout,gene screening,genomics,bioinformatics,yeast two-hybrid and overexpression.Our results will lay the foundation to prevent and control of rice blast.Firstly,we carried out a preliminary analysis of the fifteen ESCRT family genes of M.oryzae(MoVps27,MoHsel,Mo Vps23,MoVps28,MoVps22,MoVps25,MoVps36,MoVps20,MoVps32,MoVps24,MoVps2,MoCHMP7,Mo Vtal,Mo Vps4,Mo Vps60),including the expression analysis during the growth and development stage of Magnaporthe oryzae,the location of these genes and the interaction between ESCRT family members.The results of qPCR assays showed that the MoVps27,MoVps32,MoVtal and MoVps60 showed high expression in the hypha induced by starvation.MoVps27,MoVps23,MoVps28,MoVps22,MoVps20,MoVps32,MoVps24,MoVps2 and MoCHMP7 showed low expression during the conidiogenesis of M.oryzae.MoHse1,MoVps22,MoVps25,MoVps36,MoVps20,MoVps24,MoVps4 and MoVps60 showed high expression in the process of the formation of appressorium of M.oryzae.MoVps27,MoVps23,MoVps28,MoVps36,MoVps2 and MoCHMP7 showed high expression in the process of pathogenicity of M.oryzae.In addition,GFP-MoVtal was co-located with mCherry-MoAtg8 by the observation of confocal fluorescence microscope.Yeast two-hybrid test showed that MoVps20 had interaction with MoVps32,and they were the memebers of ESCRT-?.Furthermore,we constructed the knockout vectors of the fifteen genes of the ESCRT family,and knocked out these genes by Agrobacterium-mediated transformation method.Finally,we obtained three gene deletion mutants ?MoVps27,?MoCHMP7 and ?MoVta1,And then the three mutants were rescued,and the complementation strains were assigned as MoVps27C,MoCHMP7C and MoVta1C.The phenotypic analysis of these three gene deletion mutants were performed,and the results as follows,The growth proportion of ?Mo Vps27 was significantly lower than that of the wild type Guyll,the proportion of decline was as to 29%,and the color of colony was brownish yellow and the aerial mycelium was very thin.The growth rate of?MoCHMP7 was at least 15%lower than that of wild type Kj201,and the aerial mycelium became thinner.?MoVta1 growth rate also decreased significantly,its colony color was obvious brownish yellow,and aerial hyphae was thinner than that of?MoVps27.?MoVps27,?MoCHMP7 and ?MoVta1 had almost no conidia on the conidiophore,and the conidiophore differentiating from mycelium became fewer,indicating ESCRT genes MoVps27 and MoCHMP7 were involved in the process of the growth and development and asexual reproduction of M.oryzae.MoVps27 and MoCHMP7 were also involved in the stress response of M.oryzae to external stress.The mating assays showed that the genes MoVps27 and MoVtal respectively was unnecessary for the sexual reproduction of M.oryzae.The pathogenicity assays on barley leaves showed that the ?MoVps27,AMoCHMP7 and ?MoVtal mutant lost pathogenicity completely,indicating that these three genes were involved in the process of pathogenesis of M.oryzae.Overexpression of the MoVps27 gene in Guyll,the phenotypes were similar as the ?MoVps27 mutant,such as redued in growth and conidaiton,lost pathogenicity on barley.Therefore,Vps27,CHMP7 and Vtal could deemed as the potential drug targets for the control of rice blast.