Secretome Analysis Of Appressorium Development In Magnaporthe Oryzae

Rice blast,caused by the filamentous ascomycete fungus Magnaporthe oryzae,is one of the most serious diseases that causes global rice production.The appressorium is a specialized cellular structure formed by Magnaporthe oryzae before penetrating plant tissue.This appressorium stage is a key period for the rice blast fungus to infect rice.Secreted proteins have been described as pathogenicity factor,play an important role at different infection stages of Magnaporthe oryzae.However,the current study of Magnaporthe oryzae secreted proteins are largely unknown,especially during appressorium development stage.The study to survey the appressorium development secretome in Magnaporthe oryzae,can be used to further our understanding of the early interaction between Magnaporthe oryzae and rice.1.In this study,in vitro Magnaporthe oryzae secretome were investigated using shotgun technology.Differentially expressed secretome of appressorium development in M.oryzae was perfromed bylabel-free mass spectrometry-based protein quantification technologies of appressorium development secretome.The major results were as follow:1.We examined the secretome of M.oryzae by mimicking the early stages of infection in vitro using PVDF membrane.The spore germination,germ-tube growth,appressorium formation,mycelium formation was successfully observed.The high quality extraction of M.oryzae secreted proteins purified by ultrafiltration.It suggested that PVDF membranes may be used as suitable platforms for developing the secretome of M.oryzae early during the infection process.2.The large-scale identification of M.oryzaeappressorium secretome at a development phase(8 h)and a maturation phase(24 h)wer performed by using shotgun technology.The result showed that 2766 secreted proteins were identified.Among of them,there were 278 classical secretory proteins,664 non-classical secretory proteins,and 1824 unknown secretion pathway secretory proteins.Predictive analysis of protein function showed that there were 1921 functional annotation secreted proteins.Identified proteins were mainly related to signal pathway,infection structure formation,secondary metabolism,reactive oxygen species detoxifying proteins,and proteins with unknown function.Moreover,CAZymes analysis showed that 32 secreted proteins belonged to CAZymes.CAZymes domain analysis showed that 23 preteins were predicted to be CWDEs.The GO analysis showed that a total of 2456 secreted proteins were annotated into 51 GO functional subcategories.The KEGG pathway annotation analysis showed that 1519 secreted proteins were annotated into 247 K EGG pathways.3.M.oryzaesecretome at different appressorium development stagewas performedby using label-free technology.The results showed that:(1)1702 secreted proteins were identified atthe development phase(8 h),and 1604 at the maturation phase(24 h).(2)A total of 340 secreted proteins were differentially expressed atthe two different appressorium development stage.Among them,there were 162 specific expression secreted proteins(93 preteins in development phase,69 proteins in maturation phase),and 178 differentially expressed proteins.4.Bioinformatics analysis was performed to compare the differentially expressed secreted proteins attwo different appressorium developmentstages.The results showed that(1)Identified proteins were mainly related to signal transduction,lipid metabolism,hydrolytic activity,energy metabolism and protein synthesis at appressorium development phase(8 h).At maturation phase(24 h),the secreted proteins were mainly related to cell wall degradation,reactive oxygen species detoxifying proteins,energy metabolism and protein synthesis.(2)The GO enrichment analysis showed that the main biological process of different stages of conidial germination are involved in ribosome biogenesis,proteasomal ubiquitin-independent protein catabolic process,ribonucleo protein complex biogenesis and other process.The main molecular functions are involved in structural molecule activity,structural constituent of ribosome,threonine-type endopeptidase activity and other process.The main cellular component are involved in proteasome core complex,proteasome complex,endopeptidase complex and other process.(3)The KEGG enrichment analysis showed that the differe ntially expressed secreted proteins mainly involved in Neomycin,kanamycin and gentamicin biosynthesis,ribosome,proteasome,starch and sucrose metabolism,citrate cycle(TCA cycle),HIF-1 signaling pathway.