Secretome Analysis Of Early Stage Of Conidia Germination In Fusarium Oxysporum F.sp.cubense Race 4

Banana Fusarium wilt,caused by Fusarium oxysporum f.sp.cubense(Foc),is one of the most devastating disease of banana.According to the range of harmful banana varieties,Foc can be divided into four races.Among of them,the race 4(Foc4)can infect almost all banana varieties.At present,the pathogenicity mechanism of Foc is still unclear.Secreted proteins as one of pathogenicity factor,play an important role in the infection process of Foc.Therefore,the study of protein expresssion profiles secreted by Foc4 can help to understand the pathogenicity mechanism of Foc4,and provide a new views of the control of banana wilt.In this study,we simulated the banana-Foc4 interaction in vitro,and analyzed the differential expression of secretome using non-labeled protein quantification technology during early stage of conidia germination in Foc4 induced by banana tissue extracts.The functional information of differentially expressed proteins in Foc4 was predicted by bioinformatics technology.The major results are as follows:1.In order to seclect the most appropriate culture medium for Foc4 conidia germination,banana tissue extracts were added to different culture mediums.The results showed that a higher conidial germination rate in NCM-A4 medium was observed,which could reduce interference of exogenous protein.Therefore,NCM-A4 medium was considered as the culture medium appropriate for Foc4 secretome analysis.In this culture medium,the growth of Foc4 conidia had three typical stages,including conidia swell period or germination tube formate period,germination tube elongate period and mature mycelium formate period.Among of them,7 h and 10 h are critical periods for tube germinate and mature mycelium formate,respectively.2.Differentially expressed secretome of Foc4 conidia were analyzed using lab-free technology at two critical germination periods induced by banana tissue.The results showed that 743 secreted proteins were differentially expressed.Among of them,there were 167 classical secretory proteins(22.5%),254 non-classical secretory proteins(34.2%)and 322 unknown secretion pathway secretory proteins(43.3%).3.Differentially expressed secreted proteins of Foc4 conidia induced by banana tissue extracts were analysed in 7 h and 10 h,respectively 522 secreted proteins were differentially expressed at 7 h,and 196 secreted proteins were differentially expressed at 10 h.Differentially expressed secreted proteins at two critical periods were analyzed,showing that compared with 7 h,190 secreted proteins were differentially expressed induced by banana tissue at 10 h,191 secreted proteins were differentially expressed uninduced at 10 h.4.Bioinformatics analysis was performed to compare the d ifferentially expressed secreted proteins induced by banana tissue extracts and the control at two critical period respectively.(1)At 7 h,the GO enrichment analysis showed that the main biological process of 522 differentially expressed secreted proteins are involved in lipid metabolic process,organelle localization,cellular lipid metabolic process.The main molecular functions are involved in purine ribonucleotide binging,purine nucleoside binging and ATP binding.CAZymes analysis showed that 45 secreted proteins belonged to CAZymes and most of them were GH family.CAZymes domain analysis showed that 24 proteins were predicted to be CWDEs.(2)At 10 h,the GO enrichment analysis showed that the main biological process of 196 differentially expressed secreted proteins are involved in positive regulation of DN A metabolic process,regulation of actin filament polymerization,positive regulation of hydrolase activity.The main molecular functions are involved in molecular function regulator,enzyme regulator activity,succinate dehydrogenase activity.The KEGG enrichment analysis showed that secondary metabolites biosynthesis and tyrosine metabolism pathways were significantly affected.CAZymes analysis showed that 13 secreted proteins belonged to CAZymes and most of them were GH family.CAZymes domain analysis showed that 9 proteins were predicted to be CWDEs.5.Bioinformatics analysis was performed to compare the d ifferentially expressed secreted proteins induced by banana tissue extracts at 10 h with the secreted proeins at 7 h.The GO enrichment analysis showed that the main biological process of secreted proteins are involved in tubulin complex assembly,alkaloid metabolic process,programmed cell death.The main molecular functions are involved in tubulin binding,succinate dehydrogenase activity,oxidoreductase activity.The KEGG enrichment analysis showed that secondary metabolites biosynthesis and tyrosine metabolism pathways were significantly affected.