Functional Analysis Of Hydrophobic Surface Binding Protein A Gene In Fusarium Oxysporum F.sp. Cubense Race 4

Fusarium oxysporum f.sp.cubense(Foc)is a typical necrotrophic pathogenic fungus,which can infect bananas and causes banana wilt.Currently,it has been threatening the healthy and sustainable development of global banana industry.Identifying Foc’s pathogenic genes and exploring its pathogenic mechanism can provide theoretical basis and practical basis for formulating strategies for sustainable control of banana wilt.Secreted proteins,as an important class of bioactive molecules in the interaction between pathogenic fungi and host plants,play an important role in a variety of physiological and pathogenic processes.The Foc4 secretion proteome has been studied in our laboratory and we found a unique protein,Hydrophobic surface binding protein A(HSBA),which was significantly up-regulated after banana tissue induction.therefore,we speculated that it might be related to the pathogenicity of Foc4.In this study,the knockout mutant was obtained by using the gene knockout technology.Phenotype,penetration ability,and pathogenicity assay of the mutant were analyzed to verify that the function of Fo HSBA gene in growth,development,and pathogenicity.Mechanism of lost pathogenicity of mutants was determined by analyzing the expression levels of pathogenicity-related genes such as growth-development-related genes,cutinase genes,and chitin synthesis genes in mutant strains.The specific results are as follows:(1)The expression of HSBA gene(Fo HSBA)in Foc4 was verified and analyzed by using Quantitative Real-time PCR technology.The results showed that the expression level of Fo HSBA in Foc4 was significantly up-regulated under the conditions of banana tissue induction,which was consistent with the results of the secreted proteome and speculated that it might be related to the pathogenicity of Foc4.(2)Using the spilt-marker method to knock out the HSBA gene of Foc4.Ninety hygromycin-resistant transformants were obtained by PEG mediated protoplast transformation.The knock-out mutant ΔFohsba-43 was identified by PCR,Southern blotting and RT-PCR techniques.A random insertion method was used to obtain a complemented strain ΔFohsba-com of the mutant ΔFohsba-43.(3)It was found that the growth rate of △Fohsba on the PDA plate was significantly lower than the wild type;aerial mycelia was sparse;the spore production was reduced;and the proportion of miniconidia was increased by comparing the morphology and mycelial growth of the mutant to Foc4 wild type and ΔFohsba-com.(4)Through the study of the sensitivity of the mutants to cell wall stress factors,it was found that the mutants were not significantly inhibited on PDA medium containing Congo Red than the wild-type and complementary strains;while the degree of suppression of the mutants was not significantly different from that of the wild-type and complement strains on PDA medium containing CFW.(5)Through the penetration ability and colonization ability testing of mutants andΔFohsba-com,it was found that the mutants could not penetrate cellophane and the colonization ability on tomatoes was weakened.(6)Using real-time quantitative PCR technology,the expression of △Fohsba growth-related genes,chitin synthesis genes and cutinase genes were analyzed.The results showed that The expression levels of the growth-related genes FOIG＿02194,FOIG＿07247and FOC4＿g10010682;chitin synthesis genes Fochs3 and Fochs V;cutinase genes FOC4＿g10002722,FOIG＿00223 and FOIG＿08972 genes are reduced to varying degrees at various stages of growth and development of mutant strains.It further illustrates that Fo HSBA gene affects the pathogenicity-related functions of banana wilt pathogen growth and development,cell wall composition and penetration ability.