Functional Analysis Of Flh2 And Mon1 Gene In Fasurium Oxysporum F.sp.cubense

Banana wilt is a serious soil-transmitted vascular fungal disease caused by Fusarium oxysporum f.sp.cubense(Foc),which has caused huge economic losses to the banana industry at home and abroad.In the prevention and control of banana wilt,domestic and foreign are mainly starting from the aspects of physical control,chemical control,biological control,disease resistance breeding and comprehensive prevention and control,have done many research and exploration.but Still not very effective in solving the problem of banana wilt disease prevention and control.Strengthening the pathogenic mechanism of Fusarium oxysporum f.sp.is of great significance for the development of a long-lasting effective banana wilt control strategy.Based on the clues obtained from our previous proteomics studies of Fusarium oxysporum sp.cubense race 4(Foc4),it was found that the vacuolar fusion protein mon1 gene was up-regulated during the inoculation of Brazilian banana by Foc4,suggesting that the mon1 gene may be Played a role in the pathogenesis in Foc4.In addition,prediction of effector genes combined with gene transposon.it was found that the flh2 effector gene is also located in a characteristic subregion of the causative chromosome and presumably associated with the pathogenicity of Fusarium oxysporum.In this study,a gene knockout linear homologous fragment was constructed by overlapping PCR,to construct a complementary vector that the PCT74 plasmid was modified twice with EZ fusion homologous recombinase,the hygromycin resistance gene in the PCT74 plasmid was replaced with the neomycin G418 resistance gene,and the green fluorescent marker gene in the PCT74 plasmid was replaced with the complementary gene fragment.The protoplast transformation was carried out by PEG-mediated method.Knocked out the mon1 and flh2 genes in Foc4 wild-type strain through homologous recombination method and was respectively complemented.The phenotype and pathogenicity of the knockout mutants and complement mutants were then analyzed.The results are as follows:Compared with the wild type strain.the mutant strains of △mon1 colony showed a blue-white color and grew slowly,the mycelium became thinner,the branch decreased and the sporulation decreased,Also,the resistance to exogenous oxygen stress,capacity of cell wall penetration and cellulose utilization of the mutant strains decreased largely.Pathogenicity test showed the mutant strains exhibited a significantly weakened banana infection.It indicates that the mon1 gene plays an important role in the growth,sporulation and pathogenicity of Foc4.Compared with the wild type strain,the mutant strains of △flh2 colony showed pure white color,grew faster,the mycelial morphology was basically the same as the wild type,the sporulation amount increased,the resistance to exogenous oxygen stress,cell wall penetration ability and utilization of cellulose basically the same as the wild type.Pathogenicity test showed the mutant strains exhibited a significantly weakened banana infection.It indicated that flh2 gene had no negative effect on the growth and development of the strain,which may affect the pathogenicity of Fusarium oxysporum by other means.By back-mutating the flh2 gene and the mor1 knockout,the phenotype and pathogenicity of the restorer strain were basically the same as those of the wild type.