Knockout And Function Analysis Of Two Pathogenicity-Related Genes In Fusarium Oxysporum F.sp.cubense

Fusarium wilt of banana caused by the soil-borne fungus Fusarium oxysporum f.sp.cubense?FOC?is one of the most destructive diseases of banana.Among them race 4 can infect almost all current varieties of banana and induce the most destructive threats on banana product worldwide.So far,there is still lack of effective measures to control the disease.Studying on the function of the pathogenicity related genes of FOC4 will help to understand its pathogenesis,and thus to provide a theoretical basis for the development of control strategies of the disease.Based on our previous laboratory's screening,we found a mutant named W2987with significantly reduced virulence.In this mutant,T-DNA inserts between two genes.In order to find out the function of these two genes,we cloned the two genes FOIG₀1729 and FOIG₀1730 which may be affected by the T-DNA insertion.By knocking out the two genes respectly and phenotypic analysis,functions with pathogenicity of the two genes were studied.The main results are as follows:1.Blast and phylogenetic analysis show that of the two genes FOIG₀1729 and FOIG₀1730 code hypothetical proteins.Gene FOIG₀1729 coded protein show high similarty with another two homologous proteins of Fusarium oxysporum,but it can not be found in other species.It illustrated that FOIG₀1729 is a new gene and conservative in Fusarium.Gene FOIG₀1730 coded protein blast with other homologous protein of Fusarium closely,and showed some homologous with Neurospora with very low similarity.It also can not be found in other species.The results suggested that FOIG₀1730 is a new gene and conservative in Fusarium.2.The two genes FOIG₀1729 and FOIG₀1730 were deleted respectly using PEG-mediated protoplast transformation.Through PCR and Southern blot analysis,gene knockout mutants were verified.Furthermore the target gene expression in knockout mutants was verified by q RT-PCR.The results showed that the two target genes were successfully deleted in the knockout mutants.3.The FOIG₀1729 and FOIG₀1730 knockout mutants showed no significant differences with the wild type strain XJZ2 on the morphology and growth rate of the colony.Oxidative stress response analysis results showed that mutants are not sensitive to H₂O₂.Our mutants are also insensitive to the cell wall perturbing agents?calcofluor white and Congo red?.Furthermore,compared with the wild type strain,the sporulation of the two knids of mutants is reduced,and spore heat resistance of the mutants is also decreased.4.Compared with WT,the mutants can't produce clearing halos.The result showed the mutants have no toxic to E.coli.The expression of the gene related to beauvericin synthesis was detected by q RT-PCR assay in FOIG₀1729 and FOIG₀1730 gene knockout mutants.We found that the expression of kivr was significantly down-regulated in both two genes knockout mutants,which is the beginning site of the synthesis of beauvericin.Therefore,these two genes are closely related to the synthesis of beauvericin.5.Compared with the wild type strain,deletion of FOIG₀1729 and FOIG₀1730 both led to decline in virulence to the banana host,but FOIG₀1729knockout mutants showed less pathogenicity than FOIG₀1730 knockout mutants.In summary,FOIG₀1729 and FOIG₀1730 are closely related to the pathogenicity of F.oxysporum.Deletion of these two genes results in significant reduction of the conduction of spores and the activity of spores,and affects on the formation of mycotoxins.