Construction Of Acetylome And Functional Analysis Of Pathogenicity Factor FocSGE1 In Fusarium Oxysporum F. Sp. Cucumerinum

Fusarium oxysporum f.sp.cucumerinum(Foc)is a widespread soilborne plant pathgen which cause Fusarium wilt of cucumber.Our previous study found that Foc mild virulence stain foc-3b was significantly increased after serial passage on the resistant cucumber cultivar,and an induced variant Ra-4 with enhanced virulence was isolated.In this study,we constructed the differentially regulated acetylomes between virulence-enhanced variant and mild virulence strains,and between parasitic process and vegetative growth.From these acetylomes some potential virulence related proteins with Kac modification were identified.We chose FocSGE1 from these proteins and studied its regulation function in pathogenicity of Foc.In the previous study we found that FocSIX11 could regulate virulence of Foc,and combined with this result we studied the downstream regulation pathway of FocSGE1.The main results are as follows:1.Construct the differentially regulated acetylomes between virulence-enhanced variant and mild virulence strains of Foc,and between parasitic process and vegetative growth.From these acetylomes some potential virulence related proteins such as SGE1 homologous protein,MAPK regulator,fusaric acid(FA)biosynthesis and cytochrome P450 enzyme were identified,and many Kac proteins of Foc were involved in response to host defense,which showed a potential regulation pathway of increased virulence.2.Study the virulence regulation function of FocSGE1 and its K116 acetylation site.By knockout,complementation of FocSGE1 gene and functional mutation of K116 acetylation site strains,we found that the knockout and functional mutant strains were basically consistent with the wild-type strain foc-3b in terms of vegetative growth,conidiation and stress tolerance,but the virulence of knockout and K116 Q mutant strains were significantly reduced.In addition,the complementation strain did not show any significantly difference.3.Study the downstream regulation pathway of FocSGE1.EMSA showed that the FocSIX11 upstream DNA sequence AAACTTAA could be binded by FocSGE1.The secretion function of FocSIX11 was studied by yeast invertase experiment.By quantitative PCR it showed that the expression of FocSGE1 after inoculation was regulated by its K116 acetylation site,and the expression of FocSGE1 reduced nearly 90% in K116 R and K116 Q mutation strains.However,the expression of FocSIX11 was regulated by FocSGE1 and its K116 acetylation site at 6 h after inoculation;in this time point the expression of FocSIX11 significantly decreased in knockout,K116 R and K116 Q mutantion strains.There was a compensatory highly increase in FocSIX11 expression after 24 h of inoculation,and in this stage there was little difference in expression of Focsix11 among all strains.This study preliminarily elucidated the regulation pathway of FocSGE1 in Foc.The deacetylation of K116 site on FocSGE1 lead the regulation of virulence in Foc.The expression of FocSIX11 would be decreased at the early stage after inoculation,and made the virulence of Foc reduce if K116 site could not be deacetylated.Although complete deacetylation of K116 site also decreased the expression of FocSIX11 at the early stage after inoculation,it might enable Foc to turn on or enhance the expression of other virulence factors,thus making up for the loss of virulence caused by the decreased expression of FocSIX11.These results of this study will provide a foundation of research for the virulence regulation of acetylation in Foc,and also provide a research direction for the pathogenesis and enhancement mechanism of the virulence in Foc.