Study On The Molecular Mechanism Of FgPDK1 Regulating Fusarium Graminearum In Response To Salt Stress

Fungi have developed a serious of regulatory networks,including HOG(high osmolarity glycerol)pathway,in response to osmotic stress.However,little is known about the upstream regulators for these networks.In this study,physiological,biochemical,and genetic approaches were applied to investigate the involvement of FgPDKl(Fusarium graminearum pyruvate dehydrogenase kinase 1),a key gene in mitochondrial energy metabolism,in the modulation of osmotic regulatory networks in F.graminearum under salt stress.Basically,the following results were obtained.1.AFgPDK1(FgPDK1 knock-out mutant)was more sensitive to salt stress than that of wild type(standard strain PH-1).NaCI at different concentrations significantly inhibited the growth of PH-1 in both dose-and time-dependent manners.The EC50 of NaC1 for PH-1 growth was 0.8 M.NaC1 at 0.8 M remarkably inhibited conidial germination of PH-1.The expression of FgPDK1 in PH-1 was significantly enhanced under NaCI treatment,with maximal enhancement occurring at 0.8 M of NaC1.Compared to PH-1 under treatment of NaCI at 0.8 M,the growth of ?FgPDK1 decreased significantly while the growth of ?FgPDK1-C(parental FgPDK1 complemented strain)remained unchanged.Observation from SEM(scanning electronic microscope)indicated that ?FgPDK1 showed more serious irregular shrinkage of hyphae than that of PH-1 under treatment of NaCI at 0.8 M.2.FgPDK1 was able to maintain ROS(reactive oxygen species)homeostasis in F.graminearum under NaCI stress.Physiological and histochemical approaches were used to investigate the effect of NaCI(0.8 M)on oxidative injury,ROS accumulation,and cell death in the hyphae of PH-1,?FgPDKl1,and ?FgPDK1-C.Compared to PH-1 and ?FgPDK1-C,more ROS and MDA content as wells as dead cells were detected in the hyphae of ?FgPDK1.Then the expression of genes encoding anti-oxidative enzymes was analyzed.NaCI(0.8 M)treatment resulted in the decrease in CAT(catalase)expression in three strains while AFgPDK1 showed significantly lower expression level of CAT than PH-1 and ?FgPDK1-C.NaCI(0.8 M)treatment induced the expression of SOD(superoxide dismutase)in three strains while AFgPDK1 showed significantly lower expression level of SOD than PH-1 and?FgPDK1-C.These results suggested that FgPDKl played important roles in the maintenance of high expression level of anti-oxidative genes,which further help to scavenge over-accumulated ROS under salt stress.3.FgPDK1 regulated membrane integrity and glycerol accumulation in F.graminearum under NaCl stress.Compared to PH-1 and ?FgPDK1-C,NaCl(0.8 M)treatment resulted in more electrolyte leakage and higher glycerol accumulation in?AFgPDK1 However,the expression levels of FgGDP1 and FgGDP2,two genes encoding for glycerol synthase,were significantly lower in the hyphae of ?FgPDK1 than that of PH-1 and AFgPDKl-C,which were opposite to the change of glycerol content.In fungi,glycerol can be transported out of cells by membrane protein Fpsl that is negatively regulated by Rgcl.Further studies indicated that?FgPDK1 showed higher expression level of FgRgcl and lower expression level of FgFpsl than that of PH-1 and ?FgPDK1-C under NaCl stress.Therefore,glycerol accumulation in?FgPDKl may be resulted from the blockade of glycerol efflux under NaCl stress.4.FgPDKl modulated HOG pathway in F.graminearum during high osmotic adaption.First,expression of the key genes in positive regulatory HOG pathway was studied.NaC1(0.8 M)treatment stimulated the expression of FgHOGl and its upstream positive regulator FgPbs2 in three strains.However,the expression levels of these two genes were lower in AFgPDK1 than in PH-1 and ?FgPDKl-C,which were consistent in the expression patterns of FgGDP1 and FgGDP2.These results suggested that HOG pathway was suppressed in ?FgPDK1 under salt stress.Next,the effect of FgPDKl deletion on the negative regulatory HOG pathway was investigated.NaCl(0.8 M)treatment resulted in the decrease in the expression of FgSlnl and FgSSKl and the increase in the expression of FgSSK2(negatively regulated by FgSSK1)in three strains.The expression levels of FgSlnl andFgSSK1 were higher in?FgPDKl than in PH-1 and ?FgPDKl-C,which were opposite to the expression pattern of FgSSK2.These results suggested that the negative regulatory HOG pathway was activated in AFgPDK1 under salt stress,which further resulted in the suppression of HOG pathway.5.FgPDK1 modulated ionic balance in F.graminearum in response to salt stress.Compared to PH-1 and ?FgPDK1-C,NaC1(0.8 M)treatment resulted in higher Na+content,lower K+ content,and higher Na/K in hyphal cells of ?FgPDK1.Compared to PH-1 and ?FgPDK1-C,?FgPDK1 showed higher expression level of FgENA1(Na+ influx channel).However,one Na+ efflux channel gene(FgNhal)and three K+efflux channel genes(FgTOKl,FgTrk1,and FgNHXl)showed opposite expression patterns.These results suggested that FgPDK1 was able to maintain intracellular ionic homeostasis by modulating different ionic channels in F.graminearum under salt stress.In sum,this study revealed that FgPDK1 was a vital regulator of salt stress adaption by modulating ROS homeostasis,HOG pathway,and ionic balance in F.graminearum,which provided new evidences to further understand the molecular regulatory mechanism of osmotic adaption in fungi.