Functional Analyses Of Key Genes Involved In Stress Tolerance And Nitrogen Metabolism In Fusarium Graminearum

Fusarium graminearum is a major phytopathogen which causes Fusarim head blight(FHB),a devastating disease on wheat.Every year,this fungal species can cause a considerable yield loss on crops.In addition,it can produce several harmful mycotoxins which threaten human and animal's health.The genome sequence of F.graminearum have been completed,and these data provide a significant important resource for deeply studying gene function of F.graminearum.In this study,we selected candidate genes involved in stress tolerance from a gene chip with F.graminearum strain 5035.A homologous recombination gene knockout strategy was used to delete these genes.Futher more,by investigating the sensitivity of these deletion mutants to S76-3 metabolites,Sorbitol,NaCl,SDS and Congo red,we found two key genes that played important roles in stress tolerance and these two genes were futher characterized for their functionality.In addition,eight genes involved in nitrogen metabolism in F.graminearum strain 5035 were investigated.Main results are as follows:1.After successful deletion of 45 genes,two genes(FgCYK3,FgDOP1)were found to significantly mediate stress tolerance.Compared to wild type(WT)5035,?FgCYK3 was more sensitive to S76-3 metabolites and NaCl,while ?FgDOP1 was more sensitive to all of five compounds used.These two genes have different characters and functions.1)FgCyk3 is a homologue to cytokinesis protein 3(Cyk3)in Saccharomyces cerevisiae.In this study,the colony morphology of ?FgCYK3 was no obviously different from that of wild type 5035 on Patato dextrose agar medium(PDA)and minimal medium(MM).But ?FgCYK3 had abnormal conidia and reduced conidiation capacity.Its pathogenicity was also reduced on wheat spike.These results indicated that FgCYK3 plays an important role in conidium morphogenesis,conidiation and pathogenicity in F.graminearum.2)FgDopl is a homologue to Doplp in S.cerevisiae and DopA in Aspergillus nidulans.In this study,?FgDOP1 deletion mutant grew more slowly than wild type 5035 on PDA and MM.And ?FgDOP1 had abnormal conidia and reduced conidiation ability and germination capacity.Its pathogenicity was also reduced on wheat seedling.These results indicated that FgDOPl plays an important role in hyphal growth,conidium morphogenesis,conidiation and pathogenicity in F.graminearum.2.In nitrogen metabolism pathways,different genes have different functions.1)For genes that are involved in secondary nitrogen metabolism,nitrate or nitrite transporter deletion mutant ?Fg00416,nitrate reductase deletion mutant ?Fg01947 and nitrite reductase deletion mutant ?Fg08402 grew normally on PDA,but they can not grow on MM which contains nitrate as its only nitrogen resource.Formamidase deletion mutant ?Fg09042 grew normally on PDA and MM,but when MM omitting nitrate and containing formamide as its only nitrogen resource,?Fg09042 did not grow.These results indicated that there is only one metabolism pathway to assimilate each of secondary nitrogen resources and there are not functional overlapping genes in these pathways.2)For genes that are involved in preferred nitrogen metabolism,NAD-specific glutamate dehydrogenase deletion mutant ?Fg04117,NADP-specific glutamate dehydrogenase deletion mutant ?Fg07174,?Fg04117-?Fg07174 double deletion mutant and two glutamine synthetase deletion mutants ?Fg10264,?Fg11697 grew normally on PDA and MM.These results indicated that there are at least two pathways involved in preferred nitrogen metabolism,and there are functional overlapping genes in each pathway.3)The pathogenicity of all gene deletion mutants were also assayed.The results indicated that except ?Fg01947 and ?Fg11697,the remaining six gene deletion mutants had significant reduced pathogenicity on wheat spike.In summary,these results provide new information on the development of controlling strategies for F.graminearum and on identifaction of new genes for functional analyses for this fungal pathogen.