Metabolic Profile Of The Kinome And Functional Analysis Of Adenylyl Cyclase-associated Protein FgCap1 In Fusarium Graminearum

Fusarium head blight,caused by Fusarium graminearum,is one of the most destructive diseases of wheat in world wheat-growing regions.In addition to causing dramatical yield losses,F.graminearum produces the trichothecene mycotoxin deoxynivalenol(DON).In previous study,96 protein kinases mutants were generated and 28 of them had no orthologs in yeast including 12 had no obvious phenotypes examined.Genes deletion mutants with no phenotypes might caused metabolites changes by affected the intercellular metabolic pathways.The central goal of metabolomics is to analysis metabolites changes in a certain environment condition with genetic influence thus metabolomics could connect metabolites changes with genotype and phenotype.In this study,we analyzed changes in the metabolic profiles of 93 kinase mutants by gas chromatography/mass spectrometry(GC/MS)analysis.All the mutants were cultured in minimal medium(MM)and complete medium(CM)for 15d.Vegetative hyphae were harvested by filtration through Miracloth and ground in liquid nitrogen.The resulting samples were then extracted with methanol and derivalized with N-trimethylsilylimidazole/trimethylchlorosilane(TMSI/TMCS)or methyl chloroformate(MCF).In MM,more linoleic acid,sorbitol and sucrose and less alabitol were detected in CM.Moreover,mutants with no obvious metabolic profile changes in MM showed changes in metabolites types and concentration in CM.These results indicated metabolites changes were influenced by both gene modification and environment conditions.The three MAPK cascades pathway were important for plant infection and other biological process in F.graminearum.In both medium,FgHogl cascade mutants clustered together and showed reduce in arabitol and sorbitol.FgMgvl cascade mutants clustered with FgGpmkl cascade mutants in MM.Interestingly,FgMgvl cascade mutants with other 11 mutants clustered together and showed significantly increase in arabitol and decrease in turanose.Further study showed FgMgvl cascade mutants affected the Glycerolipid metabolism,Pentose and glucuronate interconversions and Galactose metabolism.Good separation was detected between mutants with no defect with wild type strain PH-1 and mutants with no perithecium formation by OPLS-DA analysis indicating a clear difference between the metabolic profile of these two groups and 14 metabolites with VIP values over 1 were considered contributing most to the separation.Three mutants from the 12 mutants had no orthologs with no phenotype showed clear metabolites changes and these mutants mainly affected carbohydrate metabolism.Accurate definition of their metabolic functions needed more data.The expression of trichothecene biosynthesis(TRI)genes and DON production are mainly regulated by the cyclic adenosine monophosphate-protein kinase A(cAMP-PKA)pathway and two pathway-specific transcription factors(TRI6 andTRI10).Interestingly,deletion mutants of TRI6 show reduced expression of several components of cAMP signalling,including the FgCAP1 adenylate-binding protein gene that has not been functionally characterized in F.graminearum.In this study,we show that FgCap1 interacts with Fac1 adenylate cyclase and that deletion of FgCAP1 reduces the intracellular cAMP level and PKA activity.The Fgcapl deletion mutant is defective in vegetative growth,conidiogenesis and plant infection.It also shows significantly reduced DON production and TRI gene expression,which can be suppressed by exogenous cAMP,indicating a PKA-dependent regulation of DON biosynthesis by FgCap1.The wild-type,but not tri6 mutant,shows increased levels of intracellular cAMP and FgCAPl expression under DON-producing conditions.Furthermore,the promoter of FgCAPl contains one putative Tri6-binding site that is important for its function during DON biosynthesis,but is dispensable for hyphal growth,conidiogenesis and pathogenesis.In addition,FgCap1 shows an actin-like localization to the cortical patches at the apical region of hyphal tips.Phosphorylation of FgCapl at S353 was identified by phosphoproteomics analysis.The S353A mutation in FgCAP1 has no effect on its functions during vegetative growth,conidiation and DON production.However,expression of the FgCAP1S353A allele fails to complement the defects of the Fgcapl mutant in plant infection,indicating the importance of the phosphorylation of FgCapl at S353 during pathogenesis.Taken together,our results suggest that FgCAPl is involved in the regulation of DON production via cAMP signalling and subjected to a feedback regulation by TRI6,but the phosphorylation of FgCapl at S353 is probably unrelated to the cAMP-PKA pathway because the S353A mutation only affects plant infection.