Regulation Of β₂ Tubulin And Its Carbendazim-Resistant Substitutions To Deoxynivalenol Biosynthesis In Fusarium Graminearum

Fusarium head blight（FHB）caused by a filamentous fungus Fusarium graminearum is a devastating wheat disease worldwide.The disease not only drastically reduces yield and quality,mycotoxins,particularly deoxynivalenol（DON）,secreted by the pathogen in infested grains also threaten human and animal health.In the past four decades,carbendazim and other benzimidazole fungicides have been used to control FHB in China.However,our previous researches showed that carbendazim-resistance in pathogen populations emerged in the field of East China.In addition,amino acid substitutions inβ₂tubulin at codons 167,198 or 200 significantly increase DON biosynthesis in F.graminearum.Furthermore,previous results indicated that the increased DON biosynthesis are associated with the up-regulation of Fghxk1 and down-regulation of FgIDH1,FgIDH2,FgIDH3.To illuminates the underlying molecular mechanism between carbendazim-resistant substitutions inβ₂tubulin and elevated DON biosynthesis,affinity capture-mass spectrometry（ACMS）,Co-immunoprecipitation（Co-IP）,bimolecular fluorescence complementation（Bi FC）and split luciferase complementation assay（SLCA）were used for screen and identify Fgβ₂-interacting proteins in current study.This study characterized the biological functions of key proteins interacted with Fgβ₂and revealed the molecular mechanism associated withβ₂tubulin,its carbendazim-resistant substitutions,and DON biosynthesis in F.graminearum.The main results are as below:1.Analysis and identification of Fgβ₂-interacting proteinsAs one of the important cytoskeleton components,microtubules（MTs）not only participate in cell polarity,mitosis,motility and vesicle traffic,but regulate various metabolic pathways.Previous studies showed that the expression of various metabolic genes are changed in carbendazim-resistant strains,which also led to increased DON biosynthesis,but the molecular mechanism remains unclear.In order to illustrate the molecular mechanism under carbendazim-resistant substitutions and elevated DON biosynthesis,we screened and identified Fgβ₂-interacting proteins via ACMS and SLCA.The results showed that 267 and 160 proteins were obtained in TBI and YEPD media,respectively.Among them,48 proteins existed in both two media.Gene ontology（GO）analysis revealed that most of the Fgβ₂-interacting proteins belong to“metabolism”and“genetic information processing”pathways.The proteins in“metabolism”pathway were mainly involved in amino acid metabolism,oxidative phosphorylation,EMP and TCA cycle.The proteins in“genetic information processing”pathway were mainly involved in translation.To verify the expression pattern of Fgβ₂-interacting proteins,q RT-PCR assays were applied to examine the transcriptional levels of selected proteins in TBI and YEPD media.In TBI media,the expression levels of EMP-and amino acid metabolism-related protein-encoding genes significantly increased,whereas the expression of those involved in TCA cycle significantly decreased,and no expression differences were detected in translation-related genes.Furthermore,SLCA was performed to verify the interaction between Fgβ₂and these candidate proteins.As expected,majority of proteins involved in translation,amino acid metabolism,EMP and TCA cycle interacted with Fgβ₂physically,suggesting thatβ₂tubulin and its carbendazim-resistant substitutions may regulate the functions of these proteins,and further participate in DON biosynthesis.Combined with previous results,the proteins associated with EMP and TCA cycle were selected for further study.2.Carbendazim-resistant substitutions impair the interaction between Fgβ₂and FgIDH3The q PCR results showed that the expression levels of several key genes involved in TCA cycle were down-regulated in F167Y mutant nt167,including FgIDH1,FgIDH2,FgIDH3,whereas the production of acetyl-CoA increased significantly.Results further demonstrated that exogenous acetyl-CoA treatment could accelerate DON biosynthesis in F.graminearum.Subcellular localization assays showed that FgIDH1 was localized in the vacuoles,whereas FgIDH2 and FgIDH3 were localized in the mitochondria.Although theΔFgIDH1,ΔFgIDH2 andΔFgIDH3 mutants showed no obvious defects in vegetative growth,their virulence levels were significantly reduced.When cultured in GYEP medium for 3 days,abundant swollen hyphal structures associated with DON biosynthesis were observed inΔFgIDH2 andΔFgIDH3 when compared to the wild-type progenitor.In addition,DON production and Tri5 expression were increased inΔFgIDH2 andΔFgIDH3in comparison to the wild type.Furthermore,the increases were found in acetyl-CoA and pyruvic acid production inΔFgIDH2 andΔFgIDH3.DON production,Tri5 expression,acetyl-CoA and pyruvic acid production were further analyzed in otherβ₂tubulin carbendazim-resisitant substitution mutants.Remarkable increases were observed in each carbendazim-resisitant mutant,indicating that the increased DON biosynthesis is connected with substitutions inβ₂tubulin conferring carbendazim-resistance.In addition,the interaction between Fgβ₂and FgIDH3 was demonstrated by Co-IP,Bi FC and SLCA.Interestingly,the interaction intensity between Fgβ₂and FgIDH3 was impaired by carbendazim-resistant substitutions,which resulted in decreased FgIDH3 expression and mitochondrial isocitrate dehydrogenase activity.This further brought about the accumulation of upstream metabolites citric acid.This study further demonstrated that carbendazim treatment could disrupt the interaction between Fgβ₂and FgIDH3,which promotes DON biosynthesis in F.graminearum in the field.Taken together,these results showed that FgIDH2 and FgIDH3 participated in DON biosynthesis.By disrupting the interaction between Fgβ₂and FgIDH3,carbendazim-resistant substitutions facilitate acetyl-CoA production and DON biosynthesis in carbendazim-resistant strains.3.Carbendazim-resistant substitutions impair the interaction between Fgβ₂and FgGPIGlucose-6-phosphate isomerase（GPI）is a key enzyme which controls the second step of glycolysis.Gene deletion assay revealed that FgGPI is involved in vegetative growth,asexual development,environmental stresses response and virulence.In addition,the expression of key genes involved in EMP and TCA cycle were altered,which resulted in glucose metabolism disturbance and pyruvic acid biosynthesis reduction,and eventually led to impairment in DON biosynthesis.Furthermore,the interaction between Fgβ₂and FgGPI was demonstrated by Co-IP assay,suggesting that carbendazim-resistant substitutions could impair the interaction intensity between Fgβ₂and FgGPI,which lead to increased expression of FgGPI.However,carbendazim does not interfere with the binding between Fgβ₂and FgGPI.These results suggested that FgGPI regulates fungal development processes,environmental stresses response,virulence and DON biosynthesis in F.graminearum.The study further revealed that carbendazim-resistant substitutions could impair the binding between FgGPI and Fgβ₂,leading to increased FgGPI expression and DON production in carbendazim-resistant strains.4.MT-assisted mechanism for toxisome assemblyA recent study revealed that the spatial distribution and the metabolic activity of purine biosynthesis multienzyme complex,the purinosome,is regulated by MT network in HeLa cells.In addition,the trichothecene biosynthetic complex,known as the toxisome was recently proposed in F.graminearum.As a components of eukaryotic cytoskeleton,actin plays a vital role in toxisome formation.However,the connection between MT and toxisome remain unknown.To investigate the association between MT and toxisome,fungal cells were treated with a MT inhibitor carbendazim.As results showed,colocalization was observed between MT network and toxisome,and carbendazim treatment could disrupt toxisome assembly in F.graminearum.In addition,carbendazim treatment significantly inhibited expression of Fg Tri1 and subsequently DON biosynthesis.These outcomes showed that MT participates in toxisome assembly.Furthermore,when the connection between toxisome and each MT subunit was investigated,toxisome failed to assemble in?Fgɑ₁and?Fgβ₂mutants.Western blot assays indicated low expression of Tri1 in?Fgɑ₁and?Fgβ₂mutants.In addition,DON production also drastically decreased in?Fgɑ₁and?Fgβ₂.Moreover,Fg Tri1 interacted with Fgɑ₁and Fgβ₂but not Fgɑ₂and Fgβ₁.Subcellular localization and SLCA assay suggested that Fgɑ₁binds with Fgβ₂and formɑ₁-β₂heterodimer in F.graminearum.Taken together,these results support a model ofɑ₁-β₂heterodimer interacting with Tri1 and serving as a scaffold to further regulate toxisome assembly and DON biosynthesis in F.graminearum.In conclusion,the interactions between Fgβ₂and FgIDH3 or FgGPI could regulate acetyl-CoA and DON biosynthesis in F.graminearum,deletion of FgIDH3 or disrupting the interaction between Fgβ₂and FgIDH3 could accelerate DON biosynthesis.The interaction region between Fgβ₂and FgIDH3 is similar to carbendazim,therefore carbendazim-resistant substitutions not only disturb the affinity between carbendazim and Fgβ₂,but the interaction between Fgβ₂and FgIDH3.In addition,sublethal carbendazim could disrupt the binding between Fgβ₂and FgIDH3,which resulted in increased DON biosynthesis in F.graminearum.On the other hand,high concentration of carbendazim could impairɑ₁-β₂heterodimer formation and inhibit toxisome formation in carbendazim-sensitive strains.However,high concentration of carbendazim has no impact inɑ₁-β₂heterodimer formation but could disturb the interaction between Fgβ₂and FgIDH3,which resulted in increased DON biosynthesis in carbendazim-resistant strains.