The Functions Of G Proteins In Fusarium Graminearum And Their Regulation On Signal Transduction

Fusarium head blight(FHB),caused by Fusarium graminearum,is one of the most destructive diseases of wheat and barley in many wheat-growing regions.In addition to causing severe yield losses,F.graminearum produces the trichothecene mycotoxin deoxynivalenol(DON)in infected grains.The formation of primary infection source,the initiation of infection and DON production depend on the recognition of stimuli from the environment and the host.G-protein-coupled signal pathway is a conserved signal transduction pathway in eukaryotes.It consists of G-protein-coupled receptors,heterotrimeric G protein composed of α,β and γ subunits,downstream signal pathways and various signal regulatory proteins.In a previous study,we systematically characterized the function of the F.graminearum GPCRs family in vegetative growth,sexual reproduction and plant infection,but the downstream signal transduction pathways of them remains to be further investigated.G proteins associate with GPCRs and activate downstream pathways,therefore play a critical role in intracellular signaling transduction.The G proteins of F.graminearum are composed of α,β and γ subunits.The Gα subunits are encoded by three genes GPA1,GPA2 and GPA3,while Gβ and Gγ are encoded by GPB1 and GPG1 respectively.To investigate the functions and downstream pathways of F.graminearum G proteins,genes encoding these multiple subunits of G proteins were deleted.In compared with the wild type,the Δgpa1 mutant was blocked in sexual reproduction,and reduced in germination and pathogenicity.In addition,the Δgpa1 mutant was less sensitive to oxidative and cell wall stress.Bacteria mpf3 was easier to adhere to the Δgpa1 mutant than the wild type,indicating that GPA1 is not only required for abiotic stress response,but also plays a critical role in bacterial-fungal interaction.To determine the downstream pathways of GPA1,Tp EY and Tp GY phosphorylation assays were conducted.Gpmk1 MAPK signaling was the major downstream target of Gpa1,but the deletion of GPA1 also affected the activation of Mgv1 and Hog1 pathway.Unlike Δgpa1 mutant,Δgpa2 mutant was only slightly defected in vegetative growth and sexual reproduction.Nevertheless,the pathogenicity was significantly reduced in the absence of GPA2.As the only Gβ subunit coding gene,Gpb1 played an important role in vegetative growth,sexual reproduction and pathogenicity of F.graminearum.Although the deletion mutants of the GPA1,GPA2 and GPB1 led to various defects,overexpression of them showed no obvious effects.To further investigate the exact mechanism of G protein in the regulation of downstream pathways,three dominant active forms of Gpa1 were introduced into the Δgpa1 mutant.Defects in the vegetative growth,sexual development and pathogenicity were observed.Interestingly,different active forms of G protein caused the activation of different downstream signal pathways.Strains with a glycine-toarginine mutation at position 42 activated the Gpmk1 pathway,an arginine-to-cysteine mutation at position 178 activated the Hog1 cascade,and a gluta mine-to-leucine mutation at position 204 activated the Mgv1 pathway.Therefore,the distinct functions of G protein in signaling transduction was likely associated with its different activated forms.Interestingly,we also identified a G-like protein named Rack1 in F.graminearum.The Δrack1 mutant showed severe defects in growth,sexual development and plant infection.By screening the rack1-interacting proteins using pull down assays.Six kinase of MAPK pathways(including MAPKKK,MAPKK and MAPK),had potential interaction with Rack1,suggested Rack1 was likely involved in the cross-talk of different MAPK signalings.A total of four phosphorylation sites were identified in Rack1,indicated Rack1 was subjected to posttranslational modifications.Taken together,G protein signaling play a central role in signaling sensing and intracellular transduction.Deletion of G protein-encoded genes resulted in defects in vegetative growth,sexual development and pathogenicity.Different interacting proteins and active forms of G proteins deter mine the accurate activation of downstream signaling.Our work will not only improve our understanding of fungal sensing and cell signaling during sexual reproduction,but also lead to the development of novel strategies for disease control.