| Fusarium head blight(FHB)caused by Fusarium graminearum(teleomorph:Gibberella zeae)is the most devastating fungal disease.In addition to the high economic impact of FHB,the infected cereals are often contaminated with deoxynivalenol which causes food pollution,severely threats to world food safety.Soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNAREs)proteins are a large family of proteins that play a central role in membrane fusion.Our previous study has demonstrated that FgVam7,which encodes a SNARE protein involving in vesicle trafficking,plays crucial roles in growth,asexual/sexual development,deoxynivalenol(DON)production and pathogenicity in F.graminearum.In current study,the affinity purification approach was used to identify FgVam7-interacting proteins to explore its regulatory mechanisms during vesicle trafficking.Furthermore,we characterized the biological function of interaction proteins.A better understanding the mechanism of FgVam7 how involved in vesicle trafficking pathway of F.graminearum,and main results are showed as follows:1.The affinity purification approach was used to identify FgVam7-interacting proteinsOur previous study has demonstrated that FgVam7,which encodes a SNARE protein involving in vesicle trafficking,plays crucial roles in growth,asexual/sexual development,deoxynivalenol production and pathogenicity in F.graminearum.However,it is not clear how FgVam7 mediates vesicle trafficking and vacuole fusion,and which proteins function together with FgVam7 during these processes.Here,the affinity purification approach was used to identify FgVam7-interacting proteins to explore its regulatory mechanisms during vesicle trafficking.Analysis the results of the FgVam7 affinity purification protein spectrum,a total of 21 proteins were detected.We performed yeast two hybrid(Y2H)to assay one to one interaction.The results showed that:13 of 21 proteins was interacted;8 of 21 proteins no interacted.These proteins could divided into vesicle trafficking,autophagy process,primary metabolism and hypothetical protein.These results suggested that FgVam7 may be involved in biochemical function,and regulate the development and virulence in F.graminearum.Because FgVam7 involving in vesicle trafficking,plays crucial roles in growth,asexual/sexual development,deoxynivalenol production and pathogenicity in F.graminearum.In order to better underatand the mechanism of how FgVam7 mediates vesicle trafficking and vacuole fusion,we selected the related proteins of vesicle trafficking to study.2.FgVps39 played important roles in development and pathogenicity of F.graminearum.We analyzed the candidate affinity proteins of FgVam7 by yeast two hybrid,the result showed that the interaction between FgVps39 and FgVam7.HOPS complex that is involved in the late endosome to vacuole transportation.The △mutant generated by targeted deletion was significantly reduced in vegetative growth and asexual development.It failed to produce sexual spores and was defective in plant infection and DON production.Further cellular localization and cytological examinations suggested that FgVps39 is involved in vesicle trafficking from early/late endosomes to vacuoles in F.graminearum.Additionally,the △Fgvps39 mutant was defective in vacuole morphology and autophagy,and it was delayed in endocytosis.In addition,the yeast two hybrid and GST pull down assays,FgVam7,FgVps39,and FgSsol interacted with each other as a complex.Our results demonstrate that FgVam7 interacts with FgVps39 and FgSsol to form a unique complex,which is involved in vesicle trafficking and modulating the proper development of infection-related morphogenesis in F.graminearum.3.FgVps41 is required for both membrane and protein trafficking dependent fungal development and plant infection in F.graminearum.We analyzed the candidate affinity proteins of FgVam7 by yeast two hybrid,the result showed that the interaction between FgVps41 and FgVam7.Targeted gene deletion proved that FgVps41 is important for vegetative growth,asexual/sexual development,conidial morphology,plant infection,and DON production.Further cellular localization and cytological observed suggested FgVps41 plays a critical role in vesicle trafficking from early/late endosomes to the vacuole membrane,is recruited to prevacuolar compartments(PVCs)and the vacuole membrane by interacting with FgRab7,and is subsequently phosphorylated by vacuolar type I casein kinase FgYck3.These results indicate that FgVps41 is important for signal transduction mediated by vesicle trafficking,thereby controlling the development of infection-related morphogenesis in F.graminearum.4.T-SNARE protein FgPep12 regulates vegetative development and virulence by interact with FgVam7 in F.graminearumWe analyzed the candidate affinity proteins of FgVam7 by yeast two hybrid,the result showed that the interaction between FgPep12 and FgVam7.Targeted gene deletion proved that FgPep12 plays an important role in vegetative hyphae developmet,asexual/sexual reproduction,virulence and DON production.Cellular localization and cytological examined suggested FgPep12 localized in early/late endosome and vacuole,indicated that FgPep12 involved in membrane fusion.These results suggested that FgPep12 was involved in vesicle trafficking,and regulated development and pathogenicity of F.graminearum... |
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