Expression Pattern And Functions Of Small RNA And SYP122 In Plants Response To Sclerotinia Sclerotiorum Infection

Sclerotinia stem rot disease of rapeseed,which was caused by Sclerotinia sclerotiorum(Lib.)de Bary,is a worldwide disease.The disease could cause destructive yield loss.However,the mechanism of the resistance of plants against sclerotinia remains unclear.Therefore,further exploring for resistance enhancing genes in plants,understanding of the interaction mechanisms between plant and S.sclerotiorum,and dissection of the molecular mechanisms associated resistance should facilitate the development of efficient management strategies against Sclerotinia rot disease.Based on this purpose,this study explored the plant resistant molecular mechanisms against Sclerotinia rot disease from the plant endogenous non-coding small RNA pathway and resistance related protein,respectively.Small RNA is a 20-40 nts noncoding RNA molecule.It involved in multitude regulation of cellular processes.Successful activation of the RNAi pathway involves the deployment of Argonaute(AGO)protein,Dicer-like protein(DCL)and RNA-dependent RNA polymerase(RDR).These efficient and indispensable components of the RNAi pathway have not been identifyed and characterized in one of the most important oil crops,B.napus.In this study,28 BnAGO,eight BnDCL and 13 BnRDR genes were identified in B.napus.We subsequently analyzed their structures,conserved domains and chromosomal localization.By Real time quantitative analysis,15 BnAGO genes exhibited much higher expression levels in flowers and siliques than leaves,suggesting that they might play tissue-specific roles in B.napus.Additionally,BnAGO2(BnAGO2a and BnAGO2b),BnAGO3(BnAGO3a and BnAGO3b)and BnAGO5(BnAGO5a and BnAGO5b)were strongly induced in both susceptible(84039)and resistant(Zhongshuang9)cultivars during infection by the pathogen S.sclerotiorum,whereas BnAGO7(BnAGO7a,BnAGO7b and BnAGO7c)were significantly suppressed,suggesting that these genes are very likely involved in different defense responses.Interestingly,Arabidopsis AGO2 and AGO7 showed consistent expression with aforementioned rapeseed cultivars.Further genetic analysis demonstrated that ago2 and ago 7 were more susceptible to S.sclerotiorum than WT plants.Furthermore,We identified a new miRNA ath-miRin38-3P by deep sequencing S.sclerotiorum and mock treatments of A.thaliana Col-0 and analyzed its target gene AT3G03820.In conclusion,our results provide useful information for further study of biological function of a miRNA and its targets in Arabidopsis-Sclerotinia interaction and new ideas for resistant breeding in the future.Plants are exposed to thousands of pathogens in their lives.This has necessitated the plants to develop efficient mechanisms that involve timely adjust intracellular vesicles transport system to transport resistance related materials.Increasing evidence has shown that SNARE(soluble N-ethyl-maleimide sensitive factor attachment protein receptor)protein play an important role in plants immune responses by mediating vesicle transport and fusion.In this study,we provided detailed characterization of a SNARE protein from our early screening data.We found an Arabidopsis Qa-SNARE gene SYP122(AT3G52400)was highly induced by pathogen S.sclerotiorum.We found that overexpression SYP122 leaded to more resistant to S.sclerotiorum,whereas the SYP122 T-DNA insertion mutant SALK₀08617 exhibited more susceptible than the wild type,indicating that SYP122 is required for plant defense against S.sclerotiorum infection.YFP-SYP122 fusion protein is predominatly localized on the plasma membrane.We used time-lapse microscopy with a great time resolution to document the dynamics of YFP-SYP122 and revealed that SYP122 plays an important role in mediating exocytosis by vesicles fusion with plasma membranes.By using fluorescence recovery after photobleaching(FRAP)analysis and brefeldin A(BFA)treatment,we confirmed that SYP122 is involved in continual vesicular trafficking to the plasma membranes.In addition,we demonstrated that C-terminal transmembrane domain of SYP122 is required for the correct localization and disease resistance.Thus,our results suggested that SYP122 functions in antifungal immunity might by mediating continuous extracellular transport to modulate exocytosis of antimicrobial proteins.This study not only sheds light to molecular mechanism underlying SNARE-based membrane trafficking or fusion events contributing host immunity during Arabidopsis-S.sclerotiorum interaction,but also provide a theoretical basis for improving plant resistance to S.sclerotiorum,breeding and utilization of resistance varieties.