Screening Of The Host Factors Interacting With VPg Of Sugarcane Streak Mosaic Virus By Yeast Two-Hybrid System

Sugarcane(Saccharum spp.Hybrid)is one of the most important sugar crops around the world,but also an important energy crop.In the sugarcane production,sugarcane mosaic disease(SMD)generally occurs in the sugarcane field of the world,which is one of the most serious viral diseases,seriously affecting the yield and quality of sugarcane.Sugarcane mosaic virus(SCMV),sorghum mosaic virus(SrMV)and sugarcane mosaic virus(SCSMV)is the main pathogen of SMD.They belong to the family of Potyviridae,but SCMV and SrMV belong to the genus of potato virus Y(Potyvirus),and SCSMV virus belongs to Poacevirus.At present,the pathogenesis of SMD is less reported,especially the research on the molecular mechanism of SCSMV infection is still blank.In order to investigate the interaction between sugarcane and SCSMV,a yeast two hybrid cDNA library of sugarcane leaves infected by SCSMV was constructed in this study.The yeast two-hybrid system(Y2HS)was used to screen the cDNA Library by using SCSMV-VPg protein as bait.At the same time,the interaction between SCSMV-VPg and sugarcane transcription elongation factor C subunit(ScELC)was studied and the role of ScELC during SCSMV infection was explored preliminarily.The results are as follows:(1)A yeast two hybrid cDNA library of sugarcane leaves infected by SCSMV was constructed by using Gateway.The study for the screening of sugarcane host factors interaction with SCSMV lays the foundation and will facilitate sugarcane breeding and germplasm identification that focuses on SCSMV resistance.(2)The coding sequences of HC-Pro,P3N-PIPO,CP and VPg from SCSMV were cloned by PCR and the yeast two hybrid bait vector pGBKT7-Hc-Pro,pGBKT7-P3N-PIPO,pGBKT7-CP and pGBKT7-VPg were successfully constructed.The bait vectors were not self-activating activity and toxicity for the yeast strain Y2Hgold,and can be used to screen the sugarcane cDNA library.(3)The sugarcane cDNA library was screened by using SCSMV-VPg protein as bait and yeast two-hybrid system(Y2HS).The nine positive clones were initially obtained,and bioinformatics analysis showed these genes are involved in transcriptional regulation,basal metabolism,photosynthesis and stress resistance.(4)The candidate positive clone ScELC was selected to study.ScELC and SCSMV,SCMV,SrMV of VPg were proved to exist interaction relationship by using yeast two-hybrid(Y2H)and bimolecular fluorescence complementation(BiFC).The tissue specific expression of ScELC gene show it was expressed in all tissues of sugarcane plants of the seedling and mature stages,which were the highest of expression levels in leaf of sugarcane seedling and root of sugarcane maturity,respectively.The subcellular localization of ScELC protein was studied.It was located in the cytoplasm and nucleus.In order to study the role of ScELC during SCSMV infection,the impact of SCSMV infection on the expression of ScELC was analyzed by Real-time qPCR,and the interaction between ScELC and sugarcane translation initiation factor eIF4E(SceIF4E1,SceIF4E2 and SceIF4E3)were studied.The results showed the transcript level of ScELC was significantly increased after inoculation with SCSMV.The results of Y2H and BiFC showed ScELC interacted only with SceIF4E2,indicating ScELC and SceIF4E2 may have some relevance in the process of SCSMV infection.