Characterization Of V2 Protein Encoded By Cotton Leaf Curl Multan Virus

Cotton leaf curl Multan virus(CLCuMuV)is an important whitefly-transmitted ge minivirus and the epidemics of the virus causes devastating losses to local horticultura l and economical crops worldwide.CLCuMuV,which was first reported in Multan of Pakistan,and spread to other parts of Pakistan and India,causes great losses to the l ocal cotton production.In China,CLCuMuV was first reported in Guangzhou in 2006 and spread to Guangxi,Fujian,Hainan,Jiangsu and the surrounding provinces in the following several years,which caused devastating losses to the production of local h orticultural and economical crops,including Hibiscus rosa-sinensis,Abelmoschus escule ntus,Malvaiscus arboreus and Gossypium hirsutum.CLCuMuV is a monopartite begomovirus in family Geminiviridae,which contains,a single-stranded circular DNA genome and associated with a betasatellite.DNA con tains 6 ORFs encoding V1(CP),V2,C1(Rep),and C2(TrAP),C3(REn)and C4 p rotein and betasatellite encodes one protein(?C1).Geminivirus-encoded V2 protein wa s reported as an important protein with diverse functions associated with symptoms in duction,virus movement,and suppression of gene silencing.For CLCuMuV,earlier st udies showed that V2 protein from a Pakistan isolate of CLCuMuV acts as a viral su ppressor of post-transcriptional gene silencing and involves in viral pathogenicity.Ho wever,there were no reports on the function of V2 protein from Chinese CLCuMuV isolates.So,we designed a series of experiments based on a Chinese CLCuMuV iso late,including subcellular localization,pathogenicity,and the interaction between V2 p rotein and host factors,to clarify the role of V2 protein in the interaction between vi rus and host plants and the possible mechanism of CLCuMuV epidemic in China.To characterize V2 protein encoded by CLCuMuV occurred in China,a pair of primer was designed to amplify the full length of V2 gene using CLCuMuV-infected Hibiscus rosa-sinensis from Jiangsu province as a template.The full length of V2 gen e was cloned and sequenced.Amino acid sequence analysis revealed that V2 protein in this study shared the highest identities with isolates from China and lower identitie s with isolates from Pakistan and India,indicating that V2 protein encoded by CLCu MuV isolated from China was different from those from Pakistan and India and resea rch based on V2 protein encoded by Chinese isolate was more important for the furt her dissection of the pathogenicity of Chinese CLCuMuV and the epidemic of CLCu MuV in China.Thus,the isolate from Jiangsu was selected for further study.As V2 protein was reported essential for virus infection in many begomoviruses,to clarify the subcellular localization of V2 protein encoded by Chinese CLCuMuV is olate,V2 gene was cloned into a 35S:YFP vector to obtain YFP tagged V2 protein(V2-YFP)and transiently expressed in the Nicotiana benthamiana by agrobacterium i nfiltration.The subcellular localization of V2 protein was observed by confocal micro scope and the result showed that strong GFP fluorescence signal was observed around the nucleus and at the cell periphery(cytoplasm),and some punctate fluorescent s pots were also observed in the cytoplasm.The expression of V2 gene and V2 protein(fused with YFP)in the infiltrated N.benthamiana leaves was confirmed by RT-PC R and western blotting,respectively.These results demonstrated that the V2 protein en coded by CLCuMuV was localized around the nucleus and at the cell periphery(cyto plasm),and V2 protein could form aggregates in the cytoplasm as well.To the best of our knowledge,this is the first report about the subcellular localization of V2 pr otein encoded by CLCuMuV,and it will be helpful to the further research on the fu nction of V2 protein.Numerous researches showed that begomovirs-encoded proteins could interact with each other protein encode by virus to fulfil multiple functions during the process of v irus infecting host plants.To identify the potential viral proteins interacted with V2 p rotein,six genes(V1,V2,C1,C2,C3,and C4)encoded by CLCuMuV was cloned a nd subsequently transferred to the Y2 H vectors,respectively.The results of Y2 H expe riments showed that there were no interaction between V2 protein and the other 5 pr oteins(V1,C1,C2,C3,and C4),but V2 protein could interact with itself,which sug gesting V2 protein might form different multimers to perform different functions.In o rder to further investigate on the way of V2 protein self-interaction,3 truncated muta nts of V2 protein were obtained according to the results of online motif scan analysis and their effects on the interaction with V2 protein were tested using Y2 H assay.The results revealed that the motif of 1 to 78 aa of V2 protein is essential for its self-interaction.During the process of virus infection,host factors are also playing an important role.To clarify the possible host factors interacted with V2 protein,the full length of a ribosomal protein gene(RPS20)encoded by cotton was cloned and sequenced base d on the previous research.Sequence analysis revealed that the protein belongs to 40 S ribosomal protein.Then RPS20 was subcloned to Y2 H vectors and the interaction between V2 protein and RPS20 was tested by Y2 H assay.The result revealed there was a weak interaction between V2 and RPS20,which indicated V2 protein might re gulate RPS20 through direct interaction during the process of virus infection.In this study,V2 protein of a CLCuMuV isolate from Jiangsu was selected for s ubcellular localization,pathogenicity and interaction analysis.The results showed V2 p rotein was localized around the nucleus and cytoplasm.V2 protein could interact with itself and 1-78 aa was essential motif for the self-interaction.A host factor,RPS20,c ould interact with V2 protein.These results provided a preliminary understanding to t he function of CLCuMuV-encoded V2 protein and laid a foundation for further resear ches on the pathogenesis of CLCuMuV.