Study On Interactions Of Anti-virus Protein Y3 And TMV CP In Vivo

Tobacco mosaic virus(TMV)is RNA virus that has varied hosts other than the tobacco plant,including more than 310 kinds of plants.Therefore,there is an urgent need to seek antiviral agents to control TMV.It is particularly green and effective to explore the natural and biological products for the control of tobacco mosaic virus,which is related to the environmental pollution and crop's phytotoxicity caused by some chemical agents.For nearly 10 years,many scholars have found that plants or fungal extracts have resistance to TMV.Protein Y3,isolated and purified from the edible mushroom Coprinus comatus,furnishes resistance to the tobacco mosaic virus(TMV).Y3 has a certain effect to degrade the viral capsid in vitro,inhibiting the synthesis of viral proteins,so that Y3 can inhibit the infection of the virus.To further verify that there is an interaction of Y3 with CP in tobacco cell,in this study,we used the bimolecular fluorescence complementation technique(BiFC)to detect the interaction of proteinY3 and CP.Firstly,the y3 gene cDNA sequence were introduced into the Bi FC carrier pSPYNE(R)173,CP gene cDNA sequence were ligated with the BiFC carrier pSPYCE(M)-CP,so the recombinant plasmid pSPYNE(R)173-y3 and pSPYCE(M)-CP were successfully constructed,then they were transformed into Agrobacterium tumefaciens GV3101 and introduced into tobacco leaf epidermal cells by infiltration injection.The fluorescence signal was observed by laser confocal microscopy,indicating that Y3 protein interacted with TMV CP in tobacco cells.In order to study the interactive site of Y3 protein and TMV CP,the related functional domain of Y3 protein was analyzed by protein sequence analysis software.It was found that the position at 23 th to 26 th amino acid was CK2 phosphorylation site,Serine(TCA)of the Y3 protein fused to the fluorescent vector pSPYNE(R)173 was changed to alanine(GCA),then the obtained mutant and the fusion vector pSPYCE(M)-CP were introduced into tobacco leaf epidermal cells.The fluorescence signal was observed by laser confocal microscopy,showing that the mutation did not affect the interaction between Y3 protein and TMV CP.The above results indicate that theY3 protein indeedly interacts with the TMV CP in the tobacco cell,and the only mutations of the CK2 phosphorylation site do not prevent the Y3 protein from interacting with the TMV CP,which has laid a good foundation for further study of its site of interaction and a clear mechanism of action.