Characteristics Of CGMMV CP And Its Interaction With Viral Proteins And Host Factors

Cucumber green mottle mosaic virus(CGMMV)is an important an important member of the tobacco mosaic virus,it can infect watermelons,cucumbers and many other crops.The epidemics of this virus caused devastating losses to cucurbit crops recently in China,which had restricted the development of melon crops production.It is reported that many coat proteins(CPs)of tobamovirus play an important role in virus assembly,infectivity and pathogenicity.However,there are still few reports on CGMMV at present.Although CGMMV is a member of tobamovirus,it has low homology and different hosts compared with other members of tobamovirus such as tobacco mosaic virus(TMV),a typical representative of tobamovirus.To reveal the CP function of CGMMV,a CGMMV isolate from Jiangsu Province in 2012 was used in this study.Its CP was characterized andstudied on self-interaction and interaction with movement protein(MP),which could lay a foundation for further research of the CP function and its interaction mechanism with host.To study the characteristics of the CP protein encoded by CGMMV,total RNA was extracted from diseased tissues,and the complete sequence of CP gene was cloned by RT-PCR..Sequence analysis showed that the CP gene comprised 486 nucleotides which encoded a 17.5 kDa protein.Sequence comparison revealed that CP genes of CGMMV isolated from Jiangsu in 2012 had very high sequence similarity with CP gene sequence of other isolates,with some point mutations to a few isolates only.It indicated that the CP protein was relatively conserved(especially between the domestic isolates),and the isolate from Jiangsu were well represented as the research object.For the relevance of a virus protein and its subcellular location,we constructed YFP tagged CP(CP-YFP)and transiently expressed in Nicotiana benthamiana by Agrobacterium infiltration.The subcellular localization of CP was observed by confocal microscope,which showed strong GFP fluorescence signal in nucleus and cytoplasm.It demonstrated that the CP encoded by CGMMV was localized in the nucleus and cytoplasm.As a series of important life activities are mostly accomplished within the cytoplasm,such as replication,transcription and assembly of tobamovirus,this result indicated CP might play different roles in different subcellular position during the process of CGMMV infecting host plant.In the process of interaction between viruses and plants,to successfully infect a host plant,virus proteins are involved in multiple stages of the infection process.So many studies have shown that virus proteins usually have multiple functions,and virus proteins often achieve these functions in many ways,such as the protein interaction.Among the members of tobamovirus,CP is also a multifunctional protein.To analyze the possible interaction mode of CGMMV CP,the CP and MP genes were cloned into yeast vectors,including pGAD-CP,pGBK-CP,pGAD-MP and pGBK-MP.The interactions between CP and CP,CP and MP were studied by yeast two-hybrid system,which showed that all the yeast strains co-transformed with pGAD-CP and pGBK-CP,pGAD-CP and pGBK-MP,pGBK-CP and pGAD-MP,could not grow normally on defective culture medium.These experimental results indicated that there was no self-interaction of CGMMV CP,and no interaction between CP and MP.In the interaction between virus and the plant,,virus proteins can also act with host factors that related to the process of defense to facilitate its infection in addition to its own function.To further reveal host-associated factors that interact with CP proteins,we used yeast two-hybrid system to screen cucumber cDNA library wh CGMMV CP as a bait protein,and finally obtained 28 candidate factors.Their nucleotide sequences were annotated by BLAST(4 of them have no annotated information)and found that they encoded 9 proteins,containing DnaJ?NAD?GAPD?XET and so on.This was the first report of host factors that interacted with CGMMV CP and CP function analysis.The main results were as follows:After analyzing the acquired candidate factors,we chose the xyloglucan endotransglucosylase gene(csXET),for further study.The csXET gene contained only partial fragments in the candidate factors obtained from the cucumber cDNA library,so we extracted cucumber RNA and cloned the entire gene sequence of csXET.Results showed its full-length was 897 bp,encoding a protein with a molecular weight of 33.4 kDa which contained a conserved domain of GH16-XET.Cluster analysis showed that the protein had the highest homology to the remaining cucumber XET,and followed by watermelon.Protein interactions between CP and csXET were confirmed by yeast two-hybrid system.which showed that csXET could interact with CP.Because XET participates in the process of plant lignocellulose degradation and has an effect on the ductility of plant cell wall,it is presumed that CP and its interaction with csXET might be related to the dwarfism of host plants caused by CGMMV infection.