The Interaction Between The Coat Protein Of Cucumber Green Mottle Mosaic Virus And Proteins Of Nicotiana Benthamiana

Cucumber green mottle mosaic virus (CGMMV), the type member of the genus Tobamovirus, is one of the most important causal agent of diseases affecting Cucurbitaceae crops and is repeatedly epidemic in China, leading to huge economic losses. The biological functions of virus-encoded proteins and management of virus were studied during the past years. But virus-host interactions were little known. So it was essential to study the interaction between coat protein of virus-encoded and Nicotiana benthamiana.In this study, total RNA was extracted from leaves of cucumber infected by CGMMV. CGMMV-CP gene was amplified by RT-PCR and then inserted into the vector pMD18-T. After the sequence was verified, the gene was inserted into pDBleu, creating the recombinant yeast plasmid pDB-CP. The recombinant plasmid pDB-CP was transformed into yeast strain MaV203 and the transformants were used to make transformation-competent yeast cells. Then these cells were transformed with plasmids of the Nicotiana benthamiana cDNA library.The transformants were plated on different dropout nutrient mediums andβ-galactosidase activity was detected. The result was that only one positive clone homologous to ribosomal protein L14 of Nicotiana benthamiana was obtained.In order to indentify whether full-length ribosomal protein L14 of Nicotiana benthamiana could interact with CP, the RPL14 gene was inserted into the vector pDEST22, creating the recombinant yeast plasmid pDEST22-RPL14. The recombinant plasmid pDB-CP and pDEST22-RPL14 were transformed into yeast strain MaV203. The transformants were plated on different dropout nutrient mediums andβ-galactosidase activity was detected. The result showed that CP could interact with ribosomal protein L14. And, the interaction between CP and RPL14 was verified by Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation(co-IP). The cellular localization studies showed that RPL14 accumulated in nucleus and cytoplasmic granule and CP accumulated in cytoplasm and nucleus.