| Cucurbit chlorotic yellows virus(CCYV)(genus Crinivirus,family Closteroviridae)infects cucumber,melon and other melon crops,causing serious economic losses.As a newly discovered virus,its current studies mainly concentrated in the genome sequence,molecular detection and vector transmission,Tne mechanism of interaction between virus and host and the movement protein of CC YV are unclear.The silencing repressor and the movement protein are key proteins for virus infection.Identifying the movement protein encoded by the virus and studying the host factors that interact with the silencing repressor will help to clarify the infection mechanism of the virus.Therefore,this study is carried out from the following two aspects,and the main results are as follows:Preliminary work in the laboratory has identified the interaction between P22 and RPS21 in yeast.In order to further verify the in vivo interaction,we used bimolecule fluorescence complementary(BIFC)and co-localization technology to verify the in vivo interaction between P22 and RPS21.By constructing the deletion mutant of RPS21 and transforming the agrobacterium to infiltrate the tobacco,the nuclear localization signal was determined to be 91-145 amino acids.The interaction domain of RPS21 was defined as N-terminal 1-145 amino acid by yeast co-transformation and BIFC.The interaction domain of P22 was defined by yeast co-transformation as 68-128 amino acids and 129-188 amino acids.The yeast co-transformation results of RPS21 and the other nine viral proteins encoded by CCYV(P4.9,p1-6,p2-6,P9,P26,P59,CP,CPm,and HSP70)showed that RPS21 also interacted with P4.9,p2-6,and P59.In order to verify the interaction between RPS21 and P22 against the activity of silencing suppressor P22,PGD3mycRPS21,PGD3flagP22 and PGDGFP were used to co-infiltrate the Nicotiana benthamiana.PGD3mycGUS was used as controls.Fluorescence intensity analysis and Western Blot were performed 3 days later.The results showed that in the presence of RPS21,the fluorescence intensity of GFP decreased,and the expression level of GFP protein decreased significantly.The expression level of P22 was detected by semi-quantitative RT-PCR,and the results showed that the mRNA level of P22 decreased in the presence of RPS21.By constructing GFP expression vectors of viral proteins,we determined that the subcellular localization of the ten viral proteins encoded by CCYV were all nuclear and cytoplasmic localization,the fluorescent cells of P4.9-GFP、P1-6-GFP、P9-GFP、HSP70-GFP was significantly more than any other fusion protein expression,P4.9,P1-6 and P9 were selected for further study,by co-infiltrating with PGDGFP and taking PGD3mycGUS as the control,the results showed that P4.9,P1-6 and P9 all promoted the diffusion of GFP fluorescence to the surrounding environment;aniline blue staining showed that P4.9 could co-locate with the plasmodesmata;endoplasmic reticulum and golgi body co-localizing results showed that P4.9 could co-localize with endoplasmic reticulum,but not with golgi body. |
