Analyzing The Interaction Between Tomato Mosaic Virus Coat Protein And Tobacco Ferredoxin I Or IP-L

Tomato mosaic virus (ToMV), a member of the Tobamovirus genus, has a positive-sense single-stranded RNA genome of about 6400 nucleotides in length, which encodes three non-structural proteins and a 17.6-kDa coat protein (CP). ToMV can infect tomato via juice and seed dissemination, causing severe influence on the quality and yield of tomato. Over the past decade, researchers conducted a variety of studies to investigate the biologic and molecular biologic characteristics of ToMV and have accumulated abundant information for the prevention and control of this virus. However, the underlying interaction mechanism between ToMV and host plants was not completely understood. Previous work showed that ferredoxin I (Fd I) and IP-L can interact in vitro with ToMV CP. In view of this point, the interactions of ToMV CP with Tobacco Fd I and IP-L were investigated in this study to gain better understanding on the molecular pathogenic mechanism of ToMV.This paper is an extension of our previous work, in which we demonstrated that ToMV CP and ferredoxin I (FdⅠ) could interact with each other. In this study, yeast two-hybrid technique was adopted to further indentify the minimum interaction binding domain between ToMV CP and Fd I. Firstly, three Fd I deletion mutants (i.e., Fd I△N47, Fd I△N71 and Fd I△N 112) were constructed to yeast expression vector pGADT7. Then, the interactions between pGBKT7-CP and the above-mentioned deletion mutants were analyzed with the yeast two-hybrid system. The results manifested that all the three proteins could interact with pGBKT7-CP. In addition, the interaction degree between Fd△N112 and pGBKT7-CP was weaker relative to those of Fd△N47 and Fd△N71 with pGBKT7-CP. At the same time, the interactions between full length Fd I and the recombinant plasmid pGBKT7-CP△N31, pGBKT7-CP△N54 and pGBKT7-CP△N106 that were preserved in our laboratory were detected by using the yeast two-hybrid system, respectively. The results suggested that all the three deletion mutants could interact with Fd I. In sum, we identified the minimum combining domain for the interaction of ToMV CP and Fd I, bothα-helical domains (residues 54) of ToMV CP and the C-terminal helical region (residues 32) of Fd I are required for the interaction.To determine whether the interaction between ToMV CP and Fd I is specific or not, the interactions between Fd I and the CP of Tobacco mosaic virus (TMV, Cucumber mosaic virus(CMV), Potato virus X (PVX) and Tobacco rattle virus (TRV) preserved in our laboratory were determined by using the yeast two-hybrid system, respectively. Then, the interactions between TMVCP, CMV CP, PVX CP, TRV CP and Fd I were determined by using the yeast two-hybrid system, respectively. The results showed that all the CPs could interact with Fd I, suggesting the non-specific interaction characteristic between ToMV CP and Fd I.To further examine the interaction between ToMV CP and IP-L in tobacco body, three plant expression plasmids, i.e., pSPYCE-35S-Fd, pSPYNE-35S-CP and pSPYCE-35S-IP-L were constructed. Bimolecular fluorescence complementation (BiFC) technique was used to detect the interaction of these plasmids with ToMV CP. The results showed that Fd I and IP-L can interact with ToMV CP in the living cells of tobacco leaves. To detect the expression of host protein in plant after infected by virus, recombinant plasmid pEGX-IP-L was constructed by ligating IP-L gene into the expression vector pEGX-6p-1 and then transferred into-E. coil BL21. The soluble GST-IP-L protein with molecular weight 42.8kDa was successfully expressed in E. coil BL21 induced with 1.0 mrnol·L-1 IPTG at 37℃. Rabbit was immunized with the expressed target peptide as antigen to get an antiserum. The titer of the obtained antiserum was determined to be 1/1 600. Western blot analysis indicated that this antiserum had a high specificity for detecting the expression of virus protein in host plants.