RNA Silencing Mediated By PVY~N CP Gene Is Suppressed By P25 In Transgenic Tobacco

RNA silencing is a defence mechanism involved in degradation of exogenous nucleic acid via sequence-specific interactions. To counteract this, many kinds of viruses have evolved suppressor proteins that help them infect host successfully. PVX is a typical member of Potato virus X (PVX) category, which often results in degradation of Potato. The p25 is movement protein encoded by the virus RNA, which determine the damage degree from the virus infection. Many recent researches showed that p25 was associated with Potato virus X movement, long-distance spreading and the occurrence of system -symptom. The p25 mediated the spread of the virus in plants by the interaction of the movement protein and plant cellularity. Otherwise p25 is the suppressor of RNA-mediated gene silencing. In this study, the recombinant binary vector of pBI121-p25 was transferred into tobacco via Agrobacterium tumefaciens transformation.p25 transgenic plants were hybridized with PVY~N CP transgenic plants, and then the progenies with the p25 and CP transgenes were selected. By Northern blot and virus resistance analysis, we studied the influence of p25 stably expressed in tobacco on RNA silencing-mediated virus resistance. The effect of p25 on plasmodesmata diameter in transgenic tobacco was also detected using transmission scanning electron microscope. The main results are as the following:1. p25 gene was cloned and then constructed into XbaI-SacI sites of plant expression vector pBI121.The recombinant binary vector pBI121-p25 was transferred into tobacco via Agrobacterium tumefaciens-mediated transformation.Western blot analysis indicated that movement proteins were successfully translated in transgenic plantlets.2. The electron microscopic assay suggested that the diameter of plasmodesmata was increased by 3-6 folds in PVX-p25 transgenic plants compared with that of wild-type plants. This result shows that p25 expressed in transgenic plants performs the same function as viral p25. All of them can enhance the size exclusion limit of plasmodesmata. 3. Using RNA-mediated virus resistant transgenic tobaccos as paternal lines, which were hybridized with p25 stably expressed transgenic tobaccos,the hybrid progenies with PVX p25 and PVY~N CP transgenes were obtained.4. Double-transgenic plants inoculated by PVY~N showed symptom in the seventh day post-incubation. ELISA analysis suggested that high-titer PVY~N could be detected in the double-transgenic plants inoculated by PVY~N. We demonstrated that the stably expressed p25 lead double-transgenic plants to lose their virus resistance.5. Northern blot analysis indicated that the level of PVY~N CP RNA of double-transgenic plants was higher than that of paternal lines presenting PTGS. In this study, we demonstrated that the stably expressed p25 in the double-transgenic plants could suppress RNA silencing, and the virus resistance losing of double-transgenic plants was strongly related to RNA silencing suppression by p25.