Engineering the 3'-UTR of Tobacco Vein Mottling Virus to Confer Cross-Protection Against Potyviruses.

Tobacco vein mosaic virus (TVMV) is a member of the family Potyviridae. We explored if engineering the 3' noncoding RNA would alter viral infection symptoms and viral infectivity of TVMVwt, which could be used for cross-protection. We engineered an infectious clone of TVMV mutant virus pLX-TVMV58 (TVMV58), which contains a 58-nt 'AT' fragment in the 3'-untranslated region (UTR) of the full-length cDNA infectious clone pLX-TVMVwt (TVMVwt), using a one-step assembly method. We explored whether the mechanism of the attenuated symptoms in Nicotiana benthamiana caused by TVMV58 compared with that by TVMVwt is linked to the stem structure of TVMV58 3'-UTR. The viral infectivity of the mutant virus TVMV58mutA (with three loops in the structure of TVMV58 3'-UTR) was slightly higher than TVMV58, whereas the viral infectivity of the mutant virus TVMV58mutB (with two loops in the structure of TVMV58 3'-UTR) decreased significantly. However, we observed that the mutant virus TVMV58mutC (with no loop in the structure of TVMV58 3'-UTR) or TVMVmutTG lost infectivity. The infectivity of TVMV58 and its mutants was independent of the RNA silencing mechanism, which was supported by a similar infection pattern in NbDCL-silenced plants. Moreover, TVMV58 showed high cross-protection against PVY-Ros1 (potato virus Y isolate carrying the Rosea1 gene) and TuMV-GFP (turnip mosaic virus expressing GFP) in N. benthamiana, but showed no cross-protection effect against tobacco mosaic virus (TMV) infection.