Restriction-ligation-independent production of a TVCV infectious clone and a TVCV-based gene expression vector

Abstract

Transgenic expression of proteins in plants is central to research and biotechnology, and, often, it is desirable to obtain this expression without altering the nuclear or plastid genomes. Thus, expression vectors based on plant viruses that infect multiple cells are useful; furthermore, they are also advantageous for studies of the life cycle of the virus itself. Here, we report the development of an expression vector based on a Turnip vein-clearing virus (TVCV), a tobamovirus known to easily infect two model plants, Nicotiana benthamiana, and Arabidopsis thaliana. Avoiding restriction digestion, we utilized a restriction-ligation-independent cloning approach to construct an infectious cDNA clone of TVCV from the viral RNA and then to convert this clone to a gene expression vector adapted for Gateway-based recombination cloning for transgene insertion. The functionality of the resulting vector, designated pTVCV-DEST, was validated by the expression of an autofluorescent reporter transgene following agroinoculation of the target plant.