Potatovirus X and Tobacco mosaic virus-based vectors compatible with the Gateway™ cloning system

Abstract

Virus-based expression vectors are important tools for high-level production of foreign proteins and for gene function analysis through virus induced gene silencing. To exploit further their advantages as fast, high yield replicons, a set of vectors was produced by converting and adapting Potato virus X (PVX) and Tobacco mosaic virus (TMV)-based vectors to allow easy cloning of foreign sequences by the Gateway™ cloning system. Target genes were cloned efficiently by recombination and successfully expressed in Nicotiana benthamiana following inoculation by Agrobacterium (agroinfection). Using green fluorescent protein (GFP) as marker, high-level expression with both PVX-GW and TMV-GW vectors was confirmed. A Gateway inserted phytoene desaturase gene ( pds) fragment in PVX-GW and TMV-GW vectors (PVX-GW-PDS and TMC-GW-PDS), induced gene silencing of the endogenous pds gene in N. benthamiana as evidenced by chlorotic leaves. The PVX-GW vector was adapted further by cloning the GFP gene upstream of the Gateway sequences, allowing the easy production of GFP fusions after recombination of a target gene. Subcellular localization of resulting GFP fusion was validated by recombining and expressing the coat protein gene from Tomato chlorotic mottle virus, revealing its nuclear localization. A PVX-GW transient expression assay of a nucleocapsid protein gene fragment of Tomato spotted wilt virus and of a single chain antibody against this protein was shown to confer effective resistance to TSWV infection.