Modification of Tobacco rattle virus RNA1 to Serve as a VIGS Vector Reveals That the 29K Movement Protein Is an RNA Silencing Suppressor of the Virus

Abstract

Tobacco rattle virus (TRV) has a bipartite, positive-sense single-stranded RNA genome and is widely used for virus-induced gene silencing (VIGS) in plants. RNA1 of TRV that lacks the gene for the cysteine-rich 16K silencing-suppression protein infects plants systemically in the absence of RNA2. Here, we attempted to engineer RNA1 for use as a VIGS vector by inserting heterologous gene fragments to replace 16K. The RNA1 vector systemically silenced the phytoene desaturase (PDS) gene, although less efficiently than when the original VIGS vector system was used, which consists of wild-type RNA1 and engineered RNA2 carrying the heterologous gene. Infectious RNA1 mutants with a dysfunctional 16K suppressed silencing and enhanced transgene expression in green fluorescent protein-transgenic Nicotiana benthamiana following inoculation by agroinfiltration, unlike mutants that also lacked 29K, a movement protein (MP) gene. The 30K MP gene of Tobacco mosaic virus complemented in cis the movement defect but not the silencing suppression functions of TRV 29K. Silencing suppression by 29K occurred in the context of RNA1 replication but not in an agroinfiltration assay which tested 29K alone for suppression of sense-mediated silencing. Both 29K and 16K were needed to avoid necrotic symptoms in RNA1-infected N. benthamiana. The results shed new light on virulence factors of TRV.