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.

Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants

“…TRV RNA1 contains coding sequences for a replicase and two RNAi suppressors, namely 16K, the main suppressor of RNAi [ 34 – 36 ], and 29K that also acts as a viral cell-to-cell and long distance movement protein [ 37 ]. TRV RNA1 can infect plants systemically without RNA2, but RNA2 enhances accumulation of RNA1 in plant tissues by an unknown mechanism [ 37 ]. TRV-M1 is a TRV RNA1 replicon lacking the coding sequence of 16K and is only weakly protected against RNAi [ 37 ].…”

“…TRV RNA1 can infect plants systemically without RNA2, but RNA2 enhances accumulation of RNA1 in plant tissues by an unknown mechanism [ 37 ]. TRV-M1 is a TRV RNA1 replicon lacking the coding sequence of 16K and is only weakly protected against RNAi [ 37 ]. One half in the full-grown leaves of N .…”

“… ( A ) Full-grown leaves of N . benthamiana were co-infiltrated with an Agrobacterium tumefaciens strain expressing PPR3 and a strain expressing the replicon TRV-M1 of Tobacco rattle virus (TRV) RNA1, which lacks the 16K gene coding for an RNAi suppressor [ 76 ], or were infiltrated only with the A . tumefaciens strain expressing TRV-M1 (control).…”

Virus-induced gene silencing is a versatile tool for unraveling the functional relevance of multiple abiotic-stress-responsive genes in crop plants

“…For example, viral vectors have been modified to improve silencing efficiency. Recently, the RNA1 component of the bipartite TRV-vector was modified to serve as a VIGS vector which can infect plants systemically in the absence of RNA2 (Deng et al, 2013). This vector was developed by partially removing the 16K cysteine rich protein.…”

Virus-Based MicroRNA Silencing in Plants

“…TRV-based vectors (Liu et al, 2002b) have been widely applied as virus-induced gene silencing (VIGS) to knock down gene expression in various plant species (Bachan and Dinesh-Kumar, 2012), and they have also been successfully modified for the expression of foreign genes in plants ( MacFarlane and Popovich, 2000). TRV, like all successful viruses, can escape host RNA interference defense and infect host plants systemically because it encodes two weak genesilencing suppressors (Martín-Hernández and Baulcombe, 2008;Deng et al, 2013). However, TRV induces only very mild symptoms in many host plants (Ratcliff et al, 2001), and it does not cause global deregulation of the miRNA regulatory pathway (Martínez-Priego et al, 2008).…”