Protocol

Virus-Induced Gene Silencing as a Tool for Delivery of dsRNA into Plants

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA

¹Corresponding author (savithramma.dinesh-kumar{at}yale.edu)

INTRODUCTION

The inherent RNA silencing mechanism in plants has been effectively manipulated as a tool for the targeted down-regulation of genes. Numerous methods have been employed to initiate this homology-based RNA degradation process, but all rely on the activity of double-stranded RNAs (dsRNAs) corresponding to the gene of interest. Virus-induced gene silencing (VIGS) has gained acceptance as the tool of choice for transient induction of silencing. It involves creation of engineered viruses carrying sequences corresponding to the host gene to be silenced. Infection leads to synthesis of viral dsRNA, an intermediate step in viral replication. This activates the anti-viral RNA silencing pathway, resulting in down-regulation of the host gene transcript. While several VIGS vectors have been developed, the Tobacco Rattle Virus (TRV) provides the most robust results in terms of efficiency, ease of application, and absence of disease symptoms. Engineered TRV vectors carrying host-derived segments are transformed into Agrobacterium tumefaciens, which is then introduced into the plant. This protocol outlines a simple procedure for introducing the TRV-based binary vectors pTRV1 and pTRV2 into solanaceous plants such as Nicotiana benthamiana and Solanum lycopersicon (tomato), as well as Arabidopsis.