Analysis of Antiviral Resistance Signaling Pathways by Virus-Induced Gene Silencing in Nicotiana benthamiana.

Owing to the absence of antiviral chemicals, strategies to deploy antiviral resistance in plants are desirable. Deciphering antiviral resistance mechanisms has been extensively promoted by virus-induced gene silencing (VIGS) technique, which decreases the transcriptional level of the gene of interest via RNA silencing machinery triggered by the partial gene fragment inserted into a viral vector. This technique has contributed to addressing the function of a number of host genes, which are involved in signaling pathways leading to Resistance (R) protein-mediated resistance and the viral disease symptoms such as systemic necrosis. Here we describe the general VIGS protocol and its tips to analyze antiviral resistance mechanism using Tobacco rattle virus (TRV)-based VIGS vector in tobacco plant Nicotiana benthamiana. In most cases, the knockdown of host genes by TRV vector is highly efficient and uniform at the whole plant level but is not associated with any severe symptoms. Using this method together with inoculation of a challenge virus and transient overexpression of a viral elicitor or another host signaling component by agroinfiltration, as well as measurement of hallmarks of antiviral responses, we can address the role of the host factors and the epistatic relationship between several host factors in the resistance signaling pathway against plant virus infection. The protocol described here using the highly susceptible host N. benthamiana to a variety of plant viruses provides the opportunity to study resistance mechanisms underlying many host-virus interactions.