Protocol for triggering plant gene silencing by viral delivery of short RNA

Virus-induced gene silencing (VIGS) leverages viral vectors to deliver 200-400-nt inserts targeting specific genes. Here, we present a protocol for triggering gene silencing by virus-delivered short RNA inserts (vsRNAi). We describe steps for designing 32-nt vsRNAi that simultaneously target Nicotiana benthamiana homeologous genes involved in chlorophyll biosynthesis and assembling them into JoinTRV, a vector system based on tobacco rattle virus (TRV). This approach results in robust gene silencing phenotypes, including visible leaf yellowing and reduced chlorophyll levels. For complete details on the use and execution of this protocol, please refer to García et al.¹.