Engineering Encapsidated TRV1 as a Complete VIGS Platform

Tobacco rattle virus (TRV) is a bipartite single-stranded RNA virus that encodes a replicase, movement protein, and silencing suppressor on TRV1 and a capsid protein on TRV2. Researchers typically insert target silencing sequences into TRV2 and coexpress this with TRV1 to achieve virus-induced gene silencing (VIGS). However, TRV1 does not require TRV2 for mobility or replication within a plant host. With this knowledge, we engineer TRV1 alone as a self-replicating RNA (srRNA) that moves systemically throughout plants for targeted gene repression of up to 89%. As TRV1 is encapsidated in trans by the capsid protein encoded on TRV2, we demonstrate the ability to encapsidate our TRV1 srRNAs for application to target plants by coexpression of the capsid protein off a nonviral expression vector. The subsequent encapsidated TRV1 srRNA can then be harvested and applied to new plants by using a simple spray-on application. Since the RNA for the capsid does not accompany the TRV1 srRNA, our srRNAs are incapable of spreading from plant to plant after the initial application. Minimal to no phenotypic penalties were observed when we used our spray-on srRNA approach. To our knowledge, this is the first demonstration of engineering a sprayable TRV1-based srRNA that is highly capable of repressing target genes. As TRV has a broad host range and our encapsidated srRNAs are unlikely to persist in the environment, we envision that this platform can be used for targeted gene silencing in agriculture.