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.