Enhancement of virus‐induced gene silencing through viral‐based production of inverted‐repeats

Summary Plant virus‐based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology‐dependent RNA degradation mechanism. This phenomenon, called virus‐induced gene silencing (VIGS), has potential as a powerful reverse‐genetics tool in functional genomic programmes through transient, loss‐of‐function screens. Here, we describe a method to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production, a critical step in the VIGS response. Incorporation of 40–60 base direct inverted‐repeats into a plant viral vector generates RNA molecules that form dsRNA hairpins. A tobacco mosaic virus (TMV)‐based vector carrying such inverted‐repeats, homologous to a green fluorescent protein ( gfp ) transgene or an endogenous phytoene desaturase ( pds ) gene, generated a stronger and more pervasive VIGS phenotype than constructs carrying corresponding cDNA fragments in sense or antisense orientation. Real‐time RT‐PCR indicated that there was up to a threefold reduction in target mRNA accumulation in the tissues where VIGS was triggered by constructs carrying inverted‐repeats compared to those where it was triggered by sense or antisense constructs. Moreover, an enhanced VIGS pds phenotype was observed using a different vector, based on barley stripe mosaic virus, in the monocotyledonous host barley. This demonstrates that VIGS can be significantly improved through the inclusion of small inverted‐repeats in plant virus‐based vectors, generating a more robust loss‐of‐function phenotype. This suggests that dsRNA formation can be a limiting factor in the VIGS phenomenon.