Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats.
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.
Lacomme
Christophe
C
Programme of Cell-to-Cell Communication, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK. clacom@scri.sari.ac.uk
Hrubikova
Katarina
K
Hein
Ingo
I
D001483:Base Sequence; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D017343:Genes, Plant / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D001467:Hordeum / Q000235:genetics; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D012330:RNA, Double-Stranded / Q000235:genetics; D018749:RNA, Plant / Q000032:analysis / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics; D014027:Tobacco Mosaic Virus / Q000235:genetics* / Q000502:physiology*; D019076:Transgenes / Q000235:genetics
D012330:RNA, Double-Stranded; D018749:RNA, Plant