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GrainGenes Reference Report: TPJ-34-543

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Reference	TPJ-34-543
Title	Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats
Journal	Plant Journal
Year	2003
Volume	34
Pages	543-553
Author	Lacomme C Hrubikova K Hein I
Abstract	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
Keyword	[ Hide all but 1 of 43 ] antisense arabidopsis barley barley stripe mosaic virus base cdna degradation dna double stranded rna dsrna fragments gene gene silencing genomic gfp green fluorescent protein hairpin host interference inverted-repeat mechanism molecule molecules mosaic virus nicotiana benthamiana orientation phytoene phytoene desaturase protein reduction rt-pcr sequence suppression tissue tobacco tobacco mosaic virus tool transgene transient vector vectors virus virus-induced gene silencing

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