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

Christophe Lacomme, Katarina Hrubikova, Ingo Hein

Research output: Contribution to journalArticlepeer-review

140 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)543-53
Number of pages11
JournalPlant Journal
Volume34
Issue number4
DOIs
Publication statusPublished - 9 May 2003

Keywords

  • Base Sequence
  • Gene Expression Regulation, Plant
  • Gene Silencing
  • Genes, Plant/genetics
  • Genetic Vectors/genetics
  • Hordeum/genetics
  • Molecular Sequence Data
  • Plants, Genetically Modified
  • RNA, Double-Stranded/genetics
  • RNA, Plant/analysis
  • Repetitive Sequences, Nucleic Acid/genetics
  • Tobacco/genetics
  • Tobacco Mosaic Virus/genetics
  • Transgenes/genetics

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