Efficient virus‐induced gene silencing in Arabidopsis using a ‘one‐step’ TYMV‐derived vector

Abstract

). † These authors contributed equally to this work. SummaryVirus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants. This technique exploits recombinant viral vectors harbouring fragments of plant genes in their genome to generate double-stranded RNAs that initiate homology-dependent silencing of the target gene. Several viral VIGS vectors have already been successfully used in reverse-genetics studies of a variety of processes occurring in plants. Here, we show that a viral vector derived from Turnip yellow mosaic virus (TYMV) has the ability to induce VIGS in Arabidopsis thaliana, accession Col-0, provided that it carries an inverted-repeat fragment of the target gene. Robust and reliable gene silencing was observed when plants were inoculated simply by abrasion with intact plasmid DNA harbouring a cDNA copy of the viral genome, thus precluding the need for in vitro transcription, biolistic or agroinoculation procedures. Our results indicate that a 76 bp fragment is sufficient to cause gene silencing in leaves, stems and flowers, and that the TYMV-derived vector also has the ability to target genes expressed in meristematic tissues. The VIGS vector described here may thus represent an ideal tool for improving high-throughput functional genomics in Arabidopsis.

The Development of an Efficient Multipurpose Bean Pod Mottle Virus Viral Vector Set for Foreign Gene Expression and RNA Silencing

“…These results are consistent with those using ALSV in soybean, where the 5# end PDS sense insertion had little PDSsilencing effect (Igarashi et al, 2009). Similar results were also reported in Arabidopsis, where sense insertion in TYMV had minimal effect on GUS transgene and PDS silencing (Pflieger et al, 2008). Here, we found that the antisense orientation generally resulted in stronger silencing phenotypes.…”

“…This difference is likely due to the fact that pBPMV-GFP-BAR is larger than pBPMV-GFP2; thus, we do not expect the double gene expression construct to replicate and move as efficiently and quickly as the smaller single gene expression construct. While the biolistic inoculation is highly efficient, inoculation by direct DNA rubbing may make use of the BPMV vectors even more facile, as was reported for Soybean mosaic virus (SMV; Seo et al, 2009) in soybean and Turnip yellow mosaic virus (TYMV; Pflieger et al, 2008) in Arabidopsis (Arabidopsis thaliana). We evaluated direct DNA rubbing of soybean seedlings for two constructs: pBPMV-P19, which expresses the heterologous TBSV P19 RNA-silencing suppressor; and the RNA-silencing construct pBPMV-PDS-3R, which contains an endogenous PDS gene 3# ORF antisense insertion in the BPMV RNA2.…”

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants

“…Expression of a hairpin-loop dsRNA structure generated a stronger silencing than expression of the corresponding sense or anti-sense cDNA (Waterhouse et al, 1998;Smith et al, 2000;De Buck et al, 2001). It has been reported that insertion of direct inverted-repeat sequences of target genes that could fold as dsRNA hairpin structures strongly enhance VIGS from Tobacco mosaic virus, BSMV (Lacomme et al, 2003), and Turnip yellow mosaic virus (Pflieger et al, 2008). This is true for pFoMV-sgmediated VIGS.…”

<i>Peanut stunt virus</i>-induced gene silencing in white lupin (<i>Lupinus albus</i>)

“…In addition to the insert size and position of target genes, the effect of the cloning orientation of the foreign gene was investigated using white lupin plants. No clear differences were detected between sense and antisense orientations in this study (Figure 4), although antisense fragments have been shown to be more effective for silencing in other VIGS systems (Igarashi et al 2009;Pflieger et al 2008;Zhang et al 2010). …”