Potato virus X-induced gene silencing in leaves and tubers of potato

Abstract

Virus induced gene silencing (VIGS) is increasingly used to generate transient loss-of-function assays and has potential as a powerful reverse-genetics tool in functional genomic programs as a more rapid alternative to stable transformation. A previously described potato virus X (PVX) VIGS vector has been shown to trigger silencing in the permissive host Nicotiana benthamiana. This paper demonstrates that a PVX-based VIGS vector is also effective in triggering a VIGS response in both diploid and cultivated tetraploid Solanum species. We show that systemic silencing of a phytoene desaturase gene is observed and maintained throughout the foliar tissues of potato plants and was also observed in tubers. Here we report that VIGS can be triggered and sustained on in vitro micropropagated tetraploid potato for several cycles and on in vitro generated microtubers. This approach will facilitate large-scale functional analysis of potato expressed sequence tags and provide a noninvasive reverse-genetic approach to study mechanisms involved in tuber and microtuber development.

Figure 1.

PVX VIGS vector accumulates in a range of Solanum species. a, Schematic representation of a PVX vector in the pGREEN0000 binary plasmid: 35S, 35S promoter of cauliflower mosaic virus; RdRp (165K), PVX 165K RNA-dependent RNA polymerase; TGB 25K, 8K, and 12K, PVX triple gene block movement proteins; CP, viral coat protein gene; NOS, nopaline synthase transcriptional terminator; LB and RB, left and right T-DNA border sequences. Schematic representation of Solanum tuberosum and Nicotiana benthamiana full-length pds cDNAs (black line). The pds cDNA region from S. tuberosum cloned into PVX is represented as a dark gray box. The corresponding region from N. benthamiana is boxed in light gray, sequence stretches of more than 21 nucleotides identical to S. tuberosum are represented as dark gray rectangles. Arrows indicate the position of the primers used for RT-PCR and real time RT-PCR. b, Western-blot analysis of PVX CP protein accumulation in N. benthamiana, S. bulbocastanum, and S. tuberosum cvs in inoculated and systemic upper uninoculated leaves at 15 dpi. c, Nucleotide alignment of pds cDNA region cloned into PVX from S. tuberosum (PDS-St) with N. benthamiana (PDS-Nb). Boxes indicate the identical sequences stretches of more than 21 nucleotides.

Figure 2.

PVX.PDS_AS triggers VIGS in diploid and tetraploid Solanum species. Photobleaching phenotypes observed by 21 dpi on tetraploid S. tuberosum cv Bintje (a and b) and diploid S. bulbocastanum (d) and PVX.GFP control infected plant (c). Close-up on photobleached leaves of S. tuberosum cvs Bintje (f), Stirling (g), Desiree (h), S. bulbocastanum (j), N. benthamiana (l), and on symptomless PVX.GFP infected leaf of cv Bintje (e) and S. bulbocastanum (i). Uninfected N. benthamiana leaf (k).

Figure 3.

Molecular and biochemical characterization of pds VIGS in diploid and tetraploid Solanum species. a, RT-PCR of S. tuberosum cv Bintje pds-silenced and control plants in response to challenge with PVX constructs. Both RT-PCR products corresponding to endogenous pds and ubiquitin mRNAs have been assessed. PCR conditions ranging from 20 to 50 amplification cycles were tested in both cases. Presented here are 30 cycles corresponding to the log-linear phase of amplified PCR product in nonsilenced tissues (challenged PVX.GFP construct). NTC, nontemplate control; replicates are leaves from three different challenged plants. b, Real-time RT-PCR determination of normalized relative amounts of pds mRNA levels in silenced and control plants challenged with PVX.PDS_AS or PVX.GFP constructs (21 dpi). Sampled leaves were cut in half for either RNA extraction (real-time RT-PCR) or phytoene isolation (HPLC analysis). Values are expressed in percentage of normalized pds mRNA related to PVX.GFP control. Values represent the means of at least three leaves from different plants per construct per experiment ±se. For each sample, real-time RT-PCR was carried out in triplicate. c, HPLC analysis of phytoene accumulation induced by PVX.PDS_AS VIGS vector. Typical HPLC profile from S. tuberosum cv Bintje and N. benthamiana is presented here. As before, silenced leaves from plant challenged with PVX.PDS_AS and corresponding leaves from PVX.GFP plants were sampled at 21 dpi. Arrows indicate peaks for both 15 cis-phytoene and all trans-phytoene. d, Quantification of phytoene accumulation in silenced and control Solanum species and N. benthamiana. Values for 15 –cis-phytoene and trans-phytoene are HPLC area units (arbitrary units) and represent the means of at least three leaves from different plants per construct per experiment (se < 20%).

Figure 4.

Systemic pds silencing in tubers and in vitro propagated plants and microtubers. a, Photobleaching phenotypes observed on in vitro propagated S. tuberosum cv Desiree after 3 subcultures (representing 12 weeks of in vitro propagation post-challenge with PVX.PDSas, middle section) or 1 subculture (4 weeks post-challenge with PVX.PDS_AS, right section). PVX.GFP control infected plant is shown on the left section. b, Schematic representation of in vitro culture and generation of in vitro grown microtubers. Time scale in week post-challenge with PVX construct is presented. c, Real-time RT-PCR determination of normalized relative amounts of pds mRNA levels in silenced and control leaves from in vitro grown S. tuberosum cv Desiree plants challenged with PVX.PDS_AS or PVX.GFP constructs. Leaves were harvested after 4 weeks post challenge (28 dpi, subculture 1) or after 12 weeks post challenge (more than 80 dpi, subculture 3). As before, sampled leaves were cut in half for either RNA extraction (real-time RT-PCR) or phytoene isolation (HPLC analysis). Values are expressed in percentage of normalized pds mRNA related to PVX.GFP control. Values represent the means of at least three leaves from five different in vitro plants per construct per experiment ±se. For each sample, real-time RT-PCR was carried out in triplicate. d, Quantification of phytoene accumulation in leaves of in vitro S. tuberosum cv Desiree challenged with PVX.PDS_AS or PVX.GFP. Phytoene accumulation was monitored in norflurazon-treated in vitro plants. Values for 15 cis-phytoene and trans-phytoene are HPLC area units (arbitrary units) and represent the means of at least three leaves from different plants per construct per experiment (se < 20%). e, Quantification of phytoene accumulation in tubers (approximately 15 weeks after challenge with PVX constructs) from silenced (PVX.PDS_AS challenged) and control (PVX.GFP challenged) S. tuberosum cv Desiree and Stirling. Values for 15 cis-phytoene and trans-phytoene are HPLC area units (arbitrary units) and represent at least ten tubers from at least 2 different plants per construct per experiment (se < 20%). f, Quantification of phytoene accumulation in in vitro generated microtubers from silenced and control S. tuberosum cv Desiree, after 8 weeks (subculture 1) or 12 weeks (subculture 2) initial challenge with PVX constructs. Values for 15 cis-phytoene and trans-phytoene are HPLC area units (arbitrary units) and represent 15 to 20 microtubers generated from 4 to 6 different in vitro grown plants per construct per experiment (se < 20%).