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. 2016 Mar 16:6:23082.
doi: 10.1038/srep23082.

RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases

Lydia J R Hunter  1   2 , Samuel F Brockington  1 , Alex M Murphy  1 , Adrienne E Pate  1 , Kristina Gruden  3 , Stuart A MacFarlane  2 , Peter Palukaitis  2   4 , John P Carr  1
Affiliations

RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases

Lydia J R Hunter et al. Sci Rep. .

Abstract

Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.

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Figures

Figure 1
Figure 1. Phylogenetic analysis of plant RDR genes.
(a) Phylogeny of the potato RDR genes with other plant RDRs. White open circles are clade names, black circles are inferred gene duplications, red boxes are Solanum tuberosum sequences and grey boxes are Arabidopsis thaliana sequences. The S. tuberosum RDR sequences found on the Potato Genome Sequencing Consortium (PGSC) database are accompanied by their PGSC coding-sequence number. (b) Rosid-specific RDR7 phylogeny. This enlarged section of (a) shows the Rosid species that possess RDR7 gene sequences.
Figure 2
Figure 2. RTqPCR analysis of StRDR1 expression after salicylic acid (SA) treatment of Pentland Dell potato plants.
Samples were taken at 0 hours (immediately before treatment), 24 hours and 48 hours after SA or control (water) treatment. Control-treated Pentland Dell plants were sprayed with water and the control sample at 0 hours was used as the reference. Cyclophilin was used as the reference gene for the RTqPCR analysis. Significant increases in StRDR1 transcript accumulation were seen at 24 hours and 48 hours after SA treatment (p < 0.005; *). Error bars represent standard error of the mean.
Figure 3
Figure 3. Generation of StRDR1-depleted transgenic potato lines.
PCR of plant genomic DNA to verify the presence of the StRDR1-specific hairpin constructs RNAi1 (a) or RNAi2 (b) in the transformed potato lines. PCR used a primer complementary to a sequence located in the 35S promoter of the T-DNA insert and primers specific to either RNAi 1 and 2, to amplify regions of 535 bp and 550 bp, respectively. DNA from non-transformed (NT) plants and a no template PCR control (negative: -ve) were included in the analysis and control PCR reactions were carried out using primers specific for β-tubulin (expected product of 480 bp). (c) StRDR1 accumulation is efficiently decreased in a potato lines expressing the RNAi1 (Line 1.3) and RNAi2 construct (Line 2.5) (for other examples see Supplementary Fig. S3). Accumulation levels of transcripts of StRDR2 (d) and StRDR6 (e) were not diminished in plants of transgenic lines expressing either RNAi1 or RNAi2 to inhibit StRDR1 expression. Error bars represent standard error of the mean.
Figure 4
Figure 4. TMV infection and StRDR1 transcript accumulation in StRDR1-depleted transgenic potato plants.
(a) RTqPCR was used to determine the relative level of TMV coat protein (CP) RNA (a) in inoculated leaves of non-transformed (NT) and transgenic Line 1.3 plants at 7 days post-inoculation (dpi). RNA was also extracted from mock-inoculated leaves. In each of three independent experiments, TMV CP RNA accumulation in transgenic samples was normalized to that in the non-transformed control. In only one experiment was any significant increase in CP accumulation noted (p = 0.007: *) (b) RTqPCR was used to measure accumulation, relative to that mock-inoculated non-transformed plant, of RDR1 in mock-inoculated and TMV-inoculated plants at 7 dpi in inoculated, mock-inoculated leaves and upper non-inoculated leaves. Error bars represent standard error of the mean.
Figure 5
Figure 5. Accumulation of PVX and PVYO RNA in StRDR1-depleted transgenic potato plants.
RTqPCR was used to determine the relative levels of PVX (a) and PVYO (b) RNA in inoculated leaves of non-transformed (NT) and transgenic Line 1.3 plants at 7 and 10 days post-inoculation, respectively. RNA was also extracted from mock-inoculated leaves. In each of three independent experiments, viral RNA accumulation in transgenic samples was normalized to that in the non-transformed control. Error bars represent standard error of the mean.
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