A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco

Abstract

Two major classes of small noncoding RNAs have emerged as important regulators of gene expression in eukaryotes, the short interfering RNAs (siRNAs) associated with RNA silencing and endogenous micro-RNAs (miRNAs) implicated in regulation of gene expression. Helper component-proteinase (HC-Pro) is a viral protein that blocks RNA silencing in plants. Here we examine the effect of HC-Pro on the accumulation of siRNAs and endogenous miRNAs. siRNAs were analyzed in transgenic tobacco plants silenced in response to three different classes of transgenes: sense-transgenes, inverted-repeat transgenes, and amplicon-transgenes. HC-Pro suppressed silencing in each line, blocking accumulation of the associated siRNAs and allowing accumulation of transcripts from the previously silenced loci. HC-Pro-suppression of silencing in the inverted-repeat- and amplicon-transgenic lines was accompanied by the apparent accumulation of long double-stranded RNAs and proportional amounts of small RNAs that are larger than the siRNAs that accumulate during silencing. Analysis of these results suggests that HC-Pro interferes with silencing either by inhibiting siRNA processing from double-stranded RNA precursors or by destabilizing siRNAs. In contrast to siRNAs, the accumulation of endogenous miRNAs was greatly enhanced in all of the HC-Pro-expressing lines. Thus, our results demonstrate that accumulation of siRNAs and miRNAs in plants can be differentially regulated by a viral protein. The fact that HC-Pro affects the miRNA pathway raises the possibility that this pathway is targeted by plant viruses as a means to control gene expression in the host.

Fig 1.

HC-Pro suppression of IR- and amplicon-induced RNA silencing prevents the accumulation of siRNAs and results in accumulation of a new size class of small RNAs. (A) Diagram of the transgene loci in lines 6b5, T4, and 155 as predicted by DNA blot analysis. 35S indicates the position of the cauliflower mosaic virus 35S promoter, and nos indicates the position of the nopaline synthase terminator. Arrows indicate the direction of transcription in line T4 based on the predicted arrangement of the 35S promoters (52). The amplicon transgene in line 155 encodes a PVX complementary DNA in which the coat protein gene is replaced by the GUS locus. The PVX viral ORFs encode RdRp and three movement proteins, p25 (25), p12 (12), and p8 (8) (23). (B) RNA gel blot analysis of small RNAs from silenced transgenic lines and lines in which silencing has been suppressed by HC-Pro. Lanes 1–4 and 10 show small RNAs from IR-silenced tobacco line T4 (lanes 1 and 2; lane 1 is a longer exposure of lane 2), sense transgene-silenced tobacco line 6b5 (lanes 3 and 10), and an amplicon-silenced tobacco line 155 (lane 4). Lanes 5–7, 9, and 11 show small RNAs from an IR line expressing HC-Pro (T4 × HC-Pro, lane 5), a sense transgene line expressing HC-Pro (6b5 × HC-Pro, lanes 6 and 11), and an amplicon line expressing HC-Pro (155 × HC-Pro, lane 7; lane 9 is a shorter exposure of lane 7). The probe was ³²P-labeled RNA corresponding to the sense strand of the 3′ 700 nt of the GUS-coding sequence and detects anti-sense strand GUS RNAs. The migration of 21-, 23-, and 26-nt DNA oligomers is shown in lane 8. EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control. Low molecular weight RNA (20 μg) was loaded in each lane, except for lanes 7 and 9 (155 × HC-Pro), in which 5 μg was loaded, and lane 11, in which 240 μg was loaded. (C) RNA gel blot analysis of small RNAs from the same samples shown in B. The probe was ³²P-labeled RNA corresponding to the anti-sense strand of the 3′ 700 nt of the GUS-coding sequence and detects sense-strand, GUS small RNAs. EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control. Low molecular weight RNA (20 μg) was loaded, except for lanes 6 and 7 (155 × HC-Pro), in which 7 μg was loaded. (D) The 5′ phosphorylation status of the 25- to 27-nt larger small RNAs. Small RNAs were isolated from the HC-Pro-amplicon transgenic line (155 × HC-Pro) and treated with CIP and polynucleotide kinase (kinase) as indicated, and sizes of the resulting RNAs were analyzed by RNA gel blot analysis. The migration of 21-, 23-, and 26-nt DNA oligomers is indicated.

Fig 2.

HC-Pro suppression of both IR and amplicon but not sense transgene-induced RNA silencing results in the accumulation of full-length GUS dsRNA. (A) RNA gel blot showing the level of GUS RNA before and after RNase A digestion in silenced lines T4 (lanes 1 and 2), 155 (lanes 3 and 4), 6b5 (lanes 5 and 6), and a GUS-expressing control line T19 (lanes 7 and 8). Total RNA (25 μg) was digested for each plant line. EtdBr staining of 25S rRNA is shown as a loading control. (B) RNA gel blot showing the level of GUS RNA before and after RNase A digestion in plant lines T4 × HC-Pro (lanes 1–3), 155 × HC-Pro (lanes 4–6), 6b5 × HC-Pro (lanes 7–9), and the GUS expressing line T19 × HC-Pro (lanes 10–12). The position of GUS viral RNA and subgenomic RNAs (sgRNAs) is indicated. Total RNA (25 μg) was digested for each plant line. Heat refers to boiling the samples immediately before RNase A digestion to denature dsRNA. EtdBr staining of 25S rRNA is shown as a loading control. (C) RNA gel blot showing the level of GUS mRNA before and after RNase A digestion in silenced line 6b5 (lanes 1–3) and the unsilenced line 6b5 × HC-Pro (lanes 4–6). Total RNA (100 μg) was digested for each plant line, and 10 μg of total RNA was used for the untreated sample. The heat control is described in B. EtdBr staining of 25S rRNA is shown as a loading control.

Fig 3.

HC-Pro expression leads to increased miRNA accumulation in tobacco. (A) RNA gel blot analysis of 20 μg of small RNAs from leaf tissue of the silenced line 6b5 (lanes 1, 3, 5, 7, and 9), the HC-Pro-expressing line 6b5 × HC-Pro (lanes 2, 4, 6, and 8), and the GUS-expressing control line T19 (lane 10). The specific probe used to detect each miRNA is noted (miR167, miR164, miR156, and miR171). The migration of 21- and 25-nt DNA oligomers is shown on the left, and EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control. (B and C) RNA gel blot analysis of miR167 and miR164 miRNAs, extracted from flower (F), leaf (L), and stem (St) tissue of a control line Xanthi (lanes 1–3) and the HC-Pro-expressing line X-27-8 (lanes 4–6). The migration of 21- and 25-nt DNA oligomers is shown on the left, and EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control.