Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses

Abstract

RNA silencing is an evolutionarily conserved surveillance system that occurs in a broad range of eukaryotic organisms. In plants, RNA silencing acts as an antiviral system; thus, successful virus infection requires suppression of gene silencing. A number of viral suppressors have been identified so far; however, the molecular bases of silencing suppression are still poorly understood. Here we show that p19 of Cymbidium ringspot virus (CymRSV) inhibits RNA silencing via its small RNA-binding activity in vivo. Small RNAs bound by p19 in planta are bona fide double-stranded siRNAs and they are silencing competent in the in vitro RNA-silencing system. p19 also suppresses RNA silencing in the heterologous Drosophila in vitro system by preventing siRNA incorporation into RISC. During CymRSV infection, p19 markedly diminishes the amount of free siRNA in cells by forming p19-siRNA complexes, thus making siRNAs inaccessible for effector complexes of RNA-silencing machinery. Furthermore, the obtained results also suggest that the p19-mediated sequestration of siRNAs in virus-infected cells blocks the spread of the mobile, systemic signal of RNA silencing.

Figure 1

p19 binds silencing-generated 21-nt RNAs in planta. Extracts prepared from upper systemically infected leaves of CymRSV- or Cym19stop-inoculated plants at 6 dpi were immunoprecipitated with either α-p19 or α-CP (control) antibody. Inputs and eluates of IPs were analysed with Northern (A) and Western blotting (B, C). In vitro transcribed internally labelled positive-strand RNA of the CymRSV CP ORF was used as a probe for Northern blot analyses. As a size marker, γ-³²P-ATP-labelled synthetic 21-nt ssRNA was applied. Protein blots were probed with α-p19 (B) and α-CP (C) antibodies, respectively. * indicates p19-bound RNAs shorter than 21 nt, which were generated artificially during the IP. (D) p19-bound 21-nt RNAs are double stranded. Extract of leaves of CymRSV-infected plants first separated on a gel-filtration column at 4°C to reduce nonspecific degradation of p19-bound RNA. p19 containing peak fractions were used to perform IP as in (A). Eluates of IPs were loaded onto a 15% polyacrylamide containing native 1 × TBE gel and analysed with Northern blotting. In vitro transcribed internally labelled positive-strand RNA of CymRSV was used as a probe. Duplexes of 21-nt (synthetic siRNA) and 19-nt (‘blunt' duplex) RNA oligonucleotides and single-stranded 21- and 19-nt synthetic RNA oligonucleotides were used as size markers. Oligonucleotides were labelled with γ-³²P-ATP. The complementary strands of the duplexes were phosphorylated with ATP before annealing.

Figure 2

Characterisation of p19-bound 21-nt dsRNAs. (A) Analyses of p19-bound 21-nt RNAs by quantitative Northern blotting using increasing amounts of a synthetic 21-nt oligonucleotide complementer to the positive strand of CymRSV and RNA prepared from α-p19 IP (Figure 1D). An internally labelled positive strand of CymRSV was used as a probe. (B) Quantification of data obtained in (A). Closed circles, concentration standards; open circle, RNA isolated from α-p19 IP. The line shows the linear fit of the standards calculated with the computer program Microcal Origin 5.00. (C) 21-nt dsRNA from α-p19 IP driving the degradation of the cognate target RNA in the in vitro RNA-silencing system. A final concentration of 16.38–262.2 nMp19-bound 21-nt dsRNAs was added to the reactions. For target RNA, we used the full-length CymRSV₁₋₄₇₃₃ transcript at 100 pM concentration. dsRNA (5 nM) corresponding to the full-length CymRSV was used as a positive control. For negative control, the same system except GFP target RNA at 200 pM and dsGFP350 at 3 nM in lane 2 was used.

Figure 3

Determination of the apparent dissociation constant (K_a) of p19-GST recombinant protein. (A) Representative gel mobility shift assay carried out with 0.17–43.75 nM of p19-GST and a constant amount of (0.144 nM) radioactively labelled synthetic siRNA. (B) Representative plot of direct binding of p19-GST to siRNAs. The curve is best fitted to the indicated sets of data with the computer program Microcal Origin 5.00. The apparent dissociation constant (K_a) is estimated as the concentration of the protein required to give 50% saturation. Note that we do not know the percent of active p19-GST in our protein extract; therefore, we can calculate the apparent dissociation constant (K_a).

Figure 4

p19 suppresses RNA silencing in the heterologous in vitro RNA-silencing system. (A) p19 inhibits RNA silencing in the Drosophila in vitro system in a dose-dependent manner. In the RNA-silencing reaction, dsGFP350 dsRNA was used as inducer at 3 nM concentration and radioactively labelled full-length (725 nt) GFP transcript (200 pM) was added as a target RNA to the Drosophila embryo lysate. In vitro reactions were supplemented with various concentrations of CymRSV p19 expressed as a GST fusion protein (p19) or GST (87.5–2800 nM). Target degradation was monitored on a 1.2% agarose gel. RNA-silencing activity was indicated by loss of target RNA and appearance of the shorter degradation products (degraded target). (B) Effect of p19 on DICER activity in vitro. Radioactively labelled dsGFP350 was added to Drosophila embryo lysate at 3 nM concentration and p19 was used at various concentrations (21.87–2800 nM). dsRNA processing to siRNA was analysed on an 8% polyacrylamide gel. As a size marker, the Decade Marker System (Ambion) was used. (C) p19 inhibits RNA silencing by sequestering siRNAs in vitro. siRNA duplex (siGFP161) at 20 nM was used to induce the degradation of labelled ssGFP350 target RNA (100 pM) in the in vitro RNA-silencing system. Reactions were supplemented with various amounts of p19 (5.46–1400 nM final concentration). Inhibition of RNA-silencing activity is indicated by loss of the 74 base in the size 5′ end cleavage product. As a size marker, the Decade Marker System (Ambion) was used. (D) p19 cannot counteract with active RISC complex. Degradation of the ssGFP350 target RNA (lane 1) was induced by 20 nM siRNA (siGFP161, lane 2), or 500 mM ss siRNA (GFP161AS, lane 3). As indicated by the ±sign, 700 nM of p19 or GST was added simultaneously with siGFP161 or siGFP161AS to inhibit RNA silencing (lanes 4, 6, 7, 9). In lanes 5 and 8, p19 or GST was added 20 min after the addition of siGFP161. Inhibition of RNA silencing activity is indicated by loss of the 74 base in the size 5′ end cleavage product.

Figure 5

siRNAs and p19 are present in the same high molecular weight chromatography fractions of virus-infected plant extracts. The extracts prepared from systemic leaves of CymRSV- or Cym19stop-infected N. benthamiana plants were size separated by the Superdex-200 gel-filtration column, and then fractions were tested for the presence of siRNAs and for p19. (A) Northern blot of RNAs isolated from a half volume of gel-filtration fractions of extract from CymRSV-infected plants. (B) Western blot of the other half of the same fractions shown in panel (A) were probed with an α-p19 antibody. (C) Northern blot of RNA isolated from gel-filtration fractions of Cym19stop-infected plants. RNA gel blots were probed with radioactively labelled in vitro CymRSV transcript. γ-³²P-ATP-labelled 21-nt synthetic RNA oligo was used as a size marker (M) for RNA gel blots. The elution position of protein molecular weight markers for all panels is shown in (C). 669 kDa, thyroglobulin; 440 kDa, ferritin; 150 kDa, aldolase; 66 kDa, bovine serum albumin; 29 kDa, carbonic anhydrase.