Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1

Abstract

Plant viruses encode RNA silencing suppressors (VSRs) to counteract the antiviral RNA silencing response. Based on in-vitro studies, several VSRs were proposed to suppress silencing through direct binding of short-interfering RNAs (siRNAs). Because their expression also frequently hinders endogenous miRNA-mediated regulation and stabilizes labile miRNA* strands, VSRs have been assumed to prevent both siRNA and miRNA loading into their common effector protein, AGO1, through sequestration of small RNA (sRNA) duplexes in vivo. These assumptions, however, have not been formally tested experimentally. Here, we present a systematic in planta analysis comparing the effects of four distinct VSRs in Arabidopsis. While all of the VSRs tested compromised loading of siRNAs into AGO1, only P19 was found to concurrently prevent miRNA loading, consistent with a VSR strategy primarily based on sRNA sequestration. By contrast, we provide multiple lines of evidence that the action of the other VSRs tested is unlikely to entail siRNA sequestration, indicating that in-vitro binding assays and in-vivo miRNA* stabilization are not reliable indicator of VSR action. The contrasted effects of VSRs on siRNA versus miRNA loading into AGO1 also imply the existence of two distinct pools of cellular AGO1 that are specifically loaded by each class of sRNAs. These findings have important implications for our current understanding of RNA silencing and of its suppression in plants.

Figure 1

Effect of P38 expression on SUC:SUL plants. (A) Phenotypes of wild-type (SUC:SUL) or P38-expressing (SSxP38#1 and #2) SUC:SUL plants. (B) Northern analysis of SUL siRNA (@SUL), miRNA (@173, @390, @159) and trans-acting siRNA (@255, @TAS3) accumulation in wild-type or P38-expressing plants. SUC:SUL plants carrying the double dcl3/dcl4 (SSxdcl3/4) mutations are used as a control for DCL2-dependent 22 nt-long siRNA accumulation. Ethidium bromide staining of ribosomal RNAs (rRNAs) is used as loading control of the gel. Figure source data can be found with the Supplementary data.

Figure 2

Effect of P38 on silencing factors or miRNA targets accumulation. (A) Accumulation of endogenous AGO1, DCL3, DCL4, DRB4 and the miRNA targets DCL1 and CIP4 was assessed by protein blot analysis of wild-type (SS) or P38-expressing (P38) SUC:SUL plants. Arrows indicate the specific band as opposed to crossreacting bands detected by the antibodies used. Equal loading was verified by Coomassie staining of the membrane after western blotting. (B) Quantitative real-time PCR of the miRNA or trans-acting siRNA targets accumulation in the same plants as described in (A). The small RNA that targets each of these endogenous mRNA is indicated under brackets. mRNA levels were normalized to that of Actin2 (At3g18780) and then to the WT plants which were arbitrarily set to 1. Error bars represent standard deviation from two independent experiments involving triplicate PCRs each.

Figure 3

Effect of P38 on small RNA loading into AGO1. Immunoprecipitation experiments were conducted in wild-type or P38-expressing SUC:SUL plants using an AGO1-specific antibody. Total RNA extracted from the respective IPs was subjected to northern analysis using the indicated probes by sequential rounds of probing and stripping the same membrane. The presence of AGO1 in each IP was confirmed by protein blot analysis (data not shown). rRNA: loading control. Figure source data can be found with the Supplementary data.

Figure 4

Companion cell-specific expression of P38 does not suppress cell-to-cell SUL-silencing movement. (A) Leaf phenotypes of companion cell-specific (#1 and #2) or ectopic (#5) expression of P38 in SUC:SUL transgenics. (B) In-situ hybridization of P38 mRNA in transversal sections of leaves depicted in (A). (C) Immunoprecipitation experiments were conducted using an AGO1-specific antibody in control plants (SS), blend of transgenic SUC:P38 T0 or selected T1 line (#1). The presence of AGO1 in each IP was confirmed by protein blot analysis (upper panel). Total RNA extracted from the respective IPs was subjected to northern analysis using the indicated probes.

Figure 5

Differential effect of several VSRs on siRNA and miRNA loading into AGO1. (A) Protein blot analysis of AGO1 accumulation in wild-type or transgenics CHS-RNAi plants expressing various VSRs (namely, TBSV P19, TuMV Hc-Pro, PCV P15 or P15ΔN6 and TCV P38). (B) RNA gel blot analysis of VSRs (upper panel) and Chalcone synthase expression (lower panel) in transgenics CHS-RNAi plants. (C) RNA gel blot analysis of CHS siRNA (@CHS) or miRNA accumulations in total RNA and AGO1 immunoprecipitated fractions from lines characterized in (A) and (B). (D) Immunoprecipitation experiments were conducted in VSR-expressing lines to assess the loading of endogenous inverted repeat-derived siRNA (@IR71) in AGO1. Figure source data can be found with the Supplementary data.

Figure 6

Effect of P19, Hc-Pro, P15 and P38 expression on silencing factors or miRNA targets accumulation. (A) Quantitative real-time PCR of miRNA or trans-acting siRNA targets accumulation in wild-type or VSR-expressing transgenic plants. The small RNA that targets each of these endogenous mRNA is indicated under brackets. mRNA levels were normalized to that of Actin2 (At3g18780) and then to the WT plants which were arbitrarily set to 1. Error bars represent standard deviation from two independent experiments involving triplicate PCRs each. (B) Accumulation of endogenous AGO1, DCL3, DCL4, DRB4 and the miRNA targets DCL1, AGO2 and CIP4 was assessed by protein blot analysis in the same plants as described in (A). Arrows indicate the specific band as opposed to crossreacting bands detected by the antibodies used. Control western blots showing the specificity of the various antibodies are available in Supplementary Figure S2. Equal loading was verified by Coomassie staining of the membrane after western blotting.