A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation

Abstract

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

FIG. 1.

Genomic map locations of srPSTVds and PSTVd siRNAs. The single-nucleotide difference between PSTVd^Int (A) and PSTVd^IntU257A (B) is indicated by the arrowhead. The patterns of PSTVd siRNAs produced in vitro are shown in panel C. Blue and red bars represent plus and minus strands, respectively. Dashed lines delineate the boundaries among the five structural domains (see the text for details). T_L, left-terminal domain; T_R, right-terminal domain.

FIG. 2.

Viral suppressors do not affect accumulation of srPSTVds or genomic PSTVd. (A) Transgenic N. benthamiana expressing GFP was inoculated with PSTVd. Infected leaves were infiltrated with Agrobacterium carrying the respective suppressors. Five days later, agroinfiltrated areas were collected for RNA gel blot analysis of srPSTVd, siGFP, genomic PSTVd, and GFP mRNA accumulations. (B) N. benthamiana protoplasts were cotransfected with PSTVd and various suppressors. Accumulations of srPSTVd and PSTVd were examined at 3 days postinoculation. Cotransfections of GFP, dsGFP, and suppressors were performed to confirm suppression activities of the suppressors. CP, TCV coat protein; P19, TBSV P19; HCPro, TEV HC-Pro. C-PSTVd, circular genomic PSTVd; L-PSTVd, linear unit-length PSTVd. RA, ratio of RNA accumulation levels obtained from three biological replicates. rRNA bands in PAGE stained with ethidium bromide served as a loading control. C-PSTVd was used for RA assessment.

FIG. 3.

Processing of structured, linear plus-PSTVd RNAs into srPSTVds by Arabidopsis DCL(s). (A) srPSTVds of ∼21 nt accumulated in PSTVd-transfected Arabidopsis protoplasts. (B) Partially purified Arabidopsis DCLs process miR319 precursor RNAs and PSTVd RNAs into small RNAs of ∼21 nt but not ssGFP RNA. L-PSTVd, linear unit-length PSTVd.

FIG. 4.

Replication of PSTVd is resistant to induced RNA silencing in N. benthamiana protoplasts. (A) PSTVd accumulates at similar levels in the absence or presence of differing amounts of siRNAs. The siRNA sequence corresponds to positions 194 to 215 of the PSTVd genome. (B) PSTVd accumulates at similar levels in the presence of 5 μg of different siRNAs synthesized based on srPSTVds. (C) Accumulation of GFP mRNA decreases with increasing amounts of GFP siRNAs. (D) PSTVd accumulates at similar levels in the absence or presence of differing amounts of dsRNAs. C-PSTVd, circular genomic PSTVd; L-PSTVd, linear unit-length PSTVd. RA, ratio of RNA accumulation levels obtained from three biological replicates. rRNA bands in PAGE stained with ethidium bromide served as a loading control. C-PSTVd was used for RA assessment.

FIG. 5.

srPSTVds are functional in RISC-mediated cleavage of a target RNA. (A) Schematic view of mRNAs produced from DNA constructs used in this experiment. The GFP:Sensor construct contains an ∼35-nt sensor sequence that is complementary to the most abundant srPSTVds. The arrows denote the primers used for RT-PCR analyses. (B) The DNA constructs were introduced by agroinfiltration into noninfected or PSTVd-infected tomato leaves. Three to 4 days after infiltration, GFP protein expression was examined with a fluorescence microscope. (C) RNA levels were determined by RT-PCR. Three biological replicates gave similar results.

FIG. 6.

PSTVd does not possess silencing suppressor activities or elicit endogenous silencing suppressor activities. (A) Silencing against GFP was induced in N. benthamiana protoplasts (upper panel) or in transgenic N. benthamiana (lower panel). PSTVd infection did not suppress the induced silencing, whereas cotransfection of TBSV P19 suppressed silencing. (B) Quantitative comparison of GFP expression in the protoplasts with different treatments. The level of GFP expression without any treatment (control) is set to 1.0. The expression levels with different treatments are expressed relative to this control value. Averages for three biological replicates are presented with standard deviations.

FIG. 7.

PSTVd secondary structure is resistant to RISC-mediated cleavage. N. benthamiana protoplasts were transfected with PVX vectors to express various GFP:PSTVd fusion constructs (full-length, right-half, and lower-half; see the text for details) in the cytoplasm. Silencing was induced by dsGFP or dsPSTVd as indicated. Shown are the GFP protein levels (A) and RNA levels (B). The two bands in the RNA gel blot represent genomic and subgenomic RNAs of PVX. (C) Schematic view of RNA structures of respective fusion constructs. Gray and black lines represent GFP mRNA and PSTVd RNA, respectively. RA, ratio of RNA accumulation levels obtained from three biological replicates. rRNA bands in PAGE stained with ethidium bromide served as a loading control.