Replication of a pathogenic non-coding RNA increases DNA methylation in plants associated with a bromodomain-containing viroid-binding protein.

Viroids are plant-pathogenic molecules made up of single-stranded circular non-coding RNAs. How replicating viroids interfere with host silencing remains largely unknown. In this study, we investigated the effects of a nuclear-replicating Potato spindle tuber viroid (PSTVd) on interference with plant RNA silencing. Using transient induction of silencing in GFP transgenic Nicotiana benthamiana plants (line 16c), we found that PSTVd replication accelerated GFP silencing and increased Virp1 mRNA, which encodes bromodomain-containing viroid-binding protein 1 and is required for PSTVd replication. DNA methylation was increased in the GFP transgene promoter of PSTVd-replicating plants, indicating involvement of transcriptional gene silencing. Consistently, accelerated GFP silencing and increased DNA methylation in the of GFP transgene promoter were detected in plants transiently expressing Virp1. Virp1 mRNA was also increased upon PSTVd infection in natural host potato plants. Reduced transcript levels of certain endogenous genes were also consistent with increases in DNA methylation in related gene promoters in PSTVd-infected potato plants. Together, our data demonstrate that PSTVd replication interferes with the nuclear silencing pathway in that host plant, and this is at least partially attributable to Virp1. This study provides new insights into the plant-viroid interaction on viroid pathogenicity by subverting the plant cell silencing machinery.

Satellite RNA-derived small interfering RNA satsiR-12 targeting the 3' untranslated region of Cucumber mosaic virus triggers viral RNAs for degradation.

RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. The most frequently cloned satsiRNA, satsiR-12, was predicted to imperfectly match to Cucumber mosaic virus (CMV) RNAs in the upstream area of the 3' untranslated region (3' UTR). Moreover, an artificial satsiR-12 (asatsiR-12) mediated cleavage of a green fluorescent protein (GFP) sensor construct harboring the satsiR-12 target site. asatsiR-12 also mediated reduction of viral RNAs in 2b-deficient CMV (CMVΔ2b)-infected Nicotiana benthamiana. The reduction was not observed in CMVΔ2b-infected RDR6i plants, in which RDR6 was silenced. Following infection with 2b-containing CMV, the reduction in viral RNAs was not observed in plants of either genotype, indicating that the asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b protein. Our results suggest that satsiR-12 targeting the 3' UTR of CMV RNAs triggered RDR6-dependent antiviral silencing. Competition experiments with wild-type CMV RNAs and anti-satsiR-12 mutant RNA1 in the presence of 2b and satRNA demonstrate the inhibitory effect of the 2b protein on the satsiR-12-related degradation of CMV RNAs, revealing a substantial suppressor function of the 2b protein in native CMV infection. Our data provide evidence for the important biological functions of satsiRNAs in homeostatic interactions among the host, virus, and satRNA in the final outcome of viral infection.

RNA-dependent RNA polymerase 1 from Nicotiana tabacum suppresses RNA silencing and enhances viral infection in Nicotiana benthamiana.

Endogenous eukaryotic RNA-dependent RNA polymerases (RDRs) produce double-stranded RNA intermediates in diverse processes of small RNA synthesis in RNA silencing pathways. RDR6 is required in plants for posttranscriptional gene silencing induced by sense transgenes (S-PTGS) and has an important role in amplification of antiviral silencing. Whereas RDR1 is also involved in antiviral defense in plants, this does not necessarily proceed through triggering silencing. In this study, we show that Nicotiana benthamiana transformed with RDR1 from Nicotiana tabacum (Nt-RDR1 plants) exhibits hypersusceptibility to Plum pox potyvirus and other viruses, resembling RDR6-silenced (RDR6i) N. benthamiana. Analysis of transient induction of RNA silencing in N. benthamiana Nt-RDR1 and RDR6i plants revealed that Nt-RDR1 possesses silencing suppression activity. We found that Nt-RDR1 does not interfere with RDR6-dependent siRNA accumulation but turns out to suppress RDR6-dependent S-PTGS. Our results, together with previously published data, suggest that RDR1 might have a dual role, contributing, on one hand, to salicylic acid-mediated antiviral defense, and suppressing, on the other hand, the RDR6-mediated antiviral RNA silencing. We propose a scenario in which the natural loss-of-function variant of RDR1 in N. benthamiana may be the outcome of selective pressure to maintain a high RDR6-dependent antiviral defense, which would be required to face the hypersensitivity of this plant to a large number of viruses.