Replicating Potato spindle tuber viroid mediates de novo methylation of an intronic viroid sequence but no cleavage of the corresponding pre-mRNA.

In plants, Potato spindle tuber viroid (PSTVd) replication triggers post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) of homologous RNA and DNA sequences, respectively. PTGS predominantly occurs in the cytoplasm, but nuclear PTGS has been also reported. In this study, we investigated whether the nuclear replicating PSTVd is able to trigger nuclear PTGS. Transgenic tobacco plants carrying cytoplasmic and nuclear PTGS sensor constructs were PSTVd-infected resulting in the generation of abundant PSTVd-derived small interfering RNAs (vd-siRNAs). Northern blot analysis revealed that, in contrast to the cytoplasmic sensor, the nuclear sensor transcript was not targeted for RNA degradation. Bisulfite sequencing analysis showed that the nuclear PTGS sensor transgene was efficiently targeted for RdDM. Our data suggest that PSTVd fails to trigger nuclear PTGS, and that RdDM and nuclear PTGS are not necessarily coupled.