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ABSTRACT Viroids are single‐stranded circular noncoding RNAs that mainly infect crops. Upon infection, nuclear‐replicating viroids engage host DNA‐dependent RNA polymerase II for RNA‐templated transcription, which is facilitated by a host protein TFIIIA‐7ZF. The sense‐strand and minus‐strand RNA intermediates are differentially localised to the nucleolus and nucleoplasm regions, respectively. The factors and function underlying the differential localisation of viroid RNAs have not been fully elucidated. The sense‐strand RNA intermediates are cleaved into linear monomers by a yet‐to‐be‐identified RNase III‐type enzyme and ligated to form circular RNA progeny by DNA ligase I (LIG1). The subcellular compartment for the ligation reaction has not been characterised. Here, we show that LIG1 and potato spindle tuber viroid (PSTVd) colocalise near the nucleolar region inNicotiana benthamianaprotoplasts. The colocalised region is also the highly condensed region of sense‐strand PSTVd RNA, indicating that PSTVd RNA and LIG1 form a biomolecular condensate for RNA processing. This finding expands the function of biomolecular condensates to the infection of subviral pathogens. In addition, this knowledge of viroid biogenesis will contribute to exploring thousands of viroid‐like RNAs that have been recently identified. 

We developed a new method for simultaneously visualizing RNAs and proteins in plant cells. This method works well for endogenous as well as infectious RNAs and their cognate binding proteins.