Discovery of novel hammerhead, twister, and DVRz-associated circular RNAs in Vitaceae, Solanaceae, and Rosaceae.

Viroids and viroid-like exogenous circular RNAs (ex-circRNAs) are small infectious RNA agents capable of autonomous replication within host cells, independent of any helper virus. In this study, we analyzed rRNA-depleted transcriptomic data sets from members of the Vitaceae, Solanaceae, and Rosaceae families to explore the diversity of these subviral elements. Using both homology-based and alignment-free ribozyme detection methods, we recovered 16 known viroids and identified 13 novel viroid-like RNAs from plant metatranscriptomic data sets. Among these, we report two new hammerhead ribozyme-containing viroid-like RNAs-CGHVd-RNA in Physalis peruviana and GPLMVd-RNA in Vitis vinifera-that display distinct secondary structures and limited sequence similarity to known viroids, supporting their classification as new species within the Avsunviroidae family. We also identified a symmetric twister ribozyme-containing ex-circRNA in peach, with validated in vitro self-cleavage activity. Additionally, three ambi-like circular RNAs were detected in grapevine and peach, encoding divergent RNA-dependent RNA polymerases, hairpin (HP), and delta virus ribozymes (DVRz). Finally, a novel Obelisk-like RNA with unique structural features was discovered in the Cape gooseberry. These results reveal the hidden complexity and broad host range of plant-associated viroid-like RNAs and suggest the existence of previously unrecognized subviral lineages with distinct evolutionary origins.IMPORTANCEThis study reveals remarkable diversity in plant-associated subviral RNA populations, identifying 13 novel viroid-like RNAs alongside 16 known viroids. Notable discoveries include new hammerhead ribozyme-containing species-level ex-circRNAs from Cape gooseberry and grapevine, a symmetric twister ribozyme ex-circRNA in peach, and, surprisingly, ambi-like circular RNAs with RNA-dependent RNA polymerases. These findings significantly expand our understanding of the complexity and evolutionary diversity of plant-associated subviral RNAs.