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The simplest known plant pathogens are the viroids. Because of their non-coding single-stranded circular RNA genome, they depend on both their sequence and their structure for both a successful infection and their replication. In the recent years, important progress in the elucidation of their structures was achieved using an adaptation of the selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) protocol in order to probe viroid structures in solution. Previously, SHAPE has been adapted to elucidate the structures of all of the members of the family  Avsunviroidae , as well as those of a few members of the family  Pospiviroidae . In this study, with the goal of providing an entire compendium of the secondary structures of the various viroid species, a total of thirteen new  Pospiviroidae  members were probed in solution using the SHAPE protocol. More specifically, the secondary structures of eleven species for which the genus was previously known were initially elucidated. At this point, considering all of the SHAPE elucidated secondary structures, a classification system for viroids in their respective genera was proposed. On the basis of the structural classification reported here, the probings of both the  Grapevine latent viroid  and the  Dahlia latent viroid  provide sound arguments for the determination of their respective genera, which appear to be  Apscaviroid  and  Hostuviroid , respectively. More importantly, this study provides the complete repertoire of the secondary structures, mapped in solution, of all of the accepted viroid species reported thus far. In addition, a classification scheme based on structural hallmarks, an important tool for many biological studies, is proposed.

Classification of the Pospiviroidae based on their structural hallmarks