Abstract

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.

Highlights

  • Viroids are plant pathogens found in many agriculturally important cultures [1]

  • The primary goal of this study was to generate the secondary structures of all known viroids species based on probing data at a single nucleotide resolution in solution

  • The adoption of a relatively fast protocol using fluorescently labelled oligonucleotides, and the capillary electrophoretic analysis of the cDNA resulting from the modified RNA templates, made the goal achievable

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Summary

Introduction

Viroids are plant pathogens found in many agriculturally important cultures [1]. They are simple pathogens composed of a single-stranded circular, non-coding RNA genome of 246 to 401 nucleotides (nt). A total of thirty-two distinct species are generally accepted as existing by the scientific community, all of which can be classified into two families [2]. The first of these, the Avsunviroidae, is a small group composed of four species. All possess a hammerhead self-cleaving motif and replicate in the chloroplast through a symmetrical rolling circle mechanism. The type species of this family is the Avocado sunblotch viroid. The second, the Pospiviroidae, includes twenty-eight members that replicate in the nucleus via an asymmetric rolling

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