Although they induce symptoms in plants similar to those accompanying virus infections, viroids have unique structural, functional, and evolutionary characteristics. They are composed of a small, nonprotein-coding, single-stranded, circular RNA, with autonomous replication. Viroid species are clustered into the families Pospiviroidae and Avsunviroidae, whose members replicate (and accumulate) in the nucleus and chloroplast, respectively. Viroids replicate in three steps through an RNA-based rolling-circle mechanism: synthesis of longer-than-unit strands catalyzed by host RNA polymerases; processing to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes; and circularization. Within the initially infected cells, viroid RNA must move to its replication organelle, with the resulting progeny then invading adjacent cells through plasmodesmata and reaching distal parts via the vasculature. To carry out these movements, viroids must interact with host factors. The mature viroid RNA could be the primary pathogenic effector or, alternatively, viroids could exert their pathogenic effects via RNA silencing.

The mutation rates of viroids, plant pathogens with minimal non-protein-coding RNA genomes, are unknown. Their replication is mediated by host RNA polymerases and, in some cases, by hammerhead ribozymes, small self-cleaving motifs embedded in the viroid. By using the principle that the population frequency of nonviable genotypes equals the mutation rate, we screened for changes that inactivated the hammerheads of Chrysanthemum chlorotic mottle viroid . We obtained a mutation rate of 1/400 per site, the highest reported for any biological entity. Such error-prone replication can only be tolerated by extremely simple genomes such as those of viroids and, presumably, the primitive replicons of the RNA world. Our results suggest that the emergence of replication fidelity was critical for the evolution of complexity in the early history of life.

Natural hammerhead ribozymes are mostly found in some viroid and viroid‐like RNAs and catalyze their cis cleavage during replication. Hammerheads have been manipulated to act in trans and assumed to have a similar catalytic behavior in this artificial context. However, we show here that two natural cis ‐acting hammerheads self‐cleave much faster than trans ‐acting derivatives and other reported artificial hammerheads. Moreover, modifications of the peripheral loops 1 and 2 of one of these natural hammerheads induced a >100‐fold reduction of the self‐cleavage constant, whereas engineering a trans ‐acting artificial hammerhead into a cis derivative by introducing a loop 1 had no effect. These data show that regions external to the central conserved core of natural hammerheads play a role in catalysis, and suggest the existence of tertiary interactions between these peripheral regions. The interactions, determined by the sequence and size of loops 1 and 2 and most likely of helices I and II, must result from natural selection and should be studied in order to better understand the hammerhead requirements in vivo .

Summary: How viroids, tiny non‐protein‐coding RNAs (∼250–400 nt), incite disease is unclear. One hypothesis is that viroid‐derived small RNAs (vd‐sRNAs; 21–24 nt) resulting from the host defensive response, via RNA silencing, may target for cleavage cell mRNAs and trigger a signal cascade, eventually leading to symptoms. Peach latent mosaic viroid (PLMVd), a chloroplast‐replicating viroid, is particularly appropriate to tackle this question because it induces an albinism (peach calico, PC) strictly associated with variants containing a specific 12–14‐nt hairpin insertion. By dissecting albino and green leaf sectors of Prunus persica (peach) seedlings inoculated with PLMVd natural and artificial variants, and cloning their progeny, we have established that the hairpin insertion sequence is involved in PC. Furthermore, using deep sequencing, semi‐quantitative RT‐PCR and RNA ligase‐mediated rapid amplification of cDNA ends (RACE), we have determined that two PLMVd‐sRNAs containing the PC‐associated insertion (PC‐sRNA8a and PC‐sRNA8b) target for cleavage the mRNA encoding the chloroplastic heat‐shock protein 90 (cHSP90), thus implicating RNA silencing in the modulation of host gene expression by a viroid. Chloroplast malformations previously reported in PC‐expressing tissues are consistent with the downregulation of cHSP90, which participates in chloroplast biogenesis and plastid‐to‐nucleus signal transduction in Arabidopsis. Besides PC‐sRNA8a and PC‐sRNA8b, both deriving from the less‐abundant PLMVd (−) strand, we have identified other PLMVd‐sRNAs potentially targeting peach mRNAs. These results also suggest that sRNAs derived from other PLMVd regions may downregulate additional peach genes, ultimately resulting in other symptoms or in a more favorable host environment for viroid infection.

Peach latent mosaic viroid (PLMVd), the causal agent of peach latent mosaic disease, has been sequenced and found to be a circular RNA molecule of 337 nucleotide residues, which adopts a branched conformation when it is folded in the model of lowest free energy. PLMVd exhibits limited homologies with other viroids and some satellite RNAs, but it does not have any of the central conserved sequences characteristic of the subgroups of typical viroids. However, a segment of approximately one-third of the PLMVd sequence has the elements required to form in the RNAs of both polarities the hammerhead structures proposed to act in the in vitro self-cleavage of avocado sunblotch viroid (ASBVd) and some satellite RNAs. Plus and minus partial- and full-length RNA transcripts of PLMVd containing the hammerhead structures displayed self-cleavage during transcription and after purification as predicted by these structures. These data are consistent with the high stability of the PLMVd hammerhead structures, more similar to the corresponding structures of some satellite RNAs than to those of ASBVd, and indicate that the self-cleavage reactions of PLMVd are most probably mediated by single hammerhead structures. Our results support the inclusion of PLMVd in a viroid subgroup represented by ASBVd, whose members are characterized by their ability to self-cleave in vitro, and probably in vivo, through hammerhead structures. A consensus phylogenetic tree has been obtained suggesting that PLMVd, together with ASBVd, may represent an evolutionary link between viroids and viroid-like satellite RNAs.