Conserved Motifs and Domains in Members of Pospiviroidae.

In 1985, Keese and Symons proposed a hypothesis on the sequence and secondary structure of viroids from the family Pospiviroidae: their secondary structure can be subdivided into five structural and functional domains and "viroids have evolved by rearrangement of domains between different viroids infecting the same cell and subsequent mutations within each domain"; this article is one of the most cited in the field of viroids. Employing the pairwise alignment method used by Keese and Symons and in addition to more recent methods, we tried to reproduce the original results and extent them to further members of Pospiviroidae which were unknown in 1985. Indeed, individual members of Pospiviroidae consist of a patchwork of sequence fragments from the family but the lengths of fragments do not point to consistent points of rearrangement, which is in conflict with the original hypothesis of fixed domain borders.

Advances in Viroid-Host Interactions.

Viroids are small, single-stranded, circular RNAs infecting plants. Composed of only a few hundred nucleotides and being unable to code for proteins, viroids represent the lowest level of complexity for an infectious agent, even below that of the smallest known viruses. Despite the relatively small size, viroids contain RNA structural elements embracing all the information needed to interact with host factors involved in their infectious cycle, thus providing models for studying structure-function relationships of RNA. Viroids are specifically targeted to nuclei (family Pospiviroidae) or chloroplasts (family Avsunviroidae), where replication based on rolling-circle mechanisms takes place. They move locally and systemically through plasmodesmata and phloem, respectively, and may elicit symptoms in the infected host, with pathogenic pathways linked to RNA silencing and other plant defense responses. In this review, recent advances in the dissection of the complex interplay between viroids and plants are presented, highlighting knowledge gaps and perspectives for future research.

Can artificial neural replicators be useful for studying RNA replicators?

Here, I discuss the usefulness of the application of special artificial neural systems - neural replicators - to study viroids - small pathogens that are short replicating RNA sequences. Using special representations of nucleotide sequences in the form of two sequences with binary components - these two sequences are incomplete representations of the same nucleotide sequence - I show that these neural systems of different sizes are replicated in a special way on them. This allows us to extract some useful information about viroids and their structure, motifs, and relationships. This study is only the first attempt to use neural replicators to analyze genetic data.

Dendrobium viroid, a new monocot-infecting apscaviroid.

Viroids are small circular RNA molecules which have been found to infect many dicot species. Only coconut cadang-cadang viroid and coconut tinangaja viroid have been reported so far to infect a monocot (coconut). Data mining in silico has proven an efficient approach to identify new viruses/viroids, and a systematic screen of public transcriptomic data revealed a 648 nucleotides (nt) sequence potentially representing a novel viroid-like RNA in a transcriptome shotgun assembly from Dendrobium officinale. This sequence contained two central conserved regions (CCRs) characteristic of members of the genus Apscaviroid, indicating that the viroid-like RNA is 324 nt in length. The infectivity of dimeric RNA transcripts generated by in vitro transcription of a synthetic cDNA, was demonstrated by directly injecting into the stems of young Dendrobium officinale plants. The presence of this novel viroid, tentatively designated as Dendrobium viroid (DVd), in the inoculated plants was confirmed by 2D-PAGE together with northern hybridization. DVd is predicted to have a rod-like secondary structure containing a CCR and a terminal conserved region (TCR), and phylogenetic analysis shows that it groups with the known members of the genus Apscaviroid. It is most closely related to citrus viroid V (56% nt identity). A field survey revealed a low DVd incidence (0.96%) in Dendrobium species in China. To our best knowledge, DVd is the only viroid known to infect orchids and the third one from monocotyledonous plants.

Current overview on viroid-host interactions.

Viroids are one of the most enigmatic highly structured, circular, single-stranded RNA phytopathogens. Although they are not known to code for any peptide, viroids induce visible symptoms in susceptible host plants that resemble those associated with many plant viruses. It is known that viroids induce disease symptoms by direct interaction with host factors; however, the precise mechanism by which this occurs remains poorly understood. Studies on the host's responses to viroid infection, host susceptibility and nonhost resistance have been underway for several years, but much remains to be done in order to fully understand the complex nature of viroid-host interactions. Recent progress using molecular biology techniques combined with computational algorithms, in particular evidence of the role of viroid-derived small RNAs in the RNA silencing pathways of a disease network, has widened the knowledge of viroid pathogenicity. The complexity of viroid-host interactions has been revealed in the past decades to include, but not be limited to, the involvement of host factors, viroid structural complexity, and viroid-induced ribosomal stress, which is further boosted by the discovery of long noncoding RNAs (lncRNAs). In this review, the current understanding of the viroid-host interaction has been summarized with the goal of simplifying the complexity of viroid biology for future research. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Apple chlorotic fruit spot viroid: a putative new pathogenic viroid on apple characterized by next-generation sequencing.

Viroid-like symptoms were observed in 2016 on apple fruits of the cultivar "Ilzer Rose" in southern Burgenland, Austria. Preliminary molecular biological investigations indicated that the symptoms were caused by a new unknown viroid. Therefore, new primers were designed, and the whole genome sequence of the viroid (354 nt) was determined by next-generation amplicon sequencing using the Illumina MiSeq® platform (San Diego, California, USA). The viroid secondary structure has a rod-like conformation and contains conserved regions (the TCR, CCR upper strand, and CCR lower strand) that are characteristic of members of the genus Apscaviroid. Based on our results and the demarcation criteria for viroids, the tentatively named "apple chlorotic fruit spot viroid" should be considered a putative new member of the genus Apscaviroid.

Molecular variability of apple hammerhead viroid from Italian apple varieties supports the relevance in vivo of its branched conformation stabilized by a kissing loop interaction.

In the absence of protein-coding ability, viroid RNAs rely on direct interactions with host factors for their infectivity. RNA structural elements are likely involved in these interactions. Therefore, preservation of a structural element, despite the sequence variability existing between the variants of a viroid population, is considered a solid evidence of its relevant role in vivo. In this study, apple hammerhead viroid (AHVd) was first identified in the two apple cultivars 'Mela Rosa Guadagno' (MRG) and 'Agostinella' (AG), which are cultivated since long in Southern Italy, thus providing the first solid evidence of its presence in this country. Then, the natural variability of AHVd viroid populations infecting MRG and AG was studied. The sequence variants from the two Italian isolates shared only 82.1-87.7% sequence identity with those reported previously from other geographic areas, thus providing the possibility of exploring the impact of this sequence divergence on the proposed secondary structure. Interestingly, all the AHVd sequence variants considered in this study preserved a branched secondary structure stabilized by a kissing-loop interaction, resembling the conformation proposed previously for variants from other isolates. Indeed, most mutations did not modify the proposed conformation because they were co-variations, conversions of canonical into wobble base-pairs, or vice versa, as well as changes mapping at loops. Importantly, a cruciform structural element formed by four hairpins, one of which is implicated in the proposed kissing-loop interaction, was also preserved because several nucleotide changes actually resulted into two, three and up to five consecutive co-variations associated with other changes that did not affect the secondary structure. These data provide very strong evidence for the relevance in vivo of this cruciform structure which, together with kissing-loop interaction, likely contribute to further stabilizing the branched AHVd secondary structure.

In silico survey of the central conserved regions in viroids of the Pospiviroidae family for conserved asymmetric loop structures.

Viroids are the smallest replicative pathogens, consisting of RNA circles (∼300 nucleotides) that require host machinery to replicate. Structural RNA elements recruit these host factors. Currently, many of these structural elements and the nature of their interactions are unknown. All Pospiviroidae have homology in the central conserved region (CCR). The CCR of potato spindle tuber viroid (PSTVd) contains a sarcin/ricin domain (SRD), the only viroid structural element with an unequivocal replication role. We assumed that every member of this family uses this region to recruit host factors, and that each CCR has an SRD-like asymmetric loop within it. Potential SRD or SRD-like motifs were sought in the CCR of each Pospiviroidae member as follows. Motif location in each CCR was predicted with MUSCLE alignment and Vienna RNAfold. Viroid-specific models of SRD-like motifs were built by superimposing noncanonical base pairs and nucleotides on a model of an SRD. The RNA geometry search engine FR3D was then used to find nucleotide groups close to the geometry suggested by this superimposition. Atomic resolution structures were assembled using the molecular visualization program Chimera, and the stability of each motif was assessed with molecular dynamics (MD). Some models required a protonated cytosine. To be stable within a cell, the pKa of that cytosine must be shifted up. Constant pH-replica exchange MD analysis showed such a shift in the proposed structures. These data show that every Pospiviroidae member could form a motif that resembles an SRD in its CCR, and imply there could be undiscovered mimics of other RNA domains.

Parsimonious Scenario for the Emergence of Viroid-Like Replicons De Novo.

Viroids are small, non-coding, circular RNA molecules that infect plants. Different hypotheses for their evolutionary origin have been put forward, such as an early emergence in a precellular RNA World or several de novo independent evolutionary origins in plants. Here, we discuss the plausibility of de novo emergence of viroid-like replicons by giving theoretical support to the likelihood of different steps along a parsimonious evolutionary pathway. While Avsunviroidae-like structures are relatively easy to obtain through evolution of a population of random RNA sequences of fixed length, rod-like structures typical of Pospiviroidae are difficult to fix. Using different quantitative approaches, we evaluated the likelihood that RNA sequences fold into a rod-like structure and bear specific sequence motifs facilitating interactions with other molecules, e.g., RNA polymerases, RNases, and ligases. By means of numerical simulations, we show that circular RNA replicons analogous to Pospiviroidae emerge if evolution is seeded with minimal circular RNAs that grow through the gradual addition of nucleotides. Further, these rod-like replicons often maintain their structure if independent functional modules are acquired that impose selective constraints. The evolutionary scenario we propose here is consistent with the structural and biochemical properties of viroids described to date.

Viability and genetic stability of potato spindle tuber viroid mutants with indels in specific loops of the rod-like secondary structure.

Maintenance of the rod-like structure of potato spindle tuber viroid (PSTVd), which contains over 20 loops and bulges between double-stranded helices, is important for viroid biology. To study tolerance to modifications of the stem-loop structures and PSTVd capacity for mutation repair, we have created 6 mutants carrying 3-4 nucleotides deletions or insertions at three unique restriction sites, EagI, StyI and AvaII. Differences in the infectivity of these in vitro generated PSTVd mutants can result from where the mutations map, as well as from the extent to which the secondary structure of the molecule is affected. Deletion or insertion of 4 nucleotides at the EagI and StyI sites led to loss of infectivity. However, mutants with deletion (PSTVd-Ava-del) or insertion (PSTVd-Ava-in) of 3 nucleotides (221GAC223), at the AvaII site (loop 20) were viable but not genetically stable. In all analyzed plants, reversion to the wild type PSTVd-S23 sequence was observed for the PSTVd-Ava-in mutant a few weeks after agroinfiltration. Analysis of PSTVd-Ava-del progeny allowed the identification of 10 new sequence variants carrying various modifications, some of them having retained the original three nucleotide deletion at the AvaII site. Interestingly, other variants gained three nucleotides in the deletion site but did not revert to the original wild type sequence. The genetic stability of the progeny PSTVd-Ava-del sequence variants was evaluated in tomato leaves (early infection) and in both leaves and roots (late infection), respectively.

Identification of a viroid-like RNA in a lychee Transcriptome Shotgun Assembly.

In the course of a systematic screen of plant transcriptomic data, the sequence of a novel viroid-like RNA was identified in four contigs in a recent Transcriptome Shotgun Assembly (TSA) from lychee in China. Portions of this sequence are closely related to the central conserved region (CCR) and terminal conserved region (TCR) present in members of the genus Apscaviroid, sequences that are important criteria for viroid classification. RT-PCR with two divergent adjacent primers amplified the full 304 nucleotide sequence of this viroid-like RNA which can be folded into the rod-like secondary structure that is a typical feature of viroids in the family Pospiviroidae. Northern-blot hybridization following denaturing PAGE and sequential polyacrylamide gel electrophoresis (sPAGE) revealed the presence of both circular and linear forms of the viroid-like RNA in lychee leaf tissue. Phylogenetic analysis indicates that this RNA groups with known members of the genus Apscaviroid with a maximum pairwise sequence identity of <62%. Field surveys revealed the presence of this viroid-like RNA in most lychee samples collected in China; however, it was not found in the accessions in a germplasm collection of lychees that originated from a wide range of other countries. None of the four herbaceous hosts and one woody host tested were susceptible to infection with the lychee viroid-like RNA via agroinoculation and RNA inoculation. The provisional name lychee viroid-like RNA (LVd-like RNA) is proposed for this tentative new species in the genus Apscaviroid.

The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins.

Avocado sunblotch viroid (ASBVd), the type member of the family Avsunviroidae, replicates and accumulates in chloroplasts. Whether this minimal non-protein-coding circular RNA of 246-250 nt exists in vivo as a free nucleic acid or closely associated with host proteins remains unknown. To tackle this issue, the secondary structures of the monomeric circular (mc) (+) and (-) strands of ASBVd have been examined in silico by searching those of minimal free energy, and in vitro at single-nucleotide resolution by selective 2'-hydroxyl acylation analysed by primer extension (SHAPE). Both approaches resulted in predominant rod-like secondary structures without tertiary interactions, with the mc (+) RNA being more compact than its (-) counterpart as revealed by non-denaturing polyacryamide gel electrophoresis. Moreover, in vivo SHAPE showed that the mc ASBVd (+) form accumulates in avocado leaves as a free RNA adopting a similar rod-shaped conformation unprotected by tightly bound host proteins. Hence, the mc ASBVd (+) RNA behaves in planta like the previously studied mc (+) RNA of potato spindle tuber viroid, the type member of nuclear viroids (family Pospiviroidae), indicating that two different viroids replicating and accumulating in distinct subcellular compartments, have converged into a common structural solution. Circularity and compact secondary structures confer to these RNAs, and probably to all viroids, the intrinsic stability needed to survive in their natural habitats. However, in vivo SHAPE has not revealed the (possibly transient or loose) interactions of the mc ASBVd (+) RNA with two host proteins observed previously by UV irradiation of infected avocado leaves.

Molecular diversity among viroids infecting chrysanthemum in India.

Association of Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) with the Chrysanthemum plants exhibiting severe stunting, distinct yellow leaf mottling, and chlorosis was detected in the main chrysanthemum-growing regions of India. Sequence analysis of 90 cDNA clones obtained for CSVd and CChMVd, representing the chrysanthemum-growing regions of India, revealed the high degree of sequence variation throughout the genome under natural conditions. Additionally, all the analyzed CChMVd clones revealed the presence of UUUC in the tetraloop, a signature of symptomatic variants in susceptible cultivars. Phylogenetic analysis revealed that Indian CSVd is closely related to European isolates from ornamentals, whereas CChMVd clustered along with the isolates reported from the East Asian countries.

Self-assembly Controls Self-cleavage of HHR from ASBVd (-): a Combined SANS and Modeling Study.

In the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the Avsunviroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (-) RNAs thus generated are processed by cleavage to unit-length where ASBVd (-) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (-) and its derived 79-nt HHR (-). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (-) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (-) though with a remoter region from an extension of the HI domain.

Structure and Associated Biological Functions of Viroids.

Mature viroids consist of a noncoding, covalently closed circular RNA that is able to autonomously infect respective host plants. Thus, they must utilize proteins of the host for most biological functions such as replication, processing, transport, and pathogenesis. Therefore, viroids can be regarded as minimal parasites of the host machinery. They have to present to the host machinery the appropriate signals based on either their sequence or their structure. Here, we summarize such sequence and structural features critical for the biological functions of viroids.

Elucidation of the structures of all members of the Avsunviroidae family.

Viroids are small single-stranded RNA pathogens which cause significant damage to plants. As their nucleic acids do not encode for any proteins, they are dependant solely on their structure for their propagation. The elucidation of the secondary structures of viroids has been limited because of the exhaustive and time consuming nature of classic approaches. Here, the method of high-throughput selective 2'-hydroxyl acylation analysed by primer extension (hSHAPE) has been adapted to probe the viroid structure. The data obtained using this method were then used as input for computer-assisted structure prediction using RNA structure software in order to determine the secondary structures of the RNA strands of both (+) and (­) polarities of all Avsunviroidae members, one of the two families of viroids. The resolution of the structures of all of the members of the family provides a global view of the complexity of these RNAs. The structural differences between the two polarities, and any plausible tertiary interactions, were also analysed. Interestingly, the structures of the (+) and (­) strands were found to be different for each viroid species. The structures of the recently isolated grapevine hammerhead viroid-like RNA strands were also solved. This species shares several structural features with the Avsunviroidae family, although its infectious potential remains to be determined.To our knowledge, this article represents the first report of the structural elucidation of a complete family of viroids.

Viroids: survivors from the RNA world?

Because RNA can be a carrier of genetic information and a biocatalyst, there is a consensus that it emerged before DNA and proteins, which eventually assumed these roles and relegated RNA to intermediate functions. If such a scenario--the so-called RNA world--existed, we might hope to find its relics in our present world. The properties of viroids that make them candidates for being survivors of the RNA world include those expected for primitive RNA replicons: (a) small size imposed by error-prone replication, (b) high G + C content to increase replication fidelity, (c) circular structure for assuring complete replication without genomic tags, (d) structural periodicity for modular assembly into enlarged genomes, (e) lack of protein-coding ability consistent with a ribosome-free habitat, and (f) replication mediated in some by ribozymes, the fingerprint of the RNA world. With the advent of DNA and proteins, those protoviroids lost some abilities and became the plant parasites we now know.

Virulence determination and molecular features of peach latent mosaic viroid isolates derived from phenotypically different peach leaves: a nucleotide polymorphism in L11 contributes to symptom alteration.

Symptoms of chlorosis along leaf edges (chlorosis-edge), along leaf veins (chlorosis-vein) and yellowing on peach leaves have been observed for a long history in the field, while the pathological factor(s) responsible for these symptoms remained unknown. Peach latent mosaic viroid (PLMVd) was detected in the leaves collected from three unique phenotypic peach trees showing above mentioned symptoms. The obtained PLMVd isolates were subjected to population structure analyses and biological assays to evaluate their pathogenicity on peach seedlings in an effort to elucidate the relationship between the PLMVd and the symptoms observed on peach trees in China. In addition, molecular features of PLMVd isolates were analyzed to obtain some insight into the structure-function relationships of this viroid. The results revealed that the symptoms of chlorosis-edge and yellowing were indeed incited by PLMVd, and a direct link between the nucleotide polymorphisms and the symptoms of yellowing and chlorosis-edge was established, i.e. residue U338 responsible for the yellowish symptom and C338 responsible for the chlorosis-edge symptom. This study provides an additional proof to endorse a previous proposal that PLMVd pathogenicity determinants reside in L11. The illustrative etiology of the disease, visualization of the symptoms progression and identification of the unique single nucleotide polymorphism possibly involved in the symptom induction will significantly increase understanding of the pathogenic mechanisms of PLMVd and will help in designing control strategies for the resulting disease.

Plant viroids: isolation, characterization/detection, and analysis.

When Diener discovered Potato spindle tuber viroid in 1971 (Diener, Virology 45:411-428, 1971), only a limited number of techniques were available for plant virus detection and purification. Biological assays using indicator hosts showing characteristic symptoms of infection and able to support high levels of viroid replication played a critical role in viroid detection and characterization. Polyacrylamide gel electrophoresis (PAGE) was the first molecular technique to be used for the rapid (2-3 days) identification of viroid-infected plants. Because it is the only diagnostic method that is sequence-independent, PAGE under denaturing conditions continues to play a key role in the identification of new viroids. Starting in the early 1980s, dot blot hybridization began to replace PAGE for routine viroid diagnosis. The first diagnostic protocols based on reverse transcription-polymerase chain reaction (RT-PCR) appeared approximately 10 years later, and much effort has subsequently been devoted to simplifying the sample preparation procedure and identifying group-specific primer pairs. This chapter describes four simple, easy-to-follow protocols-two involving PAGE and two others based on enzymatic amplification of viroid cDNAs-that currently play key roles in viroid discovery and characterization.