Viroids, Satellite RNAs and Prions: Folding of Nucleic Acids and Misfolding of Proteins.

Theodor ("Ted") Otto Diener (* 28 February 1921 in Zürich, Switzerland; † 28 March 2023 in Beltsville, MD, USA) pioneered research on viroids while working at the Plant Virology Laboratory, Agricultural Research Service, USDA, in Beltsville. He coined the name viroid and defined viroids' important features like the infectivity of naked single-stranded RNA without protein-coding capacity. During scientific meetings in the 1970s and 1980s, viroids were often discussed at conferences together with other "subviral pathogens". This term includes what are now called satellite RNAs and prions. Satellite RNAs depend on a helper virus and have linear or, in the case of virusoids, circular RNA genomes. Prions, proteinaceous infectious particles, are the agents of scrapie, kuru and some other diseases. Many satellite RNAs, like viroids, are non-coding and exert their function by thermodynamically or kinetically controlled folding, while prions are solely host-encoded proteins that cause disease by misfolding, aggregation and transmission of their conformations into infectious prion isoforms. In this memorial, we will recall the work of Ted Diener on subviral pathogens.

Cellular roadmaps of viroid infection.

Viroids are single-stranded circular noncoding RNAs that infect plants. According to the International Committee on Taxonomy of Viruses, there are 44 viroids known to date. Notably, more than 20 000 distinct viroid-like RNA sequences have recently been identified in existing sequencing datasets, suggesting an unprecedented complexity in biological roles of viroids and viroid-like RNAs. Interestingly, a human pathogen, hepatitis delta virus (HDV), also replicates via a rolling circle mechanism like viroids. Therefore, knowledge of viroid infection is informative for research on HDV and other viroid-like RNAs reported from various organisms. Here, we summarize recent advancements in understanding viroid shuttling among subcellular compartments for completing replication cycles, emphasizing regulatory roles of RNA motifs and structural dynamics in diverse biological processes. We also compare the knowledge of viroid intracellular trafficking with known pathways governing cellular RNA movement in cells. Future investigations on regulatory RNA structures and cognate factors in regulating viroid subcellular trafficking and replication will likely provide new insights into RNA structure-function relationships and facilitate the development of strategies controlling RNA localization and function in cells.

"Pathomorphogenic" Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco.

Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.

Mining metatranscriptomes reveals a vast world of viroid-like circular RNAs.

Viroids and viroid-like covalently closed circular (ccc) RNAs are minimal replicators that typically encode no proteins and hijack cellular enzymes for replication. The extent and diversity of viroid-like agents are poorly understood. We developed a computational pipeline to identify viroid-like cccRNAs and applied it to 5,131 metatranscriptomes and 1,344 plant transcriptomes. The search yielded 11,378 viroid-like cccRNAs spanning 4,409 species-level clusters, a 5-fold increase compared to the previously identified viroid-like elements. Within this diverse collection, we discovered numerous putative viroids, satellite RNAs, retrozymes, and ribozy-like viruses. Diverse ribozyme combinations and unusual ribozymes within the cccRNAs were identified. Self-cleaving ribozymes were identified in ambiviruses, some mito-like viruses and capsid-encoding satellite virus-like cccRNAs. The broad presence of viroid-like cccRNAs in diverse transcriptomes and ecosystems implies that their host range is far broader than currently known, and matches to CRISPR spacers suggest that some cccRNAs replicate in prokaryotes.

Unraveling the mystery of viroid nuclear import.

Viroids are closed-circular infectious RNAs that are known to infect plants. Despite their small noncoding genome, they have the ability to cause disease. The nuclear import mechanism of nucleus replicating viroids is not well understood. Ma et al. have recently highlighted the route of viroid entry into the nucleus.

Novel Viroid-Like RNAs Naturally Infect a Filamentous Fungus.

To date, viroids have been found to naturally infect only plants, resulting in substantial losses for some crops. Whether viroids or viroid-like RNAs naturally infect non-plant hosts remains unknown. Here the existence of a set of exogenous, single-stranded circular RNAs, ranging in size from 157 to 450 nucleotides, isolated from the fungus Botryosphaeria dothidea and nominated B. dothidea RNAs (BdcRNAs) is reported. BdcRNAs replicate autonomously in the nucleus via a rolling-circle mechanism following a symmetric pathway. BdcRNA infection induces symptoms, because BdcRNAs can apparently modulate, to different degrees, specific biological traits (e.g., alter morphology, decrease growth rate, attenuate virulence, and increase or decrease tolerance to osmotic and oxidative stress) of the host fungus. Overall, BdcRNAs have genome characteristics similar to those of viroids and exhibit pathogenic effects on fungal hosts. It is proposed that these novel fungus infecting RNAs should be termed mycoviroids. BdcRNA(s) may be considered additional inhabitants at the frontier of life in terms of genomic complexity, and represent a new class of acellular entities endowed with regulatory functions, and novel epigenomic carriers of biological information.

A Novel Self-Cleaving Viroid-Like RNA Identified in RNA Preparations from a Citrus Tree Is Not Directly Associated with the Plant.

Viroid and viroid-like satellite RNAs are infectious, circular, non-protein coding RNAs reported in plants only so far. Some viroids (family Avsunviroidae) and viroid-like satellite RNAs share self-cleaving activity mediated by hammerhead ribozymes (HHRzs) endowed in both RNA polarity strands. Using a homology-independent method based on the search for conserved structural motifs of HHRzs in reads and contigs from high-throughput sequenced RNAseq libraries, we identified a novel small (550 nt) viroid-like RNA in a library from a Citrus reticulata tree. Such a viroid-like RNA contains a HHRz in both polarity strands. Northern blot hybridization assays showed that circular forms of both polarity strands of this RNA (tentatively named citrus transiently-associated hammerhead viroid-like RNA1 (CtaHVd-LR1)) exist, supporting its replication through a symmetric pathway of the rolling circle mechanism. CtaHVd-LR1 adopts a rod-like conformation and has the typical features of quasispecies. Its HHRzs were shown to be active during transcription and in the absence of any protein. CtaHVd-LR1 was not graft-transmissible, and after its first identification, it was not found again in the original citrus source when repeatedly searched in the following years, suggesting that it was actually not directly associated with the plant. Therefore, the possibility that this novel self-cleaving viroid-like RNA is actually associated with another organism (e.g., a fungus), in turn, transiently associated with citrus plants, is proposed.

A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription.

Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.

A circular RNA vector for targeted plant gene silencing based on an asymptomatic viroid.

Gene silencing for functional studies in plants has been largely facilitated by manipulating viral genomes with inserts from host genes to trigger virus-induced gene silencing (VIGS) against the corresponding mRNAs. However, viral genomes encode multiple proteins and can disrupt plant homeostasis by interfering with endogenous cell mechanisms. To try to circumvent this functional limitation, we have developed a silencing method based on the minimal autonomously-infectious nucleic acids currently known: viroids, which lack proven coding capability. The genome of Eggplant latent viroid, an asymptomatic viroid, was manipulated with insertions ranging between 21 and 42 nucleotides. Our results show that, although larger insertions might be tolerated, the maintenance of the secondary structure appears to be critical for viroid genome stability. Remarkably, these modified ELVd molecules are able to induce systemic infection promoting the silencing of target genes in eggplant. Inspired by the design of artificial microRNAs, we have developed a simple and standardized procedure to generate stable insertions into the ELVd genome capable of silencing a specific target gene. Analogously to VIGS, we have termed our approach viroid-induced gene silencing, and demonstrate that it is a promising tool for dissecting gene functions in eggplant.

Rolling circle RNA synthesis catalyzed by RNA.

RNA-catalyzed RNA replication is widely considered a key step in the emergence of life's first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here, we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. We observe sustained RCS by a triplet polymerase ribozyme beyond full-length circle synthesis with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularization. Thus, all steps of a viroid-like RNA replication pathway can be catalyzed by RNA alone. Finally, we explore potential RCS mechanisms by molecular dynamics simulations, which indicate a progressive build-up of conformational strain upon RCS with destabilization of nascent strand 5'- and 3'-ends. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

Many organisms today rely on a trio of molecules for their survival: DNA, to store their genetic information; proteins, to conduct the biological processes required for growth or replication; and RNA, to mainly act as an intermediary between DNA and proteins. Yet, how these inanimate molecules first came together to form a living system remains unclear. Circumstantial evidence suggests that the first lifeforms relied to a much greater exrtent on RNA to conduct all necessary biological processes. There is no trace of this 'RNA world' today, but molecular 'fossils' may exist in current biology. Viroids, for example, are agents which can infect and replicate inside plant cells. They are formed of nothing but a circular strand of RNA that serves not only as genetic storage but also as ribozymes (RNA-based enzymes). Viroids need proteins from the host plant to replicate, but scientists have been able to engineer ribozymes that can copy complex RNA strands. This suggests that viroid-like replication could be achieved using only RNA. Kristoffersen et al. put this idea to the test and showed that it is possible to use RNA enzymatic activity alone to carry out all the steps of a viroid-like copying mechanism. This process included copying a viroid-like RNA circle with RNA, followed by trimming the copy to the right size and reforming the circle. These two latter steps could be carried out by a ribozyme that could itself be encoded on the RNA circle. A computer simulation indicated that RNA synthesis on the circle caused increasing tension that could ease some of the barriers to replication. These results increase our understanding of how RNA copying by RNA could be possible. This may lead to developing molecular models of a primordial RNA-based replication, which could be used to investigate early genetic systems and may have potential applications in synthetic biology.

Adaptation of a filter paper method for RNA template preparation for the detection of avocado sunblotch viroid by reverse transcription qPCR.

An easy, rapid and inexpensive method of preparing RNA template for a reverse transcription qPCR assay for avocado sunblotch viroid (ASBVd) is described. This method depends on the principle of reversible binding of viroid RNA to filter paper under different concentrations of monovalent cation. Lysis buffers containing either sodium chloride or lithium chloride were compared, and 1.5 M lithium chloride was shown to be optimal for the adsorption of the viroid RNA to the filter paper. The extraction method was validated using field samples and equivalent yields of viroid RNA were obtained using this method and either a commercial RNA extraction kit or a dsRNA chromatography method. The filter paper method of RNA extraction is ideally suited for the large-scale surveillance for ASBVd.

Rapid diagnostic detection of tomato apical stunt viroid based on isothermal reverse transcription-recombinase polymerase amplification.

Tomato apical stunt viroid (TASVd) is a serious threat to tomato plants that can cause a considerable yield loss. In the present study, two isothermal molecular diagnostic assays based on reverse transcription-recombinase polymerase amplification (RT-RPA) utilizing the AmplifyRP® platform for plant pathogen detection were developed. The results of this research demonstrated distinct specificity of both developed assays, AmplifyRP® Acceler8™ and AmplifyRP® XRT, expressed in the absence of any cross-reaction activity to all total RNA extracts obtained from plants infected with other pospiviroids. The RT-RPA assays detected viroid RNA in 81- and 27-fold dilutions of the original TASVd-infected crude extract for AmplifyRP® Acceler8™ and AmplifyRP® XRT, respectively. The sensitivity tests in serial water dilutions showed the ability of AmplifyRP® Acceler8™ and AmplifyRP® XRT to detect 8 and 80 fg of pure TASVd RNA transcript, respectively. The influence of crude extract on viroid RNA transcript detection was also examined and a decrease of sensitivity of approximately 100-fold for both RT-RPA assays was revealed. To our knowledge, this is the first report describing development of RT-RPA assays to detect TASVd in plants using the AmplifyRP® platform that can be further employed both in laboratory conditions and in the field for on-site diagnosis.

ViroidDB: a database of viroids and viroid-like circular RNAs.

We introduce ViroidDB, a value-added database that attempts to collect all known viroid and viroid-like circular RNA sequences into a single resource. Spanning about 10 000 unique sequences, ViroidDB includes viroids, retroviroid-like elements, small circular satellite RNAs, ribozyviruses, and retrozymes. Each sequence's secondary structure, ribozyme content, and cluster membership are predicted via a custom pipeline optimized for handling circular RNAs. The data can be explored via a purpose-built user interface that features visualizations, multiple sequence alignments, and a portal for downloading bulk data. Users can browse the data by sequence type, taxon, or typo-tolerant search of metadata fields. The database is freely accessible at https://viroids.org.

Structural basis of RNA polymerase inhibition by viral and host factors.

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.

Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli.

RNA interference (RNAi) is a natural mechanism for protecting against harmful genetic elements and regulating gene expression, which can be artificially triggered by the delivery of homologous double-stranded RNA (dsRNA). This mechanism can be exploited as a highly specific and environmentally friendly pest control strategy. To this aim, systems for producing large amounts of recombinant dsRNA are necessary. We describe a system to efficiently produce large amounts of circular dsRNA in Escherichia coli and demonstrate the efficient insecticidal activity of these molecules against Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), a highly damaging pest of corn crops. In our system, the two strands of the dsRNA are expressed in E. coli embedded within the very stable scaffold of Eggplant latent viroid (ELVd), a small circular non-coding RNA. Stability in E. coli of the corresponding plasmids with long inverted repeats was achieved by using a cDNA coding for a group-I autocatalytic intron from Tetrahymena thermophila as a spacer. RNA circularization and large-scale accumulation in E. coli cells was facilitated by co-expression of eggplant tRNA ligase, the enzyme that ligates ELVd during replication in the host plant. The inserted intron efficiently self-spliced from the RNA product during transcription. Circular RNAs containing a dsRNA moiety homologous to smooth septate junction 1 (DvSSJ1) gene exhibited excellent insecticide activity against WCR larvae. Finally, we show that the viroid scaffold can be separated from the final circular dsRNA product using a second T. thermophila self-splicing intron in a permuted form.

Effect of VIRP1 Protein on Nuclear Import of Citrus Exocortis Viroid (CEVd).

Before replicating, Pospiviroidae viroids must move into the plant nucleus. However, the mechanisms of viroid nuclear import are not entirely understood. To study the nuclear import of viroids, we established a nuclear import assay system using onion cell strips and observed the import of Alexa Fluor-594-labeled citrus exocortis viroid (CEVd). To identify the plant factors involved in the nuclear import of viroids, we cloned the Viroid RNA-binding Protein 1 (VIRP1) gene from a tomato cultivar, Seokwang, and heterologously expressed and purified the VIRP1 protein. The newly prepared VIRP1 protein had alterations of amino acid residues at two points (H52R, A277G) compared with a reference VIRP1 protein (AJ249595). VIRP1 specifically bound to CEVd and promoted its nuclear import. However, it is still uncertain whether VIRP1 is the only factor required for the nuclear import of CEVd because CEVd entered the plant nuclei without VIRP1 in our assay system. The cause of the observed nuclear accumulation of CEVd in the absence of VIRP1 needs to be further clarified.

Integrated Proteo-Transcriptomic Analyses Reveal Insights into Regulation of Pollen Development Stages and Dynamics of Cellular Response to Apple Fruit Crinkle Viroid (AFCVd)-Infection in Nicotiana tabacum.

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

Evaluation of screening platforms for virus-like particle production with the baculovirus expression vector system in insect cells.

Recombinant protein and virus-like particle (VLP) production based on the baculovirus expression vector system is fast, flexible, and offers high yields. Independent from the product, a multitude of parameters are screened during process development/optimisation. Early development acceleration is a key requirement for economic efficiency, and µ-scale bioreactor systems represent an attractive solution for high-throughput (HTP) experimentation. However, limited practical knowledge is available on the relevance and transferability of screening data to pilot scales and manufacturing. The main goal of the present study was to evaluate a HTP µ-bioreactor platform with respect to its aptitude as a screening platform mainly based on transferability of results to benchtop bioreactors representing the conventional production regime. Second question was to investigate to what extent the online sensors of the µ-bioreactor contribute to process understanding and development. We demonstrated that transferability of infection screening results from the HTP µ-bioreactor scale to the benchtop bioreactor was equal or better than that from shaker cultivation. However, both experimental setups turned out to be sub-optimal solutions that only allowed for a first and rough ranking with low relevance in the case of absolute numbers. Bioreactor yields were up to one order of magnitude higher than the results of screening experiments.

Citrus exocortis viroid causes ribosomal stress in tomato plants.

Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.

High-Throughput Sequencing Analysis of Small RNAs Derived from Coleus Blumei Viroids.

Characterization of viroid-derived small RNAs (vd-sRNAs) is important to understand viroid-host interactions; however, vd-sRNAs belonging to the genus Coleviroid are yet to be identified and characterized. Herein, we used coleus plants singly infected with coleus blumei viroid (CbVd)-1, -5, or -6 and doubly infected with CbVd-1 and -5 to identify and analyze their vd-sRNAs. We found sense and antisense vd-sRNAs for CbVd-1, -5 and -6, and 22-nt vd-sRNAs were the most abundant; moreover, the 5'-terminal nucleotides (nts) of CbVd-1, -5, and -6 were biased toward U and C, and sRNAs derived from these three viroids were unevenly distributed along their genomes. We also noted that CbVd-5 and -6 share a fragment that forms the right half of the rod-like secondary structure of these viroids, which implied that they generated almost the same type of vd-sRNAs. This finding indicated that vd-sRNA biogenesis is mainly determined by the primary sequence of their substrates. More importantly, we found two complementary vd-sRNAs (22 nt) that were generated from the central conserved region (CCR) of these three viroids, suggesting an important role of CCR in vd-sRNA biogenesis. In conclusion, our results provide novel insight into the biogenesis of vd-sRNAs and the biological roles of CCR.