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.

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.

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.

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.

"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.

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.

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.

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.

Combined Activity of DCL2 and DCL3 Is Crucial in the Defense against Potato Spindle Tuber Viroid.

Viroids are self replicating non-coding RNAs capable of infecting a wide range of plant hosts. They do not encode any proteins, thus the mechanism by which they escape plant defenses remains unclear. RNAi silencing is a major defense mechanism against virus infections, with the four DCL proteins being principal components of the pathway. We have used Nicotiana benthamiana as a model to study Potato spindle tuber viroid infection. This viroid is a member of the Pospiviroidae family and replicates in the nucleus via an asymmetric rolling circle mechanism. We have created knock-down plants for all four DCL genes and their combinations. Previously, we showed that DCL4 has a positive effect on PSTVd infectivity since viroid levels drop when DCL4 is suppressed. Here, we show that PSTVd levels remain decreased throughout infection in DCL4 knockdown plants, and that simultaneous knockdown of DCL1, DCL2 or DCL3 together with DCL4 cannot reverse this effect. Through infection of plants suppressed for multiple DCLs we further show that a combined suppression of DCL2 and DCL3 has a major effect in succumbing plant antiviral defense. Based on our results, we further suggest that Pospoviroids may have evolved to be primarily processed by DCL4 as it seems to be a DCL protein with less detrimental effects on viroid infectivity. These findings pave the way to delineate the complexity of the relationship between viroids and plant RNA silencing response.

Alterations of the viroid regions that interact with the host defense genes attenuate viroid infection in host plant.

Understanding in intimate details how the viroid interaction with host's defense genes is a cornerstone for developing viroid resistant plants. In this present study, small RNAs (sRNA) derived from Potato spindle tuber viroid (PSTVd) were studied in silico in order to detect any interactions with the serine threonine kinase receptor, a transmembrane protein that plays a role in disease resistance in plants. Using molecular biology techniques, it was determined that PSTVd infection negatively affects at least three serine threonine kinase receptors as well as with three other genes that are known to be involved in the overall development of the tomato plants. The transient expression of these putative PSTVd-sRNAs, using the microRNA sequence as a backbone, in tomato plants induced phenotypes similar to viroid infection. Mutants created by altering the sequence of PSTVd in these regions failed to infect the tomato plant. The data presented here illustrates the importance of these regions in viroid survival, and suggests a possible avenue of exploration for the development of viroid resistant plants.

Functional analysis of a viroid RNA motif mediating cell-to-cell movement in Nicotiana benthamiana.

Cell-to-cell trafficking through different cellular layers is a key process for various RNAs including those of plant viruses and viroids, but the regulatory mechanisms involved are still not fully elucidated and good model systems are important. Here, we analyse the function of a simple RNA motif (termed 'loop19') in potato spindle tuber viroid (PSTVd) which is required for trafficking in Nicotiana benthamiana leaves. Northern blotting, reverse transcriptase PCR (RT-PCR) and in situ hybridization analyses demonstrated that unlike wild-type PSTVd, which was present in the nuclei in all cell types, the trafficking-defective loop19 mutants were visible only in the nuclei of upper epidermal and palisade mesophyll cells, which shows that PSTVd loop19 plays a role in mediating RNA trafficking from palisade to spongy mesophyll cells in N.benthamiana leaves. Our findings and approaches have broad implications for studying the RNA motifs mediating trafficking of RNAs across specific cellular boundaries in other biological systems.

Circular RNAs with hammerhead ribozymes encoded in eukaryotic genomes: The enemy at home.

A new family of non-autonomous retrotransposons with self-cleaving hammerhead ribozymes, the so called retrozymes, has recently been found encoded in diverse plant genomes. These retroelements can be actively transcribed, and their RNAs accumulate in the cells as abundant non-coding circular RNAs (circRNAs) of small size (600-1000 nt). Related circRNAs with self-cleaving ribozymes had already been described in plants, and belong to a group of infectious RNA agents with an uncertain origin: the viroids and viroid-like satellites of plant RNA viruses. These pathogenic circRNAs show many structural similarities with retrozyme circRNAs, and both have been found to occur in flowering plants as heterogeneous RNA molecules of positive and negative polarities. Taking all these data together, we hypothesize that circRNAs encoded by genomic retrozymes could have given origin to infectious circRNAs with self-cleaving ribozymes. Moreover, we propose that retrozymes in time could have evolved from the ancient family of Penelope-like retroelements, which also harbour hammerhead ribozymes. Putative retrozyme sequences with hammerhead ribozymes have been detected as well in metazoan genomes, opening the door to a common occurrence of circRNAs with self-cleaving motifs among eukaryotes.

Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A "naked" rod-like conformation similar but not identical to that observed in vitro.

With a minimal (250-400 nt), non-protein-coding, circular RNA genome, viroids rely on sequence/structural motifs for replication and colonization of their host plants. These motifs are embedded in a compact secondary structure whose elucidation is crucial to understand how they function. Viroid RNA structure has been tackled in silico with algorithms searching for the conformation of minimal free energy, and in vitro by probing in solution with RNases, dimethyl sulphate and bisulphite, and with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), which interrogates the RNA backbone at single-nucleotide resolution. However, in vivo approaches at that resolution have not been assayed. Here, after confirming by 3 termodynamics-based predictions and by in vitro SHAPE that the secondary structure adopted by the infectious monomeric circular (+) RNA of potato spindle tuber viroid (PSTVd) is a rod-like conformation with double-stranded segments flanked by loops, we have probed it in vivo with a SHAPE modification. We provide direct evidence that a similar, but not identical, rod-like conformation exists in PSTVd-infected leaves of Nicotiana benthamiana, verifying the long-standing view that this RNA accumulates in planta as a "naked" form rather than tightly associated with protecting host proteins. However, certain nucleotides of the central conserved region, including some of the loop E involved in key functions such as replication, are more SHAPE-reactive in vitro than in vivo. This difference is most likely due to interactions with proteins mediating some of these functions, or to structural changes promoted by other factors of the in vivo habitat.

Changes in the DNA methylation pattern of the host male gametophyte of viroid-infected cucumber plants.

Eukaryotic organisms exposed to adverse conditions are required to show a certain degree of transcriptional plasticity in order to cope successfully with stress. Epigenetic regulation of the genome is a key regulatory mechanism allowing dynamic changes of the transcriptional status of the plant in response to stress. The Hop stunt viroid (HSVd) induces the demethylation of ribosomal RNA (rRNA) in cucumber (Cucumis sativus) leaves, leading to increasing transcription rates of rRNA. In addition to the clear alterations observed in vegetative tissues, HSVd infection is also associated with drastic changes in gametophyte development. To examine the basis of viroid-induced alterations in reproductive tissues, we analysed the cellular and molecular consequences of HSVd infection in the male gametophyte of cucumber plants. Our results indicate that in the pollen grain, accumulation of HSVd RNA induces a decondensation of the generative nucleus that correlates with a dynamic demethylation of repetitive regions in the cucumber genome that include rRNA genes and transposable elements (TEs). We therefore propose that HSVd infection impairs the epigenetic control of rRNA genes and TEs in gametic cells of cucumber, a phenomenon thus far unknown to occur in this reproductive tissue as a consequence of pathogen infection.

Specific argonautes selectively bind small RNAs derived from potato spindle tuber viroid and attenuate viroid accumulation in vivo.

The identification of viroid-derived small RNAs (vd-sRNAs) of 21 to 24 nucleotides (nt) in plants infected by viroids (infectious non-protein-coding RNAs of just 250 to 400 nt) supports their targeting by Dicer-like enzymes, the first host RNA-silencing barrier. However, whether viroids, like RNA viruses, are also targeted by the RNA-induced silencing complex (RISC) remains controversial. At the RISC core is one Argonaute (AGO) protein that, guided by endogenous or viral sRNAs, targets complementary RNAs. To examine whether AGO proteins also load vd-sRNAs, leaves of Nicotiana benthamiana infected by potato spindle tuber viroid (PSTVd) were agroinfiltrated with plasmids expressing epitope-tagged versions of AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, AGO7, AGO9, and AGO10 from Arabidopsis thaliana. Immunoprecipitation analyses of the agroinfiltrated halos revealed that all AGOs except AGO6, AGO7, and AGO10 associated with vd-sRNAs: AGO1, AGO2, and AGO3 preferentially with those of 21 and 22 nt, while AGO4, AGO5, and AGO9 additionally bound those of 24 nt. Deep-sequencing analyses showed that sorting of vd-sRNAs into AGO1, AGO2, AGO4, and AGO5 depended essentially on their 5'-terminal nucleotides, with the profiles of the corresponding AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Furthermore, agroexpression of AGO1, AGO2, AGO4, and AGO5 on PSTVd-infected tissue attenuated the level of the genomic RNAs, suggesting that they, or their precursors, are RISC targeted. In contrast to RNA viruses, PSTVd infection of N. benthamiana did not affect miR168-mediated regulation of the endogenous AGO1, which loaded vd-sRNAs with specificity similar to that of its A. thaliana counterpart. Importance: To contain invaders, particularly RNA viruses, plants have evolved an RNA-silencing mechanism relying on the generation by Dicer-like (DCL) enzymes of virus-derived small RNAs of 21 to 24 nucleotides (nt) that load and guide Argonaute (AGO) proteins to target and repress viral RNA. Viroids, despite their minimal genomes (non-protein-coding RNAs of only 250 to 400 nt), infect and incite disease in plants. The accumulation in these plants of 21- to 24-nt viroid-derived small RNAs (vd-sRNAs) supports the notion that DCLs also target viroids but does not clarify whether vd-sRNAs activate one or more AGOs. Here, we show that in leaves of Nicotiana benthamiana infected by potato spindle tuber viroid, the endogenous AGO1 and distinct AGOs from Arabidopsis thaliana that were overexpressed were associated with vd-sRNAs displaying the same properties (5'-terminal nucleotide and size) previously established for endogenous and viral small RNAs. Overexpression of AGO1, AGO2, AGO4, and AGO5 attenuated viroid accumulation, supporting their role in antiviroid defense.

Replicating Potato spindle tuber viroid mediates de novo methylation of an intronic viroid sequence but no cleavage of the corresponding pre-mRNA.

In plants, Potato spindle tuber viroid (PSTVd) replication triggers post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) of homologous RNA and DNA sequences, respectively. PTGS predominantly occurs in the cytoplasm, but nuclear PTGS has been also reported. In this study, we investigated whether the nuclear replicating PSTVd is able to trigger nuclear PTGS. Transgenic tobacco plants carrying cytoplasmic and nuclear PTGS sensor constructs were PSTVd-infected resulting in the generation of abundant PSTVd-derived small interfering RNAs (vd-siRNAs). Northern blot analysis revealed that, in contrast to the cytoplasmic sensor, the nuclear sensor transcript was not targeted for RNA degradation. Bisulfite sequencing analysis showed that the nuclear PTGS sensor transgene was efficiently targeted for RdDM. Our data suggest that PSTVd fails to trigger nuclear PTGS, and that RdDM and nuclear PTGS are not necessarily coupled.

Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase.

Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246-401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNase presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA ligase 1, converting it to an RNA ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd monomeric linear replication intermediate opened at position G95-G96 and containing 5'-phosphomonoester and 3'-hydroxyl terminal groups. Moreover, we also show a decreased PSTVd accumulation and a reduced ratio of monomeric circular to total monomeric PSTVd forms in Nicotiana benthamiana Domin plants in which the endogenous DNA ligase 1 was silenced. Thus, in a remarkable example of parasitic strategy, viroids reprogram for their replication the template and substrate specificity of a DNA-dependent RNA polymerase and a DNA ligase to act as RNA-dependent RNA polymerase and RNA ligase, respectively.

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.

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.