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Ricardo Flores - One of the best experts on this subject based on the ideXlab platform.
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ICTV Virus Taxonomy Profile: Pospiviroidae.
The Journal of general virology, 2021Co-Authors: Francesco Di Serio, Teruo Sano, Georgios Vidalakis, Jacobus Th J Verhoeven, Jaroslav Matousek, Robert A. Owens, Vicente Pallás, John W. Randles, Ricardo FloresAbstract:Members of the family Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA-RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/Pospiviroidae.
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symptomatic plant viroid infections in phytopathogenic fungi a request for a critical reassessment
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: P Serra, Alberto Carbonell, Selma Gagozachert, Beatriz Navarro, Shifang Li, Francesco Di Serio, Ricardo FloresAbstract:Since their discovery (1), viroids—small (∼250 to 430 nt), non–protein-coding, circular RNAs—are thought to infect and cause disease only in plants (2); thus, the report that they infect and incite symptoms in filamentous phytopathogenic fungi (3) is surprising. Viroids are classified into two families (4). Members of the Pospiviroidae , including potato spindle tuber viroid (PSTVd) (1, 5), replicate in the nucleus through an RNA–RNA rolling-circle mechanism catalyzed by host enzymes (RNA polymerase, RNase, and RNA ligase). Members of the Avsunviroidae , like peach latent mosaic viroid (PLMVd) (6), form hammerhead ribozymes (HHRz) that functionally substitute the RNase during replication in chloroplasts (4). The host range of the second family is restricted to plant species (or relatives) in which the viroids were described. Unexpectedly, Wei et al. (3) report that seven viroids, including PLMVd and avocado sunblotch viroid (ASBVd) (both of the Avsunviroidae ), infect Nicotiana benthamiana , a known host for only some members of … [↵][1]1To whom correspondence may be addressed. Email: rflores{at}ibmcp.upv.es or francesco.diserio{at}ipsp.cnr.it. [1]: #xref-corresp-1-1
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Viroid pathogenesis: a critical appraisal of the role of RNA silencing in triggering the initial molecular lesion.
FEMS microbiology reviews, 2020Co-Authors: Ricardo Flores, Sonia Delgado, Beatriz Navarro, Pedro Serra, Francesco Di SerioAbstract:The initial molecular lesions through which viroids, satellite RNAs and viruses trigger signal cascades resulting in plant diseases are hotly debated. Since viroids are circular non-protein-coding RNAs of ∼250-430 nucleotides, they appear very convenient to address this issue. Viroids are targeted by their host RNA silencing defense, generating viroid-derived small RNAs (vd-sRNAs) that are presumed to direct Argonaute (AGO) proteins to inactivate messenger RNAs, thus initiating disease. Here, we review the existing evidence. Viroid-induced symptoms reveal a distinction. Those attributed to vd-sRNAs from potato spindle tuber viroid and members of the family Pospiviroidae (replicating in the nucleus) are late, non-specific and systemic. In contrast, those attributed to vd-sRNAs from peach latent mosaic viroid (PLMVd) and other members of the family Avsunviroidae (replicating in plastids) are early, specific and local. Remarkably, leaf sectors expressing different PLMVd-induced chloroses accumulate viroid variants with specific pathogenic determinants. Some vd-sRNAs containing such determinant guide AGO1-mediated cleavage of mRNAs that code for proteins regulating chloroplast biogenesis/development. Therefore, the initial lesions and the expected phenotypes are connected by short signal cascades, hence supporting a cause-effect relationship. Intriguingly, one virus satellite RNA initiates disease through a similar mechanism, whereas in the Pospiviroidae and in plant viruses the situation remains uncertain.
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Direct visualization of the native structure of viroid RNAs at single-molecule resolution by atomic force microscopy
RNA biology, 2019Co-Authors: Miguel Moreno, Ricardo Flores, Luis Vázquez, A. López-carrasco, J. A. Martín-gago, Carlos BrionesAbstract:Viroids are small infectious, non-protein-coding circular RNAs that replicate independently and, in some cases, incite diseases in plants. They are classified into two families: Pospiviroidae, composed of species that have a central conserved region (CCR) and replicate in the cell nucleus, and Avsunviroidae, containing species that lack a CCR and whose multimeric replicative intermediates of either polarity generated in plastids self-cleave through hammerhead ribozymes. The compact, rod-like or branched, secondary structures of viroid RNAs have been predicted by RNA folding algorithms and further examined using different in vitro and in vivo experimental techniques. However, direct data about their native tertiary structure remain scarce. Here we have applied atomic force microscopy (AFM) to image at single-molecule resolution different variant RNAs of three representative viroids: potato spindle tuber viroid (PSTVd, family Pospiviroidae), peach latent mosaic viroid and eggplant latent viroid (PLMVd and ELVd, family Avsunviroidae). Our results provide a direct visualization of their native, three-dimensional conformations at 0 and 4 mM Mg2+ and highlight the role that some elements of tertiary structure play in their stabilization. The AFM images show that addition of 4 mM Mg2+ to the folding buffer results in a size contraction in PSTVd and ELVd, as well as in PLMVd when the kissing-loop interaction that stabilizes its 3D structure is preserved.
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ICTV Virus Taxonomy Profile: Avsunviroidae.
The Journal of general virology, 2018Co-Authors: Francesco Di Serio, Teruo Sano, Georgios Vidalakis, Jacobus Th J Verhoeven, Jaroslav Matousek, Robert A. Owens, Vicente Pallás, John W. Randles, Ricardo FloresAbstract:Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA–RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at http://www.ictv.global/report/avsunviroidae.
Gerhard Steger - One of the best experts on this subject based on the ideXlab platform.
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Viroid quasispecies revealed by deep sequencing.
RNA biology, 2016Co-Authors: Joseph R. J. Brass, Jaroslav Matousek, Robert A. Owens, Gerhard StegerAbstract:Viroids are non-coding single-stranded circular RNA molecules that replicate autonomously in infected host plants causing mild to lethal symptoms. Their genomes contain about 250-400 nucleotides, depending on viroid species. Members of the family Pospiviroidae, like the Potato spindle tuber viroid (PSTVd), replicate via an asymmetric rolling-circle mechanism using the host DNA-dependent RNA-Polymerase II in the nucleus, while members of Avsunviroidae are replicated in a symmetric rolling-circle mechanism probably by the nuclear-encoded polymerase in chloroplasts. Viroids induce the production of viroid-specific small RNAs (vsRNA) that can direct (post-)transcriptional gene silencing against host transcripts or genomic sequences. Here, we used deep-sequencing to analyze vsRNAs from plants infected with different PSTVd variants to elucidate the PSTVd quasipecies evolved during infection. We recovered several novel as well as previously known PSTVd variants that were obviously competent in replication and identified common strand-specific mutations. The calculated mean error rate per nucleotide position was less than [Formula: see text], quite comparable to the value of [Formula: see text] reported for a member of Avsunviroidae. The resulting error threshold allows the synthesis of longer-than-unit-length replication intermediates as required by the asymmetric rolling-circle mechanism of members of Pospiviroidae.
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Viroid quasispecies revealed by deep sequencing
2016Co-Authors: Joseph R. J. Brass, Jaroslav Matousek, Robert A. Owens, Gerhard StegerAbstract:Viroids are non-coding single-stranded circular RNA molecules that replicate autonomously in infected host plants causing mild to lethal symptoms. Their genomes contain about 250–400 nucleotides, depending on viroid species. Members of the family Pospiviroidae, like the Potato spindle tuber viroid (PSTVd), replicate via an asymmetric rolling-circle mechanism using the host DNA-dependent RNA-Polymerase II in the nucleus, while members of Avsunviroidae are replicated in a symmetric rolling-circle mechanism probably by the nuclear-encoded polymerase in chloroplasts. Viroids induce the production of viroid-specific small RNAs (vsRNA) that can direct (post-)transcriptional gene silencing against host transcripts or genomic sequences. Here, we used deep-sequencing to analyze vsRNAs from plants infected with different PSTVd variants to elucidate the PSTVd quasipecies evolved during infection. We recovered several novel as well as previously known PSTVd variants that were obviously competent in replication and identified common strand-specific mutations. The calculated mean error rate per nucleotide position was less than 5 × 10−3, quite comparable to the value of 2.5 × 10−3 reported for a member of Avsunviroidae. The resulting error threshold allows the synthesis of longer-than-unit-length replication intermediates as required by the asymmetric rolling-circle mechanism of members of Pospiviroidae.
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viroid specific small rna in plant disease
RNA Biology, 2012Co-Authors: Christian Hammann, Gerhard StegerAbstract:Viroids are the smallest autonomous infectious nucleic acids known today. They are non-coding, unencapsidated, circular RNAs with sizes ranging from 250 to 400 nucleotides and infect certain plants. These RNAs are transcribed by rolling-circle mechanisms in the plant host’s nuclei (Pospiviroidae) or chloroplasts (Avsunviroidae). Since viroids lack any open reading frame, their pathogenicity has for a long time been a conundrum. Recent findings, however, show that viroid infection is associated with the appearance of viroid-specific small RNA (vsRNA). These have sizes similar to endogenous small interfering RNA and microRNA and thus might alter the normal gene expression in the host plant. In this review we will summarize the current knowledge on vsRNA and discuss the current hypotheses how they connect to the induced symptoms, which vary dramatically, depending on both the plant cultivar and the viroid strain.
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Viroids: The Smallest Known Infectious Agents Cause Accumulation of Viroid-Specific Small RNAs
From Nucleic Acids Sequences to Molecular Medicine, 2012Co-Authors: Jaroslav Matousek, Detlev Riesner, Gerhard StegerAbstract:Viroids are plant-infectious, noncoding, unencapsidated, circular RNAs ranging in size from 250 to 400 nucleotides that are transcribed in a rolling-circle mechanism either in nuclei (Pospiviroidae) or in chloroplasts (Avsunviroidae) of plant hosts. The pathogenic effect caused by viroids is still an enigma: Potato spindle tuber viroid (PSTVd), the type strain of Pospiviroidae, causes typical symptoms in tomato plants, but the severity of symptoms depends on the tomato cultivar; different strains of PSTVd, which vary in sequence by a few mutations from each other, induce symptoms from very mild up to necrosis upon infection of a cultivar. According to recent findings, viroids cause the accumulation of viroid-specific small RNAs (vsRNA) similar in size to small interfering (siRNA) and miRNAs, but they do escape the cytoplasmic silencing mechanism. In this chapter, we will discuss these findings and hypotheses on the biogenesis of viroid-specific small RNAs and connections to symptom induction.
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Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop.
Virology, 2006Co-Authors: Nadine Kolonko, Gerhard Steger, Oliver Bannach, Katja Aschermann, Michaela Moors, Michael Schmitz, Detlev RiesnerAbstract:Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified by affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta.
Francesco Di Serio - One of the best experts on this subject based on the ideXlab platform.
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ICTV Virus Taxonomy Profile: Pospiviroidae.
The Journal of general virology, 2021Co-Authors: Francesco Di Serio, Teruo Sano, Georgios Vidalakis, Jacobus Th J Verhoeven, Jaroslav Matousek, Robert A. Owens, Vicente Pallás, John W. Randles, Ricardo FloresAbstract:Members of the family Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA-RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/Pospiviroidae.
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symptomatic plant viroid infections in phytopathogenic fungi a request for a critical reassessment
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: P Serra, Alberto Carbonell, Selma Gagozachert, Beatriz Navarro, Shifang Li, Francesco Di Serio, Ricardo FloresAbstract:Since their discovery (1), viroids—small (∼250 to 430 nt), non–protein-coding, circular RNAs—are thought to infect and cause disease only in plants (2); thus, the report that they infect and incite symptoms in filamentous phytopathogenic fungi (3) is surprising. Viroids are classified into two families (4). Members of the Pospiviroidae , including potato spindle tuber viroid (PSTVd) (1, 5), replicate in the nucleus through an RNA–RNA rolling-circle mechanism catalyzed by host enzymes (RNA polymerase, RNase, and RNA ligase). Members of the Avsunviroidae , like peach latent mosaic viroid (PLMVd) (6), form hammerhead ribozymes (HHRz) that functionally substitute the RNase during replication in chloroplasts (4). The host range of the second family is restricted to plant species (or relatives) in which the viroids were described. Unexpectedly, Wei et al. (3) report that seven viroids, including PLMVd and avocado sunblotch viroid (ASBVd) (both of the Avsunviroidae ), infect Nicotiana benthamiana , a known host for only some members of … [↵][1]1To whom correspondence may be addressed. Email: rflores{at}ibmcp.upv.es or francesco.diserio{at}ipsp.cnr.it. [1]: #xref-corresp-1-1
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Viroid pathogenesis: a critical appraisal of the role of RNA silencing in triggering the initial molecular lesion.
FEMS microbiology reviews, 2020Co-Authors: Ricardo Flores, Sonia Delgado, Beatriz Navarro, Pedro Serra, Francesco Di SerioAbstract:The initial molecular lesions through which viroids, satellite RNAs and viruses trigger signal cascades resulting in plant diseases are hotly debated. Since viroids are circular non-protein-coding RNAs of ∼250-430 nucleotides, they appear very convenient to address this issue. Viroids are targeted by their host RNA silencing defense, generating viroid-derived small RNAs (vd-sRNAs) that are presumed to direct Argonaute (AGO) proteins to inactivate messenger RNAs, thus initiating disease. Here, we review the existing evidence. Viroid-induced symptoms reveal a distinction. Those attributed to vd-sRNAs from potato spindle tuber viroid and members of the family Pospiviroidae (replicating in the nucleus) are late, non-specific and systemic. In contrast, those attributed to vd-sRNAs from peach latent mosaic viroid (PLMVd) and other members of the family Avsunviroidae (replicating in plastids) are early, specific and local. Remarkably, leaf sectors expressing different PLMVd-induced chloroses accumulate viroid variants with specific pathogenic determinants. Some vd-sRNAs containing such determinant guide AGO1-mediated cleavage of mRNAs that code for proteins regulating chloroplast biogenesis/development. Therefore, the initial lesions and the expected phenotypes are connected by short signal cascades, hence supporting a cause-effect relationship. Intriguingly, one virus satellite RNA initiates disease through a similar mechanism, whereas in the Pospiviroidae and in plant viruses the situation remains uncertain.
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ICTV Virus Taxonomy Profile: Avsunviroidae.
The Journal of general virology, 2018Co-Authors: Francesco Di Serio, Teruo Sano, Georgios Vidalakis, Jacobus Th J Verhoeven, Jaroslav Matousek, Robert A. Owens, Vicente Pallás, John W. Randles, Ricardo FloresAbstract:Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA–RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at http://www.ictv.global/report/avsunviroidae.
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Viroid RNA turnover: characterization of the subgenomic RNAs of potato spindle tuber viroid accumulating in infected tissues provides insights into decay pathways operating in vivo
Nucleic acids research, 2015Co-Authors: Sofia Minoia, Sonia Delgado, Beatriz Navarro, Francesco Di Serio, Ricardo FloresAbstract:While biogenesis of viroid RNAs is well-known, how they decay is restricted to data involving host RNA silencing. Here we report an alternative degradation pathway operating on potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids (family Pospiviroidae). Northern-blot hybridizations with full- and partial-length probes revealed a set of PSTVd (+) subgenomic (sg)RNAs in early-infected eggplant, some partially overlapping and reaching levels comparable to those of the genomic circular and linear forms. Part of the PSTVd (+) sgRNAs were also observed in Nicotiana benthamiana (specifically in the nuclei) and tomato, wherein they have been overlooked due to their low accumulation. Primer extensions of representative (+) sgRNAs failed to detect a common 5' terminus, excluding that they could result from aborted transcription initiated at one specific site. Supporting this view, 5'- and 3'-RACE indicated that the (+) sgRNAs have 5'-OH and 3'-P termini most likely generated by RNase-mediated endonucleolytic cleavage of longer precursors. These approaches also unveiled PSTVd (-) sgRNAs with features similar to their (+) counterparts. Our results provide a mechanistic insight on how viroid decay may proceed in vivo during replication, and suggest that synthesis and decay of PSTVd strands might be coupled as in mRNA.
José-antonio Daròs - One of the best experts on this subject based on the ideXlab platform.
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Viroid Replication: Rolling-Circles, Enzymes and Ribozymes
Viruses, 2009Co-Authors: Ricardo Flores, Alberto Carbonell, Diego Molina-serrano, María-eugenia Gas, María-Ángeles Nohales, Selma Gago, Marcos De La Peña, José-antonio DaròsAbstract:Viroids, due to their small size and lack of protein-coding capacity, must rely essentially on their hosts for replication. Intriguingly, viroids have evolved the ability to replicate in two cellular organella, the nucleus (family Pospiviroidae) and the chloroplast (family Avsunviroidae). Viroid replication proceeds through an RNA-based rolling-circle mechanism with three steps that, with some variations, operate in both polarity strands: i) synthesis of longer-than-unit strands catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase, in both instances redirected to transcribe RNA templates, ii) cleavage to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes embedded in both polarity strands, while in the family Pospiviroidae the oligomeric RNAs provide the proper conformation but not the catalytic activity, and iii) circularization. The host RNA polymerases, most likely assisted by additional host proteins, start transcription from specific sites, thus implying the existence of viroid promoters. Cleavage and ligation in the family Pospiviroidae is probably catalyzed by an RNase III-like enzyme and an RNA ligase able to circularize the resulting 5' and 3' termini. Whether a chloroplastic RNA ligase mediates circularization in the family Avsunviroidae, or this reaction is autocatalytic, remains an open issue.
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Monomeric Linear RNA of Citrus Exocortis Viroid Resulting from Processing In Vivo Has 5′-Phosphomonoester and 3′-Hydroxyl Termini: Implications for the RNase and RNA Ligase Involved in Replication
Journal of virology, 2008Co-Authors: María-eugenia Gas, Carmen Hernández, Ricardo Flores, Diego Molina-serrano, José-antonio DaròsAbstract:Members of the family Pospiviroidae, like Citrus exocortis viroid (CEVd), replicate through an RNA-based asymmetric rolling-circle mechanism in which oligomeric plus-strand [(+)] RNA intermediates are cleaved to monomeric linear (ml) RNA and then circularized. Here we show, by rapid amplification of 5' and 3' cDNA ends and in vitro ligation assays, that ml CEVd (+) RNA resulting from cleavage of a dimeric transcript transgenically expressed in Arabidopsis thaliana contains 5'-phosphomonoester and 3'-hydroxyl termini. The nature of these termini and the double-stranded structure previously proposed as the substrate for cleavage in vivo suggest that a type III RNase catalyzes cleavage and an RNA ligase distinct from tRNA ligase promotes circularization.
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citrus viroid v molecular characterization and synergistic interactions with other members of the genus apscaviroid
Virology, 2008Co-Authors: P Serra, C. J. Barbosa, Ricardo Flores, José-antonio Daròs, N DuranvilaAbstract:Abstract Studies on Atalantia citroides , a citrus relative, revealed the existence of a viroid not described previously. The new viroid has a GC-rich genome of 293–294 nucleotides and contains the central conserved region characteristic of members of the genus Apscaviroid , and the terminal conserved region present in this and other genera of the family Pospiviroidae . The secondary structure of minimum free energy predicted for the new viroid is a rod-like conformation with 68.7% paired nucleotides and showing sequence identities with other viroids always lower than 90%, the conventional limit that separates different species within a given genus. Infectivity assays showed that the new viroid induces mild but characteristic symptoms on the indicator Etrog citron. Co-inoculation of CVd-V with either Citrus bent leaf viroid or Citrus viroid III , two other members of the genus Apscaviroid infecting citrus, disclosed synergistic interactions manifested in enhanced leaf symptoms and very pronounced dwarfing. Viroid titers, however, remained unaltered in co-infected plants. Possible mechanisms underlying the observed synergistic effects are discussed. According to its molecular and biological properties and its unusual ability to replicate in A. citroides , the new viroid, tentatively named Citrus viroid V (CVd-V), should be considered a new species of the genus Apscaviroid.
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Structure and Evolution of Viroids
Origin and Evolution of Viruses, 2008Co-Authors: Nuria Duran-vila, José-antonio Daròs, Santiago F. Elena, Ricardo FloresAbstract:ABSTRACT Viroids are minimal RNA replicons composed by a single-stranded and highly structured circular small RNA able to infect plants and induce diseases. Viroids lack protein-coding capacity and are therefore parasites of their host transcription machinery. The small size, circularity, high G+C content, and presence of hammerhead ribozymes in some viroids, suggest their evolutionary origin in the RNA world. Phylogenetic reconstructions and structural and biological properties support a classification into two families—Pospiviroidae and Avsunviroidae—whose members replicate in the nucleus and chloroplast, respectively. Viroids may have a common origin with a class of small satellite RNAs with which they share structural similarities and a rolling circle replication mechanism involving hammerhead ribozymes. Inoculation with infectious viroid-cDNAs results in progenies readily accumulating genetic variation, with Avsunviroidae populations being more diverse than Pospiviroidae populations. Moreover, assuming that the fitness of a haplotype is determined by its ability to fold into the secondary structure of minimum free energy, the estimated per site deleterious mutation rate in the Avsunviroidae is 10-fold higher than in the Pospiviroidae. The dissimilar nuclear and chloroplastic RNA polymerases mediating replication in both families may influence their mutation rates, particularly when transcribing atypical RNA templates. Both families also differ in their structural robustness against mutation, with the Pospiviroidae rod-like structures being more robust than the Avsunviroidae branched structures (and the redundant variants of a specific viroid being more robust than their non-redundant counterparts). Chimeric viroids might have emerged from recombination between co-infecting viroids during transcription by a “jumping” RNA polymerase. Viroids polymorphic populations can be described by the quasispecies model of molecular evolution, and one of its main tenets (that a slow replicator outcompetes a faster one provided that the former is more robust against mutation) has been experimentally proven. Hosts play an important role in shaping the structure of viroid populations. Specific domains of the viroid secondary structure are responsible for symptom expression. Depending on their phylogenetic proximity, interactions between co-infecting viroids may result in interference (cross-protection) or synergism, both of which may be governed by RNA silencing mechanisms that may have shaped viroid structure and evolution as well. Wild plant species serve as symptomless reservoirs of certain viroids, while their spread and persistence in cultivated species is associated with agricultural practices.
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Analysis of viroid replication.
Methods in molecular biology (Clifton N.J.), 2008Co-Authors: Ricardo Flores, Carmen Hernández, María-eugenia Gas, Diego Molina, José-antonio DaròsAbstract:Viroids, as a consequence of not encoding any protein, are extremely dependent on their hosts. Replication of these minimal genomes, composed exclusively by a circular RNA of 246-401 nt, occurs in the nucleus (family Pospiviroidae) or in the chloroplast (family Avsunviroidae) by an RNA-based rolling-circle mechanism with three steps: (1) synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases recruited and redirected to transcribe RNA templates, (2) cleavage to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3) circularization through an RNA ligase or autocatalytically. This consistent but still fragmentary picture has emerged from a combination of studies with in vitro systems (analysis of RNA preparations from infected plants, transcription assays with nuclear and chloroplastic fractions, characterization of enzymes and ribozymes mediating cleavage and ligation of viroid strands, dissection of 5' terminal groups of viroid strands, and in situ hybridization and microscopy of subcellular fractions and tissues), and in vivo systems (tissue infiltration studies, protoplasts, studies in planta and use of transgenic plants expressing viroid RNAs).
Robert A. Owens - One of the best experts on this subject based on the ideXlab platform.
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ICTV Virus Taxonomy Profile: Pospiviroidae.
The Journal of general virology, 2021Co-Authors: Francesco Di Serio, Teruo Sano, Georgios Vidalakis, Jacobus Th J Verhoeven, Jaroslav Matousek, Robert A. Owens, Vicente Pallás, John W. Randles, Ricardo FloresAbstract:Members of the family Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA-RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/Pospiviroidae.
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ICTV Virus Taxonomy Profile: Avsunviroidae.
The Journal of general virology, 2018Co-Authors: Francesco Di Serio, Teruo Sano, Georgios Vidalakis, Jacobus Th J Verhoeven, Jaroslav Matousek, Robert A. Owens, Vicente Pallás, John W. Randles, Ricardo FloresAbstract:Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA–RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at http://www.ictv.global/report/avsunviroidae.
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Viroid quasispecies revealed by deep sequencing.
RNA biology, 2016Co-Authors: Joseph R. J. Brass, Jaroslav Matousek, Robert A. Owens, Gerhard StegerAbstract:Viroids are non-coding single-stranded circular RNA molecules that replicate autonomously in infected host plants causing mild to lethal symptoms. Their genomes contain about 250-400 nucleotides, depending on viroid species. Members of the family Pospiviroidae, like the Potato spindle tuber viroid (PSTVd), replicate via an asymmetric rolling-circle mechanism using the host DNA-dependent RNA-Polymerase II in the nucleus, while members of Avsunviroidae are replicated in a symmetric rolling-circle mechanism probably by the nuclear-encoded polymerase in chloroplasts. Viroids induce the production of viroid-specific small RNAs (vsRNA) that can direct (post-)transcriptional gene silencing against host transcripts or genomic sequences. Here, we used deep-sequencing to analyze vsRNAs from plants infected with different PSTVd variants to elucidate the PSTVd quasipecies evolved during infection. We recovered several novel as well as previously known PSTVd variants that were obviously competent in replication and identified common strand-specific mutations. The calculated mean error rate per nucleotide position was less than [Formula: see text], quite comparable to the value of [Formula: see text] reported for a member of Avsunviroidae. The resulting error threshold allows the synthesis of longer-than-unit-length replication intermediates as required by the asymmetric rolling-circle mechanism of members of Pospiviroidae.
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Viroid quasispecies revealed by deep sequencing
2016Co-Authors: Joseph R. J. Brass, Jaroslav Matousek, Robert A. Owens, Gerhard StegerAbstract:Viroids are non-coding single-stranded circular RNA molecules that replicate autonomously in infected host plants causing mild to lethal symptoms. Their genomes contain about 250–400 nucleotides, depending on viroid species. Members of the family Pospiviroidae, like the Potato spindle tuber viroid (PSTVd), replicate via an asymmetric rolling-circle mechanism using the host DNA-dependent RNA-Polymerase II in the nucleus, while members of Avsunviroidae are replicated in a symmetric rolling-circle mechanism probably by the nuclear-encoded polymerase in chloroplasts. Viroids induce the production of viroid-specific small RNAs (vsRNA) that can direct (post-)transcriptional gene silencing against host transcripts or genomic sequences. Here, we used deep-sequencing to analyze vsRNAs from plants infected with different PSTVd variants to elucidate the PSTVd quasipecies evolved during infection. We recovered several novel as well as previously known PSTVd variants that were obviously competent in replication and identified common strand-specific mutations. The calculated mean error rate per nucleotide position was less than 5 × 10−3, quite comparable to the value of 2.5 × 10−3 reported for a member of Avsunviroidae. The resulting error threshold allows the synthesis of longer-than-unit-length replication intermediates as required by the asymmetric rolling-circle mechanism of members of Pospiviroidae.
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In silico prediction and validation of potential gene targets for pospiviroid-derived small RNAs during tomato infection.
Gene, 2015Co-Authors: Katia Avina-padilla, Rosemarie W. Hammond, Robert A. Owens, Octavio Martínez De La Vega, Rafael F. Rivera-bustamante, Juan Pablo Martínez-soriano, Jean-philippe Vielle-calzadaAbstract:Viroids are small, covalently closed, circular non-coding RNA pathogens of flowering plants. It is proposed that the symptoms of viroid pathogenesis result from a direct interaction between the viroid genomic RNA and unknown host plant factors. Using a comparative genomic approach we took advantage of the detailed annotation of the Arabidopsis thaliana (Arabidopsis) genome to identify sequence homologies between putative viroid-derived small RNAs (vd-sRNAs) and coding regions in the plant genome. A pool of sequence homologies among 29 species of the Pospiviroidae family and the Arabidopsis genome was analyzed. Using this strategy we identified putative host gene targets that may be involved in symptom expression in viroid-infected plants. In this communication, we report the in silico prediction and the experimental validation of pospiviroid-derived sRNAs conserved in the lower strand of the pathogenicity domain of seven viroid species infecting tomato; those vd-sRNAs targeted for cleavage the host mRNA encoding a conserved tomato WD40-repeat protein (SolWD40-repeat; SGN_U563134). Analysis of SolWD40-repeat expression indicated that this gene is down-regulated in tomato plants infected with tomato planta macho viroid (TPMVd). Furthermore, 5' RLM-RACE revealed that the SolWD40-repeat mRNA is cleaved at the predicted target site showing complementarity to a corresponding TPMVd-sRNA identified in silico. Our approach proved to be useful for the identification of natural host genes containing sequence homologies with segments of the Pospiviroidae genome. Using this strategy we identified a functionally conserved gene in Arabidopsis and tomato, whose expression was modified during viroid infection in the host genome; regulation of this gene expression could be guided by vd-sRNA:mRNA complementarity, suggesting that the comparison of the Arabidopsis genome to viroid sequences could lead to the identification of unexpected interactions between viroid RNAs and their host.