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Ricardo Flores - One of the best experts on this subject based on the ideXlab platform.
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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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The predominant circular form of Avocado Sunblotch Viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins.
Journal of General Virology, 2017Co-Authors: Amparo López-carrasco, Ricardo FloresAbstract:Avocado Sunblotch Viroid (ASBVd), the type member of the family AvsunViroidae, replicates and accumulates in chloroplasts. Whether this minimal non-protein-coding circular RNA of 246–250 nt exists in vivo as a free nucleic acid or closely associated with host proteins remains unknown. To tackle this issue, the secondary structures of the monomeric circular (mc) (+) and (−) strands of ASBVd have been examined in silico by searching those of minimal free energy, and in vitro at single-nucleotide resolution by selective 2′-hydroxyl acylation analysed by primer extension (SHAPE). Both approaches resulted in predominant rod-like secondary structures without tertiary interactions, with the mc (+) RNA being more compact than its (−) counterpart as revealed by non-denaturing polyacryamide gel electrophoresis. Moreover, in vivo SHAPE showed that the mc ASBVd (+) form accumulates in Avocado leaves as a free RNA adopting a similar rod-shaped conformation unprotected by tightly bound host proteins. Hence, the mc ASBVd (+) RNA behaves in planta like the previously studied mc (+) RNA of potato spindle tuber Viroid, the type member of nuclear Viroids (family PospiViroidae), indicating that two different Viroids replicating and accumulating in distinct subcellular compartments, have converged into a common structural solution. Circularity and compact secondary structures confer to these RNAs, and probably to all Viroids, the intrinsic stability needed to survive in their natural habitats. However, in vivo SHAPE has not revealed the (possibly transient or loose) interactions of the mc ASBVd (+) RNA with two host proteins observed previously by UV irradiation of infected Avocado leaves.
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involvement of the chloroplastic isoform of trna ligase in the replication of Viroids belonging to the family avsunViroidae
Journal of Virology, 2012Co-Authors: María-Ángeles Nohales, Ricardo Flores, Diego Molinaserrano, José-antonio DaròsAbstract:Avocado Sunblotch Viroid, peach latent mosaic Viroid, chrysanthemum chlorotic mottle Viroid, and eggplant latent Viroid (ELVd), the four recognized members of the family AvsunViroidae, replicate through the symmetric pathway of an RNA-to-RNA rolling-circle mechanism in chloroplasts of infected cells. Viroid oligomeric transcripts of both polarities contain embedded hammerhead ribozymes that, during replication, mediate their self-cleavage to monomeric-length RNAs with 5′-hydroxyl and 2′,3′-phosphodiester termini that are subsequently circularized. We report that a recombinant version of the chloroplastic isoform of the tRNA ligase from eggplant (Solanum melongena L.) efficiently catalyzes in vitro circularization of the plus [(+)] and minus [(−)] monomeric linear replication intermediates from the four AvsunViroidae. We also show that while this RNA ligase specifically recognizes the genuine monomeric linear (+) ELVd replication intermediate, it does not do so with five other monomeric linear (+) ELVd RNAs with their ends mapping at different sites along the molecule, despite containing the same 5′-hydroxyl and 2′,3′-phosphodiester terminal groups. Moreover, experiments involving transient expression of a dimeric (+) ELVd transcript in Nicotiana benthamiana Domin plants preinoculated with a tobacco rattle virus-derived vector to induce silencing of the plant endogenous tRNA ligase show a significant reduction of ELVd circularization. In contrast, circularization of a Viroid replicating in the nucleus occurring through a different pathway is unaffected. Together, these results support the conclusion that the chloroplastic isoform of the plant tRNA ligase is the host enzyme mediating circularization of both (+) and (−) monomeric linear intermediates during replication of the Viroids belonging to the family AvsunViroidae.
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A chloroplastic RNA ligase activity analogous to the bacterial and archaeal 2´-5' RNA ligase.
RNA Biology, 2012Co-Authors: Diego Molina-serrano, Ricardo Flores, Jorge Marqués, María-Ángeles Nohales, José-antonio DaròsAbstract:Bacteria and archaea contain a 2’-5′ RNA ligase that seals in vitro 2’,3′-cyclic phosphodiester and 5′-hydroxyl RNA termini, generating a 2’,5′-phosphodiester bond. In our search for an RNA ligase able to circularize the monomeric linear replication intermediates of Viroids belonging to the family AvsunViroidae, which replicate in the chloroplast, we have identified in spinach (Spinacea oleracea L.) chloroplasts a new RNA ligase activity whose properties resemble those of the bacterial and archaeal 2’-5′ RNA ligase. The spinach chloroplastic RNA ligase recognizes the 5′-hydroxyl and 2’,3′-cyclic phosphodiester termini of Avocado Sunblotch Viroid and Eggplant latent Viroid RNAs produced by hammerhead-mediated self-cleavage, yielding circular products linked through an atypical, most likely 2’,5′-phosphodiester, bond. The enzyme neither requires divalent cations as cofactors, nor NTPs as substrate. The reaction apparently reaches equilibrium at a low ratio between the final circular product and the linear i...
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Processing of RNAs of the Family AvsunViroidae in Chlamydomonas reinhardtii Chloroplasts �
2006Co-Authors: Diego Molina-serrano, Ricardo Flores, Loreto Suay, María L. Salvador, José-antonio DaròsAbstract:The family AvsunViroidae comprises four Viroid species with the ability to form hammerhead ribozymes that mediate self-cleavage of the multimeric plus and minus strands resulting from replication in the chloroplast through a symmetric rolling-circle mechanism. Research on these RNAs is restricted by their host range, which is limited to the plants wherein they were initially identified and some closely related species. Here we report cleavage and ligation in transplastomic Chlamydomonas reinhardtii expressing plus- and minus-strand dimeric transcripts of representative members of the family AvsunViroidae. Despite the absence of Viroid RNA-RNA transcription, the C. reinhardtii-based system can be used to address intriguing questions about Viroid RNA processing and, in particular, about the cellular factors involved in cleavage and ligation. Species of the AvsunViroidae family, Avocado Sunblotch Viroid (ASBVd) (19), Peach latent mosaic Viroid (PLMVd) (18), Chrysanthemum chlorotic mottle Viroid (CChMVd) (23), and Eggplant latent Viroid (ELVd) (12), comprise a unique class of plant pathogens because ASBVd and PLMVd, and most likely CChMVd and ELVd, replicate in the chloroplast (13). Like nuclear Viroids (family PospiViroidae), they are formed of small
Mariechristine Maurel - One of the best experts on this subject based on the ideXlab platform.
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Self-assembly Controls Self-cleavage of HHR from ASBVd (−): a Combined SANS and Modeling Study
Scientific Reports, 2016Co-Authors: Fabrice Leclerc, Jacques Vergne, Giuseppe Zaccai, Martina Řìhovà, Anne Martel, Mariechristine MaurelAbstract:In the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the AvsunViroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (−) RNAs thus generated are processed by cleavage to unit-length where ASBVd (−) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (−) and its derived 79-nt HHR (−). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (−) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (−) though with a remoter region from an extension of the HI domain.
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Self-assembly Controls Self-cleavage of HHR from ASBVd(-): a Combined SANS and Modeling Study.
2016Co-Authors: Fabrice Leclerc, Jacques Vergne, Giuseppe Zaccai, Martina Rihova, Anne L. Martel, Mariechristine MaurelAbstract:In the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the AvsunViroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (-) RNAs thus generated are processed by cleavage to unit-length where ASBVd (-) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (-) and its derived 79-nt HHR (-). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (-) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (-) though with a remoter region from an extension of the HI domain.
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Replication of Avocado Sunblotch Viroid in the Cyanobacterium Nostoc Sp. PCC 7120
Journal of Plant Pathology & Microbiology, 2016Co-Authors: Amel Latifi, Jacques Vergne, Christophe S. Bernard, Laura Da Silva, Yannick Andéol, Amine Elleuch, Véronique Risoul, Mariechristine MaurelAbstract:Viroids are small infectious RNA molecules that replicate in plants via RNA-RNA replication processes. The molecular mechanism responsible for this replication has attracted great interest, and studies on this topic have yielded interesting biological findings on the processes in which RNA is involved. Viroids belonging to the AvsunViroidae family replicate in the chloroplasts of infected hosts. It has by now been established that chloroplasts and cyanobacteria share a common have ancestor. In view of this phylogenetic relationship, we investigated whether a member of the AvsunViroidae family could be replicated in a cyanobacterium. The results obtained here show that Avocado Sunblotch Viroid (ASBVd) RNA is able to replicate in the filamentous cyanobacterium Nostoc PCC 7120. Indeed, Northern blot hybridization showed that linear forms of “minus” polarity were detected in RNA extracted from Nostoc cells expressing ASBVd dimers of positive “polarity”, and that linear forms of “negative” polarity were detected in RNA extracted from Nostoc cells expressing ASBVd dimers of “positive” polarity. ASBVd replication does not impair the growth of Nostoc. These results provide the first evidence that a prokaryotic cell possesses all the machinery required to sustain the process of RNA-RNA replication. The data obtained here are of great importance, since they might shed light on the evolution of the cellular factors on which RNA replication processes depend.
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raman characterization of Avocado Sunblotch Viroid and its response to external perturbations and self cleavage
BMC Biophysics, 2014Co-Authors: Gaston Huibonhoa, Hussein Kaddour, Jacques Vergne, Sergei G Kruglik, Mariechristine MaurelAbstract:Background Viroids are the smallest pathogens of plants. To date the structural and conformational details of the cleavage of Avocado Sunblotch Viroid (ASBVd) and the catalytic role of Mg2+ ions in efficient self-cleavage are of crucial interest.
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Raman characterization of Avocado SunblotchViroid and its response to external perturbations and self-cleavage
BMC Biophysics, 2014Co-Authors: Gaston Hui-bon-hoa, Hussein Kaddour, Jacques Vergne, Sergei G Kruglik, Mariechristine MaurelAbstract:Background Viroids are the smallest pathogens of plants. To date the structural and conformational details of the cleavage of Avocado Sunblotch Viroid (ASBVd) and the catalytic role of Mg^2+ ions in efficient self-cleavage are of crucial interest. Results We report the first Raman characterization of the structure and activity of ASBVd, for plus and minus Viroid strands. Both strands exhibit a typical A-type RNA conformation with an ordered double-helical content and a C3′-endo/anti sugar pucker configuration, although small but specific differences are found in the sugar puckering and base-stacking regions. The ASBVd(-) is shown to self-cleave 3.5 times more actively than ASBVd(+). Deuteration and temperature increase perturb differently the double-helical content and the phosphodiester conformation, as revealed by corresponding characteristic Raman spectral changes. Our data suggest that the structure rigidity and stability are higher and the D_2O accessibility to H-bonding network is lower for ASBVd(+) than for ASBVd(-). Remarkably, the Mg^2+-activated self-cleavage of the Viroid does not induce any significant alterations of the secondary Viroid structure, as evidenced from the absence of intensity changes of Raman marker bands that, however exhibit small but noticeable frequency downshifts suggesting several minor changes in phosphodioxy, internal loops and hairpins of the cleaved Viroids. Conclusions Our results demonstrate the sensitivity of Raman spectroscopy in monitoring structural and conformational changes of the Viroid and constitute the basis for further studies of its interactions with therapeutic agents and cell membranes.
Claire Torchet - One of the best experts on this subject based on the ideXlab platform.
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Structural Analyses of Avocado Sunblotch Viroid Reveal Differences in the Folding of Plus and Minus RNA Strands
Viruses, 2014Co-Authors: Clémentine Delan-forino, Mariechristine Maurel, Jules Deforges, Lionel Benard, Bruno Sargueil, Claire TorchetAbstract:Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since Viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado Sunblotch Viroid (ASBVd), a member of the family AvsunViroidae, replicates via a symmetric OPEN ACCESS Viruses 2014, 6 490 RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo Viroid life cycle.
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structural analyses of Avocado Sunblotch Viroid reveal differences in the folding of plus and minus rna strands
Viruses, 2014Co-Authors: Clementine Delanforino, Mariechristine Maurel, Jules Deforges, Lionel Benard, Bruno Sargueil, Claire TorchetAbstract:Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since Viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado Sunblotch Viroid (ASBVd), a member of the family AvsunViroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo Viroid life cycle.
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Structural Analyses of Avocado Sunblotch Viroid Reveal Differences in the Folding of Plus and Minus RNA Strands
Viruses, 2014Co-Authors: Clémentine Delan-forino, Mariechristine Maurel, Jules Deforges, Lionel Benard, Bruno Sargueil, Claire TorchetAbstract:Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since Viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado Sunblotch Viroid (ASBVd), a member of the family AvsunViroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo Viroid life cycle.
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replication of Avocado Sunblotch Viroid in the yeast saccharomyces cerevisiae
Journal of Virology, 2011Co-Authors: Clementine Delanforino, Mariechristine Maurel, Claire TorchetAbstract:Viroids are the smallest known pathogenic agents. They are noncoding, single-stranded, closed-circular, “naked” RNAs, which replicate through RNA-RNA transcription. Viroids of the AvsunViroidae family possess a hammerhead ribozyme in their sequence, allowing self-cleavage during their replication. To date, Viroids have only been detected in plant cells. Here, we investigate the replication of Avocado Sunblotch Viroid (ASBVd) of the AvsunViroidae family in a nonconventional host, the yeast Saccharomyces cerevisiae. We demonstrate that ASBVd RNA strands of both polarities are able to self-cleave and to replicate in a unicellular eukaryote cell. We show that the Viroid monomeric RNA is destabilized by the nuclear 3′ and the cytoplasmic 5′ RNA degradation pathways. For the first time, our results provide evidence that Viroids can replicate in other organisms than plants and that yeast contains all of the essential cellular elements for the replication of ASBVd.
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Replication of Avocado Sunblotch Viroid in the Yeast
2010Co-Authors: Mariechristine Maurel, Claire TorchetAbstract:Viroids are the smallest known pathogenic agents. They are noncoding, single-stranded, closed-circular, “naked” RNAs, which replicate through RNA-RNA transcription. Viroids of the AvsunViroidae family possess a hammerhead ribozyme in their sequence, allowing self-cleavage during their replication. To date, Viroids have only been detected in plant cells. Here, we investigate the replication of Avocado Sunblotch Viroid (ASBVd) of the AvsunViroidae family in a nonconventional host, the yeast Saccharomyces cerevisiae. We demonstrate that ASBVd RNA strands of both polarities are able to self-cleave and to replicate in a unicellular eukaryote cell. We show that the Viroid monomeric RNA is destabilized by the nuclear 3 and the cytoplasmic 5 RNA degradation pathways. For the first time, our results provide evidence that Viroids can replicate in other organisms than plants and that yeast contains all of the essential cellular elements for the replication of ASBVd. Viroids are the smallest and the simplest eukaryotic known pathogens (12). They are single-stranded circular RNAs capa-ble of infecting plants and range from 246 to 401 nucleotides (nt). However, some variants of 120 or 475 nt are referenced in the subviral RNA database (47). Unlike viruses, they do not code for any protein, they are devoid of capsid or envelope
J S Semancik - One of the best experts on this subject based on the ideXlab platform.
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RNA silencing as related to Viroid induced symptom expression
Archives of Virology, 2004Co-Authors: N Markarian, S W Ding, J S SemancikAbstract:Evidence of post-transcriptional gene silencing (PTGS) in Avocado infected by Avocado Sunblotch Viroid (ASBVd), the type species of family AvsunViroidae , was suggested by detection of ASBVd-specific 22-nucleotide RNAs. PTGS was observed in infected bleached and variegated symptomatic tissues as well as symptomless carrier foliar sources and fruit with typical Sunblotch disease lesions. Tissues with the different symptom expressions, characterized by the presence of different predominant ASBVd variants, were found to induce PTGS at differential levels. Detection of the PTGS-associated small interfering RNAs (siRNAs) as well as relative concentration was also related to Viroid titer. PTGS induced in Gynura aurantiaca infected with two closely-related variants of Citrus exocortis Viroid , a member of family PospiViroidae , was not directly related to Viroid titer with initiation of symptoms.
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rna silencing as related to Viroid induced symptom expression
Archives of Virology, 2004Co-Authors: N Markarian, Haibo Li, S W Ding, J S SemancikAbstract:Evidence of post-transcriptional gene silencing (PTGS) in Avocado infected by Avocado Sunblotch Viroid (ASBVd), the type species of family AvsunViroidae, was suggested by detection of ASBVd-specific 22-nucleotide RNAs. PTGS was observed in infected bleached and variegated symptomatic tissues as well as symptomless carrier foliar sources and fruit with typical Sunblotch disease lesions. Tissues with the different symptom expressions, characterized by the presence of different predominant ASBVd variants, were found to induce PTGS at differential levels. Detection of the PTGS-associated small interfering RNAs (siRNAs) as well as relative concentration was also related to Viroid titer. PTGS induced in Gynura aurantiaca infected with two closely-related variants of Citrus exocortis Viroid, a member of family PospiViroidae, was not directly related to Viroid titer with initiation of symptoms.
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Avocado Sunblotch disease: a persistent Viroid infection in which variants are associated with differential symptoms.
The Journal of general virology, 1994Co-Authors: J S Semancik, J A SzychowskiAbstract:Variants of Avocado Sunblotch Viroid (ASBVd) of between 247 and 250 nucleotides in length have been recovered from diseased Avocado tissues. The Sunblotch syndrome covers a complex pattern of disease symptoms which are associated with infection by variants of ASBVd. The Viroid species are designated ASBVd-B, ASBVd-V and ASBVd-Sc from their association with bleached, variegated or symptomless carrier tissues respectively. Host-Viroid interactions and structural relationships among the variants suggest a transition in Sunblotch disease from a severe acute to a persistent mild form of infection.
D. N. Kuhn - One of the best experts on this subject based on the ideXlab platform.
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A Highly Sensitive Method to Detect Avocado Sunblotch Viroid for the Maintenance of Infection-Free Avocado Germplasm Collections
Viruses, 2019Co-Authors: D. N. Kuhn, Andrew D. W. Geering, Barbie Freeman, Alan H. ChambersAbstract:The United States Department of Agriculture (USDA) Agricultural Research Service (ARS) Subtropical Horticulture Research Station (SHRS) in Miami, FL holds a large germplasm collection of Avocado (Persea americana). The recent threat of infection by laurel wilt has encouraged the creation of a backup collection at a disease-free site. Creating the backup collection is complicated by infection of some trees in the germplasm collection with Avocado Sunblotch Viroid (ASBVd). Infected trees are frequently asymptomatic, necessitating the use of a molecular diagnostic assay. Although a reverse-transcription based assay already exists and has been used to assay all germplasm at the station, some trees showed inconsistent results. We have developed a more sensitive and specific assay involving pre-amplification of the entire Viroid cDNA followed by detection using real-time PCR and a TaqMan assay. A second screening of all germplasm identified additional ASBVd -infected trees and allowed us to confidently remove these trees from the station. This method enables Avocado germplasm curators to proceed with the creation of a Viroid-free backup collection.
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Avocado Sunblotch Viroid
Viroids and Satellites, 2017Co-Authors: D. N. Kuhn, Andrew D. W. Geering, Jonathan DixonAbstract:Avocado Sunblotch Viroid (ASBVd) is a 247 nucleotide, single-stranded, circular RNA that is the type species of both the genus AvsunViroid and the family AvsunViroidae. As such, it replicates in the chloroplast, utilizes a symmetrical rolling circle mechanism of replication and has distinctive hammerhead ribozyme structures that autocatalyze RNA cleavage. ASBVd has a very narrow host range, restricted in nature to Avocado. The most recognizable symptoms of infection are the sunken, yellow to purplish longitudinal scars or broad spots that appear on the surface of the fruit. Infected trees are often asymptomatic and do not display overt foliar or fruit symptoms, although fewer and marginally smaller fruit may be produced. Sequence variation is common, occurring mostly in terminal loops outside the highly complementary regions. Some sequence variants have been associated with symptoms such as leaf bleaching, leaf variegation, and symptomless carrier. ASBVd is effectively controlled through the implementation of an accredited nursery scheme that ensures nuclear and multiplication trees used to source seed and budwood are regularly tested as free from ASBVd.
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Spatial Analysis of Avocado Sunblotch Disease in an Avocado Germplasm Collection
Journal of Phytopathology, 2011Co-Authors: Raymond J. Schnell, D. N. Kuhn, Cecile L. Tondo, Michael Winterstein, Tomas Ayala-silva, John Michael MooreAbstract:Avocado Sunblotch Viroid (ASBVd) causes an important disease of Avocado, Persea americana. Symptoms of Avocado Sunblotch were first observed in the Avocado germplasm collection at the National Germplasm Repository in Miami in the early 1980s; however, the extent of infection was unknown. An ASBVd-specific reverse transcription polymerase chain reaction (RT-PCR) protocol was developed in 1996 and used to screen every tree in the collection. Surveys in 1996 and 2000 found that although 23 newly infected trees were detected, the proportion of ASBVd-positive accessions remained unchanged at 19%. However, in a 2009 survey, 50 newly infected trees were detected for an overall infection rate of 21%. Results of spatial analyses indicate that for the older plantings, the effective range of spread increased more than threefold during the 13 year span, while in the newer plantings, the pattern of infection indicates a reintroduction of the Viroid rather than natural spread. Despite strict sanitization procedures in field and greenhouse operations, ASBVd infections have increased in the USDA collection. Although genetic diversity in the collection would be reduced, eliminating all ASBVd-positive plants may be necessary to ensure that other accessions in the collection do not become infected.
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Results of the 2009 ASBVd Survey of Avocado Accessions of the National Germplasm Collection in Florida
2010Co-Authors: Cecile L. Tondo, Raymond J. Schnell, D. N. KuhnAbstract:The presence of Avocado Sunblotch Viroid (ASBVd) infection among the Avocado (Persea americana Mill.) accessions in the National Germplasm Repository at Miami (NGR–Miami) was established in previous studies. An ASBVd specific reverse transcription-polymerase chain reaction (RT-PCR) protocol was used to detect the Viroid. Surveys performed in 1996 and in 2000 found that the proportion of ASBVd positive accessions remained unchanged at 19% during that time period. The object of the current study was to assess the spread of infection, if any, and the rate and direction of transmission. For this purpose the collection was screened again for ASBVd in 2009. The germplasm collection increased from 403 to 505 trees. Fifty newly infected trees were detected. Forty-eight percent of the newly infected plants were found to be adjacent to previously infected plants, adjacent to plots from which infected plants had been removed, or adjacent to other newly infected plants that are adjacent to previously infected plants or contaminated plots. No pattern in direction of spread was discerned for non-adjacent new infections. The proportion of plants found to be positive for the Viroid in the current study is 21%. Fourteen plants previously found to be infected were found to be negative in this survey. The proportion of infected plants (historically and at present) in the current collection is 24%. The increase in ASBVd infections reinforces the importance of establishing back-up collections at different locations. Establishment of collections at the NGR in Hawaii is currently under way.
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Recovery and Indexing of Avocado Plants (Persea Americana) from Embryogenic Nucellar Cultures of an Avocado Sunblotch Viroid-Infected Tree
Plant Cell Tissue and Organ Culture, 2006Co-Authors: Isidro E. Suarez, D. N. Kuhn, Raymond A. Schnell, Richard E. LitzAbstract:Avocado ( Persea americana Mill.) plants were recovered from somatic embryos derived from the nucellus of an Avocado Sunblotch Viroid (ASBVd)-infected tree (???Vero Beach??? SE2). Embryogenic cultures were induced on semi solid medium consisting of B5 major salts, MS minor salts and organics, 45 g sucrose and 0.41 ??M picloram, and were maintained as suspension cultures in MS3:1N medium (MS containing 12 mg l^???1 NH_4NO_3, 30.3 mg l^???1 KNO_3 , 0.41 ??M picloram and (in mg l^???1) thiamine HCl, (0.4), myo-inositol (100), and sucrose (45,000). Somatic embryos developed on semi solid MS medium without growth regulators. Plants were recovered from mature somatic embryos on semi solid MS supplemented with 4.44 ??M BA and 2.89 ??M GA_3, and thereafter were maintained on semi solid MS basal medium. RT-PCR indexing of embryogenic cultures, somatic embryos and leaf tissue of regenerants demonstrated that all developmental stages were ASBVd-positive. Variation in the Viroid increased with developmental stage. Five ASBVd variants were isolated from embryogenic cultures, five ASBVd variants were isolated from somatic embryos and 33 ASBVd variants were isolated from leaves of regenerated plants. Nucellar culture cannot be considered a strategy for elimination of ASBVd from infected elite Avocado material, and appears to stimulate variation in the Viroid.
