Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing.

Proc. Natl. Acad. Sci. U.S.A.

2004

Viroids, subviral noncoding RNAs, replicate, move, and incite diseases in plants. Viroids replicate through a rolling-circle mechanism in which oligomeric RNAs of one or both polarities are cleaved and ligated into the circular monomers. Attempts to transmit viroids to Arabidopsis have failed for unknown reasons. To tackle this question, Arabidopsis was transformed with cDNAs expressing dimeric (؉) transcripts of representative species of the families Pospiviroidae and Avsunviroidae, which replicate in the nucleus and the chloroplast, respectively. Correct processing to the circular (؉) monomers was always observed, demonstrating that Arabidopsis has the appropriate RNase and RNA ligase. Northern blot hybridization also revealed the multimeric (؊) RNAs of Citrus exocortis viroid and Hop stunt viroid (HSVd) of the family Pospiviroidae, but not of Avocado sunblotch viroid of the family Avsunviroidae, showing that the first RNA-RNA transcription of the rolling-circle mechanism occurs in Arabidopsis for the two nuclear viroids and that their multimeric (؊) RNAs remain unprocessed as in typical hosts. Moreover, transgenic Arabidopsis expressing HSVd dimeric (؊) transcripts accumulated the circular (؉) monomers, although at low levels, together with the unprocessed primary transcript that served as the template for the second RNA-RNA transcription. Agroinoculation of Arabidopsis with the dimeric (؉) Citrus exocortis viroid, HSVd, and Coleus blumei viroid 1 cDNAs showed that these viroids could not move to distal plant parts, in contrast with the situation observed in their experimental hosts. Therefore, deficiencies in movement or low replication appear to be the factors limiting infectivity of some viroids in Arabidopsis.S ince Arabidopsis thaliana was adopted as the model organism for higher plants, multiple tools, resources, and experimental approaches have been developed that facilitate research with this system (1). Principal among them is the availability of the complete sequence of the Arabidopsis genome (2). Research on plant viruses has also benefited from the use of such a versatile system. In particular, studies with viruses that naturally or experimentally infect Arabidopsis have led to the identification of host factors involved in their amplification (3, 4) and movement (5, 6) and in posttranscriptional gene silencing-mediated phenomena that include disease induction (7) and plant defense responses (8, 9). However, no viroid has been reported to infect Arabidopsis so far.Viroids are small, noncoding circular RNAs of 246-401 nucleotides able to replicate autonomously in certain plants (see refs. 10-12 for reviews). Despite this minimal genome, viroids, in addition to their replication, are able to direct their intracellular, intercellular, and long-distance movement (13-15) and to activate host defense mechanisms (16-18) that in some cases are insufficient to block the induction of pathogenic effects. For these functions, viroids need to interact with multiple host factors, the identification of which wou...

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae

Daròs

Proc. Natl. Acad. Sci. U.S.A.

2004

“…Moreover, in infected avocado, the mc ASBVd (+) form is by far the most abundant viroid RNA [12][13][14]21], thus facilitating its analysis in vivo and its isolation and purification. The mc ASBVd (À) strand, although considerably less prevalent than its (+) counterpart, still reaches in planta a titre high enough to obtain the amount needed for dissection in vitro with several approaches.…”

Section: Resultsmentioning

confidence: 99%

“…The mc ASBVd (+) form was directly isolated by denaturing PAGE from viroid-enriched RNA preparations from infected tissue; we assumed that the minor contamination with its co-migrating mc (À) counterpart, probably less than 10 % [13], should not interfere with the subsequent analysis. The mc ASBVd (À) form was prepared by circularization with a wheat germ extract of the ml ASBVd (À) RNA resulting from in vitro self-cleavage of a dimeric headto-tail transcript.…”

Section: Resultsmentioning

confidence: 99%

“…ASBVd, the type member of the family Avsunviroidae, replicates in plastids (mostly chloroplasts) through a symmetric rolling-circle mechanism [12][13][14][15]. Reiterative transcription of the infecting (+) monomeric circular (mc) RNA by a nuclear-encoded polymerase (NEP) translocated into chloroplasts [16] results in oligomeric head-to-tail (À) RNA intermediates, which after self-cleaving into monomeric linear (ml) forms via cis-acting hammerhead ribozymes [17] most likely acting co-transcriptionally [18], are circularized subsequently by a chloroplastic tRNA ligase [19].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

López-Carrasco

Journal of General Virology

2017

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 (À) 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 (À) 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.

Viroids

Encyclopedia of Life Sciences

Daròs

Hernández

et al. 2011

Viroids are infectious, nonprotein‐coding, highly structured small circular ribonucleic acids (RNAs) able to replicate autonomously and induce diseases in higher plants. Viroids and viruses differ in structure, function and evolutionary origin (with the former regarded as relics of a primitive RNA world). Viroids are classified into the families Pospiviroidae and Avsunviroidae , the members of which replicate in the nucleus and chloroplast, respectively, through an RNA‐based rolling circle mechanism with three steps catalysed by: (i) host deoxyribonucleic acid (DNA)‐dependent RNA polymerases redirected to accept RNA templates, (ii) processing enzymes or, in the family Avsunviroidae , hammerhead ribozymes and (iii) RNA ligases . When infecting a cell, the viroid RNA must travel to its replication organelle, with the resulting progeny moving cell‐to‐cell through plasmodesmata and reaching distal parts through the phloem. Pathogenesis could be triggered by the replicating viroid itself, or via RNA silencing. Viroids are detected by nucleic acid‐based approaches, some can be eliminated from infected plants by meristem tip culturing, and biotechnological approaches are being developed for their control. Key Concepts: Viroids are small nonprotein‐coding RNAs that infect, replicate and induce disease in higher plants. Viroids differ from viruses in fundamental aspects that include structure, function and evolutionary origin. Viroids are classified into the families Pospiviroidae (nuclear viroids) and Avsunviroidae (chloroplastic viroids). Viroid RNAs adopt compact secondary structures additionally stabilised by elements of tertiary structure. Viroids replicate through a rolling circle mechanism with only RNA intermediates and enzyme or ribozyme processing. Different viroids have distinct host ranges and closely related viroids display cross‐protection effects when co‐infecting a common host. Viroids move intracellularly, cell‐to‐cell through plasmodesmata, and long distance through the phloem. Viroids may infect their host plants latently or induce different pathogenic alterations including death. Viroids propagate in their hosts as a population of closely related variants (quasispecies). Viroids are considered molecular fossils of the RNA world postulated to have preceded our present world dominated by DNA and proteins.