A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids

Schumacher, J.; Randles, J. W.; Riesner, Detlev

doi:10.1016/0003-2697(83)90685-1

Cited by 166 publications

(88 citation statements)

References 18 publications

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“…The method of Schumacher et al (1983), as modified by Semancik & Harper (1984), was followed. The solution of nucleic acids obtained after the cellulose chromatography was subjected to one cycle of 5 ~ polyacrylamide gel electrophoresis under non-denaturing conditions (Morris & Wright, 1975).…”

Section: Introductionmentioning

confidence: 99%

Detection of Viroid and Viroid-like RNAs from Grapevine

Flores¹,

Duran-Vila²,

Pallás³

et al. 1985

Journal of General Virology

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SUMMARYAnalysis by polyacrylamide gel electrophoresis of nucleic acid preparations, obtained from several varieties of grapevine by a procedure designed to isolate and purify viroids, revealed the presence of RNA species with some of the characteristic physical properties of viroids. Under non-denaturing conditions, a band with a mobility faster than that of citrus exocortis viroid (CEV) was detected, and under fully denaturing conditions two bands were observed, one co-migrating with the circular forms of CEV and a second migrating faster than the linear forms of this viroid. This RNA species did not hybridize with a cDNA probe to CEV. Some of the grapevine preparations were infective for Gynura aurantiaca, inducing symptoms similar to those caused by CEV, and the appearance of an RNA which had the same mobility as CEV in denaturing and non-denaturing electrophoretic systems and hybridized with cDNA to CEV. These results suggest that viroid-like and viroid RNAs can be recovered from grapevine, the former (with no detectable sequence homology to CEV) at a concentration sufficient to be observed as a physical entity in gels, and the latter (with close sequence homology to CEV) whose presence could only be revealed by bioassay. The possible involvement of these RNAs in some grapevine diseases of unknown aetiology is discussed.

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Section: Introductionmentioning

confidence: 99%

Detection of Viroid and Viroid-like RNAs from Grapevine

Flores¹,

Duran-Vila²,

Pallás³

et al. 1985

Journal of General Virology

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“…Identification of new viroids requires purification and enrichment of the naked viroid RNA by 2D gel electrophoresis before cDNA synthesis and sequencing (4,5). However, viroids generally occur at low concentrations in the infected host, making viroid discovery a challenging task for many plant pathology laboratories.…”

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confidence: 99%

Homology-independent discovery of replicating pathogenic circular RNAs by deep sequencing and a new computational algorithm

Wang

Cao

et al. 2012

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

167

105

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A common challenge in pathogen discovery by deep sequencing approaches is to recognize viral or subviral pathogens in samples of diseased tissue that share no significant homology with a known pathogen. Here we report a homology-independent approach for discovering viroids, a distinct class of free circular RNA subviral pathogens that encode no protein and are known to infect plants only. Our approach involves analyzing the sequences of the total small RNAs of the infected plants obtained by deep sequencing with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Viroid infection triggers production of viroid-derived overlapping siRNAs that cover the entire genome with high densities. PFOR retains viroid-specific siRNAs for genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, which is synthesized from circular or multimeric repeated-sequence templates during viroid replication. We show that viroids from the two known families are readily identified and their fulllength sequences assembled by PFOR from small RNAs sequenced from infected plants. PFOR analysis of a grapevine library further identified a viroid-like circular RNA 375 nt long that shared no significant sequence homology with known molecules and encoded active hammerhead ribozymes in RNAs of both plus and minus polarities, which presumably self-cleave to release monomer from multimeric replicative intermediates. A potential application of the homology-independent approach for viroid discovery in plant and animal species where RNA replication triggers the biogenesis of siRNAs is discussed. T he term "viroid" was first introduced in 1971 to describe a novel class of free RNA nonviral pathogens found in plants (1-3). Viroids are single-stranded circular RNA molecules of 246-401 nt in length and do not encode any protein. Viroid RNAs display extensive intramolecular base pairing to give rod-like or quasirod-like conformations. Replication of viroids occurs via a rolling circle mechanism by host RNA polymerases to yield head-to-tail multiple-repeat replicative intermediates. Viroids identified to date infect only plants and belong to one of the two families. Viroids in the Pospiviroidae such as potato spindle tuber viroid (PSTVd) share a five-domain model including a central conserved region (CCR) and may all replicate in the nucleus. By contrast, viroids in the Avsunviroidae such as peach latent mosaic viroid (PLMVd) lack a CCR, encode a hammerhead ribozyme in both the positive and the negative strands that self-cleaves to release monomer from the multimeric intermediates, and may all replicate in the chloroplasts (1-3).Identification of new viroids requires purification and enrichment of the naked viroid RNA by 2D gel electrophoresis before cDNA synthesis and sequencing (4, 5). However, viroids generally occur at low concentrations in the infected host, making viroid discovery a challenging task for many plant pathology laborato...

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“…The gels were silver-stained using the modified procedure of Schumacher et al. (26). The original procedure is described in (27).…”

Section: Materials and Methods Chemicals And Buffersmentioning

confidence: 99%

Structure of viroid replicative intermediates: physico-chemical studies on SP6 transcripts of cloned oligomeric potato spindle tuber viroid

Steger¹,

Tabler

Brüggemann³

et al. 1986

Nucl Acids Res

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The structure and structural transitions of transcripts of cloned oligomeric viroid were studied in physico-chemical experiments and stability calculations. Transcripts of (+) and (-) polarity, from unit up to sixfold length, were synthesized from DNA clones of the potato spindle tuber viroid (PSTV) with the SP6 transcription system. Their structural properties were investigated by optical denaturation curves, high performance liquid chromatography (HPLC), electron microscopy, sedimentation-diffusion equilibrium and velocity sedimentation. Secondary structures of the RNAs and theoretical denaturation curves were calculated using an energy optimization program. The secondary structure of lowest free energy for unit length and oligomeric transcripts is a rod-like structure similar to that of the mature circular viroids. When this structure is used as a model for calculations, there is a large degree of agreement between the theoretical and the experimental denaturation curves. At high temperatures, however, (+) strand transcripts exhibited a transition which was more stable than expected from the calculations or than was known from curves of mature viroids. This transition arises from a rearrangement of the central conserved region of viroids to a helical region of 28 stable base pairs either intermolecularly leading to bimolecular complexes, or intramolecularly giving rise to a branched secondary structure. The rearrangement could be detected by electron microscopy, HPLC, and analytical ultracentrifugation. The helical region serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes which process the oligomeric intermediates to circular mature viroids.

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A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids

Cited by 166 publications

References 18 publications

Detection of Viroid and Viroid-like RNAs from Grapevine

Detection of Viroid and Viroid-like RNAs from Grapevine

Homology-independent discovery of replicating pathogenic circular RNAs by deep sequencing and a new computational algorithm

Structure of viroid replicative intermediates: physico-chemical studies on SP6 transcripts of cloned oligomeric potato spindle tuber viroid

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