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PubMed1999

Sensitive detection of potato spindle tuber and temperate fruit tree viroids by reverse transcription-polymerase chain reaction-probe capture hybridization.

A rapid and sensitive assay for the specific detection of plant viroids using reverse transcription-polymerase chain reaction (RT-PCR) -probe capture hybridization (RT-PCR-enzyme-linked immunosorbent assay (ELISA)) was developed. The assay was applied successfully for the detection of potato spindle tuber viroid, peach latent mosaic viroid, or apple scar skin viroid from viroid infected leaf tissue. Clarified sap extract from infected leaf tissue was treated first with GeneReleaser polymeric matrix to remove inhibitors of RT-PCR reactions. Viroid cDNA was then synthesized and amplified using viroid specific primers in RT-PCR assays and the amplified viroid cDNA (amplicon) was digoxigenin (DIG) -labelled during the amplification process. The amplicon was then detected in a colorimetric hybridization system in a microtiter plate using a biotinylated cDNA capture probe. This system combines the specificity of molecular hybridization, the ease of the colorimetric protocol, and is at least 100-fold more sensitive than gel electrophoretic analysis in detecting the amplified product. Viroid cRNA may replace viroid cDNA as the capture probe. The cRNA probe was several fold more sensitive than the cDNA probe for viroid detection. Six to seven hours are needed to complete the RT-PCR-ELISA for viroid detection from infected leaf tissue.

DNA Probes↗
PubMed2004

Development of a one tube-one step RT-PCR protocol for the detection of seven viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid and Pospiviroid.

A one tube-one step RT-PCR was developed for the detection of seven viroids (Apple scar skin viroid, Apple dimple fruit viroid, Pear blister canker viroid, Hop stunt viroid, Chrysanthemum stunt viroid, Citrus exocortis viroid and Peach latent mosaic viroid) in four genera that infect eight plant species. The efficiency and specificity of this method were optimized by the use of Moloney-murine leukemia virus (M-MLV) reverse-transcriptase and HotStarTaq DNA polymerase which allowed increase sensitivity of viroid detection. The method was assessed with 56 viroid-infected field plants. The multiplex one tube-one step RT-PCR has the advantage of requiring less hands-on time to set up an assay than standard multiplex one, it also reduces the possibility of false positive tests because all steps are performed in the same tube thus, avoiding cross-contamination. The method may be used routinely for viroid detection in sanitary and certification programmes.

DNA-Directed DNA Polymerase↗
PubMed1983

Dynamics and interactions of viroids.

Viroids are single stranded circular RNA molecules of 120,000 daltons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure. The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.

Base Sequence↗
PubMed1999

Tomato chlorotic dwarf viroid: an evolutionary link in the origin of pospiviroids.

Over 40 isolates of potato spindle tuber viroid (PSTVd) have been reported from potato, other Solanum species and greenhouse tomato. These isolates have sequence similarities in the range 95-99%. A viroid which caused chlorotic leaves and severe dwarfing of plants in greenhouse tomato crops was detected. The viroid was found to hybridize readily with PSTVd probes. It migrated faster than PSTVd in return-polyacrylamide gel electrophoresis and was not amplified in RT-PCR by a primer pair based on the lower strand of the central conserved region of PSTVd. Nucleotide sequencing of the viroid indicated that it is a circular RNA of 360 nt, with less than 90% sequence similarities with PSTVd isolates. The Variable domain (V) has less than 60% and the Terminal Right domain less than 90% sequence similarity, while the remainder of the molecule has greater than 97% similarity with PSTVd. Because of its less-than 90% sequence similarities, unique V domain, lack of seed-transmission and lack of cross-protection by PSTVd, the viroid from tomato is proposed to be a distinct viroid species (tomato chlorotic dwarf viroid; TCDVd) which also differs from two viroids infecting tomato in nature. TCDVd may be an evolutionary link in the development of crop viroids, with Mexican papita viroid as the ancestral viroid.

Base Sequence↗
PubMed2004

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

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.

Arabidopsis↗
PubMed2004

Analysis of thermal stress-mediated PSTVd variation and biolistic inoculation of progeny of viroid "thermomutants" to tomato and Brassica species.

Thermal stress of PSTVd-infected Nicotiana benthamiana led to appearance of a broad PSTVd sequence distribution, where most of mutations accumulated in the left half of the viroid's secondary structure including the "pathogenicity" domain. A similar effect had been reported for hop latent viroid [Virology 287 (2001) 349]. The pool of viroid "thermomutants" progenies was transcribed into cDNA and used for biolistic inoculation of Raphanus sativa, where the PSTVd infection was detectable by reverse transcription and polymerase chain reaction (RT-PCR). Newly generated inoculum from R. sativa was used for biolistic transfer to Arabidopsis thaliana wild-type and silencing-deficient mutants bearing one of sde1, sde2, and sde3 locuses. Irrespective to A. thaliana silencing mutants, viroid levels in Brasicaceae species infected with mutated PSTVd variants were of approximately 300 times lower than it is expected for tomato. At the same time, no systemic infection of A. thaliana was achieved with the wild-type PSTVd. In Arabidopsis, a population of PSTVd, consisting of frequent and minor variants, was present and the sequence distribution differed from that of the original viroid "thermomutants"; that is, mutations were not predominantly restricted to the left half of viroid's secondary structure. At least 65% of viroid sequences from Arabidopsis library accumulated mutations in the upper conserved central region (UCCR). In addition, mutants having changes in "hairpin II" domain (C-->A transition at position 229) and in the conserved internal loop element in the left part of viroid structure (single insertion of G at position 39) were detected. All those mutants were inoculated biolistically to tomato and promoted infection especially after prolonged period of plant cultivation (50-80 days pi) when infection reached 70-90%. However, the sequence variants were unstable and reverted to the wild type and to other sequence variants stable in tomato. Our results demonstrate that heat stress-mediated production of viroid quasi-species could be of significance for viroid adaptations.

Arabidopsis↗
PubMed1996

A new sequence variant of Coleus blumei viroid 3 from the Coleus blumei cultivar 'Fairway Ruby'.

A viroid was isolated from symptomless Coleus blumei cultivar (cv) 'Fairway Ruby' plants using the bidirectional PAGE method for analysis of small circular RNA molecules. The viroid was transmitted to viroid-free plants of Coleus blumei cv. 'Scarlet Dragonfly' by mechanical inoculation. Cloning and sequencing revealed that the viroid from the Coleus cv. 'Fairway Ruby' is closely related to the Coleus blumei viroid 3-Bv (CbVd 3-Bv) isolated from the Coleus cv. 'Bienvenue'. Therefore, the new viroid sequence variant has been named Coleus blumei viroid 3-FR (CbVd 3-FR). Coleus blumei viroid 3-FR consists of 364 nucleotides, 202 G + C, 162 A + U with a GC content of 55.5%. The most stable rod-like secondary structure of this viroid has 82 G:C, 50 A:U and 10 G:U base pairs with a minimum free energy of -670.5 kJ/mol (-160.4 kcal/mol). Due to mutations that increase the stability of the rod-like secondary structure, CbVd 3-FR has a lower minimum free energy than CbVd 3-Bv (-598.2 kJ/mol; -143.1 kcal/mol). A base-exchange in the upper strand of the central domain of CbVd 3-FR may stabilize the basal helix of hairpin I.

Base Sequence↗
PubMed1999

A simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and RT-PCR.

A simple, rapid method of nucleic acid extraction on a microcentrifuge tube scale for detecting viroids is presented. Five distinct citrus viroids (CVds), chrysanthemum stunt viroid (CSVd), hop stunt viroid (HSVd), hop latent viroid (HLVd) and potato spindle tuber viroid (PSTVd) were detected in their natural host plants by hybridization using cRNA probes and reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acids (NA) were liberated from tissues by incubation in a buffer containing potassium ethyl xanthogenate (PEX) without tissue homogenization, and then precipitated with ethanol (NA-PEX). All the viroids except CVd-IV could be detected clearly in NA-PEX by hybridization. HSVd, HLVd and PSTVd could also be detected in NA-PEX by RT-PCR. Although CVds and CSVd could not be detected in NA-PEX by RT-PCR, they were detected after further purification: differential precipitation with 2-butoxyethanol and HCl treatment followed by ethanol-precipitation. In addition, PCR in the presence of tetramethylammonium chloride specifically amplified the cDNA of all five distinct CVds under the same temperature and cycle conditions. Since all the viroids could be detected in NA liberated by PEX, the amount of NA extracted by the method described here is sufficient for detecting viroids, enabling the processing of a large number of samples.

DNA, Viral↗
PubMed1986

Viroid processing: a model involving the central conserved region and hairpin I.

A model is proposed for the processing of oligomeric viroid replication intermediates into monomeric, circular progeny viroids. The model identifies a thermodynamically extremely stable base-paired configuration that partially or completely dimeric, as well as higher, viroid oligomers can assume and postulates that this structure, which involves structural features common to all viroids (the central conserved region and secondary hairpin I), is essential for precise cleavage and ligation. The model explains why recombinant plasmids containing tandem repeats of two or more viroid sequence equivalents are highly infectious when inoculated into viroid-susceptible plants, why certain plasmids containing partially duplicated viroid-specific inserts are less infectious, and why plasmids containing monomeric inserts are noninfectious or at best marginally infectious. The model also accounts for the fact that vector-derived sequences on either or both sides of the viroid sequence(s) of a restriction fragment are precisely excised and are lacking in progeny viroids.

Base Sequence↗
PubMed1982

Nucleotide sequence and secondary structure of citrus exocortis and chrysanthemum stunt viroid.

The complete nucleotide sequence of citrus exocortis viroid (CEV, propagated in Gymura) and chrysanthemum stunt viroid (CSV, propagated in Cineraria) has been established, using labelling in vitro and direct RNA sequencing methods and a new screening procedure for the rapid selection of suitable RNA fragments from limited digests. The covalently closed circular single-stranded viroid RNAs consist of 371 (CEV) and 354 (CSV) nucleotides, respectively. As previously shown for potato spindle tuber viroid (PSTV, 359 nucleotides), CEV and CSV also contain a long polypurine sequence. Maximal base-pairing of the established CEV and CSV sequences results in an extended rod-like secondary structure similar to that previously established for PSTV and as predicted from detailed physicochemical studies of all these viroids. Although the three viroid species sequenced to date differ in size and nucleotide sequence, there is 60--73% homology between them. As PSTV, CEV and CSV also contain conserved complementary sequences which are separated from each other in the native secondary structure. We postulate that the resulting 'secondary' hairpins, being formed and observed in vitro during the complex process of thermal denaturation of viroid RNA, must have a vital, although yet unknown, function in vivo. The possible origin and function of viroids are discussed on the basis of the characteristic structural features and of a considerable homology with U1a RNA found for a region highly conserved in the three viroids.

Base Sequence↗
PubMed1989

Interactions between citrus exocortis and potato spindle tuber viroids in plants of Gynura aurantiaca and Lycopersicon esculentum.

Plants of Gynura aurantiaca and tomato (Lycopersicon esculentum) were inoculated with severe strains of citrus exocortis viroid (CEV) and potato spindle tuber viroid (PSTV). The progeny of both viroids was analyzed by two systems of PAGE which allowed discrimination between CEV and PSTV on the basis of their different sizes. When inoculated separately to G. aurantiaca CEV and PSTV induced severe and very mild symptoms, respectively, with CEV accumulating to a higher level than PSTV, whereas when tomato was the host, each viroid induced severe symptoms (although the reaction caused by PSTV was more intense) with PSTV reaching a higher steady-state concentration than CEV. The simultaneous inoculation of both viroids induced in G. aurantiaca the characteristic symptomatology of CEV, which was the only viroid that could be detected, whereas the response observed in tomato was that typical of PSTV, and only this viroid could be recovered. On the other hand, when G. aurantiaca plants were first inoculated with PSTV and later challenged with CEV, part of the plants displayed the characteristic severe reaction of CEV and both viroids were present in the corresponding extracts, but the other part showed the mild symptomatology induced by PSTV, and only this viroid could be isolated. However, when the protected plants were topped the new shoots exhibited severe symptoms and only CEV could be detected. These results suggest that CEV and PSTV compete for a limiting host factor needed for their replication, transport or accumulation, with CEV and PSTV having the highest affinity for the G. aurantiaca and tomato factors, respectively.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

Viroids: the smallest and simplest agents of infectious disease. How do they make plants sick?

Viroids are autonomously replicating pathogens of higher plants that consist solely of unencapsidated, single-stranded, circular RNAs of 246-375 nucleotides. Despite their extreme simplicity, viroids cause syndromes in plants that are about as varied as those caused by plant viruses. Because viroids are not translated, their effects on plants must be a consequence of direct interaction of the viroid with host constituents. Although the molecular mechanisms of viroid pathogenesis are still unknown, analysis of molecular chimeras between viroids of different pathogenicity levels has revealed that the severity of symptoms is the result of complex interactions among three of the five viroid domains. In vitro experiments with purified mammalian protein kinase P68 have shown that the enzyme is strongly activated (phosphorylated) by viroid strains that incite moderate to severe symptoms, but far less by a mild strain. Activation of a plant homolog of P68 may be the triggering event in viroid pathogenesis.

Base Sequence↗
PubMed1998

Specific RNA self-cleavage in coconut cadang cadang viroid: potential for a role in rolling circle replication.

The rolling circle replication of the small, single-stranded viroid RNAs requires a specific processing reaction to produce monomeric RNAs that are ligated into the final circular form. For avocado sunblotch viroid, peach latent mosaic viroid, and chrysanthemum chlorotic mottle viroid, the hammerhead self-cleavage reaction is considered to provide this processing reaction. We have searched for a similar type of reaction in the 246-nt coconut cadang cadang viroid, the smallest viroid of the 24-member potato spindle tuber viroid (PSTV) group. RNA transcripts prepared from the cloned central or C domain of this viroid self-cleaved specifically after denaturation with methylmercuric hydroxide followed by incubation in the presence of spermidine but in the absence of added magnesium ions. The unique cleavage site was located in the bottom strand of the C domain within a potential hairpin structure that is conserved within members of all three subgroups of the PSTV group of viroids.

Base Sequence↗
PubMed1989

Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy.

The intracellular localization of viroids has been investigated by viroid-specific in situ hybridization and analysis by digital microscopy of the distribution of the fluorescent hybridization signals. Isolated nuclei from green leaf tissue of tomato plants infected with potato spindle tuber viroid (PSTVd) were bound to microscope slides, fixed with formaldehyde and hybridized with biotinylated transcripts of cloned PSTVd cDNA. The bound probe was detected with lissamine--rhodamine conjugated streptavidin. Nucleoli were identified by immunofluorescence using the monoclonal antibody Bv96 and a secondary FITC-conjugated antibody. In plants infected with either a lethal or an intermediate PSTVd strain, the highest intensity of fluorescence that arose from hybridization with the probe specific for the viroid (+)strand was found in the nucleoli, confirming results of previous fractionation studies. A similar distribution was found for (-)strand replication intermediates of PSTVd using specific (+)strand transcripts as hybridization probes. In order to determine if viroids are located at the surface or in the interior of the nucleoli, the distribution of the fluorescence hybridization signals was studied with a confocal laser scanning microscope (CLSM). It was shown by three-dimensional reconstruction that viroids are neither restricted to the surface of the nucleoli nor to a peripheral zone, but are instead homogeneously distributed throughout the nucleolus. The functional implications of the intranucleolar location of viroids and their replication intermediates are discussed with respect to proposed mechanisms of viroid replication and pathogenesis.

Cell Nucleus↗
PubMed1997

Peach latent mosaic viroid is locked by a 2',5'-phosphodiester bond produced by in vitro self-ligation.

Although some viroid-like satellite RNAs possess self-cleavage and self-ligation activities, we show that the peach latent mosaic viroid (PLMVd) is unique among all known viroids since it also has such activities. These catalytic activities should have important roles in the rolling circle replication of PLMVd. According to this proposed mechanism, self-cleavage of the multimeric strands occurs via hammerhead structures producing monomers possessing 2',3'-cyclic phosphate and 5'-hydroxyl termini. In the most stable predicted secondary structure for PLMVd these two extremities are juxtaposed, in order for self-ligation to occur. To establish the nature of the phosphodiester bond produced by self-ligation, we followed the classical procedure of complete enzymatic RNA hydrolysis coupled with thin layer chromatography fractionation. Using this procedure, we report that the self-ligation of PLMVd transcripts produces almost exclusively the 2',5' isomer (>96%). Primer extension assays also revealed that reverse transcriptase can read througth this 2', 5' linkage, suggesting that it does not prevent further replication of the viroid. Moreover, we have observed that this 2',5' linkage is resistant to the debranching activity contained in nuclear extracts, as well as being capable of preventing further viroid self-cleavage. Thus, if viroids do indeed self-ligate in vivo, the resulting 2', 5'-phosphodiester bond could contribute to the stability of these RNA species. Finally, an analysis of both the sequence and the structural requirements for hammerhead self-cleavage and self-ligation suggests that these two RNA processes may be interrelated. We hypothesize that the intramolecular self-ligation which produces circular conformers may contribute to the circularization step of the rolling circle replication, while the intermolecular non-enzymatic ligation is a potential mechanism for the sequence reassortment of viroids and viroid-like species.

Endoribonucleases↗
PubMed2006

Construction of a multiprobe for the simultaneous detection of viroids infecting citrus trees.

Infections with different viroid species are common among cultivated fruit trees and grapevines, and many old-clone citrus varieties contain up to five citrus viroids (CVds) within a single tree. This paper describes the construction of a CVd-Multiprobe consisting of full-length clones of Hop stunt viroid, Citrus exocortis viroid, Citrus bent leaf viroid and CVd-III. The CVd-Multiprobe was tested against RNA transcripts of the four viroids and RNA extracts from plants singly infected with CEVd or HSVd or multiply infected with different CVds. The viroids were effectively diagnosed with the DIG labeled CVd-Multiprobe when tested by Northern hybridization or dot blot analyses. The CVd-Multiprobe does not provide information on the specific viroid resulting in a positive signal. However, this should not be considered as a problem, since most citrus certification programs will discard budwood source trees infected with any of the known CVds.

Blotting, Northern↗
PubMedDate not supplied

Some properties of the viroid inducing peach latent mosaic disease.

Analysis by polyacrylamide gel electrophoresis of nucleic acid extracts from different peach samples, healthy or infected with the peach latent mosaic (PLM) disease, demonstrated the association of this disease with an RNA exhibiting the electrophoretic properties typical of circular viroid molecules. This RNA was called peach latent mosaic viroid (PLMV), since a purified preparation of it, when inoculated into GF 305 peach seedlings induced characteristic symptoms of PLM disease. PLMV was estimated to have a molecular size in the range of 330-340 bases, by comparison of its electrophoretic mobility under denaturing conditions with those of several viroid RNA. Dot-blot analysis showed that PLMV had a sequence clearly different from other viroids, including citrus exocortis viroid, apple scar skin viroid (ASSV), hop stunt viroid (HSV) and avocado sunblotch viroid. The possible significance of the limited sequence homology shared by PLMV with HSV, and especially with ASSV, is discussed.

Fruit↗
PubMedDate not supplied

Scar skin and dapple apple viroids are seed-borne and persistent in infected apple trees.

The closely related apple scar skin viroid (ASSV) and dapple apple viroid (DAV) were identified in whole seeds from infected pome fruits by hybridization of extracted nucleic acids with a 32P-labelled ASSV cRNA probe. Viroid amounts were greater in seed coats and subcoats than in seed cotyledons and embryos. ASSV or DAV was also detected in nucleic acid extracts from infected seeds, cotyledons and embryos by reverse transcription/polymerase chain reaction with viroid-cDNA-specific primers followed by Southern blot hybridization analysis of the amplified products with an ASSV cRNA probe. These results indicate that ASSV and DAV are seed-borne. ASSV and DAV were also found in the anthers, petals, receptacles, leaves, bark and roots of infected trees. The results suggest that viroid-infected trees constitute potential sources of the viroid in field spread. ASSV and DAV infections have been observed sporadically in commercial orchards in the United States and Canada and the infected trees have been eliminated. The use of viroid-free sources of seeds, seedlings, rootstocks and budwood should greatly reduce the risk of the future spread of the viroid.

Animals↗