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PubMed2006

Viroids: an Ariadne's thread into the RNA labyrinth.

Viroids are structurally, functionally and evolutionarily different from viruses. Despite their small, non-protein-encoding, single-stranded circular RNA genome, viroids can infect higher plants and cause certain diseases. Members of the two viroid families, Pospiviroidae and Avsunviroidae, have evolved to usurp the transcriptional machinery of their host nuclei and chloroplasts, respectively, in which replication proceeds through a rolling-circle mechanism involving RNA polymerization, cleavage and ligation. Remarkably, viroids subvert certain DNA-dependent RNA polymerases to transcribe RNA templates, and, in the family Avsunviroidae, post-transcriptional cleavage is catalysed by hammerhead ribozymes. Viroids are models for studying RNA evolution and for analysing RNA transport in plants, because they can move intracellularly, intercellularly through plasmodesmata and to distal parts of the plant through the vascular system. Viroids elicit RNA-silencing phenomena, which might mediate some of their biological properties, including pathogenesis. As some viroids behave as catalytic RNAs, they are regarded as remnants of the RNA world.

Base Sequence↗

PubMed1992

Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures.

Peach latent mosaic viroid (PLMVd), the causal agent of peach latent mosaic disease, has been sequenced and found to be a circular RNA molecule of 337 nucleotide residues, which adopts a branched conformation when it is folded in the model of lowest free energy. PLMVd exhibits limited homologies with other viroids and some satellite RNAs, but it does not have any of the central conserved sequences characteristic of the subgroups of typical viroids. However, a segment of approximately one-third of the PLMVd sequence has the elements required to form in the RNAs of both polarities the hammerhead structures proposed to act in the in vitro self-cleavage of avocado sunblotch viroid (ASBVd) and some satellite RNAs. Plus and minus partial- and full-length RNA transcripts of PLMVd containing the hammerhead structures displayed self-cleavage during transcription and after purification as predicted by these structures. These data are consistent with the high stability of the PLMVd hammerhead structures, more similar to the corresponding structures of some satellite RNAs than to those of ASBVd, and indicate that the self-cleavage reactions of PLMVd are most probably mediated by single hammerhead structures. Our results support the inclusion of PLMVd in a viroid subgroup represented by ASBVd, whose members are characterized by their ability to self-cleave in vitro, and probably in vivo, through hammerhead structures. A consensus phylogenetic tree has been obtained suggesting that PLMVd, together with ASBVd, may represent an evolutionary link between viroids and viroid-like satellite RNAs.

Base Sequence↗

PubMed2006

In silico predicted robustness of viroids RNA secondary structures. I. The effect of single mutations.

Viroids are plant subviral pathogens whose genomes are constituted by a single-stranded and covalently closed small RNA molecule that does not encode for any protein. Despite this genomic simplicity, they are able of inducing devastating symptoms in susceptible plants. Most of the 29 described viroid species fold into a rodlike or quasi-rodlike structure, whereas a few of them fold as branched structures. The shape of these RNA structures is perhaps one of the most characteristic properties of viroids and sometimes is considered their only phenotype. Here we use RNA thermodynamic secondary structure prediction algorithms to compare the mutational robustness of all viroid species. After characterizing the statistical properties of the distribution of mutational effects on structure stability and the wideness of neutral neighborhood for each viroid species, we show an evolutionary trend toward increased structural robustness during viroid radiation, giving support to the adaptive value of robustness. Differences in robustness among the 2 viroid families can be explained by the larger fragility of branched structures compared with the rodlike ones. We also show that genomic redundancy can contribute to the robustness of these simple RNA genomes.

Algorithms↗

PubMed1985

Complexes of viroids with histones and other proteins.

Complexes of potato spindle tuber viroid (PSTV) with nuclear proteins have been studied by in vitro reconstitution of the complexes and by isolation and characterization of in vivo complexes under non-dissociating conditions. For in vitro reconstitution, nuclear proteins were separated by SDS-gel-electrophoresis, renatured and blotted onto nitrocellulose filters, and incubated with viroid. The viroid-protein complexes were crosslinked covalently, and the viroid containing protein bands were detected by northern hybridization with a radioactive cDNA probe. The histones, a 41,000 dalton protein and to a small extent a 31,000 dalton protein were found in complexes with viroids. Raising the strength to 0.4 M NaCl destroys the complexes with the 41,000 dalton proteins but not those with the histones. From nucleoli, which are known to obtain the majority of viroids under non-dissociating conditions (Schumacher et al., (1983) EMBO J. 2, 1549-1555), a nucleosomal fraction was prepared. Viroids were found predominantly in this nucleosomal fraction. They are bound in a complex of 12-15 svedberg units.

Cell Nucleus↗

PubMed1987

Nucleotide sequence and secondary structure of apple scar skin viroid.

The complete nucleotide sequence of apple scar skin viroid(ASSV) has been established, and a probable secondary structure is proposed. A single-stranded circular ASSV RNA consists of 330 nucleotides and can assume the rodlike conformation with extensive base-pairing characteristic of all the known viroids. ASSV shows low sequence homologies with other viroids and lacks the central conserved region. These indicate that ASSV should be allocated to a separate viroid group. However, homologous sequences with potato spindle tuber viroid(PSTV) in ASSV occur in limited and scattered regions of both viroids. These homologous regions fall within the particular domains in the viroid domain model which has been previously proposed by Keese and Symons(Proc. Natl. Acad. Sci. USA. 82, 4582-4586, 1985).

Base Sequence↗

PubMed2002

Potato spindle tuber viroid strains of different pathogenicity induces and suppresses expression of common and unique genes in infected tomato.

Viroids are the smallest plant pathogens. These RNAs do not encode proteins and are not encapsidated, and yet they can replicate autonomously, move systemically, and cause diseases in infected plants. Notably, strains of a viroid with subtle differences in nucleotide sequences can cause dramatically different symptoms in infected plants. These features make viroids unique probes to investigate the role of a pathogenic RNA genome in triggering host responses. We conducted a comprehensive analysis of the differential gene expression patterns of tomato plants at various stages of infection by a mild and severe strain of Potato spindle tuber viroid (PSTVd). We also compared tomato gene expression altered by the PSTVd strains with that altered by Tobacco mosaic virus (TMV). Our analyses revealed that the two PSTVd strains altered expression of both common and unique tomato genes. These genes encode products involved in defense/stress response, cell wall structure, chloroplast function, protein metabolism, and other diverse functions. Five genes have unknown functions. Four genes are novel. The expression of some but not all of these genes was also altered by TMV infection. Our results indicate that viroids, although structurally simple, can trigger complex host responses. Further characterization of viroid-altered gene expression in a host plant should help understand viroid pathogenicity and, potentially, the mechanisms of RNA-mediated regulation of plant gene expression.

Gene Expression Regulation, Developmental↗

PubMed1991

Indian bunchy top disease of tomato plants is caused by a distinct strain of citrus exocortis viroid.

A viroid has been isolated from tomato plants affected by Indian bunchy top disease of tomato (Lycopersicon esculentum Mill.). In dot blot hybridization assays with 32P-labelled cRNA probes specific for the detection of various viroids, the Indian viroid was shown to be most closely related to the citrus exocortis viroid (CEVd). Sequence determination showed that the viroid consists of 372 nucleotides and confirmed its close relationship with CEVd. The viroid, for which we propose the acronym CEVd-t, differs from the Australian CEVd strains A and B by 36 and 47 nucleotides, respectively, and from the Spanish grapevine isolate by 52 changes. A phylogenetic analysis confirmed the closet relationship with CEVd in all structural domains, except the pathogenicity and left-terminal domains, which are closely related to the corresponding domains of the potato spindle tuber and tomato apical stunt viroids, respectively.

Base Sequence↗

PubMed1996

Identification and molecular properties of a 306 nucleotide viroid associated with apple dimple fruit disease.

A new viroid associated with an apple fruit disorder similar to, but more severe than, the dapple apple disease induced in some varieties by apple scar skin viroid (ASSVd) has been found. The new viroid, tentatively termed apple dimple fruit viroid (ADFVd), is a circular RNA of 306 nucleotides which adopts a quasi-rod-like conformation of minimum free energy. It contains the core nucleotides of the central conserved region (CCR) of the ASSVd group as well as the terminal conserved region (TCR) present in all members of the ASSVd and potato spindle tuber viroid (PSTVd) monophyletic groups. ADFVd has the highest sequence similarity with ASSVd and the 294 nucleotide citrus viroid CVd-IIIb sharing with the latter an almost identical left terminal domain. The right- and left-hand termini of ADFVd are formed by almost perfect duplications of sequences found in the CCR upper and lower strands, respectively, of PSTVd and closely related viroids.

Base Sequence↗

PubMed1989

A scheme for viroid classification.

A scheme for viroid classification is proposed based on the nature of the strictly conserved core sequence present in the central portion of the secondary structure of viroids. In this scheme, all of the known viroids can be classified as potato spindle tuber-type viroids consisting of two viroid groups and avocado sunblotch-type viroids containing one viroid group.

Base Sequence↗

PubMed2001

Control of mechanical viroid transmission by the disinfection of tables and tools.

Viroids are of practical importance as the cause of several economically significant infectious diseases. Potato spindle tuber viroid (PSTVd) causes severe yield losses in several crops, because the pathogen spreads fast within the culture. Viroids are small molecules, a few hundred nucleotides long, with a high degree of secondary structure. They do not code for any polypeptides and replicate independently of any associated plant virus. Viroids are readily transmitted by contaminated tools and tables. Furthermore PSTVd is transmitted through the pollen and true seed and can remain its infectious activity in seed for long periods. Vector transmission of PSTVd was reported to occur at low frequency. However, the mechanical transmission is the predominant factor and in this case we discuss the efficient disinfection of tools and tables as a main prophylactic trail to avoid viroid transmission. In previous studies we have tested the efficiency of several disinfectants to eliminate virus contamination. This paper demonstrates the efficient disinfection of MENNO-Florades (Menno-Chemie-GmbH, Norderstedt, Germany. A selection of different concentrations of the disinfectant and various times of incubation were applied in regard to practical use. The tests were confirmed by biological assays using suitable indicator plants, tissue print hybridization, gel electrophoresis and by nucleic acid hybridization. It was shown that PSTVd was eliminated when using the determined combination: 2% of the disinfectant while incubating for one minute or alternative 3%, 30 seconds. The possibility of viroid inactivation by a chemical method of disinfection--while plants are not affected--opens a new perspective to control viroid transmission via tools and tables.

Agriculture↗

PubMed2004

Viroids: unusual small pathogenic RNAs.

Viroids are small (about 300 nucleotides), single-stranded, circular, non-encapsidated pathogenic RNA molecules. They do not code for proteins and thus depend on plant host enzymes for their replication and other functions. They induce plant diseases by direct interaction with host factors but the mechanism of pathogenicity is still unknown. They can alter the expression of selected plant genes important for growth and development. Viroids belong to two families, the Avsunviroidae and the Pospiviroidae. Viroids of the Avsunviroidae family adopt a branched or quasi rod-like secondary structure in their native state. Members of the Pospiviroidae family adopt a rod-like secondary structure. In such native structures five structural/functional domains have been identified: central (C), pathogenicity, variable and two terminal domains. The central conserved region (CCR) within the C domain characterizes viroids of the Pospiviroidae. Specific secondary structures of this region play an important role in viroid replication and processing. Viroids of the Avsunviroidae family lack a CCR but possess self-cleaving properties by forming hammerhead ribozyme structures; they accumulate and replicate in chloroplasts, whereas members of the Pospiviroidae family have a nuclear localization. Viroid replication occurs via a rolling circle mechanism using either a symmetric or asymmetric pathway in three steps, RNA transcription, processing and ligation.

Base Sequence↗

PubMed1991

Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA.

The functional relevance of a hairpin II-containing structure of viroid RNA was studied by site-directed mutagenesis, thermodynamic calculations, experimental denaturation curves and infectivity tests. Hairpin II is formed during thermal denaturation of circular viroids or as part of a metastable structure during synthesis of viroid replication intermediates. In potato spindle tuber viroid (PSTVd), eight single-site mutations were generated in the segments which form hairpin II. From the mutated viroid cDNA clones, linear RNA transcripts of PSTVd unit length were synthesized. The relevance of hairpin II for the mechanism of denaturation was confirmed quantitatively by optical denaturation curves and temperature-gradient gel electrophoresis. Infectivity tests showed that the mutations in the core region of hairpin II reverted to the wild type sequence whereas the mutations in the peripheral regions of hairpin II remained genetically stable. These data are in accordance with the natural variance of hairpin II in other viroids of the PSTVd class. Thus, the integrity of the core of hairpin II is critical for infectivity. Hairpin II exhibits a strong similarity in sequence as well as in three-dimensional structure to certain DNA GC-clusters found in the 5'-upstream regions of some genes in man, animals, viruses and plants. A hypothesis about a function of hairpin II as a binding site for host cell transcription factors is proposed.

Base Composition↗

PubMed1981

Detection of complementary RNA intermediates of viroid replication by Northern blot hybridization.

Molecular hybridization by the Northern blot technique in combination with 125I-labeled PSTV (+) RNA and 32P-labeled PSTV cDNA as probes has been applied to detect viroid-specific sequences in healthy and viroid(PSTV)-infected tomato plants. Conditions are described which allow differentiation of (+) and (-) viroid sequences on the basis of the different thermostabilities of the corresponding hybrid molecules. By this experimental approach, it is documented that no viroid-specific DNA sequences can be detected and that viroid replication proceeds via complementary RNA intermediates. Out of the seven (-) RNA species found, six are apparently larger than the circular viroid (+) RNA and one is about the same size as the linear (+) RNA molecule.

Base Sequence↗

PubMed1988

Interference between coinoculated viroids.

Experiments were carried out to seek evidence of an interaction between two viroid RNAs introduced to tomato plants in the same inoculum. At the level of symptom expression, the severe isolate of potato spindle tuber viroid (PSTV) dominated the mild isolate. Seventy-five percent of the plants inoculated with a 100-fold excess of the mild isolate developed unattenuated symptoms of severe disease. Other experiments revealed that infectious RNA molecules transcribed from cloned DNA templates containing PSTV sequences reduced the level of hop stunt viroid (HSV) RNA present in nucleic acid extracts of plants which had been inoculated with a mixture of dimeric plus-strand transcripts of these two viroids. Plants inoculated with dual transcripts--containing two copies of PSTV linked to two copies of HSV--developed characteristic symptoms of severe PSTV. Dot hybridization demonstrated that only PSTV replicated to detectable levels in these plants. A likely interpretation of these results is that the HSV portion of the dual transcripts failed to replicate because of interference from PSTV. These results raise questions about how the process of viroid replication is related to symptom expression, and lead to suggested models for the effect of viroid-like RNAs in cells under both normal and pathogenic circumstances.

Plant Diseases↗

PubMed1988

A novel aspect of the information content of viroids.

Viroids were found to exhibit a structural periodicity characterized by repeat units of a length of 11 or 12 (potato spindle tuber viroid group and coconut cadang-cadang viroid), 60 (apple scar skin viroid) and 80 (avocado sunblotch viroid) nucleotide residues, respectively. It is suggested that structural periodicity of viroids is an indication of their protein-binding ability.

Base Sequence↗

PubMed2004

Biolistic inoculation of plants with viroid nucleic acids.

Parameters for biolistic transfer of viroid nucleic acids using a Helios Gene Gun device were assayed. The main achievement of this method is high efficiency of inoculation with linear monomeric viroid cDNAs and RNAs. This greatly facilitates the study of mutated sequence variants, viroid libraries and mixed populations. The lower limits for efficient inoculation of monomeric cDNA fragments with the sequence of potato spindle tuber viroid (PSTVd) and native PSTVd RNA as detected 21 days p.i. are in the range of 50 ng and 200 pg per tomato plant, respectively. At a higher dose, i.e. 2 ng of native RNA per plant, biolistic transfer causes drastic stunting compared to conventional mechanical inoculation, which points to higher PSTVd titers after the biolistic transfer. Infection is readily achieved with exact length monomeric RNA transcripts having 5'-triphosphate and 3'-OH termini in amounts ranging from 2 to 20 ng per plant, suggesting no need for any supplementary modifications of ends or RNA circularization. The biolistic transfer is efficient for viroid "thermomutants", which exhibit low or no infectivity with conventional mechanical inoculation with Carborundum. The biolistic inoculation is also efficient for two other members of the Pospiviroidae family, hop stunt and hop latent viroid.

Biolistics↗

PubMed2006

A novel RT-PCR approach for detection and characterization of citrus viroids.

Citrus plants are natural hosts of five viroid species and a large number of sequence variants. Because of their small size, viroids lend themselves to various RT-PCR approaches for their detection and further characterization. The one-step RT-PCR approach proposed here is based on the synthesis of viroid-cDNA by reverse transcription at 60 degrees C using a viroid specific 27-mer primer followed by standard second strand synthesis plus PCR amplification with various primer pairs. According to the primers used, full or partial length viroid-DNA is obtained. The technique avoids amplicon contamination in routine diagnosis. The suitability of the technique has been demonstrated using several nucleic acid extraction procedures and different viroid infected host species. The homogenization of tissue inside sealed plastic bags followed by nucleic acid extraction using a SDS/potassium acetate method is recommended because of its efficiency, simplicity and low cost. This extraction procedure, when coupled to the one-step RT-PCR approach, can be useful to avoid cross-contamination during routine diagnosis. A PCR strategy capable of discriminating between mild and severe strains of CEVd and identifying cachexia-inducing isolates of HSVd, is also described.

Base Sequence↗

PubMed2004

Viroids: petite RNA pathogens with distinguished talents.

Viroids are small, circular, single-stranded RNA molecules that cause several infectious plant diseases. Viroids do not encode any pathogen-specific peptides but nonetheless, the subviral pathogens replicate autonomously and spread in the plant by recruiting host proteins via functional motifs encoded in their RNA genome. During the past couple of years, considerable progress has been made towards comprehending how viroids interact with their hosts. Here, we summarize recent findings on the structure-function relationships of viroids, their strategies and mechanisms of replication and trafficking, and the identification and characterization of interacting host proteins. We also describe the impact of the RNA silencing machinery of plants on viroid RNAs and how this has started to influence our models of viroid replication and pathogenicity.

Carrier Proteins↗