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At least 145 records · Page 8Linked to original sources
PubMed2001

A novel usage of random primers for multiplex RT-PCR detection of virus and viroid in aphids, leaves, and tubers.

A multiplex reverse transcription polymerase chain reaction (m-RT-PCR) was developed for the simultaneous detection of five potato viruses and a viroid. The synthesis of cDNAs used for amplification was primed by hexanucleotides (random primers, RP). An RNA extraction procedure employing DNase I, is routinely used to isolate potato viruses and viroid (Potato virus S, PVS; Potato leafroll virus, PLRV; Potato virus X, PVX; Potato virus A and Y, PVA, PVY; and Potato spindle tuber viroid, PSTVd) from infected tissues. This extraction method produced deoxy-oligonucleotides, which in turn were used to prime the reverse transcription of RNA templates of all the viruses and the viroid. A time-course study from 15 s to 30 min showed optimal oligonucleotide generation by DNase I occurred at 10 min, an incubation time already incorporated in the extraction protocol. The presence of oligonucleotides capable of priming cDNA synthesis was also demonstrated in RNA preparations from aphids, leaves, and tubers. In order to duplicate the priming of templates by oligonucleotides, commercially available hexanucleotides were used as RP. When fragments were amplified from 5'- and 3'-ends of the random primed cDNA of PVY genome, bands of similar intensity were observed. In contrast, when two fragments (short and long) from the P1 gene region of the PVA genome were amplified, the yield of the short fragment was significantly higher in intensity than that of the long fragment in random primed cDNA. Irrespective of the origin of the primers (generated during extraction vs. commercially purchased), single or multiple viruses or the viroid were detected by amplification of random primed cDNAs present individually in the reaction or in a cDNA pool consisting of five viruses and the viroid. The cDNA produced by RP or virus specific primers (SP) was used to detect PLRV and PVY from infected tubers in a duplex reverse transcription polymerase chain reaction (d-RT-PCR). The RP cDNA gave increased detection. Comparison of RP primed cDNAs from dormant or sprouted tubers and leaves showed that for some cultivars, such as 'Shepody', leaves were more reliable for PVY and PLRV detection than the tubers, in both the d- and m-RT-PCR.

Animals↗
PubMed1999

Single-strand conformation polymorphism (SSCP) analysis as a tool for viroid characterisation.

The potential of routine single-strand conformation polymorphism (SSCP) analysis for viroid characterization has been evaluated. Electrophoresis of 311 cloned full length viroid DNA inserts recovered from a field isolate of citrus exocortis viroid (CEVd) showed shifts in the migration of the cDNA or/and hDNA strands in non-denaturing 14% polyacrylamide gels. Using a single set of electrophoresis conditions seven different groups of variants (containing one to six changes), which did not represent the overall variability among clones, were identified. The relationship between the different SSCP profiles observed among clones and the variation in their nucleotide sequences was confirmed by sequence analysis. Variations in the dimensions of the gel allowed higher resolution and therefore the detection of additional single nucleotide variations among clones initially clustered into the same group. The viroid region affected by specific changes could be established by SSCP analysis of partial viroid length DNA. The potential use of SSCP analysis as a tool to screen existing viroid populations in infected hosts prior to sequencing is discussed.

Base Sequence↗
PubMed2004

On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites.

Viroids and most viral satellites have small, noncoding, and highly structured RNA genomes. How they cause disease symptoms without encoding proteins and why they have characteristic secondary structures are two longstanding questions. Recent studies have shown that both viroids and satellites are capable of inducing RNA silencing, suggesting a possible role of this mechanism in the pathology and evolution of these subviral RNAs. Here we show that preventing RNA silencing in tobacco, using a silencing suppressor, greatly reduces the symptoms caused by the Y satellite of cucumber mosaic virus. Furthermore, tomato plants expressing hairpin RNA, derived from potato spindle tuber viroid, developed symptoms similar to those of potato spindle tuber viroid infection. These results provide evidence suggesting that viroids and satellites cause disease symptoms by directing RNA silencing against physiologically important host genes. We also show that viroid and satellite RNAs are significantly resistant to RNA silencing-mediated degradation, suggesting that RNA silencing is an important selection pressure shaping the evolution of the secondary structures of these pathogens.

Base Sequence↗
PubMed1983

Structural similarities between viroids and transposable genetic elements.

The primary structures of the tomato planta macho and tomato apical stunt viroids have been determined, and probable secondary structures are proposed. Both viroids can assume the rodlike conformation with extensive base-pairing characteristic of all known viroids. Sequence homologies between the two viroids (75%) and with members of the potato spindle tuber viroid group (73-83%) indicate that they both belong to this group. Comparative sequence analysis of all members of the group reveals striking similarities with the ends of transposable genetic elements. These similarities, the presence of inverted repeats often ending with the dinucleotides U-G and C-A, and flanking imperfect direct repeats suggest that viroids may have originated from transposable elements or retroviral proviruses by deletion of interior portions of the viral (or element) DNA.

Base Sequence↗
PubMed1992

Generation of viroid conformational isomers that are stable to incubation with magnesium ions and in a nuclear extract from tomato plants.

We identified conditions for heating and quick cooling viroid RNAs in the presence of salt which lead to the production of conformational isomers stable to incubation for at least 45 minutes at 30 degrees in the presence of magnesium ions. Elution in 0.3 M NaCl allowed the purification of an electrophoretically slow form of an in vitro transcript carrying a complete copy of the potato spindle tuber viroid RNA sequence. Slow forms of this transcript and of kinase-labeled linear viroid RNA persisted for longer than 20 minutes when incubated with a protein-rich extract prepared from the nuclei of uninfected tomato plants, although both were slowly cleaved by a nuclease present in this extract. The fast form of the transcript was highly resistant to this tomato ribonuclease. The slow form of the transcript was much more susceptible to cleavage by RNase T1 than the fast form of this RNA, suggesting that the reduced gel mobility of the slow forms results from their relatively open structure. The ability to purify viroid conformational isomers from polyacrylamide gels will facilitate biochemical studies aimed at identifying host components interacting with RNAs of the viroid replication complex, which may not all be present in the most thermodynamically favored rodlike structure of mature viroids.

Cell Nucleus↗
PubMed1981

Chrysanthemum stunt viroid: primary sequence and secondary structure.

The sequence of the 356 nucleotide residues of chrysanthemum stunt viroid (CSV) has been determined. Overlapping linear viroid fragments were obtained by partial ribonuclease digestion, radiolabelled in vitro at their 5'-ends, and sequenced using partial enzymic cleavage methods. Of the CSV sequence, 69% is contained in the published sequence of potato spindle tuber viroid (PSTV). Differences in the primary sequence of CSV and PSTV suggest that neither the positive nor putative negative strands of these two viroids code for functional polypeptide products. However, the two viroids can form similar secondary structures, implicating a role for viroid structure in replication.

Base Sequence↗
PubMed1988

Isolation of three viroids and a circular RNA from grapevines.

Analysis of nucleic acids from grapevine tissues by two-dimensional gel electrophoresis demonstrated the presence of two bands of circular RNA. The smaller RNA contained about 300 nucleotide residues and was identified as hop stunt viroid by nucleotide sequencing. The larger RNA band was a mixture of species and contained similar amounts of two components, referred to as RNA 1a and RNA 1b, and in addition a trace amount of citrus exocortis viroid (CEV) which became detectable only after inoculation of the mixture to tomato. The identity of CEV was determined by probe hybridization and nucleotide sequencing. Both RNAs 1a and 1b are distinct from CEV and have estimated sizes larger than those of CEV and other viroids reported so far. RNA 1a preparations were infectious in cucumber and in tomato and the recovered viroid had unique properties. We have provisionally named this viroid Australian grapevine viroid. Evidence for the autonomous replication of RNA 1b was not obtained.

Base Sequence↗
PubMed1991

Identification of a new viroid as the putative causal agent of pear blister canker disease.

A small RNA species with the structural and functional properties characteristic of viroids has been isolated from three different pear sources each of which induced symptoms of the pear blister canker (PBC) disease when indexed in the pear indicator A 20. A close association between this RNA and PBC disease was established, since two of the three studied sources were known to be affected by this malady only, and the viroid was not detected in healthy pear tissue. Moreover, the PBC-associated viroid (PBCVd) replicated when purified preparations were inoculated into cucumber and pear plants. PBCVd behaved in denaturing polyacrylamide gels as a circular RNA with a molecular size of approximately 315 nucleotide residues. Analysis by dot blot hybridization indicated that PBCVd shares similarities in sequence with peach latent mosaic viroid and hop stunt viroid, and to a lesser extent with apple scar skin viroid.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

A citrus exocortis viroid variant from broad bean (Vicia faba L.): infectivity and pathogenesis.

A viroid present in very low titres was isolated from symptomless field broad bean plants. It was identified as a variant of citrus exocortis viroid in the T2, V and C domains. Infection of several hosts resulted in a change in the composition of the viroid population. Serial passage through tomato and back to the host of origin, broad bean, resulted in major changes in replication efficiency, host range and pathogenicity. The unique nucleotide sequence differences identified in the original broad bean variant were not conserved after passage through alternative hosts. The effects of these sequence variations on viroid secondary structure result in nonpathogenic viroid variants which can remain unnoticed in certain plant species but may act as reservoirs of viroid disease.

Base Sequence↗
PubMed2003

A bromodomain-containing protein from tomato specifically binds potato spindle tuber viroid RNA in vitro and in vivo.

For the identification of RNA-binding proteins that specifically interact with potato spindle tuber viroid (PSTVd), we subjected a tomato cDNA expression library prepared from viroid-infected leaves to an RNA ligand screening procedure. We repeatedly identified cDNA clones that expressed a protein of 602 amino acids. The protein contains a bromodomain and was termed viroid RNA-binding protein 1 (VIRP1). The specificity of interaction of VIRP1 with viroid RNA was studied by different methodologies, which included Northwestern blotting, plaque lift, and electrophoretic mobility shift assays. VIRP1 interacted strongly and specifically with monomeric and oligomeric PSTVd positive-strand RNA transcripts. Other RNAs, for example, U1 RNA, did not bind to VIRP1. Further, we could immunoprecipitate complexes from infected tomato leaves that contained VIRP1 and viroid RNA in vivo. Analysis of the protein sequence revealed that VIRP1 is a member of a newly identified family of transcriptional regulators associated with chromatin remodeling. VIRP1 is the first member of this family of proteins, for which a specific RNA-binding activity is shown. A possible role of VIRP1 in viroid replication and in RNA mediated chromatin remodeling is discussed.

Amino Acid Sequence↗
PubMed2004

A long-distance translocatable phloem protein from cucumber forms a ribonucleoprotein complex in vivo with Hop stunt viroid RNA.

Viroids are highly structured plant pathogenic RNAs that do not code for any protein, and thus, their long-distance movement within the plant must be mediated by direct interaction with cellular factors, the nature of which is presently unknown. In addition to this type of RNAs, recent evidence indicates that endogenous RNAs move through the phloem acting as macromolecular signals involved in plant defense and development. The form in which these RNA molecules are transported to distal parts of the plant is unclear. Viroids can be a good model system to try to identify translocatable proteins that could assist the vascular movement of RNA molecules. Here, we demonstrate by use of immunoprecipitation experiments, that the phloem protein 2 from cucumber (CsPP2) is able to interact in vivo with a viroid RNA. Intergeneric graft assays revealed that both the CsPP2 and the Hop stunt viroid RNA were translocated to the scion. The translocated viroid is symptomatic in the nonhost scion, indicating that the translocated RNA is functional. The CsPP2 gene was cloned and sequenced. The analysis of its primary structure revealed the existence of a potential double-spaced-RNA-binding motif, previously identified in a set of proteins that bind to highly structured RNAs, which could explain its RNA-binding properties. The possible involvement of this phloem protein in assisting the long-distance movement of the viroid RNA within the plant is discussed.

Amino Acid Sequence↗
PubMed2003

Viroid RNA systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein VirP1.

Viroids are noncoding circular single-stranded RNAs that are propagated systemically in plants. VirP1 is a protein from tomato, which is an excellent host for potato spindle tuber viroid (PSTVd), and it has been isolated by virtue of its specific in vitro binding to PSTVd RNA. We report on the specific in vivo interaction of VirP1 with full-length viroid RNA as well as with subfragments in the three-hybrid system. The terminal right domain (TR) of PSTVd was identified as a strong interacting partner for VirP1. A weaker partner is provided by a right-hand subfragment of hop stunt viroid (HSVd), a viroid that infects tomato poorly. We present a sequence and structural motif of the VirP1-interacting subfragments. The motif is disturbed in the replicative but nonspreading R+ mutant of the TR. According to our in vivo and in vitro binding assays, the interaction of this mutant with VirP1 is compromised. We propose that the AGG/CCUUC motif bolsters recognition of the TR by VirP1 to achieve access of the viroid to pathways that propagate endogenous RNA systemic signals in plants. Systemic trafficking has been suggested for miRNA precursors, of which the TR, as a stable bulged hairpin 71 nt long, is quite reminiscent.

Base Sequence↗
PubMed1994

Inhibition of viroid infection by antisense RNA expression in transgenic plants.

Complex formation between different antisense RNAs directed against either plus-strand or minus-strand sequences of the potato spindle tuber viroid (PSTVd) was studied using temperature-gradient gel electrophoresis and immunochemical detection with an antibody specific for double-stranded RNA. Short minus-strand sequences were directed against the upper central conserved region (UCCR) of plus-strand viroid replication intermediates, a plus-strand corresponding to the left half of the rod-like secondary structure (VL+) against minus-strand replication intermediates. It was shown that antisense RNA forms complexes with the corresponding target RNA only with low yield during incubation at low (physiological) temperatures but with high yield during in vitro transcription of the target RNA when the antisense RNA is already present in the solution. The antisense RNA sequences were integrated into Solanum tuberosum L. by Agrobacterium tumefaciens transformation. Antisense RNA expression in vivo was analyzed by Northern analysis. Infection tests were performed using the transgenic potato lines in order to evaluate their degree of resistance against PSTVd infection. Although some lines showed a significant inhibition of viroid accumulation, a high variability of viroid infection in different transgenic potato lines was obtained. Since strongly infected plants were observed in all transgenic lines 6 to 8 weeks post inoculation, a threshold concentration of viroid, overcoming the antisense effect has to be assumed. When the rate of viroid accumulation was tested using agroinfection assays on leaf discs, a stronger antisense effect could be achieved.

Agrobacterium tumefaciens↗
PubMed1987

Mutational analysis of potato spindle tuber viroid reveals complex relationships between structure and infectivity.

Viroids are single-stranded, covalently closed circular RNA pathogens that can be isolated from certain higher plants afflicted with specific diseases. Their small size (246-375 nucleotides; M(r) 0.8-1.3 x 10(5)) and ability to replicate autonomously make viroids a unique model system in which to study the relationships between the structure of an RNA and its biological function. The demonstrated infectivity of certain cloned viroid cDNAs allows the use of site-specific mutagenesis techniques to probe structure-function relationships suggested by comparative sequence analysis. Several site-specific mutations that disrupt base pairing in either the native structure or secondary hairpin I destroyed the ability of potato spindle tuber viroid cDNA to initiate infection. Alterations in the terminal loops of the native structure also abolished cDNA infectivity. One pseudorevertant, a mutant cDNA containing compensating changes that restore base pairing in the native structure, was marginally infectious; a second pseudorevertant in which base pairing was restored within the stem of secondary hairpin I was not infectious. The behavior of these mutants dramatically demonstrates the effect of remarkably small structural changes on viroid infectivity and emphasizes the importance of the conserved rod-like native structure for viroid function.

Journal Article↗
PubMed1977

Comparative oligonucleotide fingerprints of three plant viroids.

5' Phosphorylation in vitro with gamma-32P-ATP and T4 phage induced polynucleotide kinase was used to obtain RNAase A and RNAase T1 fingerprints of three plant viroids: Potato spindle tuber viroid from tomato (PSTV-tom), chrysanthemum stunt viroid from cineraria (ChSV-cin) and citrus exocortis viroid from Gynura aurantiaca (CEV-gyn). These three viroids differ significantly from each other as judged from their oligonucleotide patterns. This supports the concept of individual viroid species.

Oligonucleotides↗
PubMed1978

Common structural features of different viroids: serial arrangement of double helical sections and internal loops.

The thermodynamic parameters of five different highly purified viroid "species" were determined by applying UV-absorption melting analysis and temperature jump methods. Their thermal denaturation proved to be a highly cooperative process with midpoint-temperatures (Tm) between 48.5 and 51 degrees C in 0.01 M sodium cacodylate, 1 mM EDTA, pH 6.8. The values of the apparent reaction enthalpies of the different viroid species range between 3,140 and 3,770 kJ/mol. Although the cooperativity is as high as found in homogeneous RNA double helices the Tm-value of viroid melting is more than 30 degrees C lower than in the homogeneous RNA. In order to explain this deviation, melting curves were simulated for different models of the secondary structure of viroids using literature values of the thermodynamic parameters of nucleic acids. Our calculations show that the following refinement of our earlier model is in complete accordance with the experimental data: In their native conformation viroids exist as an extended rodlike structure characterized by a series of double helical sections and internal loops. In the different viroid species 250-300 nucleotides out of total 350 nucleotides are needed to interprete the thermodynamic behaviour.

Mathematics↗
PubMed1996

Secondary structure probing of potato spindle tuber viroid (PSTVd) and sequence comparison with other small pathogenic RNA replicons provides evidence for central non-canonical base-pairs, large A-rich loops, and a terminal branch.

Using PCR and in vitro transcription, linear (non-circular) unit-length (+)strand RNA molecules of a lethal PSTVd variant were produced which are able to initiate typical disease symptoms when inoculated into tomato. Non-denaturing gel electrophoresis shows that these transcripts can adopt the same two conformations as circular PSTVd molecules, namely a fast migrating rod-like and a slowly migrating cruciform structure. The rod-like conformer of two end-labelled transcripts was probed with nucleases and dimethyl sulphate, revealing that in solution its right part is identical to computer prediction. In the left part, however, three unique features could be substantiated. (1) In the central region a UV-cross-linkable loop is closed and thus contains non-canonical base-pairs ("loop E structure"). (2) Three large "pre-melting loops" are present at 25 degrees to 37 degrees C. The structure of the leftmost one, which is A-rich and conserved in most viroids, correlates with pathogenicity. (3) Two small stem-loops instead of an unbranched structure are found at the left terminus. These hairpins can form in all "large" viroids (approximately 300 nucleotides or longer), thus placing the dodecamer conserved among these viroids, GGUUCCUGUGGU, within the upper helix and the branch junction. A large viroid from Iresine lacks one of these hairpins, whereas all "small" viroids (approximately 300 nucleotides or smaller) lack both. In several plant virus satellite RNAs and the newt satellite RNA, the motif GAUUU(U) and dodecamer remnants appear in an equivalent structure comprising two or three hairpins. Using lead- and terbium-induced cleavage of the RNA, metal binding sites were found, mostly in loops. Thus, probing of PSTVd RNA and comparison with other.

Base Composition↗
PubMed1998

Dynamic competition between alternative structures in viroid RNAs simulated by an RNA folding algorithm.

The folding pathways of viroid RNAs were studied using computer simulations by the genetic algorithm for RNA folding. The folding simulations were performed for PSTVd RNAs of both polarities, using the wild-type sequence and some previously known mutants with suggested changes in the stable or metastable structures. It is shown that metastable multihairpin foldings in the minus strand replicative intermediates are established due to the specific folding pathway that ensures the absence of the most stable rod-like structure. Simulations of the PSTVd minus strand folding during transcription reveal a metastable hairpin, formed in the left terminal domain region of the PSTVd. Despite high sequence variability, this hairpin is conserved in all known large viroids of both subgroups of PSTVd type, and is presumably necessary to guide the folding of the HPII hairpin which is functional in the minus strand. The folding simulations are able to demonstrate the changes in the balance between metastable and stable structures in mutant PSTVd RNAs. The stable rod-like structure of the circular viroid (+) RNA is also folded via a dynamic folding pathway. Furthermore, the simulations show that intermediate steps in the forced evolution of a shortened PSTVd replicon may be reconstructed by a mechanistic model of different folding pathway requirements in plus- and minus-strand RNAs. Thus the formation of viroid RNA structure strongly depends on dynamics of competition between alternative RNA structures. This also suggests that the replication efficiency of viroid sequences may be estimated by a simulation of the folding process.

Algorithms↗