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PubMed2001

Phylogenetic analysis of viroid and viroid-like satellite RNAs from plants: a reassessment.

The proposed monophyletic origin of a group of subviral plant pathogens (viroids and viroid-like satellite RNAs), as well as the phylogenetic relationships and the resulting taxonomy of these entities, has been recently questioned. The criticism comes from the (apparent) lack of sequence similarity among these RNAs necessary to reliably infer a phylogeny. Here we show that, despite their low overall sequence similarity, a sequence alignment manually adjusted to take into account all the local similarities and the insertions/deletions and duplications/rearrangements described in the literature for viroids and viroid-like satellite RNA, along with the use of an appropriate estimator of genetic distances, constitutes a data set suitable for a phylogenetic reconstruction. When the likelihood-mapping method was applied to this data set, the tree-likeness obtained was higher than that corresponding to a sequence alignment that does not take into consideration the local similarities. In addition, bootstrap analysis also supports the major groups previously proposed and the reconstruction is consistent with the biological properties of this RNAs.

Evolution, Molecular↗

PubMed1984

Nucleotide sequence of cucumber pale fruit viroid: homology to hop stunt viroid.

Double stranded cDNA of cucumber pale fruit viroid ( CPFV ) has been cloned by the method of Okayama and Berg (Mol.Cell.Biol.2,161-170 (1982] and the complete nucleotide sequence was established. The covalently closed circular molecules of single-stranded CPFV RNA consists of 303 nucleotides. The nucleotide sequence of CPFV was compared with the previously established sequence of hop stunt viroid (HSV), which consists of 297 nucleotides ( Ohno et al. Nucleic Acid Res.11,6185-6197 (1983]. CPFV differs from HSV in the nucleotide sequence at 16 positions which include 8 exchanges, 7 insertions and 1 deletion. Both viroids share about 95% sequence homology. Considering the pathogenic properties of both viroids together, it is concluded that CPFV is a cucumber isolate of HSV.

Base Sequence↗

PubMed2007

Accumulation of viroid-specific small RNAs and increase in nucleolytic activities linked to viroid-caused pathogenesis.

Strong viroid-caused pathogenesis was achieved in tomato cv. Rutgers by biolistic transfer of severe or lethal potato spindle tuber viroid (PSTVd) strains, while other tomato genotypes (e.g., Moneymaker) were tolerant. With reciprocal hybrids between sensitive and tolerant genotypes, we show that plant depression dominates over tolerance. Biolistic transfer of the most pathogenic PSTVd strain AS1 to Nicotiana benthamiana, which is considered to be a symptomless PSTVd host, led to a strong pathogenesis reaction and stunting, suggesting the presence of specific viroid pathogenesis-promoting target(s) in this plant species. Total levels of small siRNA-like PSTVd-specific RNAs were enhanced in strongly symptomatic tomato and N. benthamiana plants after biolistic infection with AS1 in comparison to the mild QFA strain. This indicates association of elevated levels of viroid-specific small RNA with production of strong symptoms. In symptom-bearing tomato leaves in comparison to controls, an RNase of approximately 18 kDa was induced and the activity of a nuclease of 34 kDa was elevated by a factor of seven in the vascular system. Sequence analysis of the nuclease cDNA designated TBN1 showed high homology with plant apoptotic endonucleases. The vascular-specific pathogenesis action is supported by light microscopic observations demonstrating a certain lack of xylem tissue and an arrest of the establishment of new vascular bundles in collapsed plants.

Amino Acid Sequence↗

PubMed1987

Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells.

A nuclear extract for the processing of oligomeric viroid RNA in vitro has been prepared from nuclei isolated from healthy potato cells grown in suspension culture. Linear RNA molecules containing concatameric units of (+) or (-) strands, respectively, of the potato spindle tuber viroid (PSTV) were synthesized in vitro with the aid of the SP6 RNA polymerase and used as substrates for processing. When oligomeric linear PSTV (+)RNAs are incubated with the nuclear extract, monomeric linear molecules are accurately excised from them, and ligated to monomeric PSTV (+)RNA circles representing the viroid proper. Oligomeric PSTV (-)RNAs are likewise processed but with a much lower efficiency. Viroid-processing operates although other nucleolytic activities are still present in the extract. These results substantiate our previous finding that oligomeric PSTV does not process autocatalytically under in vitro conditions where certain introns and other RNAs do. This is the first report of an in vitro RNA processing system derived from higher plants.

Journal Article↗

PubMed1984

A revised replication cycle for viroids: the role of longer than unit length RNA in viroid replication.

Longer than unit length plus and minus strand RNAs were detected in hop stunt viroid (HSV) infected cucumber leaf tissues by Northern blot hybridization analysis using strand-specific probes. To elucidate the role of these longer than unit length RNAs in the viroid replication cycle, we synthesized tandemly repeated plus and minus strand HSV RNAs in vitro from cloned HSV cDNA and assayed their infectivities. Two and four unit tandemly repeated plus strand RNAs were infectious, but one unit plus, and one, two and four unit minus strands were noninfectious. Taking these data into consideration, we propose a revised rolling circle model for viroid replication.

Cloning, Molecular↗

PubMed1996

Viroids proper can be distinguished from hammerhead viroids and satellite RNAs through their dinucleotide composition.

G + C-rich viroids proper exhibit a unique dinucleotide composition in that AU and UA are much less frequent than expected. Thus, evaluation of the dinucleotide pattern allows a quick discrimination between viroids proper and similar RNAs from plants, such as hammerhead-containing viroids, satellite RNAs, and defective interfering RNAs. In addition to sequence alignment, this method might be useful for the classification of a newly found small RNA replicon.

Base Composition↗

PubMed2000

A population of variants of a viroid closely related to citrus viroid-I in citrus plants.

Sequencing analyses showed a population of variants consisting of 325-330 nucleotides (nt) of a viroid closely related to citrus viroid (CVd)-I in citrus plants. These variants, for which we propose the tentative acronym CVd-I-LSS (low sequence similarity), have only 82-85% sequence similarities to CVd-I variants. A phylogenetic tree showed that the CVd-I-LSS variants formed an individual cluster that was distinct from that of the CVd-I variants, but no intermediate variants were found which could continuously connect the population of CVd-I-LSS variants with that of CVd-I variants. Citrons (Citrus medica L.) inoculated with the CVd-I-LSS seemed to show not only moderate leaf bending like citrons infected with CVd-I but also slightly more severe epinasty than citrons with CVd-I. Other biological properties of CVd-I-LSS, such as the host range, will need to be determined in order to clarify whether CVd-I-LSS is a new viroid species or a distinct strain of CVd-I. Most of the nucleotide changes between the CVd-I-LSS and CVd-I variants were found at complementary positions of the upper and lower strands within the putative pathogenic, variable, and right terminal domains, as well as in the region surrounding the central conserved region of the predicted secondary structure of CVd-I-LSS.

Base Sequence↗

PubMed1990

Construction of novel viroid chimeras containing portions of tomato apical stunt and citrus exocortis viroids.

Several novel tomato apical stunt viroid (TASV) recombinants were isolated after inoculation of tomato seedlings with monomeric viroid cDNAs. Two intraspecific recombinants were constructed by exchanging the left and right sides of the closely related Ivory Coast and Indonesian strains of TASV, and a third, interspecific, recombinant was constructed by similar manipulations involving TASV and citrus exocortis viroid (CEV) cDNAs. Characterization of these TASV recombinants by RNA protection assays and nucleotide sequence analysis of polymerase chain reaction-amplified cDNAs revealed no evidence for sequence instability. The symptoms induced by replication of the CEV-TASV chimera in tomato were milder than those induced by either TASV or the TASV chimeras and resembled those induced by the CEV isolate which provided its pathogenicity domain.

Base Sequence↗

PubMedDate not supplied

Mutational analysis of viroid pathogenicity: tomato apical stunt viroid.

A series of nucleotide substitutions within the pathogenicity domain of tomato apical stunt viroid have been evaluated for their effects upon infectivity and symptom expression. None of the 12 A----G substitutions and one C----U substitution that were examined abolished infectivity in a whole plant bioassay, and the resulting progeny were characterized by nucleotide sequence analysis of cDNAs amplified by the polymerase chain reaction. Four of the 13 substitutions gave rise to altered progeny, but the patterns of sequence changes observed were unexpectedly complex. Mutations that did not rapidly revert to the wild-type sequence are located near the right border of the pathogenicity domain, a region which shows considerable natural sequence variability. None had a detectable effect upon symptom expression. The ability to observe viroid sequence evolution in vivo may provide insight into the molecular interactions responsible for viroid host range and symptom formation.

Base Sequence↗

PubMed2001

The molecular characterization of 16 new sequence variants of Hop stunt viroid reveals the existence of invariable regions and a conserved hammerhead-like structure on the viroid molecule.

At present isolates of Hop stunt viroid (HSVd) are divided into five groups: three major groups (plum-type, hop-type and citrus-type) each containing isolates from only a limited number of isolation hosts and two minor groups that were presumed to derive from recombination events between members of the main groups. In this work we present the characterization of 16 new sequence variants of HSVd obtained from four Mediterranean countries (Cyprus, Greece, Morocco and Turkey) where this viroid had not previously been described. Molecular variability comparisons considering the totality of the sequence variants characterized so far revealed that most of the variability is found in the pathogenic and variable domains of the viroid molecule whereas both the terminal right (T(R)) and left (T(L)) domains are regions of low or no variability, respectively, suggesting the existence of constraints limiting the heterogeneity of the sequence variants. Phylogenetic analyses revealed that sequence variants belonging to the two minor recombinant subgroups are more frequent than previously thought. When the cruciform structure alternative to the typical rod-like conformation was considered it was observed that the upper part of this structure (hairpin I) was strictly conserved whereas in the lower part a reduced variability was found. The existence of a covariation in this lower part was notable. Interestingly, a hammerhead-like sequence was found within the T(R) domain of HSVd and it was strictly conserved in all the sequence variants. The evolutionary implications of the presence of this motif on the HSVd are discussed.

Base Sequence↗

PubMed1982

A reaction rate constant for avocado sunblotch viroid and its complementary DNA with application to viroid detection.

Molecular hybridization between avocado sunblotch viroid and its complementary DNA (cDNA) proceeds as if it were a bimolecular chemical reaction with a rate constant of 0.21 liters per nanomole of nucleotide per day. Computer simulations show the minimum incubation times required to detect the viroid with cDNA probes of varying sensitivity with respect to viroid concentration, and estimate the concentration of one of the reactants when the other is known.

Chemical Phenomena↗

PubMed1986

Replication of in vitro constructed viroid mutants: location of the pathogenicity-modulating domain of citrus exocortis viroid.

Sequence variants from field isolates of citrus exocortis viroid (CEV) that cause either mild or severe symptoms on tomato plants have previously been classified into two groups, A and B. These groups differ primarily in two domains, P(L) and P(R), of the proposed native structure. Infectivity studies with full-length cDNA clones of variants from each class have now directly confirmed the original correlation between Class A sequences and the severe phenotype and between Class B sequences and the mild phenotype. Direct evidence for this correlation could only be obtained by using individual sequence variants since field isolates of CEV have been shown to contain a mixture of RNA species. The construction and infectivity of chimaeric cDNA clones derived from mild and severe sequence variants of CEV has demonstrated that novel, infectious viroid molecules can be generated in vitro, and that P(L) is the pathogenicity-modulating domain. The role of the P(R) domain is not known but infectivity experiments with one chimaeric cDNA clone suggest that it may influence the efficiency of the infection or replication process of the viroid in the plant.

Journal Article↗

PubMedDate not supplied

[Determination of potato spindle tumor viroid and chrysanthenum stund viroid using biotinylated olideoxyribonucleotides].

A 26 base long oligodeoxyribonucleotide complementary to a common RNA sequence of potato spindle tuber viroid (PSTV) and chrysantemum stunt viroid (CSV) was synthesized. The 3'-end biotinylated one was used for the detection of PSTV and CSV RNA immobilized on nitrocellulose filters by nucleic acid hybridization. Visualization of hybridization results was performed by two ways, either by streptavidin-alkaline phosphatase conjugate or streptavidine and biotinylated alkaline phosphatase. It was possible to detect 0.65 ng of purified CSV and PSTV RNA. The suggested system of viroid diseases detection can be used by agricultural and horticultural enterprises.

Alkaline Phosphatase↗

PubMed1994

Gel dependence of electrophoretic mobilities of double-stranded and viroid RNA and estimation of the contour length of a viroid by gel electrophoresis.

Double-stranded (ds) RNA normally exhibits a lower electrophoretic mobility than dsDNA having the same number of base pairs. This has been attributed to its net charge density that is lower than that of B-form DNA. But we show here that dsRNA runs faster than corresponding DNA in gels containing either > or = 2.5% agarose or > or = 8% acrylamide with high crosslinking (19:1 acrylamide:N,N'-methylenebisacrylamide). However, the relative mobility of dsRNA as compared with DNA, extrapolated to 0% gel (0%T), remains constant (0.90 +/- 0.03) in all systems, in support of the charge density hypothesis. In comparison to dsRNA standards, the potato spindle tuber viroid, a small approximately 70% base-paired rod-like pathogenic RNA, is strongly retarded, presumably because of greater flexibility and/or stable curvature. Depending on the gel system, nonlinear extrapolation to 0% T leads to an apparent contour length of 140-230 bp, whereas 130 +/- 20 bp can be determined from electron micrographs and 123-126 bp from secondary structure modeling. We attribute the variation of the electrophoretic behavior of both dsRNA and viroid RNA to interactions with the gel matrix. Nevertheless, extrapolation of the apparent contour length (in bp dsRNA) determined from low-crosslinked polyacrylamide gels (2.6%C) is comparable to the determination by alternative methods.

Amino Acids↗

PubMed1992

Sequence analysis of five new field isolates demonstrates that the chain length of potato spindle tuber viroid (PSTVd) is not strictly conserved but as variable as in other viroids.

The sequence analysis of five new field isolates of potato spindle tuber viroid (PSTVd) of different virulence revealed that the length of their RNA chain is not strictly conserved to 359 nucleotides (nts), as one could have inferred from the previously sequenced PSTVd strains. It was now found that the chain length is strain-specific like in the case of practically all other viroids, and that it may vary, so far, between 356 and 360 nts. Taking our previously sequenced and least virulent mild strain PSTVd KF6-M as standard, the new mild strains PSTVd WA-M and PSTVd F-M differ from it by one or two nts. The new intermediate-severe strains PSTVd F-IS and PSTV-F 88-IS differ from the standard mild strain by eight and nine nts, respectively, whereas the new severe-lethal strain PSTVd F-SL differs in seven nts. Most of these mutations are located within the virulence-modulating (VM) region and within the variable region (VR), and only in two strains a single mutation is found in the right terminal domain.

Base Composition↗

PubMed1985

Molecular cloning of potato spindle tuber viroid (PSTV) cDNA synthesized by enzymatic elongation of PSTV-specific DNA primers: a general strategy for viroid cloning.

Different cDNAs were synthesized by primer extension from the RNA of the severe strain KF 440 of potato spindle tuber viroid (PSTV) with the aid of reverse transcriptase using three PSTV-specific DNA molecules as primers. The cDNAs were made double-stranded and cloned into plasmid pBR 322. Various overlapping subgenomic DNA fragments were prepared from these clones and recombined in two different ways. In both cases a PSTV DNA copy was obtained which represented the entire PSTV RNA genome. The sequence of the DNA of one of the resulting full-length clones was identical with the original PSTV isolate, whereas the other clone showed one nucleotide change. On the basis of these results the advantages and problems of different strategies for the molecular cloning of the circular viroid RNA genome are discussed.

Base Sequence↗

PubMed1998

Citrus viroid Ia is a derivative of citrus bent leaf viroid (CVd-Ib) by partial sequence duplications in the right terminal region.

Nucleotide sequences of group I citrus viroids Ia (CVd-Ia) and citrus bent leaf viroid (CBLVd, formerly designated CVd-Ib) isolated from citrus plants in Japan, the Philippines and China have been determined. Citrus samples in Japan and the Philippines contained CVd-Ia, which consists of 328 nucleotides(nt). Although 10 nt longer than the type CBLVd-225A in Israel they share 94% identity in overall nucleotide sequence. The Philippines sample also contained a 329-nt long CVd-Ia sequence variant, in which one base insertion and three substitutions were observed. A citrus in China contained CBLVd, which consists of 318 nt and shares 98% identity to CBLVd-225A. CVd-Ia was clearly separated from CBLVd by two 5-nt insertions located in upper (5'-AGCUG-3') and the lower (5'-CUUCU-3') strand of the right terminal region (which is also designated T2 domain) in rod-like secondary structure. Since both of the additional 5-nt sequences are similar to the adjacent sequences (5'-AGUUG-3' and 5'-CUUCU-3'), we hypothesize that CVd-Ia is a derivative of CBLVd caused by partial sequence duplications and substitutions taking place in the right terminal region.

Base Sequence↗

PubMed1986

Enhanced resolution of circular and linear molecular forms of viroid and viroid-like RNA by electrophoresis in a discontinuous-pH system.

A discontinuous-pH polyacrylamide gel electrophoresis system is described. An increase in the pH differential between the gel and the running buffer enhances the separation of low molecular weight circular and linear RNA molecules. Highly purified preparations of the circular form of viroids can be obtained with this procedure. Since all the linear RNAs of similar molecular weight migrate with the front, a relatively clean background can be obtained even when crude extracts are used. This facilitates an improved separation and identification of similarly sized viroid-like RNAs. The conditions of electrophoresis in low salt and 8 M urea also permit the effective transfer of RNA molecules directly to nylon-based membranes without any additional denaturation treatment.

Electrophoresis, Polyacrylamide Gel↗