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At least 19 recordsLinked to original sources

PubMed1989

Grapevine viroid 1B, a new member of the apple scar skin viroid group contains the left terminal region of tomato planta macho viroid.

GV1B is one of five viroids that have recently been purified from grapevines. GV1B has now been sequenced and its 363 nucleotide residues can potentially form the typical rod-like structure of viroids with 67% of nucleotides base-paired. GV1B has highest sequence similarity with grapevine yellow speckle viroid (GYSV; 73%) and has a central sequence which is conserved in GYSV and apple scar skin viroid (ASSV) which have been reported to constitute the ASSV group. Therefore, we have placed GV1B into the ASSV group. GV1B contains a direct repeat sequence at the terminal portions of its T1 and T2 regions. GV1B also contains a sequence of 69 nucleotides in the terminal portion of its T1 region which is almost identical to the corresponding region in tomato planta macho viroid (TPMV). This provides further evidence of the importance of RNA recombination in viroid evolution.

Base Sequence↗

PubMed1992

A viroid from Nematanthus wettsteinii plants closely related to the Columnea latent viroid.

A viroid was isolated from symptomless Nematanthus wettsteinii plants using the return-PAGE method for analysis of low M(r) nucleic acids. The RNA was transmitted to tomato, three cultivars of potato, and Scopolia sinensis plants by mechanical inoculation or by grafting. Infected solanaceous plants developed symptoms similar to those caused by potato spindle tuber viroid (PSTVd). The Nematanthus viroid consists of 372 nucleotides, 214 G+C, 158 A+U, with a G+C/A+U ratio of 1.35. One of seven cDNA clones showed a sequence heterogeneity (G to A) at position 73. The most stable secondary structure of this viroid has 78 G:C, 37 A:U and 11 G:U base pairs with a minimum free energy of -456.9 kJ. The viroid is closely related to the 370 nucleotide Columnea latent viroid. The Nematanthus viroid possesses regions of 100% sequence identity with six viroids belonging to the PSTVd and apple scar skin viroid groups. The viroid also replicated in tomato plants when mixed with PSTVd. Tomato plants were cross-protected against PSTVd when preinfected with the viroid from N. wettsteinii.

Base Sequence↗

PubMed1988

The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops.

A new viroid which does not seem to produce any symptoms of disease, and is therefore tentatively named hop latent viroid (HLV) was found to occur worldwide in hops. HLV proved to be infectious when mechanically inoculated onto viroid- and virus-free hops. The viroid nature of HLV was also substantiated by sequence analysis which revealed that HLV is a circular RNA consisting of 256 nucleotides, that can be arranged into the viroid-specific, rod-like secondary structure. HLV also contains the central conserved region typical for most of the presently known viroids. However HLV does not contain the viroid-specific oligo(A) stretch in the upper left part of its rod-like molecule. Because of this feature and a sequence similarity with the prototypes of the other viroid groups below 55%, HLV can be regarded as the first member of a new viroid group.

Base Sequence↗

PubMed1989

Nucleotide sequence and proposed secondary structure of Columnea latent viroid: a natural mosaic of viroid sequences.

The Columnea latent viroid (CLV) occurs latently in certain Columnea erythrophae plants grown commercially. In potato and tomato, CLV causes potato spindle tuber viroid (PSTV)-like symptoms. Its nucleotide sequence and proposed secondary structure reveal that CLV consists of a single-stranded circular RNA of 370 nucleotides which can assume a rod-like structure with extensive base-pairing characteristic of all known viroids. The electrophoretic mobility of circular CLV under nondenaturing conditions suggests a potential tertiary structure. CLV contains extensive sequence homologies to the PSTV group of viroids but contains a central conserved region identical to that of hop stunt viroid (HSV). CLV also shares some biological properties with each of the two types of viroids. Most probably, CLV is the result of intracellular RNA recombination between an HSV-type and one or more PSTV-type viroids replicating in the same plant.

Base Composition↗

PubMed1990

Australian grapevine viroid--evidence for extensive recombination between viroids.

Australian grapevine viroid (AGV, 369 residues) is a novel viroid with less than 50% sequence similarity with any known viroid. Nevertheless its entire sequence can be divided into regions, each with a high sequence similarity with segments from one of citrus exocortis, potato spindle tuber, apple scar skin, and grapevine yellow speckle viroids. AGV contains the entire central conserved region of the apple scar skin viroid group and is proposed as a member of this group. AGV appears to have originated from extensive RNA recombination involving other viroids. The vegetatively propagated grapevines which have been exposed to multiple viroid infections during their long history of cultivation may have allowed such recombination.

Base Sequence↗

PubMed1985

Infectivity and in vitro mutagenesis of monomeric cDNA clones of citrus exocortis viroid indicates the site of processing of viroid precursors.

Monomeric cDNA clones of citrus exocortis viroid (CEV) were constructed in the plasmid vector pSP6-4 and the infectivity of the clones plus in vitro-synthesized RNA transcripts determined by inoculation onto tomato seedlings. Infectivity was dependent on the site of the viroid molecule used for cloning and the orientation of the cDNA insert. Only the plus BamHI cDNA clone was infectious and produced progeny viroid with wild-type sequence at the region corresponding to the BamHI cloning site. Infectivity correlated with the terminal repetition of 11 nucleotides of viroid sequence, 5'GGATCCCCGGG 3', in the vector adjacent to the insert. The 11-nucleotide sequence lies within the highly conserved central region of viroids. Site-directed mutagenesis of a single nucleotide in the repeat at the 5'-end of the CEV insert to 5' GGATCCCC(T,A)GG 3' gave two point mutants. The two mutant CEV inserts, when excised from the vector, were not infectious. However, plasmid DNA and RNA transcripts from non-excised mutant CEV inserts were infectious. The progeny of one of these clones was examined and contained wild-type sequence. It was concluded that in vivo processing of longer-than-unit-length CEV occurs at one of three adjacent sites in the 11 nucleotide sequence and that the G nucleotide at position 97 is important for viroid replication.

Base Sequence↗

PubMed1992

Pear blister canker viroid is a member of the apple scar skin subgroup (apscaviroids) and also has sequence homology with viroids from other subgroups.

The sequence of pear blister canker viroid (PBCVd), the putative causal agent of pear blister canker (PBC) disease, has been determined. PBCVd consists of a single-stranded circular RNA of 315 nucleotide residues which assumes a branched conformation when it is folded in the model of lowest free energy. PBCVd has highest sequence similarity with grapevine 1B viroid (52.4%), but also contains sequences related to regions present in viroids that belong to different subgroups, suggesting that PBCVd could have developed from RNA recombination between viroids replicating in a common host plant. PBCVd contains almost the entire central sequence which is conserved in the members of the apple scar skin subgroup (apscaviroids) as well as a conserved sequence located in the left-terminal region of apscaviroids and pospiviroids (whose type member is potato spindle tuber viroid). A consensus phylogenetic tree has been obtained in which PBCVd and other viroids previously classified as apscaviroids appear closely related, allowing consideration of PBCVd as a new member of this subgroup.

Base Sequence↗

PubMed1992

Transcripts of the viroid central conserved region contain the local tertiary structural element found in full-length viroid.

The viroid central conserved region (CCR) is highly conserved among different viroids and is thought to be involved in viroid replication. A novel tertiary structure occurs in the CCR of native circular potato spindle tuber RNAs. To permit more detailed studies of this structural element, a small RNA oligonucleotide containing the CCR of the viroid genome was synthesized. The tertiary structure of these CCR transcripts was examined by UV-crosslinking of the RNA, followed by mapping of the crosslink using limited alkaline digestion and classical RNA secondary analysis. The CCR transcript was found to undergo UV-crosslinking between the same two bases as in full-length viroid, indicating that the tertiary structure is the same and that the CCR transcript will be useful for the affinity purification of host components.

Base Sequence↗

PubMed1989

Infectivity of chimeric viroid transcripts reveals the presence of alternative processing sites in potato spindle tuber viroid.

In an investigation of viroid replication and pathogenesis, we have assessed the effect of sequence duplication of the upper central conserved region (CCR) of the molecule on the infectivity of RNAs transcribed in vitro from partial dimers of wild-type and mutant viroid cDNAs. In one set of experiments, the relative infectivities of one monomeric potato spindle tuber viroid (PSTV) and five oligomeric SP6 transcripts [PSTV or PSTV-TASV (tomato apical stunt viroid) chimeras] were compared. With one exception, the extent of sequence duplication in the CCR, and thus the length of the so-called palindrome, does correlate with an increase in specific infectivity. In a second set of experiments, in vitro generated site-specific mutations in cloned PSTV were used as markers to determine if a cleavage/ligation at sites other than the palindrome could generate infectious molecules in vivo. The creation of a novel PSTV-TPMV (tomato planta macho viroid) chimera in these experiments provides evidence that multimeric RNAs can be processed at sites other than the CCR to yield monomeric progeny.

Base Sequence↗

PubMedDate not supplied

32P- and biotin-labelled in vitro transcribed cRNA probes for the detection of potato spindle tuber viroid and chrysanthemum stunt viroid.

Replacing nick-translated DNA probes by in vitro transcribed complementary RNA (cRNA) probes considerably increased the sensitivity of dot-blot detection tests of potato spindle tuber viroid and chrysanthemum stunt viroid. As compared to the limit of detection of 5-10 pg of viroid obtained with 32P-labelled DNA probes, cRNA probes allow the detection of less than 1 pg of pure viroid. When labelled with biotin by incorporation of biotin-labelled ribonucleotides, the cRNA probes have a limit of detection of approximately 5 pg of purified viroid.

Biotin↗

PubMed1987

The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid.

The primary structure of a grapevine viroid (GVs) isolated in Spain was determined. The sequence consisted of 369 nucleotide residues forming a circular molecule. GVs presented extensive homology with viroids of the potato spindle tuber viroid (PSTV) group, that was specially high in the case of citrus exocortis viroid (CEV) both with variants found in isolates inducing severe (92% with CEV-A) and mild (89% with CEV-DE26) symptoms on tomato. The secondary structure proposed for GVs showed that the changes in the sequence in relation to CEV-A generated modifications of the secondary structure particularly important in the left terminal (Tl), variable (V) and pathogenesis (P) viroid domains that have been postulated. Nevertheless it was noted in GVs a central core in the P domain that is conserved in the class A sequence variants characteristic of severe isolates, but not in the class B ones found in mild isolates of CEV. These observations indicate that GVs should be considered as a severe isolate of CEV from grapevine (CEV-g), a suggestion that correlates with the biological properties of CEV-g both in tomato and in Gynura aurantiaca. The presence of this central core in the P domain seems to characterize all the variants of CEV inducing severe symptoms in tomato.

Citrus↗

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↗

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↗

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↗

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↗

PubMed1991

Viroids and viroid-like satellite RNAs: a phylogenetic analysis.

With the discovery that certain RNAs possess catalytic properties (Kruger et al., 1982; Guerrier-Takada et al., 1983), the possible significance of certain small plant pathogenic RNAs, namely viroids and viroid-like satellite RNAs, as relics of precellular evolution deserves consideration. As a first step in this endeavor, I report here the results of a phylogenetic study of the computer-aligned nucleotide sequences of these RNAs that is based on the parsimony principle (Felsenstein and Sober, 1986).

Base Sequence↗