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.     

Grapevine yellow speckle viroid: structural features of a new viroid group.

A single stranded circular RNA was isolated from grapevines infected with yellow speckle disease. The RNA which we have called grapevine yellow speckle viroid (GYSV), contains 367 nucleotide residues and has the potential to form the rod-like secondary structure characteristic of viroids. GYSV has 37% sequence homology with the recently described apple scar skin viroid (ASSV; 330 residues) and has some sequence homology with the viroids in the potato spindle tuber viroid (PSTV) group. The sequence of GYSV has characteristics which fit the structural domains described for the PSTV group. However, GYSV lacks the PSTV central conserved sequence. Instead, there is a conserved sequence in the central region of GYSV and ASSV which has the potential to form a stem loop configuration and a stable palindromic structure as does the central conserved region of the PSTV group. These structural features suggest there is a different central conserved region for GYSV and ASSV. The results support the viroid nature of GYSV and its inclusion into a separate viroid group which we suggest should be represented by ASSV.     

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.     

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.     

RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct

Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (–)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.     

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.     

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.     

Hop stunt viroid: A polyphagous pathogenic RNA that has shed light on viroid–host interactions

Taxonomy Hop stunt viroid (HSVd) is the type species of the genus Hostuviroid (family Pospiviroidae). The other species of this genus is Dahlia latent viroid, which presents an identical central conserved region (CCR) but lacks other structural hallmarks present in Hop stunt viroid. HSVd replication occurs in the nucleus through an asymmetric rolling‐circle model as in the other members of the family Pospiviroidae, which also includes the genera Pospiviroid, Cocadviroid, Apscaviroid, and Coleoviroid.Physical properties Hop stunt viroid consists of a single‐stranded, circular RNA of 295–303 nucleotides depending on isolates and sequence variants. The most stable secondary structure is a rod‐like or quasi‐rod‐like conformation with two characteristic domains: a CCR and a terminal conserved hairpin similar to that of cocadviroids. HSVd lacks a terminal conserved region.Hosts and symptomsHSVd infects a very broad range of natural hosts and has been reported to be the causal agent of five different diseases (citrus cachexia, cucumber pale fruit, peach and plum apple apricot distortion, and hop stunt). It is distributed worldwide.TransmissionHSVd is transmitted mechanically and by seed.     

The viroid and viroid-like RNA database.

The viroid and viroid-like RNA database is a compilation of all natural sequences published in journals or available from the GenBank and EMBL nucleotide sequence libraries. Several information regarding these RNA species such as the position of their self-catalytic domains and the open reading frame of the human hepatitits delta virus are provided. The database also includes a determination of the likely ancestral sequence of most species and a prediction of the most stable secondary structures of these sequences. This online database is available on the World Wide Web (http://www.callisto.si.usherb.ca/[symbol: see text]jpperra ). It should provide an excellent reference point for further phylogenetic and structure-function studies of these RNA species.     

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.     

The viroid and viroid-like RNA database.

This is an online database to facilitate research on viroid, viroid-like RNAs and human hepatitis delta virus (vHDV) by presenting a large number of sequences and related data in a comprehensive and user-friendly format (e.g. position of their self-catalytic domains, open reading frame of the vHDV, prediction of the most stable secondary structures, etc.). Most of these RNA species share a common proposed replication pattern known as a DNA-independent rolling circle mechanism. Together, these species form the 'brotherhood' of the smallest known auto-replicable RNAs. This online database is available on the World Wide Web at http://www.callisto.si.usherb.ca/jpperra     

Unraveling the mystery of viroid nuclear import

Viroids are closed-circular infectious RNAs that are known to infect plants. Despite their small noncoding genome, they have the ability to cause disease. The nuclear import mechanism of nucleus replicating viroids is not well understood. Ma et al. have recently highlighted the route of viroid entry into the nucleus.     

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.     

In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus

Viroids, small single-stranded circular RNA molecules, are the smallest known infectious agents in Nature. The apparent inability of viroids to encode for proteins means that they must rely fully on host functions for their replication. The specific ultrastructural localization of viroids is fundamental to the determination of their replication strategies. In this paper the first in situ hybridization study to localize viroids within the cell at the electron microscope level is reported. Biotin-labelled RNA probes were used with subsequent detection by gold-labelled monoclonal anti-biotin antibodies to localize avocado sunblotch viroid and coconut cadang cadang viroid. Avocado sunblotch viroid was located in chloroplasts, mostly on the thylakoid membranes of cells from infected leaves of avocado (Persea americana). In contrast, coconut cadang cadang viroid was located in the nucleolus and nucleoplasm of cells of infected leaves of oil palm (Elaeis guineensis), with a higher concentration in the nucleolus. The results provide insight on the potential host RNA polymerases involved in the replication of these two viroids.