A Comparative Study of Four Viroids of Plants

The host-ranges and the reactions of particular plant hosts to inoculation with severe (s-PSTV) and mild (m-PSTV) strains of potato spindle tuber viroid, as well as with chrysanthemum stunt viroid (CSV) and cucumber pale fruit viroid (CPFV) were quite similar. Some minor differences did not exceed the limits of differences noted for the strains of the same plant viroid. Two-directional crossprotection was noted for each viroid pair when s-PSTV, m-PSTV, CSV and CPFV were tested on chrysanthemum cv., ‘Bonnie Jean’ plants. Finally, the relative mobility of RNAs of s-PSTV, m-PSTV, CSV and CPFV on 5% polyacrylamide gel was identical, no matter what extraction method from plant material was used. We postulate that these four plant viroids may be regardedas the strains of the same plant viroid “species”. On the other hand, chrysanthemum chlorotic mottle viroid (ChCMV) appeared to be a quite different plant pathogen. This viroid infected and caused symptoms only in chrysanthemum plants, and was able to infect and induce symptoms on plants which had already been infected with any other viroid studied, and it did not protect chrysanthemum cv. ‘Bonnie Jean’ plants against any of the other viroids. We were not able to locate a ChCMV-RNA band on polyacrylamide gel.     

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.     

A Low Temperature Therapy and Meristem-Tip Culture for Eliminating four Viroids from Infected Plants

Viroid-free potato and chrysanthemum plants were obtained from meristem-tips cut from potato spindle tuber viroid-infected potato plants and from chrysanthemum plants infected with chrysanthemum stunt, chrysanthemum chlorotic mottle or cucumber palefruit viroids after 6 months therapy in a growth chamber at 5 °C and 16 hours daily light of 5.000 lx intensity. Chrysanthemum plants survived quite well the conditions of therapy while potato plants grown from stem cuttings survived these conditions much worse and potato plants grown from tubers did not survive these conditions. PSTV-free plants were obtained from meristem-tips cut from sprouts grown from potato tubers infected with severe (s-PSTV) or mild (m-PSTV) strains of potato spindle tuber viroid after 6 months therapy at 6–7 °C in the dark. The tubers survived these conditions quite well. The 3 months therapy period was found too short for any plant material. The efficiency of 6 months therapy in viroid elimination varied for different viroids and different plant material from 18.5 to 80.0 %.     

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.     

Tissue and intra-cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy

Confocal laser scanning microscopy and transmission electron microscopy (TEM) were used in conjunction with in situ hybridization techniques to compare and contrast the subnuclear (ultrastructural) and tissue (histological) localizations, respectively, of citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Both these viroids, which are members of the same taxonomic subgroup of viroids, were found in the vascular tissues as well as in the nuclei of mesophyll cells of infected host plants. At the subnuclear level, however, CEV was distributed across the entire nucleus, in contrast to CCCV which was mostly concentrated in the nucleolus with the remainder distributed throughout the nucleoplasm.     

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.     

Mature monomeric forms of Hop stunt viroid resist RNA silencing in transgenic plants

Viroids, small non-coding pathogenic RNAs, are able to induce RNA silencing, a phenomenon that has been associated with the pathogenesis and evolution of these small RNAs. It has been recently suggested that viroids may resist this plant defense mechanism. However, the simultaneous degradation of non-replicating full-length viroid RNA, and the resistance of mature forms of viroids to RNA silencing, have not been experimentally demonstrated. Transgenic Nicotiana benthamiana plants expressing a dimeric form of Hop stunt viroid (HSVd) that have the capability to cleave and circularize this viroid RNA were used to address this question. A reporter construct, consisting of a full-length HSVd RNA fused to GFP-mRNA, was agroinfiltrated in these plants and its expression was suppressed. Interestingly, both circular and linear HSVd molecules were stable and able to traffic through grafts in these restrictive conditions, indicating that the mature forms of HSVd are able, in some way, to resist the RNA-silencing mechanism. The observation that a full-length HSVd RNA fused to GFP-mRNA, but not circular and/or linear viroid forms, was fully susceptible to RNA degradation strongly suggests that structures adopted by the free mature monomer protect the pathogenesis-associated forms of the viroid from RNA silencing.     

Viroids

Viroids are small, circular RNA pathogens, which infect several crop plants and can cause diseases of economic importance. They do not code for proteins but they contain a number of RNA structural elements, which interact with factors of the host. The resulting set of sophisticated and specific interactions enables them to use the host machinery for their replication and transport, circumvent its defence reactions and alter its gene expression. Although found in plants, viroids have a distant relative in the animal world: hepatitis delta virus (HDV), a satellite virus of hepatitis B virus, which has a similar rod-like structure and replicates in the nucleus of infected cells. Viroids have also a cellular relative: the retroviroids, found in some plants as independent (non-infectious) RNA replicons with a DNA copy. In this review, we summarize recent progress in understanding viroid biology. We discuss the possible role of recently identified viroid-binding host proteins as well as the recent data on the interaction of viroids with one part of the host's defence machinery, the RNA-mediated gene silencing and how this might be connected to viroid replication and pathogenicity.     

In-depth sequencing of the siRNAs associated with peach latent mosaic viroid infection

Background  

It has been observed that following viroid infection, there is an accumulation of viroid-derived siRNAs in infected plants. Some experimental results suggest that these small RNAs may be produced by the plant defense system to protect it from infection, indicating that viroids can elicit the RNA-silencing pathways. The objective of this study is to identify in the peach latent mosaic viroid (PLMVd), a model RNA genome, the regions that are most susceptible to RNA interference machinery.     

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.     

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.     

Fatal Yellowing of Oil Palms: Search for Viroids and Double-Stranded RNA

Fatal yellowing is a serious disease of still unknown origin affecting oil palms in several regions of Central and South America. In this study a search for viroids and viroid-like RNAs in oil palms was performed using two-dimensional gel electrophoresis and return gel electrophoresis of nucleic acid extracts. Although RNAs showing viroid-like gel-electrophoretic properties were detected, the presence of the known viroids was excluded by hybridization experiments using probes specific for potato spindle tuber viroid (PSTVd), coconut cadang-cadang viroid (CCCVd), or Coleus blumei viroid 1 (CbVd1). By using double-stranded RNA (dsRNA) specific monoclonal antibodies, which do not react with viroid RNA, we were able to show that oil palm RNAs, migrating like viroids are double-stranded RNA species. Since the same dsRNA pattern was found in extracts from diseased as well as from healthy oil palms, the dsRNAs can neither be part of the causative agent of fatal yellowing, nor are they associated with the disease. Their possible origin is discussed. In addition to the standard electrophoretic methods, which have been used for identification of viroids and viroid-like RNAs, we describe additional control experiments to differentiate unequivocally between circular single stranded and linear dsRNA.     

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.     

Potato spindle tuber viroid as inducer of RNA silencing in infected tomato.

Potato spindle tuber viroid (PSTVd), an RNA plant pathogen encoding no known proteins, induces systemic symptoms on tomato plants. We report detection of small RNAs of approximately 25 nucleotides with sequence specificity to PSTVd in infected plants: an indication of the presence of RNA silencing. RNA silencing, however, did not appear to be responsible for the differing symptoms induced by a mild and a severe strain of PSTVd. The unique structural and biological features of viroids make them attractive experimental tools to investigate mechanisms of RNA silencing and pathogen counterdefense.     

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.     

Spread of raspberry bushy dwarf virus by pollination, its association with crumbly fruit, and problems of control

Raspberry bushy dwarf virus (RBDV) was transmitted to raspberry seed both through the pollen and through the ovule and it infected plants pollinated with infected pollen. It did not infect plants prevented from flowering, and transmission through pollen seems to be the only method of spread in the field; in the proximity of infectors, most plants became infected during the first two or three flowering seasons. Plants containing RBDV showed no obvious symptoms, but healthy or infected flowers pollinated with infected pollen produced ‘crumbly’ fruit, containing a high proportion of aborted drupelets. RBDV was difficult to eliminate from infected raspberry by heat therapy. Raspberry cultivars that fail to become infected naturally were also immune to infection by grafting. Use of immune cultivars offers the only method of control and, because infected plants may produce crumbly fruit, future cultivars should if possible possess immunity to RBDV.     

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.