Viroids

Ricardo Flores, Instituto de Biologa Molecular y Celular de Plantas (UPV-CSIC), Valencia, Spain -Antonio Daro ` s, Instituto de Biologa Molecular y Celular de Plantas (UPV-CSIC), Valencia, Jose
Spain

Secondary article
Article Contents
. Genome Structure . Interaction with Host . Replication . Pathogenesis . Evolution . Control

ndez, Instituto de Biologa Molecular y Celular de Plantas (UPV-CSIC), Valencia, Carmen Herna
Spain

Francesco Di Serio, Instituto de Biologa Molecular y Celular de Plantas (UPV-CSIC), Valencia,

Spain

Viroids are infectious small circular RNAs able to induce specific diseases in higher plants. They differ from viruses in fundamental aspects like structure, function and evolutionary origin.

Genome Structure
Viroid genomes are exclusively formed by a singlestranded circular ribonucleic acid (RNA), ranging in size from approximately 250 to 400 nucleotides, and being, therefore, the smallest known autonomous replicons (Diener, 1971). Viroid RNAs display a characteristic high degree of self-complementarity leading to compact conformations with short single-stranded loops separated by double-stranded regions (Gross et al., 1978) that probably aord some protection against degradation by cellular nucleases. Viroids are able to infect higher plants of economic interest and to incite specic diseases in most of them (Table 1). Although the available experimental data strongly suggest that viroid RNAs, as opposed to virus genomes, do not code for any protein, they contain all the information needed for their survival and propagation. The small size of viroid genomes implies that the information embodied in them is extremely compressed and specic regions of the molecule are presumably involved in more than one function. Viroid replication, and most likely viroid movement through the infected plant, occurs via interactions with host proteins. Thus, these RNA molecules have sequence and structural motifs that enable them to exploit to their own advantage the cellular machinery whose normal functioning, when occasionally impaired, leads to the appearance of the disease. Additionally, some viroids are catalytic RNAs and supply ribozymatic activities mediating some steps of their replication cycle (see below).

Most of the known viroids belong to the rst family and are characterized by having some conserved sequence motifs, prominent among which is a central conserved region (CCR) and a rod-like secondary structure. Members of the second family, comprising only of ASBVd, Peach latent mosaic viroid (PLMVd) and Chrysanthemum chlorotic mottle viroid (CChMVd), do not have a CCR but the strands of both polarities are able to form hammerhead structures and to self-cleave through these ribozymatic activities. Within each family viroids are allocated into genera on the basis of some commonly shared motifs (Table 1) and then into species, which have a sequence similarity level of less than 90% and specic biological properties, particularly host range.

Secondary structure: structural and functional domains

Pospiviroidae members adopt a rod-like or quasi-rod-like secondary structure in vitro, but the in vivo conformation may not be necessarily the same because it can be inuenced by interactions with host proteins. However, there is experimental support, at least in some Pospiviroidae representatives, for an in vivo quasi-rod-like conformation. Based on comparative pairwise sequence similarities between PSTVd and closely related viroids, a model was proposed that divides the rod-like structure into ve structural domains (Keese and Symons, 1985) (Figure 1). In this model, now extended to all the Pospiviroidae family, the central domain (C) is anked by the pathogenic (P) and variable (V) domains, and adjacent to them are located the left terminal (TL) and right terminal (TR) domains, respectively (Figure 1a). Some of the domains have been associated with specic functions, as illustrated by the case of the P domain that received this name because nucleotide changes between severe and mild isolates of PSTVd and Citrus exocortis viroid (CEVd) map into this
1

Classification
On the basis of conserved sequence and structural motifs and phylogenetic analysis, viroids have been classied into two families, Pospiviroidae and Avsunviroidae, whose type members are Potato spindle tuber viroid (PSTVd) and Avocado sunblotch viroid (ASBVd) respectively (Table 1).

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Table 1 Viroid classication Family Pospiviroidae With CCR Without hammerhead self-cleavage Genus Pospiviroid Speciesa Potato spindle tuber viroid (PSTVd) Mexican papita viroid (MPVd) Tomato planta macho viroid (TPMVd) Chrysanthemum stunt viroid (CSVd) Citrus exocortis viroid (CEVd) Tomato apical stunt viroid (TASVd) Iresine viroid 1 (IrVd-1) Columnea latent viroid (CLVd) Hop stunt viroid (HSVd) Coconut cadang-cadang viroid (CCCVd) Coconut tinangaja viroid (CTiVd) Hop latent viroid (HLVd) Citrus viroid IV (CVd-IV) Apple scar skin viroid (ASSVd) Citrus viroid III (CVd-III) Apple dimple fruit viroid (ADFVd) Grapevine yellow speckle viroid 1 (GYSVd-1) Grapevine yellow speckle viroid 2 (GYSVd-2) Citrus bent leaf viroid (CBLVd) Pear blister canker viroid (PBCVd) Australian grapevine viroid (AGVd) Coleus blumei viroid 1 (CbVd-1) Coleus blumei viroid 2 (CbVd-2) Coleus blumei viroid 3 (CbVd-3) Avocado sunblotch viroid (ASBVd) Peach latent mosaic viroid (PLMVd) Chrysanthemum chlorotic mottle viroid (CChMVd) Sizeb 356, 359360 359360 360 354, 356 370375, 463 360, 363 370 370, 372 295303 246247, 287 301 254 256 284 329330 294, 297 306307 366368 363 318 315316 369 248, 250251 301302 361362, 364 246250 335338 398399

Hostuviroid Cocadviroid

Apscaviroid

Coleviroid

Avsunviroidae With hammerhead self-cleavage Without CCR

Avsunviroid Pelamoviroid

CCR, central conserved region. a The rst viroid in each genus is the type species. b Size in nucleotides of dierent variants.

region. Recent work, however, indicates that the situation is more complex and that other domains are also involved in triggering symptom expression (Sano et al., 1992). Within the C domain there is a CCR formed by short sequences, conserved within each Pospiviroidae genus, located in opposite positions in both strands, with those of the upper strand being anked by imperfect inverted repeats that contribute to stabilizing some transient structures that may be important in replication. Although the conserved sequences of both CCR strands are usually assumed to interact through canonical WatsonCrick base pairs (Figure 1a), there is experimental evidence indicating that this is not the case, at least in PSTVd, in which an array of noncanonical base interactions form a so-called loop E, described previously in the 5S RNA and involved in binding transcription factor IIIA and ribosomal protein L5 implicated in 5S RNA transcription and intranuclear
2

movement, respectively. The possibility exists that PSTVd could also bind these two proteins. The terminal conserved region (TCR) and the terminal conserved hairpin (TCH) (Figure 1a), are two other motifs whose conservation in sequence and relative position in the rod-like secondary structure of dierent Pospiviroidae genera argue in favour of their important functional role, so far unknown. It is interesting to note that both motifs have never been concurrently found within the same viroid RNA: the TCR is present in viroids with a size greater than approximately 300 nucleotides, whereas viroids with smaller sizes contain the TCH. The most peculiar feature of the three viroids forming the Avsunviroidae family is that they are catalytic RNAs that can self-cleave through hammerhead ribozymes (Figure 1b). From the structural point of view, ASBVd, with an usual low G + C content among viroids and a rod-

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Viroids

In HSVd and CCCVd genera

In PSTVd and ASSVd genera, and in CbVd-2 and CbVd-3

In PSTVd genus; other sequences in HSVd, CCCVd, ASSVd and CbVd-1 genera

TL TCH
C U G G G G AA UCCCC

P TCR
CNNGNGGUUCCUGUGG

C CCR
A C U U C A G G G A U C C C C G G G G A A C C U GGA G
U C G A A G U C A A G GU G G C C C A U A C AA

TR

CA

5 C C G G G G 3 U G G C C C A

A A A C C U G G A 3 U A N N N N N N N 5 C

Loop E (a)

ASBVd
GU GAAAC AG AGUC AC GUUUC GACU CU GUUUC CAAAG UC UCAG AG AGUC AC UG

PLMVd

Helix II

Helix III

5 N N N N N G A A A C N N N N N 3

3 N N N N N A G N U G N N N N N 5

3 N 5 N N Helix I N N N N N N N N C A G U

(b)

Consensus hammerhead structure

Figure 1 Structural models for viroids. (a) Rod-like secondary structure proposed for members of the Pospiviroidae family. The approximate locations of the five structural domains C (central), P (pathogenic), V (variable), and TL and TR (terminal left and right, respectively) are indicated. Nucleotide sequences of the TCH (terminal conserved hairpin), TCR (terminal conserved region) and CCR (central conserved region) are shown within boxes, together with their occurrence in different viroids. Arrows represent flanking sequences that, along with the core nucleotides of the upper CCR strand, form imperfect inverted repeats. The inset shows loop E with an S-shaped line connecting the residues linked after ultraviolet irradiation; enlarged letters refer to nucleotides that are conserved in different RNAs containing this structural motif. (b) Rod-like and branched conformations proposed for the type members of Avsunviroid and Pelamoviroid genera, respectively. Sequence conserved in all hammerhead structures are shown within boxes with dark and white backgrounds for plus and minus polarities, respectively. A consensus hammerhead structure is presented within the inset, with the arrowhead marking the self-cleavage site. In both panels N indicates nonconserved residues and continuous lines and dots between them denote canonical and noncanonical base pairs, respectively. ASBVd, Avocado sunblotch viroid; ASSVd, Apple scar skin viroid; CbVd, Coleus blumei viroid; CCCVd, Coconut cadang-cadang viroid; HSVd, Hop stunt viroid; PLMVd, Peach latent mosaic viroid; PSTVd, Potato spindle tuber viroid.

like secondary structure of lowest free energy, clearly diers from PLMVd and CChMVd, which assume a highly branched conformation in vitro (Navarro and Flores, 1997). Evidence supporting the biological signicance of this branched conformation is particularly strong in CChMVd, where some variants present compensatory

mutations preserving the existence of some of the hairpins of the predicted most stable secondary structure. These observations show that the rod-like secondary structure is not a universal property of viroids. It must also be noted that the conformation of some viroid RNAs may be

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additionally stabilized by tertiary interactions as pseudoknots, tetraloops and triple helices.

Interaction with Host

The host range of dierent viroids is very variable. For example, whereas Hop stunt viroid (HSVd) is able to infect a wide plant spectrum, each member of the Avsunviroidae family is restricted to a few closely related species. The molecular features determining the host range of viroids are unknown, but they probably depend on specic interactions of the RNA with host proteins. Recently, it has been shown that a single nucleotide substitution in PSTVd enables this viroid to infect tobacco. Interestingly, the aected position is located in the lower strand of the CCR and forms part of the loop E (Figure 1a).

Crossprotection
The viroid ability to infect a host plant may relate to previous infections by other strains of the same, or by a closely related, viroid. In fact, when a plant is preinfected with a mild viroid strain and is then challenge inoculated with a severe strain of the same viroid, the typical symptoms of the latter strain and the accumulation level of its corresponding RNA are attenuated for an indeterminate period, probably as a consequence of the competition between the two RNAs for a limiting host factor; therefore, crossprotection phenomena similar to those previously reported in viruses, also occur in viroids. However, because of the basic structural and functional dierences between viruses and viroids, and even between the two viroid families, a panoply of very distinct underlying mechanisms can be anticipated.

transmission electron microscopy, have revealed that several members of the Pospiviroidae family accumulate in the nucleus, with PSTVd and Coconut cadang-cadang viroid (CCCVd) predominantly being concentrated in the nucleoli, whereas CEVd is more uniformly distributed throughout the whole nuclear structure (Figure 2). Since recent progress indicates that a series of small nuclear and nucleolar RNAs, that play important roles in cellular metabolism, have localization signals targeting them to these subcellular compartments, a similar situation probably occurs in members of the Pospiviroidae family, although the specic nature of these signals remains to be determined. The situation is again very dierent in ASBVd, which accumulates in the chloroplast and particularly in the thylakoid membrane. In the case of PLMVd and CChMVd, the two other Avsunviroidae components, the subcellular localization has not been established due to the technical diculties derived from their very low concentration in infected tissues, but presumably they also accumulate in the chloroplast.

Replication
Viroids replicate through RNA intermediates (Grill and Semancik, 1978) and are assumed to follow a rolling circle model (Branch and Robertson, 1984). This model was proposed in view of the circular nature of the viroid molecule and the presence in infected tissues of linear oligomeric viroid RNAs of both polarities, the putative replication intermediates. The model envisages two

Viroid movement
For a successful infection the viroid RNA must be able not only to replicate but also to move within the plant. Longdistance PSTVd movement in tomato has been studied and the results indicate that the viroid follows the movement of photosynthetic products through the phloem, the same route that is also used by most plant viruses. Short-distance movement has been explored by microinjecting PSTVd into tobacco and tomato mesophyll; the viroid seems to move cell-to-cell via plasmodesmata and this movement requires some viroid-specic sequence or structural motifs. In both types of movement the viroid RNA most likely interacts with host proteins but there is no direct evidence of this.

Subcellular localization
Diverse experimental approaches, which include in situ hybridization combined with confocal laser scanning and
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Figure 2 In situ hybridization to Citrus exocortis viroid (CEVd) in tomato. Confocal micrograph of single mesophyll cell from CEVd-infected tomato leaf material showing cell nucleus with viroid signal (red/orange) and cell structure by autofluorescence (green). Reproduced by permission from Bonfiglioli et al. (1996) The Plant Journal 9: 457 465.

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alternative pathways, termed asymmetric and symmetric, with one and two operating rolling circles, respectively.

RNA pol II (?)

Chloroplastic RNA pol (?)

Rolling circle replication: asymmetric variant

Members of the Pospiviroidae family, such as PSTVd, CEVd, HSVd, and CCCVd, follow this pathway in which the infecting monomeric circular RNA (to which by convention is assigned the plus polarity) is used as a template by an RNA polymerase activity that, after several transcription rounds, leads to an oligomeric minus RNA. This concatemer is transcribed to produce an oligomeric plus RNA, which is then processed by a site-specic RNAase activity into monomeric strands that are nally circularized by an RNA ligase activity (Figure 3). PSTVd oligomeric RNAs have been detected in the nucleus, indicating that this viroid, and probably the other Pospiviroidae members, not only accumulates but also replicates in this organelle. Concerning the three enzymatic activities, the RNA polymerase involved in synthesis of plus strands is very likely the nuclear RNA polymerase II, acting on an RNA template, because replication of HSVd, PSTVd and CEVd is impeded by the low levels of aamanitin that typically inhibit this class of enzymes. The RNA polymerase mediating synthesis of the minus strands may also be the same enzyme or another nuclear RNA polymerase. On the other hand, extracts from potato nuclei are able to process in vitro a linear oligomeric PSTVd RNA into monomers and to circularize them, but the nature of the RNAase and RNA ligase presumably involved remains to be established.
Ribozyme
5 OH 2 P 3

RNA ligase (?) RNA pol II Chloroplastic RNA pol (?)

RNAase (?)
5 OH 2 P 3 5 OH

Ribozyme
2 P 3

RNA ligase +

RNA ligase (?)

(a)

(b)

Rolling circle replication: symmetric variant

In this pathway, the oligomeric minus RNA is processed to unit-length strands and ligated to the circular minus RNA that then serves as the template for the second half of the cycle. Monomeric circular ASBVd RNAs of both polarities have been found in infected tissue, forming multistranded complexes with oligomeric RNAs of the opposite polarity, supporting the idea that replication of this viroid, and probably of the other Avsunviroidae members, occurs through the symmetric pathway (Figure 3). Moreover, the ASBVd multistranded complexes have been found in chloroplasts, indicating that viroid replication occurs in this organelle, in which it also accumulates. Consistent with this view, the elongation of ASBVd plus strands is not inhibited by high levels of a-amanitin, suggesting that a chloroplastic RNA polymerase, resistant to this antibiotic, is involved. Processing of both polarity oligomeric RNAs of ASBVd, PLMVd and CChMVd occurs autocatalytically in vitro through hammerhead ribozymes (Figure 1b). Two lines of evidence suggest this is also the situation in vivo. Firstly, the monomeric plus linear CChMVd RNA extracted from infected chrysanthemum has 5- and 3-

Figure 3 Rolling circle model for replication of viroids. (a) Asymmetric and (b) symmetric variants with one and two rolling circles proposed to operate in the Pospiviroidae and Avsunviroidae families, respectively. Solid and open lines refer to plus and minus polarities, respectively, and processing sites are denoted by triangles. The enzymatic and ribozymatic activities presumably involved in the replication steps are indicated; for the activities followed by a question mark the evidence is insufficient or controversial. The linear monomeric RNAs contain most probably 5hydroxyl and 2,3-cyclic phosphate termini.

termini which are identical to those generated in vitro by the hammerhead structures; a similar situation has been observed regarding the 5-termini of some plus and minus linear ASBVd RNAs of sub- and supragenomic size. Secondly, sequence variability of PLMVd and CChMVd always preserves the stability of hammerhead structures, either because mutations are found in loops or, when occurring in helices, a second compensatory mutation restores the base pairing. Ligation of linear monomeric RNAs to the circular forms occurs autocatalytically in vitro, at least in PLMVd, but the nature of the resulting bonds, 2 5 instead of the typical 3 5, and the structure of the ligation site of one viroid-like satellite RNA that selfcleaves also through a hammerhead structure, makes the involvement of a host RNA ligase more likely. Important issues on viroid replication remain to be addressed, like the precise site of initiation of RNA synthesis. Moreover, there are conicting reports about
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the processing site of the oligomeric plus RNAs of closely related Pospiviroidae members, which has been alternatively located in the upper and lower CCR strands, and even on the underlying mechanism, with a proposal that it may also be autocatalytic (mediated by a ribozyme yet to be discovered) instead of catalysed by a host RNAase, as has been generally assumed.

Molecular models for pathogenesis

Both RNAs and proteins are potential candidates for the initial target of viroid pathogenesis. Base-pair interactions between viroids and host RNAs, such as the small nuclear RNAs and the 7S RNA, might divert them from their normal functioning and impair the cellular metabolism. Other host RNAs might also be aected if they are accidentally recognized and degraded by the hammerhead ribozymes present in the Avsunviroidae members. On the other side, cellular proteins may also be a target for viroid pathogenesis. Severe and mild isolates of PSTVd, CEVd and CChMVd dier only in a few nucleotides (approximately 1% of their sequences) but these changes may have a dramatic eect on the overall conformation or on a domain of the viroid molecule, and alter its ability to interact with one or more host proteins. In this respect a correlation has been proposed between bending of the P domain of PSTVd and symptom expression in tomato. It has also been shown that PSTVd is able to interact and activate in vitro a mammalian 68-kDa protein kinase and, interestingly, the stimulation of a severe isolate is 10-fold greater than that of a mild one. This has led to the suggestion that interaction of PSTVd with a plant homologue of this mammalian protein kinase, whose activation triggers a cascade of reactions which ultimately reduces initiation of protein synthesis, could be the primary pathogenic eect of this viroid. However, interactions of the viroid RNA, or of its replicative intermediates, with host proteins involved in viroid replication, transport or accumulation represent other possibilities. Moreover, as the two viroid families have very dierent properties, it is quite likely that there is more than one pathway leading to pathogenesis.

Pathogenesis
Despite their structural simplicity, viroids may induce macroscopic alterations comparable to those elicited by more complex pathogens such as virus, bacteria or fungi. The symptomatology provoked by viroid infections can be quite variable. On leaves, symptoms include malformations, epinasty, rugosity and chlorotic and/or necrotic spots. Stems often show internode shortening, localized necrosis on the vascular tissues and bark cracking. Deformations and colour alterations are frequent on fruits and reserve organs, and delays in foliation, owering and ripening may also be observed. The phenotypical eects can vary between dierent viroids, or between distinct isolates or hosts when considering the same viroid. Some viroids have devastating consequences, as CCCVd on the coconut palm plantations of the Philippines, whereas others incite very mild symptoms, as Grapevine yellow speckle viroid (GYSVd)-1 or GYSVd-2 on grapevine, or no symptoms at all, as Columnea latent viroid (CLVd) on Columnea. Viroid replication and symptom expression are usually favoured in plants grown at relatively high temperature and light intensity. This is reected by the fact that viroids aect mainly crops grown in tropical or subtropical areas and in greenhouses. Infection by some viroids may lead to desirable traits on certain crops, as occurs with citrus dwarng. Thus, despite their usual negative consequences, viroids may also have interesting agronomic applications.

Evolution
Viroids have a number of common structural features, like size, circularity and lack of messenger activity, with some satellite RNAs, which, in contrast to viroids, are functionally dependent on a helper RNA virus. Viroid-like satellite RNAs have self-cleaving ribozymatic domains in one or both polarity strands which play a main role during the replication cycle via a rolling circle mechanism. Recently, a new class of small plant RNAs have been described, represented so far only by the carnation small viroid-like RNA (CarSV RNA). Despite sharing structural similarities with viroid and viroid-like satellite RNAs, CarSV RNA is unique in lacking infectivity and in having a DNA counterpart, an observation that led to the proposal that the CarSV RNA and DNA are the two forms of a retroviroid-like element.

Cytopathic and biochemical effects

The macroscopic symptoms observed in viroid-infected plants are accompanied by alterations at other levels. Electron microscopy studies have shown the cytopathic eects of viroid infection, which include distortion of cell walls, accumulation of electron-dense deposits, appearance of membranous structures and disturbance of the chloroplast structure. Viroids trigger general defensive mechanisms of response to pathogens in the infected plants. At the molecular level, eects of viroid infection consist of changes in the expression of proteins, like the pathogenesis-related (PR) proteins, and alteration in the levels of hormones, like ethylene, or metabolites, like polyamines.
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Viroid quasispecies
The complex genomic structure of viroid isolates reects the extreme plasticity of these minimal pathogenic agents. Viroids do not propagate as uniform populations but as a mixture of closely related variants tting the quasispecies model proposed for RNA replicons. The high sequence heterogeneity of viroid isolates is essentially due to the accumulation of mutants emerging de novo during replication as a result of the error-prone nature of RNA polymerases. Although the frequent appearance of mutations is a main factor in viroid evolution, dierent selection mechanisms seem to inuence this process. Within the Pospiviroidae family, preservation of some sequence motifs, such as CCR, TCR and TCH, is presumably required in viable mutants, together with the maintenance of some other structural traits such as the rod-like secondary structure and the possibility of adopting certain alternative metastable conformations with a potential role during replication (Qu et al., 1993). Within the Avsunviroidae family, formation of hammerhead structures in both polarity strands seems critical for biological tness. Another important factor limiting sequence heterogeneity in PLMVd and CChMV is the preservation of a branched secondary structure and, possibly, of a pseudoknot-like interaction in PLMVd. Besides the mentioned structural constraints, some sequence changes in viroids may also be conditioned by the hosts and tissues they infect, as illustrated by certain mutations only found in CEVd variants isolated from citron or tomato, and by the segregation of specic ASBVd sequences between symptomatic and asymptomatic portions of the same leaf. Studies on the molecular evolution of individual viroid sequences have indicated that viroids can accumulate changes rapidly. Although quick generation of new quasispecies after inoculation of plants with single complementary deoxyribonucleic acid (cDNA) sequences has been reported for PSTVd and PLMVd, belonging to dierent families, the accumulation rate of sequence heterogeneity is notably higher in the latter case. It has been speculated that this could reect the involvement of dierent RNA polymerases, with distinct mutation rates, in the replication of the two viroids.

the characterization of several CEVd and CCCVd variants containing sequence duplications.

Viroid origin
There are quite a few hypotheses about the origin of viroids, suggesting, for example, that they may come from transposable elements or that they may represent escaped introns. However, the most recent and attractive proposal assumes that viroid and viroid-like satellite RNAs may be relics of precellular evolution, in line with the idea of the existence of a primitive RNA world composed of selfreplicating RNAs before the advent of DNA and proteins. Several features of these RNAs support their consideration as molecular fossils: small size, circularity (which would preclude the need of initiation and termination replicative signals), high G 1 C content (which would attenuate the eects of the low delity of primitive polymerases) and, most remarkably, the catalytic nature of some of them. After the evolution of cellular organisms, these free-living RNAs would have adopted an intracellular mode of existence, with viroids and satellite RNAs becoming dependent on the host and the helper virus, respectively. Phylogenetic analyses of the sequences of viroid and viroid-like satellite RNAs are consistent with a common origin for both groups of RNAs.

Control
As for plant viruses, viroid control measures are essentially prophylactic. Viroids are transmitted mainly by vegetative propagation and, consequently, the principal control is the use of viroid-free propagating material. For this purpose, simple and rapid methods for viroid detection are very important. Although biological tests with indicator plants have played an important role in the past, molecular methods are now taking the lead. Since viroids do not code for any protein, the enzyme-linked immunosorbent assays (ELISAs), which are based on antibodies raised against viral proteins and have been broadly used for virus detection, are not applicable to viroids that must be detected by other techniques which target the RNA itself. Prominent among these are molecular hybridization with radioactive and chemically-labelled cDNA- or cRNAspecic probes, and reverse transcription combined with the polymerase chain reaction (RT-PCR) using specic primers. Most viroids are also transmitted mechanically and the regular disinfection of pruning tools with diluted commercial bleach is recommended. Some viroids, for example ASBVd, are transmitted through seed and pollen and this must be considered when establishing nurseries that should be separated from infected areas. Only Tomato planta macho viroid (TPMVd) is known to be eciently transmitted by aphids under specic ecological conditions
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Recombination in viroids
Although mutation seems to be the main source of variability of viroid evolution, RNA recombination also plays an important role. Evidence for genetic exchanges between viroids has been inferred from the quimeric structure of some viroids, like CLVd and Australian grapevine viroid (AGVd), that suggests that they result from recombination processes between several parental sequences coinfecting the same host. Moreover, intramolecular rearrangements may also occur, as revealed from

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in which naturally infected wild solanaceous plants are cultivated near tomato elds.

Elimination of viroids from infected plants

Some viroids such as PLMVd can be removed, although with some diculty, from infected peach trees by thermotherapy (treating growing plants with hot air) followed by meristem-tip propagation, but attempts to extend this approach to eliminate other viroids like Pear blister canker viroid (PBCVd) have been unsuccessful. Since relatively high temperatures are important for viroid accumulation, PSTVd has been eliminated by extended cold treatments followed by meristem-tip culture.

replicative intermediates of viroids, or against the viroid molecules themselves, that present a dsRNA-like structure. The potential of this approach is considerable because: the same construction may render a plant resistant to a wide spectrum of viruses and viroids; the pathogen cannot escape by xing mutations because the RNAase acts against the secondary structure and not against sequence motifs; and no pathogen-derived sequences are released to the environment.

References
Branch AD and Robertson HD (1984) A replication cycle for viroids and other small infectious RNAs. Science 223: 450454. Diener TO (1971) Potato spindle tuber virus IV. A replicating low molecular weight RNA. Virology 45: 411428. Grill LK and Semancik JS (1978) RNA sequences complementary to citrus exocortis viroid in nuclei acid preparations from infected Gynura aurantiaca. Proceedings of the National Academy of Sciences of the USA 75: 896900. Gross HJ, Domdey H, Lossow C et al. (1978) Nucleotide sequence and secondary structure of potato spindle tuber viroid. Nature 273: 203 208. Keese P and Symons RH (1985) Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proceedings of the National Academy of Sciences of the USA 82: 45824586. Navarro B and Flores R (1997) Chrysanthemum chlorotic mottle viroid: unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes. Proceedings of the National Academy of Sciences of the USA 94: 1126211267. Qu F, Heinrich C, Loss P et al. (1993) Multiple pathways of reversion in viroid conservation of structural domains. EMBO Journal 12: 2129 2139. Sano T, Candresse T, Hammond RW, Diener TO and Owens RA (1992) Identication of multiple structural domains regulating viroid pathogenicity. Proceedings of the National Academy of Sciences of the USA 89: 1010410108. Sano T, Nagayama A, Ogawa T, Ishida I and Okada Y (1997) Transgenic potato expressing a double-stranded RNA-specic ribonuclease is resistant to potato spindle tuber viroid. Nature Biotechnology 15: 12901294. Yang X, Yie Y, Zhu F et al. (1997) Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes. Proceedings of the National Academy of Sciences of the USA 94: 48614865.

Biotechnological approaches for viroid control

More recently, progress in molecular biology has opened new windows for controlling viroid diseases. As already indicated, the hammerhead structures found in three viroids mediate self-cleavage in cis of the RNAs in which they are naturally embedded. But hammerhead structures can also be engineered to act in trans, an aspect with major biotechnological implications. A good example of this is the observation that transgenic potato plants expressing a ribozyme against the minus PSTVd RNA display a high resistance against PSTVd replication, thus providing the rst example of a ribozyme suppressing a viroid pathogen to an undetectable level in planta (Yang et al., 1997). Control experiments showed that transgenic potato lines expressing a catalytically inactive ribozyme were susceptible to PSTVd. Inferior resistance levels were observed in plants transformed with ribozyme constructs targeting the PSTVd plus RNA, probably because viroid minus strands accumulate to lower concentrations and/or may be more accessible to the ribozyme. Since PSTVd replicates and accumulates in the nuclei, where the transgenic constructs containing the ribozymes are synthesized, these results agree with previous observations supporting the view that the eectiveness of ribozymes is dependent on colocalization with their target RNA substrates. A second biotechnological approach for viroid control has been developed by expressing a double-stranded RNA (dsRNA)-specic RNAase from yeast in transgenic potatoes. The introduction of the transgene, although not causing any deleterious eect in the growth pattern, did induce an increased resistance to PSTVd infection in some of the transformed lines (Sano et al., 1997). The rationale behind this strategy, which has also been applied to produce virus resistant plants, is that the majority of plant viruses have single-stranded RNA genomes but replicate through intermediates containing dsRNA regions that can be recognized and degraded by the dsRNA-specic RNAase. This mechanism may also operate against the
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Further Reading
Diener TO (1979) Viroids and Viroid Diseases. New York: Wiley. Diener TO (ed.) (1987) The Viroids (The Viruses). New York: Plenum Press. Diener TO (1996) Origin and evolution of viroids and viroid-like satellite RNAs. Virus Genes 11: 119131. Ding B, Kwon MO, Hammond R and Owens R (1997) Cell-to-cell movement of potato spindle tuber viroid. Plant Journal 12: 931936. ndez C (1997) Viroids: the non-coding Flores R, Di Serio F and Herna genomes. Seminars in Virology 8: 6573. Flores R, Randles JW, Bar-Joseph M and Diener TO (1998) A proposed scheme for viroid classication and nomenclature. Archives of Virology 143: 623629.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Viroids

Maramorosch K (ed.) Viroids and Satellites: Molecular Parasites at the Frontier of Life. Boca Raton, FL: CRC Press. Riesner D (1991) Viroids: from thermodynamics to cellular structure and function. Molecular Plant Microbe Interactions 4: 122131.

Semancik JS (ed.) (1987) Viroids and Viroidlike Pathogens. Boca Raton, FL: CRC Press. Symons RH (1997) Plant pathogenic RNAs and RNA catalysis. Nucleic Acids Research 20: 26832689.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net