Microreview Viroids

Cellular Microbiology (2008) doi:10.1111/j.1462-5822.2008.01231.x

Microreview

Viroids

Efthimia Mina Tsagris,1,2* Introduction Ángel Emilio Martínez de Alba,1† Studies on viroids have led to the discovery of some of Mariyana Gozmanova1,2‡ and Kriton Kalantidis1,2 the most interesting principles of the biology of RNA: the 1Institute of Molecular Biology and Biotechnology, fact that a non-coding, non-translatable RNA can cause a Foundation for Research and Technology, disease (Diener, 1971), the extraordinary small size of their PO Box 1385, 71110 Heraklion, Greece. genome (Gross et al., 1978) and their circularity (Sänger 2Department of Biology, University of Crete, et al., 1976), which enables them to circumvent the pro- PO Box 2208, 71409 Heraklion, Greece. blems of linear genome replication such as the accurate replication of linear ends (Diener, 1989). One of the first Summary self-cleaving structures, the hammerhead ribozyme, was found in a satellite RNAvirus and a viroid RNA(Prody et al., Viroids are small, circular RNA pathogens, which 1986; Forster and Symons, 1987; Forster et al., 1987). infect several crop plants and can cause diseases Prior to the molecular characterization of the hepatitis delta of economic importance. They do not code for pro- virus (HDV), a circular (‘viroid-like’) RNA infecting human teins but they contain a number of RNA structural liver cells and associated with hepatitis B virus (Lai, 2005; elements, which interact with factors of the host. Taylor, 2006) viroids remained as an interesting, but The resulting set of sophisticated and specific inter- exotic example of a plant pathogen, investigated by some actions enables them to use the host machinery researchers in the field of plant pathology and molecular for their replication and transport, circumvent its plant virology, together with a group of biophysicists defence reactions and alter its gene expression. studying RNA structure. They recognized in this relatively Although found in plants, viroids have a distant abundant, natural RNA a perfect object to study RNA relative in the animal world: hepatitis delta virus structure transitions. Viroids have been the basis on which (HDV), a satellite virus of hepatitis B virus, which new experimental and computational methods have been has a similar rod-like structure and replicates in developed (Riesner, 1991; Steger and Riesner, 2003). the nucleus of infected cells. Viroids have also a A recent landmark of scientific breakthrough originating cellular relative: the retroviroids, found in some from viroid research is the discovery of RNA-mediated de plants as independent (non-infectious) RNA repli- novo DNA methylation, which was first described in trans- cons with a DNA copy. In this review, we summarize genic plants carrying copies of the viroid cDNA(Wasseneg- recent progress in understanding viroid biology. ger et al., 1994). Although this was an ‘artificial’ transgenic We discuss the possible role of recently identified system, it was quickly recognized that RNA-mediated DNA viroid-binding host proteins as well as the recent methylation is a mechanism that is a part of a whole battery data on the interaction of viroids with one part of the of responses that plants have towards environmental host’s defence machinery, the RNA-mediated gene changes and developmental programmes (Wassenegger, silencing and how this might be connected to viroid 2005; Henderson and Jacobsen, 2007). replication and pathogenicity. Several reviews have been published recently on viroids, showing the increasing scientific interest in these molecules as a model system and plant pathogen. In this review, we will discuss some of the most recent articles on Received 2 April, 2008; revised 20 August, 2008; accepted 25 August, viroids, and present some possible models concerning 2008. *For correspondence. E-mail [email protected]; Tel. their replication, biogenesis and evolution. (+30) 2810 394367; Fax (+30) 2810 394404. Present addresses: †Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia, Consejo Superior de Investigaciones Cientí- Basic properties and mode of replication of viroids ficas, Valencia, Spain; ‡University of Plovdiv, Department of Plant Physiology and Molecular Biology, 24, Tsar Assen St., 4000 Plovdiv Viroids have a size of ca 250–400 nucleotides. They in- Bulgaria. fect several crop plants, causing symptoms of differential

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd 2 E. M. Tsagris, Á. E. Martínez de Alba, M. Gozmanova and K. Kalantidis severity, which range from mild effects such as hardly plant organ, the phloem and its surrounding cells visible growth reduction, up to deformation, necrosis (Fig. 1B and C). For cell-to-cell and long-distance trans- or chlorosis and severe stunting (Singh et al., 2003). port, viroids use the supracellular structure of vascular Some viroid strains do not cause symptoms at all and plants, where groups of cells are connected via special seem to behave as simple RNA replicons rather than channels, called plasmodesmata. Therefore, like plant pathogens. However, symptoms depend very much viruses but unlike animal viruses, viroid RNA does not on environmental conditions and may change during have to cross the plasma membrane for long-distance infection as has also been found with plant viruses transport. It does, however, cross the borders of the (Semancik, 2003). nucleus via the nuclear pore or the chloroplast mem- Viroids do not have coding capacity and so far no branes. The precise mechanism by which this transfer viroid-coded protein has been detected in infected plants. is executed is not known. Viroids are not encapsidated. They replicate in the plant The entire replication and spread of viroids depends hosts by RNA–RNA transcription, in a ‘rolling-circle’ mode on host factors. DNA-dependent RNA polymerase II (Branch and Robertson, 1984). During replication, oligo- has been shown to replicate nuclear localized viroids meric linear RNAs of plus polarity are formed, which are (reviewed in Tabler and Tsagris, 2004), while it has been cleaved to monomers and ligated into circles (Fig. 1A). suggested that the nuclear-encoded, but chloroplast- Oligomeric forms of (-) polarity are also present as repli- localized DNA-dependent RNA polymerase (NEP) replicative intermediates. Circular molecules of minus polarity cates the chloroplastic viroids (Flores et al., 2004). have been detected in plants only in some members Viroids and HDV seem to be able to delude specific of the family Avsunviroidae, which bear self-cleaving DNA-dependent RNA polymerases of their host cells, ribozyme structures in their RNA genomes. Viroid local- offering genomic and antigenomic RNAs as a compe- ization is either nuclear (family Pospiviroidae) or chloro- titive template to the normal DNA templates of these plastic (family Avsunviroidae), where they replicate with enzymes. Or, alternatively, they just ‘remind’ to these the aid of host-encoded DNA-dependent RNA poly- enzymes their ‘RNA World’ attributes. In order to be able merases. Viroids can therefore be considered as para- to compete with the normal DNA templates, these RNA sites of the transcriptional machinery of the organelles replicons must have a competitive advantage: either a (nucleus or chloroplast), in contrast to most plant RNA high-affinity binding site, or the independence of acces- viruses, which replicate in the cytoplasm and can be sory trans-acting factors for recognition of the primitive therefore regarded as parasites of the translational ‘promoter’ by the DNA-dependent RNA polymerase, or machinery of the cell. both. In some plants, non-infectious circular RNA transcripts The viroids Potato spindle tuber viroid (PSTVd) and of similar size to viroids have been detected, which Citrus exocortis viroid (CEVd) are both replicated in contain hammerhead ribozyme structures (Darós and the nucleus, most probably by the same enzyme. PSTVd Flores, 1995; Hegedus et al., 2001). These transcripts has been shown to be transcribed in vitro and in vivo exist in the genome of their hosts as extra chromosomal by DNA-dependent RNA polymerase II (Mühlbach and or integrated concatameric DNA copies; if they replicate, Sänger, 1979; Rackwitz et al., 1981) and CEVd exists in they do so via oligomeric forms of plus and minus polarity. a complex with at least the large subunit of the DNA- As they are usually found in plants associated with a dependent RNA polymerase II (Warrilow and Symons, pararetrovirus they may use the machinery of the virus to 1999). Putative initiation sites for PSTVd have been copy and integrate their cDNA in the host’s genome. They determined, using different methods with varying results. have been named ‘retroviroids’ (Darós and Flores, 1995). Earlier work has identified nucleotide G168, localized One can speculate on whether viroids evolved from ret- at the right terminal part (Tabler and Tsagris, 1990), as roviroids, or vice versa, and what the intermediate steps transcription initiation site but nucleotides U359, C1 and have been. others (Kolonko et al., 2006) have also been identified Viroids infect the epidermis of their hosts after as potential transcription initiation sites. The methods mechanical damage of the plant cell wall. Unlike most used did not, however, allow the identification of a other plant viruses, they do not have important natural genuine 5′-triphosphate transcript. vectors from the animal kingdom (Singh et al., 2003). For Avocado sunblotch viroid (ASBVd), a viroid RNA After entry to the first cell layers of the leaves and initial pathogen replicating in the chloroplast, it has been pro- replication, they are transported to neighbouring cells, posed that a nuclear-encoded DNA-dependent RNA poly- reaching the vascular tissues, from which they travel to merase (NEP) is involved in its replication (Navarro et al., newly emerging young leaves or other sink tissues like 2000). NEP is a nuclear-encoded enzyme, which is roots, together with photoassimilates (Ding et al., 2005). transported to the chloroplast and functions there during Infected plants contain viroid RNA in the conductive chloroplast development. It is also a candidate protein

Fig. 1. A. A rolling-circle model has been proposed for replication of the circular RNA genome in order to explain the generation of oligomeric linear forms (Branch and Robertson, 1984). RNAs of sense polarity are shown in red and those of antisense polarity in blue. Processing structures are in yellow or light blue and mark the length of the linear unit. Pospiviroidae replicate in the nucleus of the plant cell. The replication cycle is asymmetric, because only circular RNAs of (+) polarity are produced. Pospiviroidae do not contain known self-cleaving structures. Avsunviroidae replicate in the chloroplast. They contain active self-cleaving and possibly ligating hammerhead ribozyme structures on the RNAs of both polarities (yellow or light blue lines). Their replication cycle is classified as symmetric, because a circular RNA of both polarities is formed. B and C. Schematic representation of systemic infection of nuclear viroids. In the circles, a cross-section of a leaf is shown (according to figs 15.1 and 21.15 of Buchanan et al., 2000). Different cell types are represented by different forms and colours. EC: epidermal cells; MC: mesophyl cells: BSC: bundle sheath cells; CC; phloem companion cells; SE: sieve elements. The nuclear membrane is represented by a dotted red circle. Black lines represent the difference in number of plasmodesmata connecting different cells. Sieve elements are degenerated cells which form the vascular tubes and do not contain nuclei. The viroid RNA is represented by open circular forms with different colours. Each colour indicates a replication cycle in the next cell (brown, orange, red, yellow, magenta), indicating possible differences in the RNA–protein complexes. For simplicity, intermediate oligomeric forms are not shown in this scheme. B. Events occurring on the first infected (or inoculated) leaf. After damage of epidermal tissues, the viroid moves slowly from cell to cell. One possibility would be that the viroid undergoes a replication cycle in each nucleus before moving to neighbouring cells. Replication in the inoculated leaf remains at low levels. C. Events occurring in the newly developing leaves. Viroid RNA has to cross the plasmodesmata from phloem tissues (SE, CC) to bundle sheath and mesophyl cells (BSC, MC). A specific structure of PSTVd has been shown to be necessary for trafficking of the RNA from phloem companion cells (CC) to bundle sheath cells (BSC) (Zhong et al., 2007) and from bundle sheath to mesophyl cells (Qi et al., 2004). Some cells may remain viroid-free due to effective defence by the silencing mechanism. for transporting the Avsunviroid RNAs into the chloro- for two chloroplast-replicating viroids have been plast. Recent findings however question whether indeed determined using an elegant method of labelling the NEP or PEP (a plastid-encoded DNA-dependent RNA newly generated transcripts with guanylyltransferase polymerase) is the replicating enzyme for avsunviroids (Navarro and Flores, 2000; Delgado et al., 2005). The (Motard et al., 2008). Regardless of which RNA poly- initiation sites for both Peach latent mosaic viroid (PMLVd) merase is involved, the initiation sites for RNA replication RNA polarities reside at a stem of the conserved

hammerhead ribozyme structure of the molecule, adjacent to the ribozyme cleavage sites (Delgado et al., 2005). A conserved hexanucleotide has been shown to be Peptides encoded No No Small and large HDAg a characteristic motif just before the initiation site, not only of the genuine viroid template, but also for an in vitro selected shorter RNA template (Motard et al., 2008) thus resembling most probably the primitive RNA promoter (Pelchat et al., 2002). Herbaceous woody (depends on viroid) woody (depends on viroid) Carnation No Human liver cells Comparing the replication steps of viroids and HDV, common but also distinct mechanisms can be identiﬁed. One of the features that all these circular RNA pathogens have in common is that they replicate in DNA-containing organelles (nucleus or chloroplasts), so that their RNA Broad/narrow (depends on viroid) Narrow Herbaceous Not infectious (Helper virus needed) Helper virus needed must therefore be transported to the corresponding organelle. HDV is larger than viroids, and contains in the e elements has been suggested earlier by Diener and antigenomic strand the open reading frame for the hepatitis delta antigen (small and large forms, HDAg) (Taylor, 2006). A homologue protein to HDAg has been reported involved and proteins involved Ribozymes involved and proteins involved to exist in the human genome. It is conceivable that HDV satellite RNA had originally a smaller size and acquired through RNA recombination the RNA encoding the HDAg (Brazas and Ganem, 1996). Reverse transcription has not been reported to be important for either viroids or HDV; Reverse transcription No Proteins No Ribozymes Yes Reverse transcriptase integration in host genome Nono Ribozymes DNA intermediate forms exist, and no integration in the genome of the host has been shown to occur. Plant retroviroids are circular RNA replicons which can be trans- mitted only through cell division; they are not pathogenic and cannot replicate autonomously (Darós and Flores, 1995). Most probably, they are reverse transcribed from a pararetrovirus reverse transcriptase (with which they RNA-templated RNA transcription DNA-dependent RNA polymerase II Nuclear-encoded chloroplastic RNA polymerase RNA polymerase II? DNA-dependent polymerase II and polymerase I are often associated). Another possibility would be that reverse transcriptase activities of endogenous retroele- ments, which are very abundant in many crop plants, participate in their replication. Retroviroids and some viroids contain hammerhead ribozymes which are Asymmetric rolling circle Replication moderolling circle Processing Host range Host type rolling circle

. (2005a) have proposed the classiﬁcation of viroids into two families, according to their primary and secondary structure, their responsible for steps during the rolling-circle replication (Côté et al., 2003). Speciﬁc Hop stunt viroid (HSVd) et al strains contain a conserved sequence element which has partial homology to the hammerhead ribozyme structure. This sequence element has not been reported to have self-cleaving activity and possibly resembles an evolution- (for PSTVd) Replication localization Chloroplast Symmetric ary link between viroids still possessing the hammerhead structure and those having lost it (Amari et al., 2001). Hepatitis delta genomic and antigenomic RNAs contain ribozymes forming other characteristic hairpin structures (Taylor, 2006). A model for the generation and evolution of circular viroids from simpler RNA molecules is presented ., 1983). The relation between viroids and HDV was indicated by several authors (reviewed in Lai, 2005; Taylor, 2006). Rod-like Nucleus/nucleolus Circular structure Y-shaped or branched (PLMVd CChMVd) Branched NucleusRod-likein Rolling Nucleus/nucleolus circle? Fig. 2A. Symmetric DNA-dependent

et al Another similarity between the plant RNA replicons and HDV is that they both infect organisms (plants and humans respectively) for which DNA methylation is an Classiﬁcation of viroids and related RNAs. Flores important epigenetic control mechanism. Viroid-like RNAs have not been found in other organisms such as inverte- Pospiviroidae Potato spindle tuber viroid 359–361 Table 1. Name Type members Size Avsunviroidae Avocado sunblotch viroid 247 Retroviroids Carnation retroviroid 275 Hepatitis delta virus 1640 subcellular localization in cells where they replicate and their mode of replication. Possible evolutionary connection of viroids and transposabl co-workers (Kiefer brates or unicellular organisms and most probably they

Fig. 2. A. A model for the generation of imperfect self-complementary helical circular RNAs. An RNA molecule has, for example, three different domains (black, green, red) and contains the structure that an RNA polymerase (represented by a light blue full circle) recognizes as a promoter. At the 3′-OH end, the structure of the RNA allows extension by the polymerase, templated by the rest of the RNA (direction of new synthesis indicated by the red arrow, complementary parts shown as dotted lines in the same colours, black, green and red). The result is an imperfect helix connected by a loop (similar to miRNA precursors). RNA–RNA transcription by DNA-dependent RNA polymerases is error-prone and results in the generation of bulged structures in the helix. This RNA acquires by recombination or through evolution a self-cleaving structure (blue line). The RNA can undergo cleavage to monomers (forward reaction), circularization or oligomerization (reverse reaction of the ribozyme). B. A possible role for Virp1. Virp1 (magenta full ellipses) is involved in import of viroid RNA (brown open circular structures) to the nucleus. Other proteins (black full circles) may participate in intercellular transport or export from the nucleus. do not exist there as pathogens (because they would Different methods have been used to isolate host proteins have been detected). interacting with the viroid RNA: direct RNA ligand screening method (Sägesser et al., 1997), cross-linking and/or biochemical isolation (Wolff et al., 1985; Darós and Recent advances in viroid research Flores, 2002) and genomic approaches, using microar- rays to identify which genes are induced and which ones Host factors involved in viroid replication and transport repressed after infection by viroids (Hammond and Zhao, In recent years, focus on viroid research has moved 2000; Itaya et al., 2002; Tessitori et al., 2007). Biochemi- on from structure–function analysis of the viroid RNAs cal identiﬁcation after cross-linking has the advantage of to the search of host factors which interact directly or isolating complexes of viroid RNA with host proteins which indirectly with viroids and facilitate their replication and exist in vivo, but transient and short-lived complexes or transport (Tabler and Tsagris, 2004; Flores et al., 2005b). complexes existing in minute amounts may escape from

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology 6 E. M. Tsagris, Á. E. Martínez de Alba, M. Gozmanova and K. Kalantidis this approach. A ‘direct’ approach of screening for RNA- orthologue genes can be suppressed in crop plants for binding proteins in cDNA libraries is a powerful method creating transgenic plants resistant to viroids. Other host (Werner et al., 1995; Sägesser et al., 1997), which has genes conferring resistance to viroids when suppressed helped to identify proteins interacting with the whole viroid or expressed have not been described and no classical RNA genome (Martínez de Alba et al., 2003), but which ‘resistance genes’ (R-genes) have been described so can potentially also be used for proteins interacting with far for these RNA pathogens. subdomains of the viroid RNA, stabilized RNA structures Using immunoprecipitation experiments, Pallás’ and or short tertiary RNA structure motifs. However, direct Owens’ groups showed that phloem protein 2 from screening methods identify interactions in another biologi- cucumber (CsPP2) is in a complex with HSVd RNA cal system and must be verified by biochemical or immu- (Gómez and Pallás, 2001; 2004; Owens et al., 2001) nological methods to confirm whether they indeed exist and can be translocated with the RNA as an infectious in planta. entity in grafting experiments between host and non-host Martínez de Alba et al. (2003) have used the direct plants. The analysis of the primary structure of CsPP2 screening method for isolation of PSTVd RNA-binding revealed the existence of a potential double-spaced proteins. One of these proteins, Viroid-binding protein 1 RNA-binding motif, previously identified in a set of (Virp1), contains nuclear localization signals, an RNA- proteins that bind to highly structured RNAs. It was pro- binding domain and a bromodomain (a protein domain posed that this phloem protein is assisting the long- found in many chromatin remodelling factors). It has been distance movement of the viroid RNA within the plant, suggested that bromodomain proteins interact with other and that this type of interaction might operate in other proteins in vivo; they bind to histones containing acety- viroid/host combinations. It will be interesting to test lated lysines. It has also been proposed that they keep whether downregulation of this protein inhibits systemic themselves and their interacting partners in the vicinity viroid translocation. of chromatin, even at phases where the nuclear mem- Ribonucleoprotein complexes were isolated and brane does not exist (during cell division); they have the characterized from Avocado sunblotch viroid (ASBVd)- ‘chromatin association mark’ (Yang, 2004). The carboxy- infected plants. In vivo cross-linking and tandem mass terminal domain of Virp1, which contains the RNA-binding spectrometry has been used to analyse the components domain, was shown to interact specifically with the right of the most abundant cross-linked protein species terminal domain of the viroid RNA in vivo and in vitro on ASBVd RNA. Two closely related chloroplast RNA- (Maniataki et al., 2003; Martínez de Alba et al., 2003). binding proteins (PARBP33 and PARBP35) have been A dual domain motif was proposed to be responsible identified. Members of this protein family are involved for the interaction (Gozmanova et al., 2003). Indeed, in stabilization, maturation and editing of chloroplast mutagenesis of one of the motifs resulted in reduction transcripts. PARBP33 behaves as an RNA chaperone of the binding interaction and mutagenesis of both that stimulates in vitro the hammerhead-mediated self- motifs destroyed the interaction down to 5%. All mutants cleavage of the multimeric ASBVd transcripts, indicating were not infectious in mechanical inoculation assays that this reaction, despite its RNA-based catalysis, is or reverted to wild-type PSTVd sequence. Kalantidis et al. assisted by proteins (Darós and Flores, 2002). (2007) recently showed that Nicotiana benthamiana plants in which expression of Virp1 is suppressed cannot Mechanism of systemic trafficking of viroid RNAs be infected with PSTVd when this is mechanically inoculated. Interestingly, protoplasts from these plants were Plants are ‘supracellular’ organisms, in which groups of also not infected with PSTVd RNA, indicating that Virp1 cells are connected by special structures called plas- has a major role in viroid infection at the cellular level. A modesmata. Viroids are a mobile, systemically spreading Virp1–GFP fusion protein was found to be localized in the RNAs; thus, they can be excellent models to study RNA nucleus, thus Virp1 is a candidate for translocating with signalling throughout the plant vasculature (Ding and the viroid RNA in the nucleus. Whether Virp1 has also a Itaya, 2007; Kalantidis et al., 2008). direct role in replication (e.g. assisting DNA-dependent PSTVd mutants with N. benthamiana acting as a host polymerase II in replication) or whether it has an addi- served in two studies which elucidated steps of systemic tional role in RNA-mediated methylation, or export of trafficking of this viroid. In the first study, the difference viroid RNA from the nucleus, is not known (Fig. 2B). From between two viroid mutants was examined, using a plant pathology view, Virp1 is a candidate resistance in situ hybridization of infected plants, and transgenic gene for nuclear-replicating viroids. As N. benthamiana N. benthamiana plants expressing the corresponding plants in which Virp1 is suppressed, when grown in tissue viroid RNAs. Mutant PSTVd-NT arose from transgenic culture and in the greenhouse, did not show any obvious tobacco plants expressing PSTVd KF440-2, a viroid that phenotype, it will be interesting to test whether Virp1 infects tomato, but does not replicate in tobacco (Wass-

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology Viroids 7 enegger et al., 1996). In contrast to KF440-2, PSTVd-NT would be targets and triggers of this defence mechanism, replicates in tobacco, although at lower levels. Mutant which is predominantly cytoplasmic. The overall structural PSTVd-NB arose from PSTVd-NT, and replicates at even similarity of viroids with the precursor transcripts of higher rates in tobacco. In situ hybridization revealed that miRNAs (the pre-miRNAs), a field emerging at the same PSTVd-NT remains restricted to phloem cells in systemic time, was recognized early (Maniataki et al., 2003). Two leaves, while PSTVd-NB is capable of exit from phloem questions arose: are viroids substrates for degradation cells in systemically infected leaves and enters the meso- by the silencing machinery and is this interaction respon- phyl of newly infected young leaves. Subsequent muta- sible for pathogenicity? tional analysis of the five different nucleotides between strain PSTVd-NT and PSTVd-NB revealed that the ‘sys- Viroid infection results in production temic trafficking’ motif consisted of two parts, with muta- of viroid-specific siRNAs tions lying in the ‘pathogenicity’ and the terminal right domains (Qi et al., 2004). Using biolistic infection on epi- Early work showed that nuclear and (surprisingly) dermal cells of young ‘sink’ leaves, Qi et al. concluded that also chloroplastic viroids are targets of the silencing the systemic trafficking of the strain is directional, from the machinery and that viroid-specific siRNAs are produced bundle sheath to the mesophyll cells and not vice versa. after infection (Itaya et al., 2001; Papaefthimiou et al., Zhong et al. (2007) created a number of PSTVd 2001; Martínez de Alba et al., 2002). In these first reports, mutants which were defective in systemic trafficking. there was no correlation observed between amount of One mutant differs in only two nucleotides from the siRNAs and severity of the viroid strain used. Interestingly, normal systemically infecting PSTVd strain, at positions the siRNAs of PSTVd were found to be localized predo- U43 and C318. If one of the nucleotides is altered to minantly in the cytoplasmic fraction (Denti et al., 2004). create from the wild-type bulge a helix, systemic traffick- Later, it was found that blotched leaf areas contain much ing is lost and the RNA cannot cross the boundary more siRNAs of ASBVd than apparently symptomless between phloem parenchyma (PP) and bundle sheath areas, and that CEVd isolates, which differed in pathoge- cells (BS) (see also Fig. 1B and C). This group analysed nicity, differed also in their siRNA abundance in the the tertiary structure and geometry of this region host plant Gynura aurantiaca (Markarian et al., 2004). of the strains, comparing them with other mutants These differences might be due to the host/viroid sys- and other viroids. They showed that this motif is, in its tems used, or to subtle differences in the experimental tertiary structure, conserved in the genus Pospiviroid, set-up. and that all systemically infecting mutants belong to Itaya et al. (2007) characterized the viroid-specific an equivalent isostericity group. They designed specific siRNAs by cloning them and found that for the genomic mutations for isosteric structures and predicted correctly RNA of (+) polarity there are mainly three ‘hot spots’ of the behaviour of these strains concerning systemic siRNAs but that the (-) polarity siRNAs are distributed spread. It will be interesting to test whether addition of more or less all over the genome of PSTVd. This implies this motif alone is capable of changing an unrelated that the predominant RNA substrate for siRNA production RNA molecule into a systemic trafficking RNA, and how is most probably not a true double stranded viroid RNA, large this RNA signal finally is. However, a recent report but the genomic (+) RNA. The (-) RNA is to a lesser from the same laboratory revealed that the mechanism extent a substrate, possibly as a replicative intermediate. of systemic movement is more complicated than antici- Interestingly, there were nearly no siRNAs found from pated and that more structural elements of the rigid the so-called ‘pathogenicity’ region, which is surprising, PSTVd RNA rod-like structure are important for systemic because mutations in this region are known to induce spread (Zhong et al., 2008). changes in pathogenicity on tomato (Schnölzer et al., 1985). siRNAs were produced also in plant protoplasts of N. benthamiana, indicating that the degradation Interaction of viroids with the host’s defence mechanism does not need the ‘supracellular’ structure of mechanisms and the interrelation with replication the whole plant. Using GFP–PSTVd fusion sequences, The mechanism of coordinated sequence-specific RNA Itaya and co-workers could show that PSTVd siRNAs are degradation was first proposed as a model in studies active in cleaving target RNAs in RISC complexes, but of transgenic plants expressing viral genes (Lindbo and the stable secondary structure of PSTVd RNA resists Dougherty, 2005). It was also recognized early that RNA- RISC-mediated cleavage. Parallel to this, Gómez and mediated mRNA degradation is an antiviral mechanism in Pallás (2007) have shown that circular forms of HSVd higher eukaryotes (Baulcombe, 2004; 2006). It was there- are not decreased in amount when translocating through fore interesting to study whether viroids, RNA pathogens a grafted transgenic tissue which produces HSVd replicating in the nucleus or in the chloroplasts of plants, siRNAs.

Machida et al. (2007) have shown that viroid siRNAs complex loaded with a specific viroid-derived siRNA might of 21 nucleotides length precede the appearance of be the cause of the symptoms. siRNAs of 24 nucleotides length in the examined period of Symptoms might be induced if viroid replication or 45 days and are consistently present throughout this time. transport recruits limiting factors of the RNA-silencing The siRNA profile may, however, differ dramatically from machinery, thus interfering with miRNA (and/or endo- leaf to leaf if a single plant is examined. The siRNA profile genous siRNA) biogenesis and function. Martin et al. of averaged samples changed during infection from size- (2007) have investigated this possibility; however, they specific, to heterogeneous and finally again to more size- could not find indications of this type of general interfer- specific. They identified an additional ‘hot spot’ of siRNA ence. Gas et al. (2007) have proposed that a Dicer-like production derived from the complementary (-) RNA RNase might be involved in viroid processing. of PSTVd, taking samples from a single leaf, which may explain why this ‘hot spot’ was not detected by Itaya et al. who had (most probably) used ‘averaged’ samples for Future directions cloning. A possible role for RNA–RNA transcription performed Martin et al. (2007) found that viroid siRNAs were by DNA-dependent RNA polymerases phosphorylated at the 5′-terminus and protected at the 3′-terminus, similar to genuine endogenous plant-derived Viroids are used as templates for DNA-dependent RNA siRNAs and miRNAs. Landry and Perreault (2005) have polymerase II (reviewed in Sänger and Tabler, 1987) and shown that (+) and (-) full-length forms of the PLMVd it has been shown that T7 phage DNA-dependent RNA can be substrates for DCL(s) activity in wheat germ polymerase can use RNA as a template too (Konarska extract. A minimal length is required for DCL binding and Sharp, 1989). DNA-dependent RNA polymerase II and activity and the best substrate contained a perfect can use its own transcript of HDV RNA as a template dsRNA helix. to produce small hairpin-shaped longer RNA transcripts (Filipovska and Konarska, 2000). In Fig. 2A, the generation of such self-complementary short RNA transcripts Viroid interaction with components of the RNA-silencing is shown. Production of such ‘aberrant’ transcripts origi- mechanism might contribute to symptom appearance nating from this type of reaction might be inhibitory to The discovery that viroids interact with the silencing the normal DNA-dependent RNA transcription of the poly- machinery (as targets and triggers) led to the hypothesis merases and in this case it should be suppressed in the that viroid-specific siRNAs might be responsible for symp- cell. Another scenario could be that this type of reaction toms caused by viroids, via RISC-mediated translational has a biological role. As genome-wide low-level basal inhibition, or cleavage of specific host mRNAs containing transcription has been shown to occur (Kapranov et al., short stretches of homologies (Tabler and Tsagris, 2004; 2007) and the fast biogenesis of miRNAs (frequent birth Flores et al., 2005b). Viroid-derived siRNAs are loaded and death of miRNA genes) has also been described on RISC complexes and might inhibit the expression of (Fahlgren et al., 2007), one could consider the possibility specific host genes. Wang et al. (2004) have shown that that de novo RNA–RNA-dependent transcription of short expression of dsRNA derived from PSTVd sequences hairpin transcripts is an underestimated part of the whole from nucleotides 16–359 can cause viroid-like symptoms genome transcriptome. This type of transcript has a struc- in tomato. This shows that in this host/viroid combination, tural similarity to the miRNA precursor transcripts, which PSTVd siRNAs may be the triggers of pathogenicity, have a length of c. 70–80 nucleotides, an imperfect stem- and that this is independent of viroid replication. Also loop secondary structure and look similar to the terminal the results of Carbonell et al. (2008) indicate the exist- parts of viroids (see also Fig. 2A). Such low-level ence of viroid-specific RISC complexes operating in ‘error-prone’ RNA–RNA transcription, in combination with infected plants, as symptoms were reduced when the reverse transcription, possibly contributes to fast adapta- viroids were co-inoculated with homologous dsRNAs tion of organisms with novel sequences. A third possibility and not with heterologous ones. Viroid symptom develop- would be that small, ‘aberrant’ RNA transcripts derived ment can be very specific, depending on host and viroid from DNA-dependent RNA polymerases play a role in genotype. This has been described for tomato/PSTVd RNA signalling in the cell. Viroid replication can serve (Schnölzer et al., 1985), tobacco/PSTVd (Qi and Ding, as a model for elucidation of the mechanism of RNA- 2003), peach/PLMVd (peach calico) (Malfitano et al., templated RNA transcription. 2003) and other host–viroid combinations. In peach calico, it has been shown that a specific hairpin structure Interaction of viroids with the silencing mechanism of PLMVd is responsible for the symptoms observed (Rodio et al., 2006; 2007). In all these cases, a RISC Many interesting questions concerning the interaction

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology Viroids 9 of viroids with the silencing mechanism remain open: Acknowledgements How (and where) do the nuclear and chloroplastic viroids We thank all our former collaborators who have been working in interfere with the (mostly cytoplasmic) operating RNA- the viroid field: P. Arabatzi, M.A. Denti, K. Karademiris, E. Mani- silencing machinery? Are viroid siRNAs produced in the ataki, E. Marinou, I. Papaefthymiou, A. Prombona, R. Sägesser, cytoplasm in the (short) transit phase of viroids during E. Stylianou and rotation students. We thank S. Tzortzakaki cell-to-cell transport? Or are viroids targeted in phases and M. Providaki for excellent technical assistance. We apologize where nuclei are disrupted (cell division) or chloroplasts to colleagues whose work was not cited due to space limitations. are first formed and increasing in size (during early deve- We are grateful to Dr Kathryn Melzak for editing the English of the manuscript. This review was supported by University of Crete lopment of meristematic and organ precursor tissues)? Grant No. 2078-2192-2161 and IMBB Grant No. 005120 In this context it is interesting to mention that biolistic (FOSRAK). For Martin. infection of cotyledons with PSTVd led to a complete ‘stalling’ of development and severe stunting of N. benthamiana infected with PSTVd (which usually is Note added in proof a symptomless host (Matousek et al., 2007). Which While this article was in proof, the following related articles were enzymes are involved in the production of viroid siRNAs published: and is there a time frame or tissue tropism? Are viroid Abraitiene, A., Zhao, Y., and Hammond, R. (2008) Nuclear siRNAs products of DCL-1 (which is in the nucleus and targeting by fragmentation of the Potato spindle tuber viroid accepts ‘imperfect’ hairpins as substrates) and/or other genome. Biochem Biophys Res Commun 368: 470–475. Di Serio, F., and Flores, R. (2008) Viroids: Molecular imple- DCL proteins (which are generally thought to accept ments for dissecting RNA trafficking in plants. RNA Biol 5: perfect dsRNA as substrate), and how does this correlate in press. http://www.landesbioscience.com/journals/rnabiology/ with their intracellular localization? An interaction between article/6638 the (nuclear) replicating viroids and the silencing machinery can be envisaged also at the level of pericentromeric repeat regulation. Pericentromeric repeats have con- References served elements (i.e. retrotransposons and similar repeat Amari, K., Gómez, G., Myrta, A., Di Terlizzi, B., and Pallás, V. elements, repeats of 5S rDNA transcriptional units), (2001) The molecular characterization of 16 new sequence but also contain spacers which are species and cultivar variants of Hop stunt viroid reveals the existence of invari- specific. Pericentromeric repeats are silenced through the able regions and a conserved hammerhead-like structure action of siRNAs from these sequences. These siRNAs on the viroid molecule. J Gen Virol 82: 953–962. Baulcombe, D.C. (2004) RNA silencing in plants. Nature 431: require the activity of an enzyme, called DNA-dependent 356–363. RNA polymerase IV, which is a fourth DNA-dependent Baulcombe, D.C. (2006) Short silencing RNA: the dark matter RNA polymerase, unique to plants (Baulcombe, 2006). of genetics? Cold Spring Harb Symp Quant Biol 71: 13–20. This protein, together with RNA dependent RNA poly- Branch, A.D., and Robertson, H.D. (1984) A replication cycle merase 2 (RDR2) and Argonaute 4 (AGO 4), two other for viroids and other small infectious RNA’s. Science 223: proteins involved in nuclear RNA silencing, leads to 450–455. endogenous siRNA production, which in turn methy- Brazas, R., and Ganem, D. (1996) A cellular homolog of hepatitis delta antigen: implications for viral replication and lates the DNA and remodels the chromatin for silencing evolution. Science 274: 90–94. (Henderson and Jacobsen, 2007). It is possible that Buchanan, B.B., Gruissem, W., and Jones, R.L. (2000) nuclear viroid replication either interferes with steps of Biochemistry and Molecular Biology of Plants. Rockville, this regulation or else uses any of its factors for own MD: American Society of Plant Physiologists. purposes. Carbonell, A., Martínez de Alba, A.E., Flores, R., and Gago, The two models of interference of viroid RNA with S. (2008) Double-stranded RNA interferes in a sequence- this regulatory and defence machinery of the host are specific manner with the infection of representative members of the two viroid families. Virology 371: 44–53. not mutually exclusive: the general interference of the Côté, F., de la Peña, M., Flores, R., and Perreault, J.P. structured viroid RNA with components of the silencing (2003) Viroids: ribozyme reactions of viroids. In Viroids. and microRNA machinery could coexist with the very Hadidi, A., Flores, R., Randles, J.W., and Semancik, J.S. specific interactions of viroid strain X with host genotype (eds). Collinwood: CSIRO Publishing, pp. 350–356. Y through a RISC loaded with a viroid-specific siRNA. Darós, J.A., and Flores, R. (1995) Identification of a The possibility that viroid RNA uses any of the proteins retroviroid-like element from plants. Proc Natl Acad Sci involved in silencing in order to complete its replication or USA 92: 6856–6860. Darós, J.A., and Flores, R. (2002) A chloroplast protein binds spread (as suggested by (Gas et al., 2007)) is the other a viroid RNA in vivo and facilitates its hammerhead- side of this interesting interaction: the viroid needs com- mediated self-cleavage. EMBO J 21: 749–759. ponents of the silencing machinery, but it is also attacked Delgado, S., Martínez de Alba, A.E., Hernández, C., and by the same machinery. Flores, R. (2005) A short double-stranded RNA motif of

Peach latent mosaic viroid contains the initiation and the Gross, H.J., Domdey, H., Lossow, C., Jank, P., Raba, M., self-cleavage sites of both polarity strands. J Virol 79: Alberty, H., and Sänger, H.L. (1978) Nucleotide sequence 12934–12943. and secondary structure of potato spindle tuber viroid. Denti, M.A., Boutla, A., Tsagris, M., and Tabler, M. (2004) Nature 273: 203–208. Short interfering RNAs specific for potato spindle tuber Hammond, R.W., and Zhao, Y. (2000) Characterization of a viroid are found in the cytoplasm but not in the nucleus. tomato protein kinase gene induced by infection by Potato Plant J 37: 762–769. spindle tuber viroid. Mol Plant Microbe Interact 13: 903– Diener, T.O. (1971) Potato spindle tuber ‘virus’. IV. A replicat- 910. ing, low molecular weight RNA. Virology 45: 411–428. Hegedus, K., Palkovics, L., Toth, E.K., Dallmann, G., and Diener, T.O. (1989) Circular RNAs: relics of precellular Balazs, E. (2001) The DNA form of a retroviroid-like evolution? Proc Natl Acad Sci USA 86: 9370–9374. element characterized in cultivated carnation species. Ding, B., and Itaya, A. (2007) Viroid: a useful model for J Gen Virol 82: 687–691. studying the basic principles of infection and RNA biology. Henderson, I.R., and Jacobsen, S.E. (2007) Epigenetic Mol Plant Microbe Interact 20: 7–20. inheritance in plants. Nature 447: 418–424. Ding, B., Itaya, A., and Zhong, X. (2005) Viroid trafficking: Itaya, A., Folimonov, A., Matsuda, Y., Nelson, R.S., and Ding, a small RNA makes a big move. Curr Opin Plant Biol B. (2001) Potato spindle tuber viroid as inducer of RNA 8: 606–612. silencing in infected tomato. Mol Plant Microbe Interact 14: Fahlgren, N., Howell, M.D., Kasschau, K.D., Chapman, E.J., 1332–1334. Sullivan, C.M., Cumbie, J.S., et al. (2007) High-throughput Itaya, A., Matsuda, Y., Gonzales, R.A., Nelson, R.S., and sequencing of Arabidopsis microRNAs: evidence for Ding, B. (2002) Potato spindle tuber viroid strains of differ- frequent birth and death of MIRNA genes. PLoS ONE 2: ent pathogenicity induces and suppresses expression of e219. common and unique genes in infected tomato. Mol Plant Filipovska, J., and Konarska, M.M. (2000) Specific HDV Microbe Interact 15: 990–999. RNA-templated transcription by pol II in vitro. RNA 6: Itaya, A., Zhong, X., Bundschuh, R., Qi, Y., Wang, Y., 41–54. Takeda, R., et al. (2007) A structured viroid RNA serves as Flores, R., Delgado, S., Gas, M.E., Carbonell, A., Molina, D., a substrate for dicer-like cleavage to produce biologically Gago, S., and De la Peña, M. (2004) Viroids: the minimal active small RNAs but is resistant to RNA-induced silenc- non-coding RNAs with autonomous replication. FEBS Lett ing complex-mediated degradation. J Virol 81: 2980–2994. 567: 42–48. Kalantidis, K., Denti, M.A., Tzortzakaki, S., Marinou, E., Flores, R., Randles, J.W., Owens, R.A., Bar-Joseph, M., Tabler, M., and Tsagris, M. (2007) Virp1 is a host protein and Diener, T. (2005a) Virus Taxonomy: Subviral Agents: with a major role in Potato spindle tuber viroid infection in Viroids. London: Elsevier Academic Press. Nicotiana plants. J Virol 81: 12872–12880. Flores, R., Hernández, C., Martínez de Alba, A.E., Darós, Kalantidis, K., Schumacher, H.T., Alexiadis, T., and Helm, J.A., and Di Serio, F. (2005b) Viroids and viroid–host inter- J.M. (2008) RNA silencing movement in plants. Biol Cell actions. Annu Rev Phytopathol 43: 117–139. 100: 13–26. Forster, A.C., and Symons, R.H. (1987) Self-cleavage of plus Kapranov, P., Willingham, A.T., and Gingeras, T.R. (2007) and minus RNAs of a virusoid and a structural model for Genome-wide transcription and the implications for the active sites. Cell 49: 211–220. genomic organization. Nat Rev Genet 8: 413–423. Forster, A.C., Jeffries, A.C., Sheldon, C.C., and Symons, Kiefer, M.C., Owens, R.A., and Diener, T.O. (1983) Structural R.H. (1987) Structural and ionic requirements for self- similarities between viroids and transposable genetic cleavage of virusoid RNAs and trans self-cleavage of viroid elements. Proc Natl Acad Sci USA 80: 6234–6238. RNA. Cold Spring Harb Symp Quant Biol 52: 249–259. Kolonko, N., Bannach, O., Aschermann, K., Hu, K.H., Moors, Gas, M.E., Hernández, C., Flores, R., and Darós, J.A. (2007) M., Schmitz, M., et al. (2006) Transcription of potato Processing of nuclear viroids in vivo: an interplay between spindle tuber viroid by RNA polymerase II starts in the left RNA conformations. PLoS Pathog 3: e182. terminal loop. Virology 347: 392–404. Gómez, G., and Pallás, V. (2001) Identification of an in vitro Konarska, M.M., and Sharp, P.A. (1989) Replication of ribonucleoprotein complex between a viroid RNA and a RNA by the DNA-dependent RNA polymerase of phage T7. phloem protein from cucumber plants. Mol Plant Microbe Cell 57: 423–431. Interact 14: 910–913. Lai, M. (2005) RNA replication without RNA-dependent RNA Gómez, G., and Pallás, V. (2004) A long-distance translocat- polymerase: surprises from hepatitis delta virus. J Virol 79: able phloem protein from cucumber forms a ribonucleopro- 7951–7958. tein complex in vivo with hop stunt viroid RNA. J Virol 78: Landry, P., and Perreault, J.P. (2005) Identification of a 10104–10110. peach latent mosaic viroid hairpin able to act as a Dicer-like Gómez, G., and Pallás, V. (2007) Mature monomeric forms of substrate. J Virol 79: 6540–6543. Hop stunt viroid resist RNA silencing in transgenic plants. Lindbo, J.A., and Dougherty, W.G. (2005) Plant pathology Plant J 51: 1041–1049. and RNAi: a brief history. Annu Rev Phytopathol 43: 191– Gozmanova, M., Denti, M.A., Minkov, I.N., Tsagris, M., and 204. Tabler, M. (2003) Characterization of the RNA motif Machida, S., Yamahata, N., Watanuki, H., Owens, R.A., and responsible for the specific interaction of potato spindle Sano, T. (2007) Successive accumulation of two size tuber viroid RNA (PSTVd) and the tomato protein Virp1. classes of viroid-specific small RNA in potato spindle tuber Nucleic Acids Res 31: 5534–5543. viroid-infected tomato plants. J Gen Virol 88: 3452–3457.

Malfitano, M., Di Serio, F., Covelli, L., Ragozzino, A., Qi, Y., Pelissier, T., Itaya, A., Hunt, E., Wassenegger, M., and Hernández, C., and Flores, R. (2003) Peach latent mosaic Ding, B. (2004) Direct role of a viroid RNA motif in mediat- viroid variants inducing peach calico (extreme chlorosis) ing directional RNA trafficking across a specific cellular contain a characteristic insertion that is responsible for this boundary. Plant Cell 16: 1741–1752. symptomatology. Virology 313: 492–501. Rackwitz, H.R., Rohde, W., and Sänger, H.L. (1981) DNA- Maniataki, E., Martínez de Alba, A.E., Sägesser, R., Tabler, dependent RNA polymerase II of plant origin transcribes M., and Tsagris, M. (2003) Viroid RNA systemic spread viroid RNA into full-length copies. Nature 291: 297– may depend on the interaction of a 71-nucleotide bulged 301. hairpin with the host protein VirP1. RNA 9: 346–354. Riesner, D. (1991) Viroids: from thermodynamics to cellular Markarian, N., Li, H.W., Ding, S.W., and Semancik, J.S. structure and function. Mol Plant Microbe Interact 4: 122– (2004) RNA silencing as related to viroid induced symptom 131. expression. Arch Virol 149: 397–406. Rodio, M.E., Delgado, S., Flores, R., and Di Serio, F. (2006) Martin, R., Arenas, C., Darós, J.A., Covarrubias, A., Reyes, Variants of Peach latent mosaic viroid inducing peach J.L., and Chua, N.H. (2007) Characterization of small calico: uneven distribution in infected plants and require- RNAs derived from Citrus exocortis viroid (CEVd) in ments of the insertion containing the pathogenicity deter- infected tomato plants. Virology 367: 135–146. minant. J Gen Virol 87: 231–240. Martínez de Alba, A.E., Flores, R., and Hernández, C. (2002) Rodio, M.E., Delgado, S., De Stradis, A., Gómez, M.D., Two chloroplastic viroids induce the accumulation of small Flores, R., and Di Serio, F. (2007) A viroid RNA with a RNAs associated with posttranscriptional gene silencing. specific structural motif inhibits chloroplast development. J Virol 76: 13094–13096. Plant Cell 19: 3610–3626. Martínez de Alba, A.E., Sägesser, R., Tabler, M., and Tsagris, Sägesser, R., Martínez, E., Tsagris, M., and Tabler, M. (1997) M. (2003) A bromodomain-containing protein from tomato Detection and isolation of RNA-binding proteins by RNA- specifically binds potato spindle tuber viroid RNA in vitro ligand screening of a cDNA expression library. Nucleic and in vivo. J Virol 77: 9685–9694. Acids Res 25: 3816–3822. Matousek, J., Kozlova, P., Orctova, L., Schmitz, A., Pesina, K., Sänger, H.L., and Tabler, M. (1987) Viroid Replication. In Bannach, O., et al. (2007) Accumulation of viroid-specific The Viroids. Diener, T.O. (ed.). New York, London: Plenum small RNAs and increase in nucleolytic activities linked to Press, pp. 117–167. viroid-caused pathogenesis. Biol Chem 388: 1–13. Sänger, H.L., Klotz, G., Riesner, D., Gross, H.J., and Motard, J., Bolduc, F., Thompson, D., and Perreault, J.P. Kleinschmidt, A.K. (1976) Viroids are single-stranded (2008) The peach latent mosaic viroid replication initiation covalently closed circular RNA molecules existing as highly site is located at a universal position that appears to be base-paired rod-like structures. Proc Natl Acad Sci USA defined by a conserved sequence. Virology 373: 362– 73: 3852–3856. 375. Schnölzer, M., Haas, B., Raam, K., Hofmann, H., and Mühlbach, H.P., and Sänger, H.L. (1979) Viroid replication is Sänger, H.L. (1985) Correlation between structure and inhibited by alpha-amanitin. Nature 278: 185–188. pathogenicity of potato spindle tuber viroid (PSTV). EMBO Navarro, J.A., and Flores, R. (2000) Characterization of the J 4: 2181–2190. initiation sites of both polarity strands of a viroid RNA Semancik, J.S. (2003) Viroids: pathogenesis. In Viroids. reveals a motif conserved in sequence and structure. Hadidi, A., Flores, R., Randles, J.W., and Semancik, J.S. EMBO J 19: 2662–2670. (eds). Collinwood: CSIRO Publishing, pp. 61–66. Navarro, J.A., Vera, A., and Flores, R. (2000) A chloroplastic Singh, R.P., Ready, K.F.M., and Nie, X. (2003) Viroids: RNA polymerase resistant to tagetitoxin is involved in biology. In Viroids. Hadidi, A., Flores, R., Randles, J.W., replication of avocado sunblotch viroid. Virology 268: 218– and Semancik, J.S. (eds). Collinwood: CSIRO Publishing, 225. pp. 30–48. Owens, R.A., Blackburn, M., and Ding, B. (2001) Possible Steger, G., and Riesner, D. (2003) Viroids: molecular involvement of the phloem lectin in long-distance viroid characteristics. In Viroids. Hadidi, A., Flores, R., Randles, movement. Mol Plant Microbe Interact 14: 905–909. J.W., and Semancik J.S. (eds). Collinwood: CSIRO Papaefthimiou, I., Hamilton, A., Denti, M., Baulcombe, D., Publishing, pp. 49–54. Tsagris, M., and Tabler, M. (2001) Replicating potato Tabler, M., and Tsagris, M. (1990) Viroid replication mecha- spindle tuber viroid RNA is accompanied by short RNA nisms. In NATO ASI Series. Fraser, R.S. (ed.). Berlin: fragments that are characteristic of post-transcriptional Springer, pp. 185–205. gene silencing. Nucleic Acids Res 29: 2395–2400. Tabler, M., and Tsagris, M. (2004) Viroids: petite RNA Pelchat, M., Grenier, C., and Perreault, J.P. (2002) pathogens with distinguished talents. Trends Plant Sci 9: Characterization of a viroid-derived RNA promoter for the 339–348. DNA-dependent RNA polymerase from Escherichia coli. Taylor, J.M. (2006) Hepatitis delta virus. Virology 344: 71– Biochemistry 41: 6561–6571. 76. Prody, G.A., Bakos, J.T., Buzayan, J.M., Schneider, I.R., and Tessitori, M., Maria, G., Capasso, C., Catara, G., Rizza, S., Bruening, G. (1986) Autolytic processing of dimeric plant De Luca, V., et al. (2007) Differential display analysis of virus satellite RNA. Science 231: 1577–1580. gene expression in Etrog citron leaves infected by Citrus Qi, Y., and Ding, B. (2003) Inhibition of cell growth and shoot viroid III. Biochim Biophys Acta 1769: 228–235. development by a specific nucleotide sequence in a non- Wang, M.B., Bian, X.Y., Wu, L.M., Liu, L.X., Smith, N.A., coding viroid RNA. Plant Cell 15: 1360–1374. Isenegger, D., et al. (2004) On the role of RNA silencing in

the pathogenicity and evolution of viroids and viral satel- Isolation of viroid-RNA-binding proteins from an expression lites. Proc Natl Acad Sci USA 101: 3275–3280. library with nonradioactive-labeled RNA probes. Biotech- Warrilow, D., and Symons, R.H. (1999) Citrus exocortis viroid niques 19: 218–222. RNA is associated with the largest subunit of RNA poly- Wolff, P., Gilz, R., Schumacher, J., and Riesner, D. (1985) merase II in tomato in vivo. Arch Virol 144: 2367–2375. Complexes of viroids with histones and other proteins. Wassenegger, M. (2005) The role of the RNAi machinery in Nucleic Acids Res 13: 355–367. heterochromatin formation. Cell 122: 13–16. Yang, X.J. (2004) Lysine acetylation and the bromodomain: Wassenegger, M., Heimes, S., Riedel, L., and Sänger, H.L. a new partnership for signaling. Bioessays 26: 1076– (1994) RNA-directed de novo methylation of genomic 1087. sequences in plants. Cell 76: 567–576. Zhong, X., Tao, X., Stombaugh, J., Leontis, N., and Ding, B. Wassenegger, M., Spieker, R.L., Thalmeir, S., Gast, F.U., (2007) Tertiary structure and function of an RNA motif Riedel, L., and Sänger, H.L. (1996) A single nucleotide required for plant vascular entry to initiate systemic traffick- substitution converts potato spindle tuber viroid (PSTVd) ing. EMBO J 26: 3836–3846. from a noninfectious to an infectious RNA for nicotiana Zhong, X., Archual, A.J., Amin, A.A., and Ding, B. (2008) tabacum. Virology 226: 191–197. A genomic map of viroid RNA motifs critical for replication Werner, R., Mühlbach, H.P., and Guitton, M.C. (1995) and systemic trafficking. Plant Cell 20: 35–47.