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Rna Silencing and Viroids 001_InvitedReview_243 25-06-2009 10:16 Pagina 243 Journal of Plant Pathology (2009), 91 (2), 243-247 Edizioni ETS Pisa, 2009 243 INVITED REVIEW RNA SILENCING AND VIROIDS M. Barba1 and A. Hadidi2 1 CRA - Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy 2 Lead Scientist Emeritus, ARS-United States Department of Agriculture Beltsville, MD 20705, USA SUMMARY intermediates, thereby serving as potent inducers of RNA silencing early in replication and as silent targets Viroids are autonomously replicating, small single- later in infection. Genetic and biochemical experiments stranded circular RNA molecules that do not code for have established a general mechanistic model for a set of proteins and may cause disease in infected, susceptible related pathways in RNA silencing and identified factors plants. Viroids have the ability to induce both RNA-me- that are required for RNA silencing in a variety of organ- diated transcriptional gene silencing (TGS) and post- isms. The process is initially triggered by long dsRNA transcriptional gene silencing (PTGS) in infected plants. precursors arising from mRNAs, endogenous trans- Viroid PTGS has also been demonstrated in a wheat posons, viruses, viral satellites, viroids or from the tran- germ extract system. A possible role of gene silencing in scription of transgenes; dsRNAs can also be introduced viroid pathogenicity and evolution has been proposed. experimentally. The dsRNA trigger is cleaved into 21-24 nucleotide duplexes, designated short-interfering RNAs Key words: Gene silencing, TGS, PTGS, viroid path- (siRNAs), by a ribonuclease III (RNAse III)-like enzyme ogenicity, viroid evolution, Citrus exocortis viroid, Potato termed Dicer in animals and Dicer-like in plants (Bern- spindle tuber viroid, Peach latent mosaic viroid, Chrysan- stein et al., 2001; Hamilton and Baulcombe, 1999; themum chlorotic mottle viroid Zamore et al., 2000). The production of siRNAs by Dicer is an ATP-dependent step (Bernstein et al., 2001; Zamore et al., 2000) and likely involves interactions with INTRODUCTION other proteins, including an Argonaute-like protein, a dsRNA binding protein, and an RNA helicase (Tabara et RNA silencing is an important mechanism of gene al., 2002). At least four Dicer homologues have been regulation in many organisms including plants. The found in both the rice and Arabidopsis genomes process can be divided into RNA-mediated transcrip- (Finnegan et al., 2003). The siRNAs produced from a tional gene silencing and post transcriptional gene si- fully dsRNA substrate by Dicer have distinctive charac- lencing. Viroids, which are the smallest known infec- teristics: they represent both polarities and have two nu- tious, circular, non-coding RNA molecules, have the cleotide 3’ overhangs with 5’ phosphate and 3’ hydroxyl ability to induce both types of RNA silencing in infect- groups (Elbashir et al., 2001a, 2001b). In another ATP- ed plants. Experimental evidence has shown that RNA dependent step (Nykanen et al., 2001), the siRNAs are silencing might mediate some of viroids biological prop- denatured and incorporated into a multi-subunit, en- erties, including pathogenesis. RNA silencing, however, donuclease protein silencing complex, the RNA-induced does not appear to be responsible for the degree of viru- silencing complex (RISC) (Hammond et al., 2000). lence of the viroid isolate or strain. Within the activated RISC, siRNAs act as guides to bring Ten years ago, knowledge of how double-stranded the complex into contact with complementary mRNAs, RNA (dsRNA) blocks gene expression through the tar- resulting in their degradation (Bernstein et al., 2001; El- geted RNA was lacking. We now have a good under- bashir et al., 2001a; Hammond et al., 2000, 2001; standing of this process, known as RNA silencing, which Zamore et al., 2000). Consequently, a plant virus can be takes place naturally in several organisms, including pro- both the inducer and the target of RNA silencing. In tozoa, fungi, plants and animals. Most known plant some organisms, including plants and nematodes, the viruses contain RNA genomes and replicate via dsRNA siRNA can serve as a primer for an RNA-dependent RNA polymerase (RdRp), thereby creating many more siRNAs (Fire et al., 1998; Baulcombe, 2007; Sijen et al., 2007). The action of an RdRp provides an amplification Corresponding author: M. Barba Fax: 39.06.8207.0246 phenomenon in the sense that only a few dsRNA mole- E-mail: marina.barba@entecra.it cules are required to degrade a much larger population 001_InvitedReview_243 25-06-2009 10:16 Pagina 244 244 RNA silencing and viroids Journal of Plant Pathology (2009), 91 (2), 243-247 of RNAs. According to this mechanism, RNA silencing strated that viroids induce PTGS (Itaya et al., 2001, is believed to play a role in the host defense against 2007; Papaefthimiou et al., 2001; Martinez de Alba et pathogen infection, as well as in inactivating the expres- al., 2002; Sano and Matsuura, 2003; Denti et al., 2004; sion of undesired host genes (Baulcombe, 2007). Wang et al., 2004; Landry et al., 2004; Landry and Per- Viroids are the smallest known autonomously repli- reault, 2005; Daros et al., 2006; Martin et al., 2007; cating pathogens that infect higher plants. They are Machida et al., 2007, 2008; Sano, 2008). small (246-401 nt), unencapsidated, circular RNA mole- Itaya et al. (2001) reported the detection of small cules and do not code for any protein, yet are able to in- RNAs of approximately 25 nucleotides with sequence duce a wide range of disease symptoms in susceptible specificity to PSTVd as an indication of the presence of plant hosts (Hadidi et al., 2003). The 29 viroid species RNA silencing. Also, Papaefthimiou et al. (2001) re- described to date can be allocated to two families, name- ported that PSTVd-specific RNAs of 22 and 23 nt of ly the Pospiviroiidae and Avsunviroidae. Members of the both polarities covering different domains of the family Pospiviroidae, whose type species is Potato spindle PSTVd molecule were detected in viroid-infected toma- tuber viroid (PSTVd), have an unbranched, lowest-free to plants, an indication of PTGS. However, they did not energy structure with a central conserved region, do not appear to be responsible for the difference in symptoms exhibit ribozyme activity, and replicate in the nucleus induced by PSTVd isolates which vary in their virulence through an asymmetric rolling-circle mechanism. Mem- (Itaya et al., 2001; Papaefthimiou et al., 2001). Machida bers of the family Avsunviroidae, where the type species et al. (2007) studied the time course of the accumula- is Avocado sunblotch viroid (ASBVd), lack a central con- tion of PSTVd-specific small RNA in infected tomato served region, exhibit ribozyme activity, replicate in the plants, reporting the presence of two size classes of chloroplast through a symmetric rolling-circle mecha- PSTVd-specific small RNAs. Subsequent nucleotide se- nism, and most members have a branched structure. quence analysis showed the presence of a previously un- In plants, RNA silencing can be divided into RNA- described cluster of small RNAs derived primarily from mediated transcriptional gene silencing (TGS) and post the negative strand of PSTVd RNA. Recent studies on transcriptional gene silencing (PTGS). RNA-mediated plants infected with PSTVd (Itaya et al., 2007; Machida TGS occurs when dsRNA with sequence homology to a et al., 2007) or Citrus exocortis viroid (CEVd) (Martin et promoter is produced, leading to de novo DNA methy- al., 2007) revealed that the majority of viroid-specific lation of the promoter region of the structural gene small RNAs are derived from some restricted regions in (Wassenegger et al., 1994), while PTGS reduces the the viroid molecule. It has been also shown that PSTVd steady-state levels of targeted host or viral cytoplasmic small RNAs are biologically active in guiding RNA-in- RNAs (Hannon, 2002). duced silencing complex (RISC)-mediated cleavage (Itaya et al., 2007). PSTVd is known to replicate and ac- cumulate in nuclei; however, PSTVd-specific siRNAs INDUCTION OF TGS IN VIROID-INFECTED PLANTS were detected in the cytoplasm but not the nuclei (Den- ti et al., 2004). The cytoplasmic localization of siRNAs That viroids could exert their pathogenicity through could be the result of either the dsRNAs are exported RNA-directed gene silencing was proposed by Sänger et and then cleaved by Dicer-like enzyme(s) or the Dicer- al. (1996). The experimental trigger of their working hy- like enzyme(s) cleavage occurs in the nucleus and pothesis was the finding that PSTVd-specific cDNA inte- siRNAs are exported, which is similar to the nuclear grated into the genome of Nicotiana tabacum. SRI be- processing of plant miRNAs (Papp et al., 2003). Alter- comes specifically methylated as soon as an autonomous natively, the monomeric circular RNA of viroid is actu- viroid RNA-directed RNA replication has taken place in ally converted in the cytoplasm by RdRp with the aid of these plants (Wassenegger et al., 1994). From these obser- siRNAs into cytoplasmic dsRNA, which is then cleaved vations it was inferred that RNA, in general, is capable of by Dicer to secondary siRNAs. inducing and directing sequence-specific de novo methy- Viroid-specific siRNAs of approximately 21 to 23 nt lation of genomic DNA. Since DNA methylation has were reported to be detected from Peach latent mosaic been associated with gene silencing, it was conceivable viroid (PLMVd)-infected and Chrysanthemum chlorotic that a viroid-induced RNA-directed DNA methylation re- mottle viroid (CChMVd)-infected plants but not from sulted in a subsequent plant gene silencing. In this case, ASBVd-infected plants (Martinez de Alba et al., 2002). PSTVd induces RNA-mediated gene silencing (TGS). Also, no significant difference in the PTGS response to strains of CChMVd of different virulence was observed as the accumulation levels of the siRNAs were essential- INDUCTION OF PTGS IN VIROID-INFECTED ly the same.
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