A new RNA virus has been identified from a sweet orange tree in southern Italy. This virus, tentatively named citrus virus A (CiVA), has a bipartite genome composed of (i) a negative-stranded (ns) RNA1, encoding the viral RNA-dependent RNA polymerase (RdRp), and (ii) an ambisense RNA2, coding for the putative movement protein (MP) and nucleocapsid protein (NP), with the two open reading frames separated by a long AU-rich intergenic region (IR) adopting a hairpin conformation. CiVA genomic RNAs and the encoded proteins resemble those of the recently discovered citrus concave gum-associated virus (CCGaV). This CCGaV, a nsRNA virus associated with the ancient citrus concave gum disease, has been proposed as the representative member of a new genus tentatively named Coguvirus. Molecular and phylogenetic analyses presented here support the classification of CiVA, and likely of other two recently described nsRNA viruses infecting plants, in this new genus. By showing that the evolutionary origin of the MP of all the putative coguviruses likely differs from that of their respective RdRp and NP, this study also provides evidence of a likely modular genome evolution for these viruses. Moreover, phylogenetic data support the proposal that, during the evolutionary history of nsRNA viruses, the plant-infecting viruses most likely emerged from an invertebrate-infecting ancestor several times as independent events. CiVA was identified in a field sweet orange tree not showing any obvious symptom and was graft-transmitted to sweet orange, grapefruit, rough lemon and Dweet tangor indicator plants that did not developed symptoms. The capacity of infecting citrus hosts of several species was also confirmed by a preliminary survey that identified orange, Mandarin, clementine and lemon trees as natural hosts of CiVA in several fields of southern Italy, again without any obvious association with specific symptoms.

DNA methylation and post-transcriptional gene silencing play critical roles in controlling infection of single-stranded (ss) DNA geminiviruses and ssRNA viroids, respectively, but both pathogens can counteract these host defense mechanisms and promote their infectivity. Moreover, a specific role of DNA methylation in viroid-host interactions is not yet confirmed. Here, using an experimental system where two nuclear-replicating agents, the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSV) and potato spindle tuber viroid (PSTVd), co-infect their common host tomato, we observed that PSTVd severely interferes with TYLCSV infectivity and accumulation, most likely as a consequence of strong activation of host DNA methylation pathways. In fact, PSTVd alone or in co-infection withTYLCSV significantly upregulates the expression of key genes governing DNA methylation in plants. Using methylation-sensitive restriction and bisulfite conversion assays, we further showed that PSTVd infection promotes a strong hypermethylation of TYLCSV DNA, thus supporting a mechanistic link with the antagonism of the viroid on the virus in co-infected tomato plants. These results describe the interaction between two nuclear-replicating pathogens and show that they differentially interfere with DNA methylation pathways.

The spread of viroids belonging to the genus Pospiviroid (family Pospiviroidae), recorded recently in ornamentals and vegetables in several European countries, calls for fast, efficient and sensitive detection methods. Based on bioinformatics analyses of sequence identity among all pospiviroids, a digoxigenin-labeled polyprobe (POSPIprobe) was developed that, when tested by dot-blot and Northern-blot hybridization, detected Potato spindle tuber viroid, Citrus exocortis viroid, Columnea latent viroid, Mexican papita viroid, Tomato planta macho viroid, Tomato apical stunt viroid, Pepper chat fruit viroid and Chrysanthemum stunt viroid. The end-point detection limits of the POSPIprobe ranged from 5(-2) to 5(-4), and from 5(-1) to 5(-3) for nucleic acid preparations obtained by phenol extraction and silica-capture, respectively, similar to those of single probes. Based on sequence identity, the POSPIprobe is expected to detect also the two pospiviroid species not tested in this study (Tomato chlorotic dwarf viroid and Iresine viroid-1). Dot-blot assays with the POSPIprobe were validated by testing 68 samples from tomato, chrysanthemum and argyranthemum infected by different pospiviroids as revealed by RT-PCR, thus confirming the potential of this polyprobe for quarantine, certification and survey programs.

Peach latent mosaic viroid (PLMVd) (Herna?ndez and Flores, PNAS USA 1992) induces distinct phenotypes in its natural host. While most isolates are symptomless in leaves, hence the term latent in the name, some incite peach mosaics (PM) of different severity and a few an extreme albinism (peach calico, PC) (Flores et al., Mol. Plant Pathol. 2006). PC- inducing variants have a characteristic insertion of 12-13 nt in one apical loop and accumulate predominantly in symptomatic but not in adjacent green leaf sectors. We have documented that two 21-nt small RNAs (PLMVd-sRNAs) -containing the insertion associated with PC- guide cleavage of the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90) as predicted by RNA silencing mediated by Argonaute 1 (AGO1), which binds specifically 21-nt sRNAs with a 5'-terminal U, like the two PLMVd-sRNAs holding the PC-associated insertion (Navarro et al., Plant J. 2012). Cleavage of the mRNA coding for cHSP90 most likely is the primary molecular lesion causing eventually PC, since this protein mediates biogenesis of chloroplasts from proplastids. To understand how general such mechanism is, we have extended these studies to PM, a question far more difficult because it implies identification of the molecular determinant, which is not associated with any specific insertion, and because in contrast with the well-defined PC phenotype, PM differs in type and severity of symptoms.Results. After some preliminary studies, we focused on a severe peach yellow mosaic (PYM) expressed in leaf sectors flanked by others green. Full-length PLMVd-cDNAs from both sectors were cloned and sequenced. From two variants, differing in one nucleotide change, we generated dimeric head-to-tail inserts in plasmids, with the corresponding in vitro transcripts being then bioassayed in GF305 peach seedlings. While one of the transcripts incited an intense PYM resembling the original field symptom, the other caused only mild alterations, thus supporting the involvement of one specific PLMVd nucleotide in this syndrome. In addition, the differential nucleotide change: i) was preserved in the progeny of the symptomatic but not in that of adjacent green sectors, ii) is absent in a set of PLMVd natural variants that replicate latently, and iii) turned a severe variant into latent when removed, and a latent variant into severe when inserted by site-directed mutagenesis. Analysis of the PLMVd-sRNAs and their potential mRNAs targets in the known peach genome, combined with RNA ligase mediated-rapid amplification of cDNA ends, have revealed that one 21-nt PLMVd-sRNA, with the PYM-associated change and two 5'-terminal Us, guide cleavage of the mRNA coding for a thylakoid protein as predicted by RNA silencing mediated by AGO1. Accumulation of this mRNA is lower in symptomatic than in asymptomatic sectors as shown by RT-qPCR, and some mutants in the gene coding for this protein, involved in photosynthesis, show yellow phenotypes akin to PYM.

By integrating next generation sequencing (NGS), bioinformatics, electron microscopy and conventional molecular biology tools, a new virus infecting kiwifruit vines has been identified and characterized. Being associated to double membrane-bound bodies in the infected tissues and having a genome composed of RNA segments, each one containing a single open reading frame in the negative polarity, this virus shows the typical features of members of the genus Emaravirus. Five genomic RNA segments were identified. Additional molecular signatures in the viral RNAs and in the proteins they encode, together with data from phylogenetic analyses, support the proposal of creating a new species in the genus Emaravirus for classifying the novel virus, which is tentatively named Actinidia chlorotic ringspot-associated virus (AcCRaV). Bioassays showed that AcCRaV is mechanically transmissible to Nicotiana benthamiana plants that, in turn, may develop chlorotic spots and ringspots. Field survey disclosed the presence of AcCRaV in four different species of kiwifruit vines in five different provinces of central and western China, and supported the association of the novel virus with symptoms of leaf chlorotic ringspots in actinidia. Data on the molecular features of small RNAs of 21-24 nt, derived from AcCRaV RNAs targeted by host RNA silencing mechanisms, are also reported and possible molecular pathways involved in their biogenesis are discussed.

Viroid RNAs are targeted by dicer-like (DCLs) enzymes, the activity of which generates viroid-derived small RNAs (vd-sRNAs) of 21-24 nucleotides (nt) in the infected tissues, thus supplying a convincing evidence of a first host anti-viroid barrier based on RNA silencing. Whether a second RNA silencing barrier, based on the RNA-induced silencing complex (RISC), may also target for degradation viroid RNAs remains controversial. The RISC core is always formed by an Argonaute (AGO) protein, which after loading endogenous or viral sRNAs, guides the complex to target for inactivation complementary RNAs (or DNAs). Using RNA immunoprecipitation with a specific antibody against the endogenous AGO1 of Nicotiana benthamiana we showed that, when infected by potato spindle tuber viroid (PSTVd), this protein is also loaded with vd-sRNAs. Moreover, agroexpression in N. benthamiana infected by PSTVd of epitope-tagged versions of nine of the ten AGOs from Arabidopsis thaliana, followed by immunoprecipitation analyses of the agroinfiltrated halos, revealed that AGO1, AGO2 and AGO3 preferentially associated with vd-sRNAs of 21 and 22 nt, while AGO4, AGO5 and AGO9 additionally bound those of 24 nt. In contrast, AGO6, AGO7 and AGO10 did not load vd-sRNAs. Deep-sequencing analysis revealed that the endogenous AGO1 of N. benthamiana and the agroexpressed AGO1, AGO2, AGO4 and AGO5 bound vd-sRNAs according to their size and 5'-terminal nucleotide, with AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Interestingly, agroexpression of AGO1, AGO2, AGO4 and AGO5 in PSTVd-infected tissue attenuated the PSTVd level, suggesting RISC-targeting of the viroid genomic RNAs or their precursors and thus supporting a role of RISC in anti-viroid defense. Moreover, PSTVd RNAs with 5'-termini compatible with degradation mediated by AGO-loaded vd-sRNA have been identified and characterized by 5'-RML-RACE in infected N. benthamiana. Further degradome analysis should provide a description of how the viroid genomic and host RNAs are targeted by AGO-loaded vd-sRNAs, thus supplying a broader view of the biological functions of vd-sRNAs.

To get an insight into the host RNA silencing defense induced by Citrus tristeza virus (CTV) and into the counter defensive reaction mediated by its three silencing suppressors (p25, p20 and p23), we have examined by deep sequencing (Solexa-Illumina) the small RNAs (sRNAs) in three virus-host combinations. Our data show that CTV sRNAs: (i) represent more than 50% of the total sRNAs in Mexican lime and sweet orange (where CTV reaches relatively high titers), but only 3.5% in sour orange (where the CTV titer is significantly lower), (ii) are predominantly of 21–22-nt, with a biased distribution of their 50 nucleotide and with those of (?) polarity accumulating in a moderate excess, and (iii) derive from essentially all the CTV genome (ca. 20 kb), as revealed by its complete reconstruction from viral sRNA contigs, but adopt an asymmetric distribution with a prominent hotspot covering approximately the 30-terminal 2,500 nt. These results suggest that the citrus homologues of Dicer-like (DCL) 4 and 2 most likely mediate the genesis of the 21 and 22 nt CTV sRNAs, respectively, and show that both ribonucleases act not only on the genomic RNA but also on the 30 co-terminal subgenomic RNAs and, particularly, on their double-stranded forms. The plant sRNA profile, very similar and dominated by the 24-nt sRNAs in the three mock-inoculated controls, was minimally affected by CTV infection in sour orange, but exhibited a significant reduction of the 24-nt sRNAs in Mexican lime and sweet orange. We have also identified novel citrus miRNAs and determined how CTV influences their accumulation

Cellular Dicer-like RNases target viroids or their double-stranded derivatives for degradation, generating 21-24 nt viroid-derived small RNAs (vd-sRNAs), which are incorporated into Argonaute proteins and guide them for sequence-specific cleavage of viroid and host mRNAs. Here, the involvement of these molecular pathways in pathogenesis has been investigated in tomato and N. benthamiana plants infected by potato spindle tuber viroid (PSTVd), a nuclear-replicating viroid causing similar symptoms (leaf curling and stunting) in both hosts.Materials and methods: Both vd-sRNAs and host microRNAs (miRNAs) were identified and quantified by high-throughput sequencing, while their potential mRNA targets were uncovered by degradome analyses. The results were validated by 5'-RML-RACE and RT-qPCR.Results: About one hundred tomato mRNAs potentially targeted for degradation by vd-sRNA were identified in the degradome. However, comparisons of data from biological replicates of healthy and infected samples reduced the potential mRNAs targets to only eight, mostly coding for proteins involved in development. RACE experiments validated these results, but RT-qPCR showed that only four of the eight mRNAs actually accumulated at lower levels in the PSTVd-infected tomato. Interestingly, comparative degradome analyses in PSTVd-infected N. benthamiana failed to uncover homologous gene (s) potentially mediating the common symptoms induced by PSTVd in this host and in tomato. Moreover, no significant modifications in the accumulation of miRNAs and their cleaved targeted mRNAs were observed in the PSTVd-infected tomato and N. benthamiana plants with respect to their healthy controls. Conclusions: This study showed that: i) cleavage of the mRNAs targeted by vd-sRNAs is not necessarily associated with the down-regulation of the corresponding gene; ii) the common symptoms induced by PSTVd in tomato and N. benthamiana do not seem to be directly linked to the effect of vd-sRNAs and/or miRNAs on host gene regulation. The implications of these findings on the mechanism of PSTVd pathogenesis will be discussed.

Viroids are infectious agents identified only in plants so far. In contrast to viruses, the genome of viroids is composed of a tiny circular RNA (250-400 nt) not coding for proteins, but containing in its compact structure all the information needed for parasitizing the transcriptional and RNA trafficking machineries of their hosts. Viroid infections are frequently accompanied by cellular and developmental disorders that ultimately result in macroscopic symptoms. The molecular events linking the structural domains of viroid RNAs with cellular and macroscopic alterations remain largely unexplored, although significant progress has been lately achieved in one specific viroid-host combination, highlighting the ability of viroids to strongly interfere with their host RNA regulatory networks. Cytopathic effects induced by nuclear-replicating viroids, which were investigated since early studies on viroids, consist in irregular proliferations of cell membranes (paramural bodies or plasmalemmasomes), cell wall distortions, and chloroplast malformations. Different alternatives have been proposed regarding how these cytological alterations may influence the onset of macroscopic symptoms. Recently, the cytopathology and histopathology incited by a chloroplast-replicating viroid have been investigated in depth, with defects in chloroplast development having been related to specific molecular events that involve RNA silencing and impairment of chloroplast ribosomal RNA maturation. On this basis, a tentative model connecting specific cytopathologic alterations with symptoms has been put forward. Here, early and more recent studies addressing this issue will be reviewed and reassessed in the light of recent advances in the regulatory roles of small RNAs.

RNA silencing is an RNA-based network regulating gene expression and defense against invasive nucleic acids in most eukaryotes, including plants. Involvement of RNA silencing in viroid-host interaction became evident when viroid derived small RNAs (vd-sRNAs) of 21-24 nt, structurally similar to host microRNAs (miRNAs) and small-interfering RNAs (siRNAs), were detected in tissues infected by nuclear and chloroplast-replicating viroids. Based on these findings, it was proposed that vdsRNAs, similarly to miRNAs, might target host mRNAs for degradation (or translation inhibition), hus leading to symptom expression in the infected plants. In the last few years, the availability of high-throughput sequencing technologies is allowing in-depth characterization of vd-sRNAs accumulating in host tissues during infection, hence contributing significantly to further dissect possible functional rolesof RNA silencing in the plant-viroid interplay. Based on these technologies, we recently characterized vd-sRNAs derived from a chloroplast replicating viroid (Peach latent mosaic viroid, PLMVd). Moreover, by semi-quantitative RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends, we have shown that two vd-sRNAs (containing the pathogenicity determinant strictly associated with an albinism) target for degradation a host mRNA, thus providing the first experimental evidence that vd-sRNAs indeed function like miRNAs. Interestingly, the targeted mRNA codes for a protein (cHSP90) involved in chloroplast biogenesis, which is the developmental pathway specifically compromised in the albino tissues infected by PLMVd variants generating the two vd-sRNAs (Navarro et al., Plant J. 2012). Altogether these data support involvement of RNA silencing in PLMVd pathogenesis and a possible more general role of vd-sRNAs in modulating host gene expression during viroid infection. To get a deeper insight into this question, we have integrated data from high-throughput sequencing of vd-sRNAs accumulating in tissues infected by chloroplast- and nuclear-replicating viroids with the respective degradome analyses. We will present data and discuss implications of the RNA degradation patterns potentially elicited by vd-sRNAs during viroid infection.

Available data on the incidence and biodiversity of pome fruit viroids in the Mediterranean basin are limited. Beforestarting a research survey to fill this gap, a tissue-printing hydridization (TPH) method to detect Apple scar skin viroid(ASSVd), Pear blister canker viroid (PBCVd) and Apple dimple fruit viroid (ADFVd) has been developed andvalidated. Afterward, TPH was used in large-scale indexing of pome fruit viroids in Bosnia and Herzegovina, Malta,Lebanon and Turkey. A total of about 1,000 trees was randomly collected and tested. Positive results obtained by TPHwere confirmed by at least one additional detection method (RT-PCR and/or Northern-blot hybridization) and viroidswere finally identified by sequencing full-length cDNA clones. PBCVd was detected in 13%, 12.4% and 5.4% of thetested pear trees in Bosnia and Herzegovina, Malta and Turkey, respectively, showing a wider diffusion of this viroidthan expected. In contrast, ASSVd was never detected and ADFVd was only found in symptomatic trees (cv. StarkingDelicious) in Lebanon, confirming a restricted presence of these viroids in the Mediterranean basin. Altogether, thesedata support the use of TPH as an easy and valuable tool for exploring pome fruit viroid spread.

Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are the five viroids known to infect naturally grapevines. We developed a multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of these five viroids and the amplification of the cDNA fragment of a host-derived mRNA (actin mRNA) as an internal positive control. Specific primers for each viroid targeted were designed by taking into account the sequence variability within and between the viroid species and tested in silico. The method was validated by testing 57 grapevine samples from Iran and showed reliability and high sensitivity. The RT-PCR-negative samples were further assayed by Northern-blot hybridization. For this, a method was developed for the simultaneous detection of three different grapevine viroids on a single hybridization membrane. In this survey, HSVd, AGVd, GYSVd-1, and GYSVd-2 were detected in 100, 95, 93, and 65% of the samples tested, respectively, confirming the wide distribution of these viroids in Iran. CEVd was not detected in any of the samples collected. Based on these results, HSVd is proposed as a positive internal control for mRT-PCR in the areas where this viroid is widespread, so as to reduce the time and costs of DNase treatment, which is required when a host-derived internal control is used. The mRT-PCR method has the potential to be used routinely for large-scale surveys and certification programs.

Spread of viroids belonging to the genus Pospiviroid (family Pospiviroidae) has been recently recorded in ornamentals and vegetables in several countries of the European Union (EU). Major concerns derive from the identification of Potato spindle tuber viroid (PSTVd) and Chrysanthemum stunt viroid (CSVd), which are quarantine pests in EU. Effectiveness of control measures against viroid spread, which are based mainly on the use of viroid-free propagation material and on the interception of infected plants, requires fast, reliable, sensitive and economic detection methods. Methods allowing simultaneous detection of several viroid species largely contribute to contain costs. In this context, a single molecular probe (polyprobe), composed of assembled sequence fragments from most viroid species belonging to Pospiviroids genus (family Pospiviroidae) appears very appropriate for detecting them by molecular hybridization because only one single transcription reaction is needed. Based on bioinformatics analyses of pospiviroid genomic RNAs, we have developed a polyprobe (POSPIprobe) that detects at least eight different pospiviroid species, including PSTVd and CSVd. Partial sequences from four different pospiviroids were selected and cloned directionally to generate a single vector that was used for synthesizing the POSPIprobe by in vitro-transcription. POSPIprobe specificity for eight pospiviroids was confirmed by Northern-blot, while its sensitivity was tested by dot-blot assays, providing similar results to that obtained using single probes separately. Dot-blot analyses with the POSPIprobe was validated by simultaneously testing 68 samples from tomato, chrysanthemum, Argyranthemum frutescens and pepper infected by different pospiviroids, confirming the high potential of this probe in quarantine, certification and survey programs. These data have been recently published in Journal of Virological Methods 2012.

La enfermedad denominada calico del melocotonero (peach calico, PC), que se manifiesta como una clorosis extrema (albinismo) en hojas, tallos y frutos, es inducida por variantes de secuencia atípicas del viroide del mosaico latente del melocotonero (Peach latent mosaic viroid, PLMVd, de la familia Avsunviroidae con replicación cloroplástica), que difieren de las variantes latentes y de aquéllas que inducen mosaico por tener una inserción característica de 12-13 nt en una posición definida del RNA genómico. El determinante patogénico asociado al PC se ha cartografiado en esta inserción específica, que adopta una conformación en horquilla. En estudios previos hemos demostrado que un tetrabucle apical UUUU es estrictamente necesario para preservar las propiedades patogénicas asociadas a este determinante molecular. En el trabajo presente hemos diseccionado las propiedades funcionales de este elemento estructural mediante mutagénesis dirigida, inoculación y observación de los síntomas expresados, y caracterización de las progenies que se acumulan en tejidos verdes y albinos de melocotoneros de semilla GF-305 inoculados con las variantes mutadas. Nuestros datos muestran que no sólo el tetrabucle apical UUUU de la inserción en horquilla, sino también su tallo y particularmente su composición nucleotídica, desempeñan un papel clave en la inducción del PC, indicando así que los requisitos de la inserción en horquilla asociada con este síndrome son más complejos de lo que incialmente se pensaba. Teniendo en cuenta que la secuenciación masiva de genotecas de pequeños RNAs de hojas sanas y afectadas por PC ha desvelado diferencias en la acumulación de algunos micro RNAs y pequeños RNAs interferentes del huésped (efectores característicos del silenciamiento mediado por RNA), la implicación de este mecanismo en la patogenicidad del PLMVd aparece como una alternativa factible.

The involvement in viroid-host interactions of RNA silencing, an RNA-based network regulating gene expression and defense against invasive nucleic acids in most eukaryotes, is supported by the accumulation in tissues infected by nucleus- and chloroplastreplicating viroids of viroid-derived small RNAs (vd-sRNAs) of 21-24 nt, structurally similar to host microRNAs (miRNAs) and small-interfering RNAs (siRNAs). Based on these findings it was proposed that vd-sRNAs, similarly to miRNAs, might target host mRNAs for degradation (or translation inhibition), thus leading to symptom expression in the infected plants. In the last few years, using high-throughput technologies, we have characterized the vdsRNAs derived from a chloroplast-replicating viroid [Peach latent mosaic viroid (PLMVd)]. Moreover, by semi-quantitative RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends, we have shown that two vd-sRNAs (containing the patogenicity determinant strictly associated with an albino phenotype) target for degradation a host mRNA, thus providing the first experimental evidence that, indeed, vd-sRNAs function like miRNAs. Interestingly, the targeted mRNA codes for a protein (cHSP90) involved in chloroplast biogenesis, which is the developmental pathway specifically compromised in the albino tissues infected by PLMVd variants generating the two vd-sRNAs (Navarro et al., 2012. The Plant Journal 70: 991-1003). Altogether these data strongly support the involvement of RNA silencing in PLMVd pathogenesis and, possibly, a more general role of vd-sRNAs in modulating host gene expression during viroid infection. For a deeper insight into this question, we have integrated data from high-throughput sequencing of vd-sRNAs accumulating in tissues infected by chloroplast- and nucleus-replicating viroids with the respective degradome analyses. Based on experimental data, the implications will be discussed of the RNA degradation patterns potentially elicited by vd-sRNAs during viroid infection.

Viroid-derived small RNAs of 21-24 nt (vd-sRNAs) accumulate in viroid-infected tissues as a consequence of the activity of Dicer-like (DCL) RNases targeting these infectious RNAs or their double-stranded derivatives. vd-sRNAs are incorporated into Argonaute (AGO) proteins, forming the core of the RNA silencing complex, and guide it to cleave viroid and host mRNAs. To further dissect these molecular pathways and to investigate their involvement in viroid pathogenesis, we applied genome-wide approaches based on high-throughput sequencing of sRNAs and degradome analyses. Two different hosts, tomato and N. benthamiana --which develop similar symptoms of leaf curling/wrinkling and stunting when infected by potato spindle tuber viroid (PSTVd)-- were examined, thus allowing comparative analyses of: i) the mRNAs targeted for cleavage by vd-sRNAs and ii) the effect of PSTVd on the accumulation of miRNAs and their cleaved targets. Results. Although degradome analyses identified about one hundred tomato mRNAs potentially targeted for degradation by vd-sRNA, data from biological replicates and comparisons of healthy and infected samples reduced the number of true targeted mRNAs --i.e. those identified with high confidence in two infected biological replicates and not in healthy controls-- to only eight mostly coding for proteins involved in development. These results were validated by RACE experiments in the three mRNAs examined. Interestingly, RT-qPCR showed that only four of the eight mRNAs actually accumulated at lower levels in the PSTVd-infected tomato, thus showing that cleavage of the mRNAs targeted by the vd-sRNAs is not necessarily associated with the down-regulation of gene expression. When the same degradome analyses were performed with healthy and PSTVd-infected N. benthamiana plants, all the identified mRNAs targeted by vd-sRNAs were different from those observed previously in tomato and, besides, they also differed in the molecular pathways they are involved. Therefore, this comparative analysis failed to pinpoint homologous gene(s), targeted by vd-sRNAs, potentially eliciting the common symptoms induced by PSTVd in tomato and N. benthamiana. Moreover, no significant modifications in the accumulation of miRNAs and their cleaved targeted mRNAs were detected in the PSTVd-infected tomato and N. benthamiana plants with respect to their healthy controls. The implications of these findings on the mechanism of PSTVd pathogenesis will be discussed.

Viroids, the smallest infectious agents endowed with autonomous replication, are tiny single-stranded circular RNAs (~250 to 400 nt) without protein-coding ability that, despite their simplicity, infect and often cause disease in herbaceous and woody plants of economic relevance. To mitigate the resulting losses, several strategies have been developed, the most effective of which include: firstly, search for naturally resistant cultivars and breeding for resistance, secondly, induced resistance by pre-infection with mild strains, thirdly, ribonucleases targeting double-stranded RNAs and catalytic antibodies endowed with intrinsic ribonuclease activity, fourthly, antisense, and sense, RNAs, fifthly, catalytic antisense RNAs derived from hammerhead ribozymes, and sixthly, hairpin RNAs and artificial small RNAs for RNA interference. The mechanisms underpinning these strategies, most of which have been implemented via genetic transformation, together with their present results and future potential, are the subject of this review.

Nucleotide modifications in RNAs were initially thought to be mainly restricted to the stable and highly abundant transfer and ribosomal RNAs (tRNAs and rRNAs). In these RNAs, modifications have been mapped by technically demanding studies that support their possible structural and/or functional roles. More recently, the improvement of RNA bisulfite sequencing protocols, originally developed for identifying C5-methylcytosine (m5C) in DNA, unveiled the widespread occurrence of m5C in many cellular coding and non-coding RNAs, thus highlighting that the previous reports on the limited presence of this modified nucleotide in cellular RNAs other than rRNAs and tRNAs were mainly due to technical limitation. Information on whether viroid RNAs contain m5C is poor and dates back to early RNase fingerprints analyses of bisulfite-treated viroid RNA preparations (Domdey et al., 1978, Nucleic Acids Res. 5, 1221-1236). These studies were performed mainly to determine the secondary structure of potato spindle tuber viroid (PSTVd) genomic RNA. Therefore, they did neither provide information at single-nucleotide resolution nor on the presence of m5C in viroid replication intermediates, particularly viroid RNAs of the complementary (-) polarity. To conclusively answer the question regarding the presence of m5C in either polarity strand, a protocol based on RNA bisulfite sequencing was developed to test at single-nucleotide resolution the viroid highly-structured RNAs, whose base-paired cytidines are particularly resistant to bisulfite conversion. This protocol was then applied to explore the presence of m5C in the (+) and (-) strands of PSTVd and avocado sunblotch viroid (ASBVd), which are members of the type-species of the families Pospiviroidae and Avsunviroidae, respectively. Results of these analyses support the absence of m5C in both strands of PSTVd and ASBVd.

An increasing number of cDNA libraries of RNAs sequenced by high-throughput technologies from several plant species is available in public databases, thus providing useful sources to screen for novel viruses through in silico tools specifically developed to this aim. In the present study, the identification of a putative novel marafivirus infecting peach, first identified by screening libraries available in public databases, is reported. Materials and methodsSmall RNA libraries from peach were retrieved from Gene Expression Omnibus (GEO) repositories; reads were assembled in de novo contigs by Velvet program (K-mer 15-17); viral sequences sharing significant identity with contigs were searched in NCBI databases by BLASTN and BLASTX; specific primers were designed in selected contigs for detecting the novel virus in peach trees and/or sequencing its genome.Results and DiscussionSeveral (44) contigs, ranging in size from 56 to 360 nt, with sequences sharing significant identity (55-95 %) with viral sequences previously reported, including grapevine red globe virus, citrus sudden death-associated virus marafivirus, oat blue dwarf virus, peach virus D, nectarine virus M, were identified in a GEO cDNA library of small RNAs (21-24 nt) generated from a non-symptomatic peach of the cv. Lovell, thus suggesting that the source plant could be infected by a novel virus, likely another marafivirus. Similar sequences were detected in symptomless peach trees of several cultivars grown in Italy, suggesting that such a virus could be quite widespread and likely not associated with a disease in peach. The molecular and structural features of the virus genome will be presented and compared with those of two recently reported marafivirus from peach (1, 2).References1. Igori et al., 2017. Archives of Virology: doi:10.1007/s00705-017-3255-y.2. Villamor et al.., 2016. Phytopathology: 106: 519-527.

Disease Note, no abstact available

Chrysanthemum stunt viroid (CSVd) is a quarantine pathogen for chrysanthemum (Dendranthema spp.) in the European countries (Plant Health Directive 2000/29/EC), because this host is severely affected, thus comes down with a disease characterized by stunting, leaf chlorosis and floral disorders. CSVd spread in Europe has efficiently been restrained so far, although several outbreaks were recorded in the past. Here we report the first occurrence of CSVd in Italy, as detected in several symptomless cultivars of Argyranthemum frutescens (marguerite daisy) by RT-PCR with specific primers and by Northern blot hybridization with a specific digoxigenin-labeled riboprobe. Viroid identity was ultimately ascertained by cloning and sequencing cDNA amplicons. Molecular characterization of CSVd isolates from six different A. frutescens cultivars disclosed viroid RNA populations with a prevalent size of 354 nt and sequences 98-100% identical to those of CSVd variants reported previously from D. grandiflora and A. frutescens. These results call for a prompt extension of surveys for assessing the presence of CSVd in symptomless A. frutescens and other ornamentals, which could constitute hidden reservoirs of this pathogen. In view of this, a tissue-printing hybridization method for detecting CSVd in A. frutescens was tested and validated.

Grapevine latent viroid (GLVd) is a new viroid recently discovered in grapevines (Vitis vinifera L.) of the variety 'Thompson Seedless', located in Xinjiang, China (Zhang et al., 2014), proposed as a new species in the genus Apscaviroid. It contains the typical structural elements of the other apscaviroids, i.e. circular genomic RNA able to assume a rod-like conformation, central and terminal conserved regions, poly-purine stretch in the pathogenicity associated P domain. Autonomous replication of GLVd in grapevine and absence of associated symptoms were confirmed by bioassays with infectious in vitro transcripts (Zhang et al. 2014). Up to now, the presence of GLVd has been reported only in grapevine of the variety 'Thompson Seedless' grown in China (Zhang et al., 2014) and in Vitis sp. collected in South Korea (Genbank accession LC163596.1, unpublished). In July 2015, a survey based on next generation sequencing (NGS) of small RNAs of the grapevine collection Grinzane-Cavour, located in Piedmont, Italy, was carried out in order to investigate the grapevine virome. Bioinformatic analyses highlighted the presence of GLVd in a pool of 10 plants collected during the survey. A unique contig of 271 bp, a genome coverage of 82.6% and an identity of 98.16% with the GLVd reference genome (KR605505.1) was obtained. This contig represented 822 (0.01%) of total reads, similar to the read percentages observed for other viroids detected by NGS (Candresse et al., 2017). The presence of GLVd as well as its circularity was confirmed by RT-PCR, using the pair of adjacent primers of opposite polarity GLVd-252F (5'-GCTCTCCAACGCCCTAA-3') and GLVd-251R (5'-ACCATTAGTCCGCACGA-3'), mapping to positions 252-268 and 235-251 of the GLVd reference (KR605505.1), respectively (Zhang et al., 2014), which amplify the full-length monomeric cDNA of the viroid. GLVd was detected in four out of the 10 plants belonging to the sequenced pool: 3 plants from 3 Vitis vinifera L. cultivars (Adissi, Rkatsiteli, and Katta Kourgan), originally from Armenia, Georgia and Uzbekistan, respectively, and 1 plant from Vitis riparia (cv. Gloire de Montpellier), originally from North America. A 330 bp genome sequence was obtained from the cultivar Vitis vinifera L. Katta Kourgan by RT-PCR amplification, cloning in the pCR(TM)-Blunt II-TOPO plasmid by Zero Blunt® TOPO® PCR Cloning Kit (Life Technologies, Carlsbad, CA) and Sanger sequencing. The sequence (Genbank accession MG770884) showed 99% identity and a single base insertion (+C226) when compared with the sequence assembled from the Illumina data, possibly reflecting sequence heterogeneity in the GLVd population. The new GLVd genomic sequence showed 97% identity with both the type sequences KR605505.1 (Chinese isolate) and LC163596.1 (Korean isolate), analogous to the sequence identity (97%) between the Chinese and Korean isolates. To the best of our knowledge, this is the first report of GLVd in Europe. As previously reported, no obvious symptom

Up to now, nearly 70 different viruses are known to infect grapevines, about half of which are associated with major diseases. By contrast, only six grapevine-infecting viroids and a viroid- like RNA, which is likely a novel viroid, have been identified. Although most of the viroids generally do not elicit severe symptoms in grapevines, some of them are the agent of diseases in certain environmental conditions or in combination with certain viruses. Moreover, some of grapevine-infecting viroids may cause severe diseases in other crops. While the identification of the five grapevine viroids dates back to the end of the 900s, Grapevine latent viroid (GLVd) and Grapevine hammerhead viroid-like RNA (GHVd) were identified only recently thanks to the application of next-generation sequencing (NGS) technologies.NGS technologies have, indeed, revolutionized the biodiversity studies and are going to deeply impact on the diagnostic protocols. NGS-based metagenomics approaches are particularly useful to investigate the plant associated viromes, offering a unique opportunity to reveal the presence of all viral agents in the investigated plants, including still unknown entities. In this work, we used an NGS-based approach in order to characterize the virome associated to a grapevine germplasm collection located in Grinzane-Cavour (Piedmont, Northern Italy), comprehending a wide diversity of current and ancient grapevine varieties. The collection includes around 400 grape cultivars from North-Western Italy, as well as regional, national and international varieties used as references, and it is among the largest and richest in materials neglected, endangered to disappear.NGS of cDNA libraries of small RNAs from about one third of the accessions in the germplasm collection was performed, and the virome was reconstructed using an ad hoc bioinformatics pipeline. Results were then validated by other independent molecular methods. Thanks to this approach, viruses routinely present in the grapevines, along with recently discovered viruses and viroids, and viral entities never described before in Italy were identified. In particular, we detected for the first time in Italy the two recently discovered viroids, GLVd and GHVd. According to our data, GLVd presence is limited to few plants in the collection, originally from extra-European territories, i.e. Armenia, Georgia, Uzbekistan and North America, whereas GHVd is more widely distributed among the grapevine accessions, including extra-European, European and Italian grapevines.

Grapevine-infecting viroids do not induce symptoms, except for Grapevine yellow speckle viroid-1 (GYSVd-1) and Grapevine yellow speckle viroid-2 (GYSVd-2), the agents of yellow speckle (YS), a disease characterized by yellow spots or flecks scattered on the leaf blade. The association of these viroids with Grapevine fanleaf virus (GFLV) is thought to elicit vein banding (VB), a syndrome characterized by chrome yellow flecks localized along the main veins and progressing into the interveinal areas of affected vines. The occurrence of these diseases and their causal agents was investigated in north-west Iran with a survey in which 137 vines were tested by multiplex RT-PCR for the presence of the five known grapevines viroids. GYSVd-1, GYSVd-2, Australian grapevine viroid (AGVd) and Hop stunt viroid (HSVd) were detected in 91%, 64%, 95%, and 100% of the tested samples, respectively, whereas Citrus exocortis viroid (CEVd) was not found. Combinations of three and four different viroids were present in most plants (88%) whereas GFLV was found in 50 samples (37%). The Iranian isolates of GYSVd-1, GYSVd-2, HSVd and AGVd showed minor molecular changes compared with the respective reference strains from grapevine. VB occurred in 22 vines infected by GYSVd-1, GYSVd-2 and GFLV, whereas YS symptoms, which occurred in 10% of the tested plants, were always shown by vines infected by GYSVd-1 and/or GYSVd-2. These findings are in line with the notion that assigns to GYSVd-1 and GYSVd-2 a role in the induction of YS and to both viroids and GFLV the genesis of VB.

Introduction and Objective. While most isolates of peach latent mosaic viroid (PLMVd) (Hernández and Flores, PNAS USA 1992) are latent in leaves, hence its name, some incite peach mosaic (PM) or albinism (peach calico, PC). PC-inducing variants have a characteristic insertion of 12-13 nucleotides and accumulate preferentially in the symptomatic but not in the surrounding green foliar sectors. We have shown previously that two 21-nt small RNAs (PLMVd-sRNAs) containing the PC-associated insertion, guide cleavage of the mRNA encoding the chloroplastic heat-shock protein as predicted by RNA silencing (Navarro et al., Plant J. 2012), with this event likely representing the primary alteration eventually resulting in symptoms. Here we have extended these studies to PM, an issue more complex because entails identification of the corresponding molecular determinant, which is not associated with a specific insertion, and because in contrast with the well-defined PC phenotype, PM differs in type and intensity of symptoms.Material and Methods. Deep sequencing was applied to host- and PLMVd-sRNAs, RT-PCR to synthesize complete PLMVd-cDNAs, semi-quantitative RT-PCR to evaluate host mRNA levels, and RNA ligase-mediated rapid amplification of cDNA ends (RACE) to map cleavage sites in mRNAs.Results. We focused on one severe yellow mosaic (peach yellow mosaic, PYM) expressed in leaf sectors interspersed with others green. Full-length PLMVd-cDNAs from both sectors were cloned and sequenced. Two clones, differing in one nucleotide substitution, served to generate dimeric head-to-tail inserts in plasmids from which in vitro transcripts were produced and bioassayed in GF-305 peach seedlings. Whereas one of the transcripts incited an intense yellow mosaic, the other caused only mild lesions, supporting the involvement of one specific PLMVd nucleotide in PYM. Additionally, the differential nucleotide substitution: i) was preserved in the progeny of symptomatic but not in that of the adjacent green sectors, and ii) is absent in a set of PLMVd variants that replicate latently. Examination in silico of the PLMVd-sRNAs and their potential mRNAs targets of peach, whose genome is completely sequenced, has shown that one PLMVd-sRNA containing the PYM-associated substitution could guide cleavage of the mRNA coding for a thylakoid protein as predicted by RNA silencing. RACE experiments indicate that that is indeed the case and that accumulation of this mRNA is lower in symptomatic than in asymptomatic sectors. Moreover, mutants in the gene coding for this thylakoid protein exhibit yellow phenotypes.Conclusions. PLMVd variants with a single nucleotide substitution elicit a yellow mosaic by cleaving, via RNA silencing, the mRNA encoding a thylakoid protein.

A novel circular DNA virus sequence has been identified through next-generation sequencing and in silico assembly of small RNAs of 21-24 nt from an apple tree grown in China. The virus genome was cloned using two independent approaches and sequenced. With a size of 2932 nt, it showed the same genomic structure and conserved origin of replication reported for members of the family Geminiviridae. However, the low nucleotide and amino acid sequence identity with known geminiviruses indicated that it was a novel virus, for which the provisional name apple geminivirus (AGV) is proposed. Rolling circle amplification followed by RFLP analyses indicated that AGV was a virus with a monopartite DNA genome. This result was in line with bioassays showing that the cloned viral genome was infectious in several herbaceous plants (Nicotiana bethamiana, Nicotiana glutinosa and Solanum lycopersicum), thus confirming it was complete and biologically active, although no symptoms were observed in these experimental hosts. AGV genome structure and phylogenetic analyses did not support the inclusion of this novel species in any of the established genera in the family Geminiviridae. A survey of 165 apple trees grown in four Chinese provinces showed a prevalence of 7.2% for AGV, confirming its presence in several cultivars and geographical areas in China, although no obvious relationship between virus infection and specific symptoms was found.

A novel virus has been identified by next-generation sequencing (NGS) in privet (Ligustrum japonicum L.) affected by a graft-transmissible disease characterized by leaf blotch symptoms resembling infectious variegation, a virus-like privet disease with an unclear aetiology. This virus, which has been tentatively named 'privet leaf blotch-associated virus' (PrLBaV), was absent in non-symptomatic privet plants, as revealed by NGS and reverse transcription-polymerase chain reaction (RT-PCR). Molecular characterization of PrLBaV showed that it has a segmented genome composed of two positive single-stranded RNAs, one of which (RNA1) is monocistronic and codes for the viral replicase, whereas the other (RNA2) contains two open reading frames (ORFs), ORF2a and ORF2b, coding for the putative movement (p38) and coat (p30) proteins, respectively. ORF2b is very probably expressed through a subgenomic RNA starting with six nucleotides (AUAUCU) that closely resemble those found in the 5?-terminal end of genomic RNA1 and RNA2 (AUAUUU and AUAUAU, respectively). The molecular signatures identified in the PrLBaV RNAs and proteins resemble those of Raspberry bushy dwarf virus (RBDV), currently the only member of the genus Idaeovirus. These data, together with phylogenetic analyses, are consistent with the proposal of considering PrLBaV as a representative of the second species in the genus Idaeovirus. Transient expression of a recombinant PrLBaV p38 fused to green fluorescent protein in leaves of Nicotiana benthamiana, coupled with confocal laser scanning microscopy assays, showed that it localizes at cell plasmodesmata, strongly supporting its involvement in viral movement/trafficking and providing the first functional characterization of an idaeovirus encoded protein.

High-throughput sequencing of small RNAs allowed the identification of a novel DNA virus in a Chinese mulberry tree affected by a disease showing mosaic and dwarfing symptoms. Rolling-circle amplification and PCR with specific primers, followed by sequencing of eleven independent full-length clones, showed that this virus has a monopartite circular DNA genome (2.95 kb) containing ORFs in both polarity strands, as reported previously for geminiviruses. A field survey showed the close association of the virus with diseased mulberries, so we tentatively named the virus mulberry mosaic dwarf-associated virus (MMDaV). The MMDaV genome codes for five and two putative proteins in the virion-sense and in the complementary-sense strands, respectively. Although three MMDaV virion-sense putative proteins did not share sequence homology with any protein in the databases, functional domains [coiled-coil and transmembrane (TM) domains] were identified in two of them. In addition, the protein containing a TM domain was encoded by an ORF located in a similar genomic position in MMDaV and in several other geminiviruses. As reported for members of the genera Mastrevirus and Becurtovirus, MMDaV replication-associated proteins are expressed through the alternative splicing of an intron, which was shown to be functional in vivo. A similar intron was found in the genome of citrus chlorotic dwarf-associated virus (CCDaV), a divergent geminivirus found recently in citrus. On the basis of pairwise comparisons and phylogenetic analyses, CCDaV and MMDaV appear to be closely related to each other, thus supporting their inclusion in a putative novel genus in the family Geminiviridae.

The complete nucleotide sequence of an Albanian isolate of grapevine leafroll-associated virus 7 (GLRaV-7-Alb) was determined. The viral genome consists of 16,404 nucleotides and has nine open reading frames (ORFs) that potentially encode proteins, most of which are typical for members of the family Closteroviridae. Only the 25-kDa (ORF8) and 27-kDa (ORF9) proteins had no apparent similarity to other viral proteins in the sequence databases. The genome structure of GLRaV-7-Alb closely resembles that of little cherry virus 1 and cordyline virus 1. In phylogenetic trees constructed with HSP70h sequences, these three viruses cluster together in a clade next to that comprising members of the genus Crinivirus, to which they are more closely related than to the clostero- and ampeloviruses. The molecular properties of these three viruses differ sufficiently from those of members of the three extant genera of the family Closteroviridae to warrant their classification in a novel genus.

High-throughput sequencing (HTS) technologies have contributed to the identification of several viroids and viroid-like RNAs, including a small (434 nt) circular RNA containing the conserved domains of hammerhead ribozymes in apple trees grown in China (Zhang et al., PLoS Pathogens 2014). Although the possible viroid nature of this RNA was anticipated, the final proof was only recently provided by bioassays confirming its autonomous replication in apple (Serra et al., Virus Res. 2018). In the same study, based on in silico modeling and co-variation analysis of viroid populations, the two polarity strands of AHVd were predicted to adopt branched conformations. Interestingly, the (+) polarity strand of AHVd, but not its (-) counterpart, was proposed to be stabilized by a kissing loop- interaction, a situation resembling that of peach latent mosaic viroid and chrysanthemum chlorotic mottle viroid (genus Pelamoviroid, family Avsunviroidae). Besides China, AHVd has been identified only in the cultivar Pacific Gale in Canada (Messmer et al., Can J. Plant Pathol. 2017).Results. In the frame of a project aimed to examine the phytosanitary status of ancient Apulian fruit tree varieties, partially purified dsRNA preparations from several fruit tree species were pooled together and analyzed by HTS (Illumina platform) for the presence of viruses and viroids. By this approach, a single contig of 303 nt sharing high sequence identity with AHVd was identified in one of the sequenced cDNA libraries. RT-PCR with AHVd- specific primers and total RNA preparations from each plant used to prepare the sequenced cDNA library allowed the identification of two apple trees of the local ancient varieties "Mela Rosa Guadagno" (MRG) and "Agostinella" (AG) as the original source plants infected by AHVd, the first report of this viroid in Italy. The natural variability of AHVd viroid populations infecting MRG and AG isolates was studied. With only five changes with respect to the master sequence, AHVd variants from MRG isolate were less variable than those from AG isolate, where polymorphic positions with respect to its master sequence were about fifty. AHVd sequence variants from the two Italian isolates were 97-93 % identical to each other, while they shared lower sequence identity with those from the China and Canada. Interestingly, most nucleotide changes in the Italian AHVd variants did not result in major modifications of the branched secondary structure recently proposed for this viroid by Serra et al. (2018). Interestingly, mutations were found in the two hairpin predicted to form a kissing- loop interaction in the AHVd (+) strand. However, nucleotide rearrangements preserved the potential kissing-loop structure, although stabilized by three instead of four interacting base pairs in the Italian isolates. Altogether, these data showing consistent co-variations preserving both the branched secondary structure and the kissing-loop interaction in AHVd variants of differe

Mulberry mosaic dwarf (MMD) disease was first recorded in 1957 and has long been endemic in China. A new viroid-like RNA was detected from mulberry trees affected by MMD and its full nucleotide sequence was determined (1). This mulberry small circular RNA (mscRNA), consisting of 356 nt, does not share significant sequence homology with other viroids and viroid-like satellite RNAs. Here we show that (+) strand of mcRNA contains a hammerhead ribozyme, while the (-) strand contains a hairpin ribozyme, resembling viroid-like satellite RNAs of nepoviruses. mscRNA transcripts of (+) and (-) strands self-cleaved in vitro as predicted by the hammerhead and hairpin structures, respectively, generating fragments with the expected 5' terminus. Circular forms of mscRNA, purified from plant extracts, and dimeric transcripts of both polarities, generated in vitro, were slash-inoculated in mulberry, hop, tomato and cucumber seedlings. Northern-blot hybridization did not detect mscRNA in the inoculated plants so far, suggesting that this circular RNA is not endowed of autonomous replication. These data, taking also into consideration that a nepovirus has been recently reported in mulberry in China (2), strongly suggest that mscRNA could be a novel viroid-like satellite RNA of a nepovirus. This hypothesis is supported by the identification, by next generation sequencing, of both mscRNA and a nepovirus in the same mulberry host. Moreover, a close association of mscRNA with MMD disease was excluded on the bases of a field survey carried out on mulberry trees affected or not affected by the disease. This data are in agreement with our recent results showing that MMD is closely associated with a new geminivirus (3).

A survey of the presence of Pear blister canker viroid (PBCVd) in pear trees in Turkey was carried out by tissue print hybridization. Four out of 74 trees (5.4%) from a pear germplasm collection tested positive and this result was confirmed by RT-PCR and sequencing. Molecular characterization of the Turkish PBCVd isolates from four different local pear cultivars, together with multiple sequence alignments and phylogenetic analyses including all PBCVd sequences reported previously, provided an insight in the sequence variability of this viroid and showed that: (i) a total of 98 polymorphic positions distributed throughout the molecule are present in PBCVd genomic RNA; (ii) some regions with presumed relevant functions are strictly preserved and, (iii) the geographic origin of the isolates and the host species apparently have a bearing on the sequence variability of the viroid. This study records the presence of PBCVd in Turkey and confirms that its geographic distribution has been underestimated, most likely because the majority of infected pear cultivars are symptomless.

The presence of Hop stunt viroid (HSVd) was detected using RT-PCR and Northern blot hybridization in five of 60 samples from symptomless mulberry trees (Morus alba) collected in Italian and Lebanese orchards in July 2010. Infection levels were c. 10% in Lebanese and 8% in Italian samples. Nucleotide alignments showed that sequences of the mulberry HSVd isolates shared 95-96% identity with those of the same viroid occurring elsewhere. In a phylogenetic tree, mulberry HSVd isolates clustered together with those of HSVd-citrus, regardless of their geographical origin. This is the first report of infection in mulberry trees by HSVd.

Peach calico (PC), an extreme chlorosis (albinism) of peach leaves, stems and fruits, is induced by sequence variants of the chloroplast-replicating Peach latent mosaic viroid (PLMVd, genus Pelamoviroid, family Avsunviroidae) harboring a characteristic insertion of 12-14 nt that assumes a hairpin conformation. Here, we show that the primary and not the secondary structure of the inserted hairpin plays a major role in PC, a finding consistent with the hypothesis that PC could be elicited by an RNA silencing mechanism. More specifically, viroid-derived small RNAs (sRNAs) of 21-24 nt generated by the host response against invading nucleic acids, may target for cleavage cell mRNAs thereby triggering a signal cascade that may result in PC. To test this hypothesis, we performed deep sequencing (Illumina) of sRNA libraries from healthy, PC-expressing and latently-infected peach leaves. Then, by semi-quantitative RT-PCR and RNA 3' ligase-mediated rapid amplification of cDNA ends (RACE) we determined that two PLMVd-derived sRNAs, containing the PC-associated insertion and exclusively accumulating in albino tissues, which are a target for cleavage the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90). These data are the first direct evidence that a viroid may modify thr host gene expression of its natural host via RNA silencing. Moreover, since cHSP90 participates in chloroplast biogenesis and plastid-to-nucleus signal transduction in Arabidopsis, down-regulation of this protein is consistent with the chloroplast malformations previously reported in PC-expressing tissues, and strongly supports the involvement of RNA silencing in PC. Whether symptom elicitation driven by RNA silencing is a particular case or reflects a general situation in viroid pathogenesis remains to be addressed. Since our study also identified many other peach mRNAs potentially targeted by PLMVdsRNAs, RNA silencing might have an additional role in plant-viroid interplay, including viroid evolution and adaptation. (A detailed account of these results has been recently published: Navarro et al., Plant Journal 70, 991-1003, 2012).

Peach latent mosaic viroid (PLMVd) displays an unusual pathogenic behavior. While consistent with its name most isolates do not incite foliar symptoms, some incite peach mosaic (PM) or an extreme albinism (peach calico, PC). PLMVdvariants inducing PC have a characteristic insertion of 12-13 nt and accumulate in symptomatic leaf sectors but not in the adjacent green sectors. Based on these two findings and on additional experimental data (including deep sequencing ofviroid-derived small RNAs) we recently proposed that PC could be mediated by RNA silencing. Extending similar analyses to PM first entails identification of the molecular determinant of PM, a question posing serious difficulties because it is not associated with a specific insertion. Moreover, in contrast with PC which is a well-defined syndrome, PM varies very much in intensity and distribution. After examining different PLMVd-PM isolates over the years, we focused on a few of them. This paper reports results obtained with one of such isolates, recovered from the peach cultivar O'Henry grown in Aragón (Spain), expressing an intense mosaic in leaf sectors intermingled with asymptomatic sectors. Previously, RNAs extracted from both sectors of the same leaves were used to generate by RT-PCR full-length PLMVd-cDNAs that were cloned and sequenced. Their multiple alignment revealed specific changes correlating with symptoms. Two clones (Ab4 and Ab7), differing in a single substitution, were used in order to produce dimeric head-to-tail inserts and then in vitro transcripts which were mechanically inoculated onto GF-305 peach seedlings. Intriguingly, while Ab4 incited strong chlorotic symptoms in most leaves, Ab7 only incited mild lesions, providing a first insight of a PLMVd nucleotide directly involved in PM. Moreover, RT-PCR amplification, cloning and sequencing showed that the differential nucleotide substitution was preserved in most of the clones from the symptomatic sectors, but no in those from the surrounding green sectors.

To date, GYSVd-2 has been found only in few countries and grapevine is the only known natural host of GYSVd-2, which has restricted distribution in world viticulture. In this study, three GYSVd-2 isolates from three distinct regions of northwest Iran were molecularly characterized. The Iranian GYSVd-2 variants were composed of 360-363 nucleotides sharing a high sequence identity of 96-100% with each other, 97-99% with the reference sequence (NC-003612) and 99-100% with other GYSVd-2 sequences. In total, six new variants were identified with most nucleotide changes with respect to the reference variant located in the terminal left (TL) domain of the proposed rod-like secondary structure. Three and five variants showed the deletion of A356 and U363 in the TL domain, respectively. Also, substitutions of G359 by U, U360 by C and C361 by U in five variants were found.In contrast, limited nucleotide changes were observed in other domains. In silico analysis showed that some of the nucleotide substitutions would affect the predicted secondary structures but possible biological roles on viroid replication and pathogenesis are unknown and would require further investigations.

Tomato is a natural host of Potato spindle tuber viroid (PSTVd) and Tomato yellow leaf curl Sardinia virus (TYLCSV), which are representative members of pospiviroids (infectious non-coding circular RNAs) and geminiviruses (single-stranded DNA viruses), respectively. While molecular events during infection have been explored separately for each one of these two nuclear replicating pathogens, plant responses during mixed infections are unknown. In this context, dissection of DNA methylation pathway is particularly interesting because it is well known that plants may methylate viral DNA to impair geminivirus infection, while whether viroids interfere with host DNA methylation pathways is unknown. Exploiting an experimental system based on PSTVd and TYLCSV co-infecting the same tomato plant, and applying qRT-PCR, methylation-sensitive restriction and bisulfite conversion assays, we found that: i) when plants were co-infected, TYLCSV infectivity and accumulation were strongly impaired, indicating an antagonistic action of PSTVd; ii) PSTVd alone or in double infection with TYLCSV significantly upregulated the expression of key genes governing DNA methylation in plants; iii) PSTVd promoted a strong hypermethylation of TYLCSV DNA in tomato plants co-infected by both pathogens, thus supporting a mechanistic link with the antagonism of the viroid on the virus during co-infection. Besides providing the first solid evidence that a viroid may interfere with host regulatory networks involved in DNA methylation, these data open new perspectives on the possible involvement of viroid-induced epigenetic changes in plant responses against other biotic and abiotic stresses.

The detection of viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs (siRNAs, 21 to 24 nucleotides [nt]) in plants infected by nuclear-replicating members of the family Pospiviroidae (type species, Potato spindle tuber viroid [PSTVd]) indicates that they are inducers and targets of the RNA-silencing machinery of their hosts. RNA-dependent RNA polymerase 6 (RDR6) catalyzes an amplification circuit producing the double-stranded precursors of secondary siRNAs. Recently, the role of RDR6 in restricting systemic spread of certain RNA viruses and precluding their invasion of the apical growing tip has been documented using RDR6-silenced Nicotiana benthamiana (NbRDR6i) plants. Here we show that RDR6 is also engaged in regulating PSTVd levels: accumulation of PSTVd genomic RNA was increased in NbRDR6i plants with respect to the wild-type controls (Nbwt) early in infection, whereas this difference decreased or disappeared in later infection stages. Moreover, in situ hybridization revealed that RDR6 is involved in restricting PSTVd access in floral and vegetative meristems, thus providing firm genetic evidence for an antiviroid RNA silencing mechanism. RNA gel blot hybridization and deep sequencing showed in wt and RDR6i backgrounds that PSTVd sRNAs (i) accumulate to levels paralleling their genomic RNA, (ii) display similar patterns with prevailing 22- or 21-nt plus-strand species, and (iii) adopt strand-specific hot spot profiles along the genomic RNA. Therefore, the surveillance mechanism restraining entry of some RNA viruses into meristems likely also controls PSTVd access in N. benthamiana. Unexpectedly, deep sequencing also disclosed in NbRDR6i plants a profile of RDR6-derived siRNA dominated by 21-nt plus-strand species mapping within a narrow window of the hairpin RNA stem expressed transgenically for silencing RDR6, indicating that minus-strand siRNAs silencing the NbRDR6 mRNA represent a minor fraction of the total siRNA population.

Control measures based on the use of viroid-free propagation material and, when possible, on the interception of infected plants, are needed to constrain the spread of viroids. Effectiveness of these defense strategies requires fast, reliable, sensitive and economically sustainable detection methods. A single molecular probe (polyprobe), composed of assembled sequence fragments derived from several viroid species has the advantage of reducing the costs of simultaneous detection of most viroid species by molecular hybridization. In fact, just one single transcription reaction is needed for synthesizing a polyprobe useful to detect different viroid species. Based on bioinformatics analyses of pospiviroid genomic RNAs, we have developed a polyprobe that can detect at least eight different viroid species belonging to Pospiviroid genus (family Pospiviroidae) (POSPIprobe), including Potato spindle tuber viroid and Chrysanthemum stunt viroid, which are quarantine pests recently intercepted in Italy. The digoxigenin-labelled polyprobe was synthesized by in vitro-transcription using as template a single vector constructed by cloning directionally partial sequences from four different pospiviroids. POSPIprobe sensitivity in dot-blot assays was similar to that obtained using single probes, and its specificity for the eight pospiviroids was further corroborated by Northern blot and tissue printing hybridization. Dot blot assays with the POSPIprobe were validated by simultaneously testing 68 samples from tomato, chrysanthemum, Argyranthemum frutescens and pepper infected by different pospiviroids, which confirmed the high potential of this probe in quarantine, certification and survey programs. Emerging phytosanitary risks due to pospiviroid spread in Europe and effectiveness of control measures will be also discussed.

How viroids, tiny non-protein-coding RNAs (~250-400 nt), incite disease is unclear. One hypothesis is that viroid-derived small RNAs (vd-sRNAs; 21-24 nt) resulting from the host defensive response, via RNA silencing, may target for cleavage cell mRNAs and trigger a signal cascade, eventually leading to symptoms. Peach latent mosaic viroid (PLMVd), a chloroplast-replicating viroid, is particularly appropriate to tackle this question because it induces an albinism (peach calico, PC) strictly associated with variants containing a specific 12-14-nt hairpin insertion. By dissecting albino and green leaf sectors of Prunus persica (peach) seedlings inoculated with PLMVd natural and artificial variants, and cloning their progeny, we have established that the hairpin insertion sequence is involved in PC. Furthermore, using deep sequencing, semi-quantitative RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends (RACE), we have determined that two PLMVd-sRNAs containing the PC-associated insertion (PC-sRNA8a and PC-sRNA8b) target for cleavage the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90), thus implicating RNA silencing in the modulation of host gene expression by a viroid. Chloroplast malformations previously reported in PC-expressing tissues are consistent with the downregulation of cHSP90, which participates in chloroplast biogenesis and plastid-to-nucleus signal transduction in Arabidopsis. Besides PC-sRNA8a and PC-sRNA8b, both deriving from the less-abundant PLMVd (-) strand, we have identified other PLMVd-sRNAs potentially targeting peach mRNAs. These results also suggest that sRNAs derived from other PLMVd regions may downregulate additional peach genes, ultimately resulting in other symptoms or in a more favorable host environment for viroid infection.

Indirect somatic embryogenesis is effective at eliminating the most important viruses affecting grapevines. Accordingly, this technique was tested as a method for eradicating two widespread viroids, Grapevine yellow speckle viroid 1 (GYSVd-1) and Hop stunt viroid (HSVd), from four grapevine cultivars. Both viroids were detected by RT-PCR in grapevine floral organs used for initiating embryogenic cultures, as well as in undifferentiated cells of embryogenic and non-embryogenic calli from anthers and ovaries. In contrast, somatic embryos differentiated from these infected calli were viroid-free, and viroids were not detected in embryo-derived plantlets even three years after their transfer to greenhouse conditions. A wider spatial distribution of HSVd than GYSVd-1 within proliferating calli was revealed by in situ hybridization, whereas no hybridization signal was detected in the somatic embryos. In addition, GYSVd-1 and HSVd were localised in the nucleus of infected cells, conclusively showing the nuclear accumulation of representative members of Apscaviroid and Hostuviroid genera, which was only an assumption, so far. Somatic embryogenesis was compared to in vitro thermotherapy, a technique routinely used for virus eradication. After thermotherapy, HSVd and GYSVd-1 were detected in all in vitro plantlets of the cultivar Roussan, and in all lines analysed after 3 years of culture in greenhouse. The high efficiency with which somatic embryogenesis may eliminate viroids and viruses from several infected grapevine cultivars, should allow to obtain virus- and viroid-free material, which would be useful not only for sanitary selection but also for basic research on plant-virus and plant-viroid interactions in grapevine.

The identification of viroid-derived small RNAs (vd-sRNAs) of 21 to 24 nucleotides (nt) in plants infected by viroids (infectious non-protein-coding RNAs of just 250 to 400 nt) supports their targeting by Dicer-like enzymes, the first host RNA-silencing barrier. However, whether viroids, like RNA viruses, are also targeted by the RNA-induced silencing complex (RISC) remains controversial. At the RISC core is one Argonaute (AGO) protein that, guided by endogenous or viral sRNAs, targets complementary RNAs. To examine whether AGO proteins also load vd-sRNAs, leaves of Nicotiana benthamiana infected by potato spindle tuber viroid (PSTVd) were agroinfiltrated with plasmids expressing epitope-tagged versions of AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, AGO7, AGO9, and AGO10 from Arabidopsis thaliana. Immunoprecipitation analyses of the agroinfiltrated halos revealed that all AGOs except AGO6, AGO7, and AGO10 associated with vd-sRNAs: AGO1, AGO2, and AGO3 preferentially with those of 21 and 22 nt, while AGO4, AGO5, and AGO9 additionally bound those of 24 nt. Deep-sequencing analyses showed that sorting of vd-sRNAs into AGO1, AGO2, AGO4, and AGO5 depended essentially on their 5?-terminal nucleotides, with the profiles of the corresponding AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Furthermore, agroexpression of AGO1, AGO2, AGO4, and AGO5 on PSTVd-infected tissue attenuated the level of the genomic RNAs, suggesting that they, or their precursors, are RISC targeted. In contrast to RNA viruses, PSTVd infection of N. benthamiana did not affect miR168-mediated regulation of the endogenous AGO1, which loaded vd-sRNAs with specificity similar to that of its A. thaliana counterpart.

Five viroid species have been reported fromgrapevine. Hop stunt viroid (HSVd) and Grapevine yellowspeckle viroid 1 (GYSVd-1) are distributed worldwide,whereas Grapevine yellow speckle viroid 2(GYSVd-2), Australian grapevine viroid (AGVd) andCitrus exocortis viroid (CEVd) are found only sporadically.However, the presence of AGVd and GYSVd-2 inseveral countries, including China, Turkey and Tunisia,suggests a wider dissemination, possibly also in Europe,where AGVd has never been found and GYSVd-2 hasbeen occasionally identified in Italy. Taking advantage ofa multiplex RT-PCR assay recently developed for detectingsimultaneously these five viroids, vines growing inItaly in commercial vineyards and germplasm collectionswere surveyed. Besides confirming the widespreadpresence of HSVd and GYSVd-1 in the field, GYSVd-2and/or AGVd were identified in two grapevine tablecultivars (Sultanina Bianca and Red Globe) from germplasmcollections. Tests extended to vines cultivated insouthern Italy confirmed the presence of both viroids,which were further characterized. No major sequencedivergences between the AGVd and GYSVd-2 variantsfrom Italy and those previously described from othercountries were observed. Phylogenetic analysis supportedthe close relationships among AGVd variants from Italy,Tunisia and Australia. To our knowledge this is the firstreport of AGVd in Europe and the first molecular characterizationof GYSVd-2 isolates from a European country.

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

A novel negative-stranded (ns) RNA virus associated with a severe citrus disease reported more than 80 years ago has been identified. Transmission electron microscopy showed that this novel virus, tentatively named citrus concave gum-associated virus, is flexuous and non-enveloped. Notwithstanding, its two genomic RNAs share structural features with members of the genus Phlebovirus, which are enveloped arthropod-transmitted viruses infecting mammals, and with a group of still unclassified phlebo-like viruses mainly infecting arthropods. CCGaV genomic RNAs code for an RNA-dependent RNA polymerase, a nucleocapsid protein and a putative movement protein showing structural and phylogenetic relationships with phlebo-like viruses, phleboviruses and the unrelated ophioviruses, respectively, thus providingintriguing evidence of a modular genome evolution. Phylogenetic reconstructions identified an invertebrate-restricted virus as themost likely ancestor of this virus, revealing that its adaptation to plants was independent from and possibly predated that of theother nsRNA plant viruses. These data are consistent with an evolutionary scenario in which trans-kingdom adaptationoccurred several times during the history of nsRNA viruses and followed different evolutionary pathways, in which genomic RNAsegments were gained or lost. The need to create a new genus for this bipartite nsRNA virus and the impact of the rapid andspecific detection methods developed here on citrus sanitation and certification are also discussed.

Viroids are tiny infectious agents of plants, consisting exclusively of a small (246-401 nucleotides) circular RNA lacking protein-coding ability. Likely mimicking structural features of cellular RNAs, they can redirect the pre-existing host transcription and RNA trafficking machineries for their own replication and spread, and by doing so they eventually elicit severe diseases. Therefore, in addition to causing phytopathological problems, viroids also offer opportunities for basic research on cellular RNA-based networks. After briefly summarizing the biological, molecular and epidemiological properties of viroids, this review focuses on relevant novelties of these infectious agents, with major emphasis on the molecular pathways presumably mediating viroid pathogenesis and on current phytosanitary emergences in Europe associated with the dissemination of viroids potentially causing severe diseases in vegetable crops.

Introduction. Chrysantemum chlorotic mottle viroid (CChMVd) (Navarro and Flores, PNAS USA 1997), a member of the genus Pelamoviroid (type member peach latent mosaic viroid, PLMVd), displays the higest mutation rate reported for any biological entity (Gago et al., Science 2009). To understand how CChMVd colonizes, evolves and incites disease in its natural host, two natural variants, one with a UUUC tetraloop tightly associated with symptoms and the other with an alternative GAAA tetraloop and asymptomatic (De la Pen?a et al., PNAS USA 1999), were assayed.Results. As anticipated, the second one induced no phenotype, and RT-PCR, cloning and sequencing of its progeny showed variants with only the GAAA tetraloop. In contrast, the first variant induced early symptoms consisting in adjacent chlorotic and green leaf sectors, with progeny analysis disclosing that variants with the UUUC tetraloop predominate in chlorotic sectors, while in green sectors they coexisted with others having one or two mutations in the pathogenic determinant. Bioassay of the latter mutant variants led to asymptomatic infections (without the UUUC tetraloop in the progeny), showing that one single substitution in this tetraloop abrogates pathogenicity. Therefore, symtomatic and non- symptomatic CChMVd variants display distinct evolutionary trajectories, with the high mutation rate of CChMVd and the ability of some variants to colonize preferentially some leaf sectors and exclude other variants, accounting most likely for the observed results. Thus, CChMVd shows a territorial behaviour, with clearly segregated populations in a single leaf. The finding that a single substitution in the UUUC tetraloop disrupts pathogenicity is consistent with the involvement of RNA silencing. To gain support for this notion, we first examined by deep sequencing the viroid-derived small RNAs (CChMV-sRNAs) generated by RNA silencing: a significant prevalence of those of 21 nt was observed, in line with previous results for PLMVd. On the other hand, the chrysanthemum transcriptome of a cultivar in which CChMVd incites clear symptoms was determined. Combining both set of data we found a 21-nt CChMV-sRNA (containing the pathogenic determinant and with two 5'- terminal Us) potentially targetting for cleavage, as predicted by RNA silencing mediated by AGO1, the mRNA of an enzyme of the Calvin's cycle, the light-independent part of photosyntesis occurring in the chloroplast stroma. RNA ligase mediated-rapid amplification of cDNA ends confirmed that this mRNA was cleaved in the expected site (between positions 10 and 11 of the complementary 21-nt CChMV-sRNA) only in symptomatic sectors. Three additional lines of evidence support that such RNA silencing mechanism mediates the CChMVd-induced primary lesion. First, RT-qPCR showed a reduced level of this mRNA in symptomatic sectors, where the complementary 21-nt CChMV-sRNA accumulates preferentially. Second, a full-length CChMVd mutated so that the mismatch a

In an attempt of identifying the causal agent of a severe citrus disease first described in the early 1930s and named citrus concave gum, a new negative stranded RNA (nsRNA) virus was identified by high-throughput sequencing (HTS). Being associated with the disease, this virus was tentatively named citrus concave-gum-associated virus (CCGaV). With a bipartite genome composed of an RNA1 encoding the RNA-dependent RNA polymerase (RdRp) in the negative strand and an ambisense bicistronic RNA2 coding for the nucleoprotein (NP) and the putative movement protein (MP), CCGaV showed structural and phylogenetic features supporting the proposal of classifying it as a representative member of a new species to be allocated in the new genus Coguvirus in the Order Bunyavirales (https://talk.ictvonline.org/files/proposals/taxonomy_proposals_plant1/m/plant01/7405). A second nsRNA virus, resembling CCGaV has been recently identified by HTS in citrus and provisionally denoted citrus bunya-like virus (CBLV). The molecular and structural features of CBLV genomic RNAs, the signatures of the proteins it encodes and phylogenetic analyses support the classification of CBLV and CCGaV in the same genus and suggest a possible modular genome evolution for these viruses. These findings, supporting the hypothesis that plant-infecting nsRNA viruses most likely have evolved from an invertebrate-infecting ancestor, several times as independent events, will be discussed. Specific methods for detecting both CCGaV and CBLV and results of a field survey on the distribution of both viruses in Southern Italy will also be presented and discussed.

El viroide del mosaico latente del melocotonero (peach latent mosaic viroid, PLMVd) (Hernández and Flores, PNAS USA, 1992) muestra un comportamiento patogénico inusual. Mientras que, de acuerdo con su nombre, la mayoría de aislados son asintomáticos en hojas, algunos inducen mosaicos (peach mosaic, PC) o un albinismo extremo (peach calico, PM). Las variantes del PLMVd que causan PC tienen una inserción característica de 12-13 nt y se acumulan preferentemente en los sectores foliares sintomáticos y no en los verdes adyacentes. Utilizando secuenciación masiva, RT-PCR semicuantitativa y amplificación rápida de extremos de cDNA mediada por RNA ligasa (RNA ligase-mediated rapid amplification of cDNA ends, RACE), hemos mostrado recientemente que dos pequeños RNAs derivados del PLMVd (PLMVd-sRNAs) conteniendo la inserción asociada al PC guían el corte de un mRNA del hospedante como predice el silenciamiento mediado por RNA (RNA silencing) (Navarro et al., Plant J. 2012). La extensión de estos análisis a la sintomatología PM conlleva la identificación del determinante(s) molecular de la misma, una cuestión que plantea serias dificultades porque no está asociado a una inserción específica y porque, en contraste con el PC que es una alteración bien definida, el PM varía mucho en intensidad y distribución. Después de examinar distintos aislados de PLMVd-PM, hemos enfocado nuestra atención sobre uno de ellos caracterizado por un intenso mosaico amarillo (peach yellow mosaic, PYM) que se expresa en sectores foliares entremezclados con otros verdes. Partiendo de RNAs de ambos sectores se generaron por RT-PCR PLMVd-cDNAs de longitud completa que fueron clonados y secuenciados. Dos clones, difiriendo en una simple sustitución nucleotídica, se emplearon para producir insertos diméricos con orientación cabeza-cola y, de ellos, transcritos in vitro que se bioensayaron. Mientras que uno de los transcritos indujo un intenso PYM, el otro solo causó lesiones suaves, proporcionando la primera pista de la implicación de un núcleotido específico en el PYM. Además: i) amplificación por RT-PCR, clonaje y secuenciación revelaron que la sustitución nucleotídica diferencial se preserva en los sectores sintomáticos, pero no así en los verdes circundantes, y ii) la sustitución nucleotídica asociada con el PYM no está presente en una serie de variantes del PLMVd que inducen infecciones asintomáticas. Un examen in silico de los PLMVd-sRNAs y de sus posibles mRNAs dianas del hospedante (del que se conoce su genoma completo), ha mostrado que un PLMVd-sRNA con la sustitución nucleotídica asociada al PYM dirigiría el corte del mRNA que codifica una proteína tilacoidal como predice el silenciamiento mediado por RNA. Los primeros experimentos para verificar dicha predicción son consistentes con la misma: la acumulación de este mRNA es menor en sectores sintomáticos que en asintomáticos.

Composed of a naked circular non-protein-coding genomic RNA, counting only a few hundred nucleotides, viroids--the smallest infectious agents known so far--are able to replicate and move systemically in herbaceous and woody host plants, which concomitantly may develop specific diseases or remain symptomless. Several viroids have been reported to naturally infect pome and stone fruit trees, showing symptoms on leaves, fruits and/or bark. However, Koch's postulates required for establishing on firm grounds the viroid etiology of these diseases, have not been met in all instances. Here, pome and stone fruit tree diseases, conclusively proven to be caused by viroids, are reviewed, and the need to pay closer attention to fulfilling Koch's postulates is emphasized.

While biogenesis of viroid RNAs is well-known, how they decay is restricted to data involving host RNA silencing. Here we report an alternative degradation pathway operating on potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids (family Pospiviroidae). Northern-blot hybridizations with full- and partial-length probes revealed a set of PSTVd (+) subgenomic (sg)RNAs in early-infected eggplant, some partially overlapping and reaching levels comparable to those of the genomic circular and linear forms. Part of the PSTVd (+) sgRNAs were also observed in Nicotiana benthamiana (specifically in the nuclei) and tomato, wherein they have been overlooked due to their low accumulation. Primer extensions of representative (+) sgRNAs failed to detect a common 5? terminus, excluding that they could result from aborted transcription initiated at one specific site. Supporting this view, 5?- and 3?-RACE indicated that the (+) sgRNAs have 5?-OH and 3?-P termini most likely generated by RNase-mediated endonucleolytic cleavage of longer precursors. These approaches also unveiled PSTVd (-) sgRNAs with features similar to their (+) counterparts. Our results provide a mechanistic insight on how viroid decay may proceed in vivo during replication, and suggest that synthesis and decay of PSTVd strands might be coupled as in mRNA.

Viroids, like RNA viruses, propagate in their hosts as populations of closely-related sequence variants (quasi-species). When assayed individually, such variants often incite distinct phenotypes but their stability along time may vary because viroids, like chrysanthemum chlorotic mottle viroid (CChMVd), display a hight mutation rate [2] that rapidly generates heterogeneous populations. The behavior of these populations is essentially determined by interference among individual components, which in viroids must occur at the RNA level. During host colonization virus populations suffer bottlenecks imposed by the host, the vector, and the territorial behavior (superinfection exclusion) of the infecting viruses, which limit the number of genomes entering and replicating in individual cells [3]. Some reports suggest that bottlenecks also operate during viroid infection, but such issue has not been further addressed. Here we have examined the evolution in time and space of two natural CChMVd variants: one containing a UUUC tetraloop strictly associated with symptoms and the other with a GAAA tetraloop and asymptomatic [1]. Inoculation with the latter induced no phenotype, with only variants with the same tetraloop in the progeny analyzed by RT-PCR, cloning and sequencing. Conversely, early symptoms incited by the first variant consisted in chlorotic and green sectors. RT-PCR, cloning and sequencing of the progeny, and deep sequencing of the viroid-derived RNAs generated by RNA silencing, showed that variants with the UUUC tetraloop predominate in chlorotic sectors, while they coexist in green sectors with others having one or two mutations in the pathogenic determinant. Cloning and bioassay of these mutant variants resulted in asymptomatic infections (with the UUUC tetraloop absent in the progeny), showing that a single substitution in the tetraloop annuls pathogenicity, a finding consistent with involvement of an RNA silencing mechanism. Therefore, pathogenic and non-pathogenic CChMVd variants exhibit distinct evolutionary patterns. The very high mutation rate of CChMVd, combined with the ability of different variants to preferentially colonize some leaf sectors and exclude other variants, may account for the observed results.

Distinct molecular mechanisms have been proposed for explaining how viroids, small non-protein-coding RNAs that infect plants, interfere with their host gene expression and elicit disease symptoms. On the one hand, by mimicking structural features of cellular RNAs, viroids may impair the host transcriptional and RNA trafficking machineries, which are usurped and redirected to facilitate their own replication and movement. On the other hand, the identification in viroid-infected plants of viroid-derived small RNAs (vd-sRNAs) of 21-24 nt, structurally similar to the small interfering RNAs that mediate RNA silencing, suggests that this RNA-based regulatory network largely conserved in eukaryotes is used by viroids for modifying host gene expression. By deep sequencing and rapid amplification of cDNA ends (RACE) we have recently shown that a host mRNA, coding for a gene involved in chloroplast development, is actually targeted for cleavage (in a sequence specific manner as predicted by RNA silencing), by two vd-RNAs accumulating in peach tissues infected by a chloroplast replicating viroid (Peach latent mosaic viroid). These results thus support a direct involvement of RNA silencing in viroid pathogenesis, particularly considering that the two vd-sRNAs map at the viroid pathogenicity determinant. Several other potential targets of vd-sRNAs have been identified in the same experimental system by bioinformatics and degradome-based analyses, suggesting a wider role of RNA silencing in plant-viroid interactions. These results highlight the power of genome-wide approaches for further dissecting the host pathways targeted

Several years ago, two cherry small circular RNAs (cscRNA1 and cscRNA2) and 10-12 double stranded RNAs (dsRNAs) of presumable viral origin were identified in sweet and sour cherry plants grown in Italy. These RNAs were found to be closely associated to a disease termed cherry chlorotic rusty spot (CCRS). The Italian CCRS disease is symptomatologically very similar to the Amasya cherry disease (ACD) described previously in Turkey, and both diseases have been closely associated with similar mycelium-like structures and with double-stranded (ds) RNAs of mycoviruses from the genera Chrysovirus, Partitivirus and Totivirus. More recently it has been realized that cherry trees from Spain affected by cherry leaf scorch (CLS), a fungal disease reported to be caused by Apiognomonia erythrostoma, family Gnomoniaceae (order Diaporthales), show a very similar symptomatology (translucent-chlorotic leaf spots evolving into rusty areas). However, although CLSand CCRS-affected trees have been associated with similar mycelia, fungal fructifications, fungal genes and mycoviral dsRNAs sharing high sequence similarity ? further supporting a close relationship between these two disorders the involvement of A. erythrostoma in CCRS has not been conclusively shown (Carrieri et al., J. Plant Pathol. 2012). Here we report that a small viroid-like RNA similar to cscRNA1 associated with CCRS is also present in CLS-affected trees, thus providing an additional link between them. Comparisons between cscRNAs from CLS and CCRS isolates have shown several common features, including sequence identity (83%), a quasi rod-like conformation with short bifurcations at both termini, and the presence of hammerhead ribozymes in both polarity strands. However, the CLS isolate lacks the recombinant molecules of smaller size (cscRNA2). Although the biological nature of cscRNAs remain to be conclusively shown, the identification of at least cscRNA1 in different cherry cultivars and geographic areas (Spain and Italy) in close association with the same mycoviral dsRNAs, strongly support the satellite nature of the cscRNAs, which could thus be the first mycovirus satellite RNAs.

As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers. Viroid genomes fold up on themselves adopting collapsed secondary structures wherein stretches of nucleotides stabilized by Watson-Crick pairs are flanked by apparently unstructured loops. However, compelling data show that they are instead stabilized by alternative non-canonical pairs and that specific loops in the rod-like secondary structure, characteristic of Potato spindle tuber viroid and most other members of the family Pospiviroidae, are critical for replication and systemic trafficking. In contrast, rather than folding into a rod-like secondary structure, most members of the family Avsunvioidae adopt multibranched conformations occasionally stabilized by kissing loop interactions critical for viroid viability in vivo. Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunvioidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae. Therefore, different RNA structures ?either global or local ? determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.

Cherry trees from Spain affected by cherry leaf scorch (CLS), a fungal disease proposed to be caused by Apiognomonia erythrostoma, show symptoms (translucent-chlorotic leaf spots evolving into rusty areas) very similar to those of cherry chlorotic rusty spot disease (CCRS) and Amasya cherry disease, reported in Italy and Turkey, respectively. The three maladies are closely associated with 10-12 double-stranded viral RNAs, and CCRS is additionally associated with two cherry small circular RNAs (cscRNA1 and cscRNA2). Here, we report that a small viroid-like RNA similar to the CCRS-associated cscRNA1 is also present in CLS-affected trees, thus extending the link between the two diseases. Both CLS and CCRS cscRNA1 elements have common features, including sequence identity (88 %), a predicted quasi rod-like conformation with short bifurcations at both termini, and the presence of hammerhead ribozymes in the strands of both polarities. However, cscRNA2, apparently derived from cscRNA1 by deletion of a short hairpin, was not detected in CLS-affected material. Although the biological nature of cscRNAs is unknown, the identification of at least cscRNA1 in different cherry cultivars and in two distinct geographic areas (Spain and Italy), always in close association with the same mycoviral dsRNAs, supports that these viroid-like RNAs could be satellite RNAs.

Despite being composed by a single-stranded, circular, non-protein-coding RNA of just 246-401 nucleotides (nt), viroids can incite in their host plants symptoms similar to those caused by DNA and RNA viruses, which have genomes at least 20-fold bigger and encode proteins. On the other hand, certain non-protein-coding plant satellite RNAs display structural similarities with viroids but for replication and transmission they need to parasitize specific helper viruses (modifying concomitantly the symptoms they induce). While phenotypic alterations accompanying infection by viruses may partly result from expressing the proteins they code for, how the non-protein-coding viroids (and satellite RNAs) cause disease remains a conundrum. Initial ideas on viroid pathogenesis focused on a direct interaction of the genomic RNA with host proteins resulting in their malfunction. With the advent of RNA silencing, it was alternatively proposed that symptoms could be produced by viroid-derived small RNAs (vd-sRNAs) -generated by the host defensive machinery- targeting specific host mRNA or DNA sequences for post-transcriptional or transcriptional gene silencing, respectively, a hypothesis that could also explain pathogenesis of non-protein-coding satellite RNAs. Evidence sustaining this view has been circumstantial, but recent data provide support for it in two cases: i) the yellow symptoms associated with a specific satellite RNA result from a 22-nt small RNA (derived from the 24-nt fragment of the satellite genome harboring the pathogenic determinant), which is complementary to a segment of the mRNA of the chlorophyll biosynthetic gene CHLI and targets it for cleavage by the RNA silencing machinery, and ii) two 21-nt vd-sRNAS containing the pathogenic determinant of the albino phenotype induced by a chloroplast-replicating viroid target for cleavage the mRNA coding for the chloroplastic heat-shock protein 90 via RNA silencing too. This evidence, which is compelling for the satellite RNA, does not exclude alternative mechanisms.

The discovery of viroids about 45 years ago heralded a revolution in Biology: small RNAs comprising around 350 nt were found to be able to replicate autonomously-and to incite diseases in certain plants-without encoding proteins, fundamental properties discriminating these infectious agents from viruses. The initial focus on the pathological effects usually accompanying infection by viroids soon shifted to their molecular features-they are circular molecules that fold upon themselves adopting compact secondary conformations-and then to how they manipulate their hosts to be propagated. Replication of viroids-in the nucleus or chloroplasts through a rolling-circle mechanism involving polymerization, cleavage and circularization of RNA strands-dealt three surprises: (i) certain RNA polymerases are redirected to accept RNA instead of their DNA templates, (ii) cleavage in chloroplastic viroids is not mediated by host enzymes but by hammerhead ribozymes, and (iii) circularization in nuclear viroids is catalyzed by a DNA ligase redirected to act upon RNA substrates. These enzymes (and ribozymes) are most probably assisted by host proteins, including transcription factors and RNA chaperones. Movement of viroids, first intracellularly and then to adjacent cells and distal plant parts, has turned out to be a tightly regulated process in which specific RNA structural motifs play a crucial role. More recently, the advent of RNA silencing has brought new views on how viroids may cause disease and on how their hosts react to contain the infection; additionally, viroid infection may be restricted by other mechanisms. Representing the lowest step on the biological size scale, viroids have also attracted considerable interest to get a tentative picture of the essential characteristics of the primitive replicons that populated the postulated RNA world.