Identification of epigenetically regulated genes involved in plant-virus interaction and their role in virus-triggered induced resistance.

BACKGROUND: Plant responses to a wide range of stresses are known to be regulated by epigenetic mechanisms. Pathogen-related investigations, particularly against RNA viruses, are however scarce. It has been demonstrated that Arabidopsis thaliana plants defective in some members of the RNA-directed DNA methylation (RdDM) or histone modification pathways presented differential susceptibility to the turnip mosaic virus. In order to identify genes directly targeted by the RdDM-related RNA Polymerase V (POLV) complex and the histone demethylase protein JUMONJI14 (JMJ14) during infection, the transcriptomes of infected mutant and control plants were obtained and integrated with available chromatin occupancy data for various epigenetic proteins and marks. RESULTS: A comprehensive list of virus-responsive gene candidates to be regulated by the two proteins was obtained. Twelve genes were selected for further characterization, confirming their dynamic regulation during the course of infection. Several epigenetic marks on their promoter sequences were found using in silico data, raising confidence that the identified genes are actually regulated by epigenetic mechanisms. The altered expression of six of these genes in mutants of the methyltransferase gene CURLY LEAF and the histone deacetylase gene HISTONE DEACETYLASE 19 suggests that some virus-responsive genes may be regulated by multiple coordinated epigenetic complexes. A temporally separated multiple plant virus infection experiment in which plants were transiently infected with one virus and then infected by a second one was designed to investigate the possible roles of the identified POLV- and JMJ14-regulated genes in wild-type (WT) plants. Plants that had previously been stimulated with viruses were found to be more resistant to subsequent virus challenge than control plants. Several POLV- and JMJ14-regulated genes were found to be regulated in virus induced resistance in WT plants, with some of them poisoned to be expressed in early infection stages. CONCLUSIONS: A set of confident candidate genes directly regulated by the POLV and JMJ14 proteins during virus infection was identified, with indications that some of them may be regulated by multiple epigenetic modules. A subset of these genes may also play a role in the tolerance of WT plants to repeated, intermittent virus infections.

Phenotypic and genomic changes during Turnip mosaic virus adaptation to Arabidopsis thaliana mutants lacking epigenetic regulatory factors.

In this study, we investigated how an emerging RNA virus evolves, interacts, and adapts to populations of a novel host species with defects in epigenetically controlled plant defense mechanisms. Mutations in epigenetic regulatory pathways would exert different effects on defense-response genes but also induce large-scale alterations in cellular physiology and homeostasis. To test whether these effects condition the emergence and subsequent adaptation of a viral pathogen, we have evolved five independent lineages of a naive turnip mosaic virus (TuMV) strain in a set of Arabidopsis thaliana genotypes carrying mutations that influence important elements of two main epigenetic pathways and compare the results with those obtained for viral lineages evolved in wild-type plants. All evolved lineages showed adaptation to the lack of epigenetically regulated responses through significant increases in infectivity, virulence, and viral load although the magnitude of the improvements strongly depended on the plant genotype. In early passages, these traits evolved more rapidly, but the rate of evolution flattened out in later ones. Viral load was positively correlated with different measures of virulence, though the strength of the associations changed from the ancestral to the evolved viruses. High-throughput sequencing was used to evaluate the viral diversity of each lineage, as well as characterizing the nature of fixed mutations, evolutionary convergences, and potential targets of TuMV adaptation. Within each lineage, we observed a net increase in genome-wide genetic diversity, with some instances where nonsynonymous alleles experienced a transient rise in abundance before being displaced by the ancestral allele. In agreement with previous studies, viral VPg protein has been shown as a key player in the adaptation process, even though no obvious association between fixed alleles and host genotype was found.

On the early identification and characterization of pear blister canker viroid, apple dimple fruit viroid, peach latent mosaic viroid and chrysanthemum chlorotic mottle viroid.

In the 90's, pear blister canker viroid (PBCVd), apple dimple fruit viroid (ADFVd), peach latent mosaic viroid (PLMVd) and chrysanthemum chlorotic mottle viroid (CChMVd) were identified and characterized in the Ricardo Flores' laboratory. In these studies, the autonomous replication of these infectious RNAs and their involvement in the elicitation of diseases in their natural hosts were also shown. Their discovery was achieved by classical approaches based on the physical purification of the viroid RNAs from polyacrylamide gels followed by the sequencing of their genomic RNAs and by bioassays to assess their autonomous replication and the fulfillment of Koch's postulates. The molecular characterization of these four viroids, including the study of their sequence variability, contributed to the establishment of the concept of quasispecies for viroids and to the development of reliable molecular diagnostic methods that have facilitated the control of the diseases they caused. Most importantly, some of these viroids became valuable experimental model systems that are still used nowadays to study structural-functional relationships in RNAs and to dissect evolutionary and pathogenic pathways underlying plant-viroid interaction. The differences between early viroid discovery strategies, relying on biological and pathogenic issues, and the current high-throughput sequencing-based approaches, that frequently allow the discovery of new viroids and viroid-like RNAs in symptomless hosts, is also discussed, clarifying why the traditional molecular and biological studies mentioned above are still required to conclusively define the nature of any novel viroid-like RNA.

Defects in plant immunity modulate the rates and patterns of RNA virus evolution.

It is assumed that host genetic variability for susceptibility to infection conditions virus evolution. Differences in host susceptibility can drive a virus to diversify into strains that track different defense alleles (e.g. antigenic diversity) or to infect only the most susceptible genotypes. Here, we have studied how variability in host defenses determines the evolutionary fate of a plant RNA virus. We performed evolution experiments with Turnip mosaic potyvirus in Arabidopsis thaliana mutants that had disruptions in infection-response signaling pathways or in genes whose products are essential for potyvirus infection. Plant genotypes were classified into five phenogroups according to their response to infection. We found that evolution proceeded faster in more restrictive hosts than in more permissive ones. Most of the phenotypic differences shown by the ancestral virus across host genotypes were removed after evolution, suggesting the combined action of selection and chance. When all evolved viral lineages were tested in all plant genotypes used in the experiments, we found compelling evidences that the most restrictive plant genotypes selected for more generalist viruses, while more permissive genotypes selected for more specialist viruses. Sequencing the genomes of the evolved viral lineages, we found that selection targeted the multifunctional genome-linked protein VPg in most host genotypes. Overall, this work illustrates how different host defenses modulate the rates and extent of virus evolution.

Molecular signatures of silencing suppression degeneracy from a complex RNA virus.

As genomic architectures become more complex, they begin to accumulate degenerate and redundant elements. However, analyses of the molecular mechanisms underlying these genetic architecture features remain scarce, especially in compact but sufficiently complex genomes. In the present study, we followed a proteomic approach together with a computational network analysis to reveal molecular signatures of protein function degeneracy from a plant virus (as virus-host protein-protein interactions). We employed affinity purification coupled to mass spectrometry to detect several host factors interacting with two proteins of Citrus tristeza virus (p20 and p25) that are known to function as RNA silencing suppressors, using an experimental system of transient expression in a model plant. The study was expanded by considering two different isolates of the virus, and some key interactions were confirmed by bimolecular fluorescence complementation assays. We found that p20 and p25 target a common set of plant proteins including chloroplastic proteins and translation factors. Moreover, we noted that even specific targets of each viral protein overlap in function. Notably, we identified argonaute proteins (key players in RNA silencing) as reliable targets of p20. Furthermore, we found that these viral proteins preferentially do not target hubs in the host protein interactome, but elements that can transfer information by bridging different parts of the interactome. Overall, our results demonstrate that two distinct proteins encoded in the same viral genome that overlap in function also overlap in their interactions with the cell proteome, thereby highlighting an overlooked connection from a degenerate viral system.

Viral Fitness Determines the Magnitude of Transcriptomic and Epigenomic Reprograming of Defense Responses in Plants.

Although epigenetic factors may influence the expression of defense genes in plants, their role in antiviral responses and the impact of viral adaptation and evolution in shaping these interactions are still poorly explored. We used two isolates of turnip mosaic potyvirus with varying degrees of adaptation to Arabidopsis thaliana to address these issues. One of the isolates was experimentally evolved in the plant and presented increased load and virulence relative to the ancestral isolate. The magnitude of the transcriptomic responses was larger for the evolved isolate and indicated a role of innate immunity systems triggered by molecular patterns and effectors in the infection process. Several transposable elements located in different chromatin contexts and epigenetic-related genes were also affected. Correspondingly, mutant plants having loss or gain of repressive marks were, respectively, more tolerant and susceptible to turnip mosaic potyvirus, with a more efficient response against the ancestral isolate. In wild-type plants, both isolates induced similar levels of cytosine methylation changes, including in and around transposable elements and stress-related genes. Results collectively suggested that apart from RNA silencing and basal immunity systems, DNA methylation and histone modification pathways may also be required for mounting proper antiviral defenses and that the effectiveness of this type of regulation strongly depends on the degree of viral adaptation to the host.

Viroid Diseases in Pome and Stone Fruit Trees and Koch's Postulates: A Critical Assessment.

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.

Engineered Functional Redundancy Relaxes Selective Constraints upon Endogenous Genes in Viral RNA Genomes.

Functional redundancy, understood as the functional overlap of different genes, is a double-edge sword. At the one side, it is thought to serve as a robustness mechanism that buffers the deleterious effect of mutations hitting one of the redundant copies, thus resulting in pseudogenization. At the other side, it is considered as a source of genetic and functional innovation. In any case, genetically redundant genes are expected to show an acceleration in the rate of molecular evolution. Here, we tackle the role of functional redundancy in viral RNA genomes. To this end, we have evaluated the rates of compensatory evolution for deleterious mutations affecting an essential function, the suppression of RNA silencing plant defense, of tobacco etch potyvirus (TEV). TEV genotypes containing deleterious mutations in presence/absence of engineered functional redundancy were evolved and the pattern of fitness and pathogenicity recovery evaluated. Genetically redundant genotypes suffered less from the effect of deleterious mutations and showed relatively minor changes in fitness and pathogenicity. By contrast, nongenetically redundant genotypes had very low fitness and pathogenicity at the beginning of the evolution experiment that were fully recovered by the end. At the molecular level, the outcome depended on the combination of the actual mutations being compensated and the presence/absence of functional redundancy. Reversions to wild-type alleles were the norm in the nonredundant genotypes while redundant ones either did not fix any mutation at all or showed a higher nonsynonymous mutational load.

Viral Fitness Correlates with the Magnitude and Direction of the Perturbation Induced in the Host's Transcriptome: The Tobacco Etch Potyvirus-Tobacco Case Study.

Determining the fitness of viral genotypes has become a standard practice in virology as it is essential to evaluate their evolutionary potential. Darwinian fitness, defined as the advantage of a given genotype with respect to a reference one, is a complex property that captures, in a single figure, differences in performance at every stage of viral infection. To what extent does viral fitness result from specific molecular interactions with host factors and regulatory networks during infection? Can we identify host genes in functional classes whose expression depends on viral fitness? Here, we compared the transcriptomes of tobacco plants infected with seven genotypes of tobacco etch potyvirus that differ in fitness. We found that the larger the fitness differences among genotypes, the more dissimilar the transcriptomic profiles are. Consistently, two different mutations, one in the viral RNA polymerase and another in the viral suppressor of RNA silencing, resulted in significantly similar gene expression profiles. Moreover, we identified host genes whose expression showed a significant correlation, positive or negative, with the virus' fitness. Differentially expressed genes which were positively correlated with viral fitness activate hormone- and RNA silencing-mediated pathways of plant defense. In contrast, those that were negatively correlated with fitness affect metabolism, reducing growth, and development. Overall, these results reveal the high information content of viral fitness and suggest its potential use to predict differences in genomic profiles of infected hosts.

2b or Not 2b: Experimental Evolution of Functional Exogenous Sequences in a Plant RNA Virus.

Horizontal gene transfer (HGT) is pervasive in viruses and thought to be a key mechanism in their evolution. On the other hand, strong selective constraints against increasing genome size are an impediment for HGT, rapidly purging horizontally transferred sequences and thereby potentially hindering evolutionary innovation. Here, we explore experimentally the evolutionary fate of viruses with simulated HGT events, using the plant RNA virus Tobacco etch virus (TEV), by separately introducing two functional, exogenous sequences to its genome. One of the events simulates the acquisition of a new function though HGT of a conserved AlkB domain, responsible for the repair of alkylation or methylation damage in many organisms. The other event simulates the acquisition of a sequence that duplicates an existing function, through HGT of the 2b RNA silencing suppressor from Cucumber mosaic virus. We then evolved these two viruses, tracked the maintenance of the horizontally transferred sequences over time, and for the final virus populations, sequenced their genome and measured viral fitness. We found that the AlkB domain was rapidly purged from the TEV genome, restoring fitness to wild-type levels. Conversely, the 2b gene was stably maintained and did not have a major impact on viral fitness. Moreover, we found that 2b is functional in TEV, as it provides a replicative advantage when the RNA silencing suppression domain of HC-Pro is mutated. These observations suggest a potentially interesting role for HGT of short functional sequences in ameliorating evolutionary constraints on viruses, through the duplication of functions.

Molecular and biological characterization of highly infectious transcripts from full-length cDNA clones of broad bean wilt virus 1.

Broad bean wilt virus 1 (BBWV-1), genus Fabavirus, has a genome composed of two single-stranded positive-sense RNAs of ∼5.8 (RNA1) and 3.4kb (RNA2). Full-length cDNA clones of both genomic RNAs (pBenR1 and pBenR2) from BBWV-1 isolate Ben were constructed under the control of the T7 promoter. In vitro derived capped transcripts were infectious in Nicotiana benthamiana, Chenopodium quinoa and Vicia faba plants. The biological activity of viral transcripts was not affected by extra bases at the 5'-terminus introduced during in vitro transcription. Virions derived from the infectious cDNA clones displayed similar viral infectivity and accumulation, as well as symptom induction as the wild-type BBWV-1 isolate.

A genetic system for Citrus Tristeza Virus using the non-natural host Nicotiana benthamiana: an update.

In nature Citrus tristeza virus (CTV), genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumors were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumors, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests (1) strong silencing or CTV RNA processing in transformed cells impairing infection progress, and (2) the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and reliable system.

Agroinoculation of Citrus tristeza virus causes systemic infection and symptoms in the presumed nonhost Nicotiana benthamiana.

Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus.

Detection and quantitation of Citrus leaf blotch virus by TaqMan real-time RT-PCR.

A real-time RT-PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of Citrus leaf blotch virus (CLBV) in citrus plants. Detection by this method was highly specific and about one thousand times more sensitive than detection by conventional RT-PCR. An external standard curve using in vitro synthesized RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range (seven logarithmic units of concentration) and very low variation coefficient values. This protocol enabled detection of as little as 100 copies of CLBV RNA in various tissues and citrus varieties infected with CLBV sources from different geographical origins. The new assay greatly improves current detection methods for CLBV and it has been most helpful for the Spanish citrus sanitation, quarantine and certification programs, and fitness evaluation of infectious cDNA clones of CLBV, useful potentially as viral vectors for citrus.

Development of a full-genome cDNA clone of Citrus leaf blotch virus and infection of citrus plants.

Citrus leaf blotch virus (CLBV), a member of the family Flexiviridae, has a ~9-kb single-stranded, positive-sense genomic RNA encapsidated by a 41-kDa coat protein. CLBV isolates are associated with symptom production in citrus including leaf blotching of Dweet tangor and stem pitting in Etrog citron (Dweet mottle disease), and some isolates are associated with bud union crease on trifoliate rootstocks, but Koch's postulates for this virus were not fulfilled. A full-genome cDNA of CLBV isolate SRA-153, which induces bud union crease, was placed under the T7 promoter (clone T7-CLBV), or between the 35S promoter and the Nos-t terminator, with or without a ribozyme sequence downstream of the CLBV sequence (clones 35SRbz-CLBV and 35S-CLBV). RNA transcripts from T7-CLBV failed to infect Etrog citron and Nicotiana occidentalis and N. benthamiana plants, whereas agro-inoculation with binary vectors carrying 35SRbz-CLBV or 35S-CLBV, and the p19 silencing suppressor, caused systemic infection and production of normal CLBV virions. Virus accumulation was similar in citron plants directly agro-infiltrated, or mechanically inoculated with wild-type or 35SRbz-CLBV-derived virions from Nicotiana, and the three sources incited the symptoms characteristic of Dweet mottle disease, but not bud union crease. Our results show that (1) virions derived from an infectious clone show the same replication, movement and pathogenicity characteristics as the wild-type CLBV; (2) CLBV is the causal agent of Dweet mottle disease but not of the bud union crease syndrome; and (3) for the first time an RNA virus could be successfully agro-inoculated on citrus plants. This infectious clone may become a useful viral vector for citrus genomic studies.

Citrus tristeza virus: a pathogen that changed the course of the citrus industry.

Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) is the causal agent of devastating epidemics that changed the course of the citrus industry. Adapted to replicate in phloem cells of a few species within the family Rutaceae and to transmission by a few aphid species, CTV and citrus probably coevolved for centuries at the site of origin of citrus plants. CTV dispersal to other regions and its interaction with new scion varieties and rootstock combinations resulted in three distinct syndromes named tristeza, stem pitting and seedling yellows. The first, inciting decline of varieties propagated on sour orange, has forced the rebuilding of many citrus industries using tristeza-tolerant rootstocks. The second, inducing stunting, stem pitting and low bearing of some varieties, causes economic losses in an increasing number of countries. The third is usually observed by biological indexing, but rarely in the field. CTV polar virions are composed of two capsid proteins and a single-stranded, positive-sense genomic RNA (gRNA) of approximately 20 kb, containing 12 open reading frames (ORFs) and two untranslated regions (UTRs). ORFs 1a and 1b, encoding proteins of the replicase complex, are directly translated from the gRNA, and together with the 5' and 3'UTRs are the only regions required for RNA replication. The remaining ORFs, expressed via 3'-coterminal subgenomic RNAs, encode proteins required for virion assembly and movement (p6, p65, p61, p27 and p25), asymmetrical accumulation of positive and negative strands during RNA replication (p23), or suppression of post-transcriptional gene silencing (p25, p20 and p23), with the role of proteins p33, p18 and p13 as yet unknown. Analysis of genetic variation in CTV isolates revealed (1) conservation of genomes in distant geographical regions, with a limited repertoire of genotypes, (2) uneven distribution of variation along the gRNA, (3) frequent recombination events and (4) different selection pressures shaping CTV populations. Measures to control CTV damage include quarantine and budwood certification programmes, elimination of infected trees, use of tristeza-tolerant rootstocks, or cross protection with mild isolates, depending on CTV incidence and on the virus strains and host varieties predominant in each region. Incorporating resistance genes into commercial varieties by conventional breeding is presently unfeasible, whereas incorporation of pathogen-derived resistance by plant transformation has yielded variable results, indicating that the CTV-citrus interaction may be more specific and complex than initially thought. A deep understanding of the interactions between viral proteins and host and vector factors will be necessary to develop reliable and sound control measures.

A real-time RT-PCR assay for detection and absolute quantitation of Citrus tristeza virus in different plant tissues.

A real-time RT-PCR assay using SYBR Green was developed for specific and reliable quantitative detection of Citrus tristeza virus (CTV) in infected plants. A general primer set designed from conserved sequences in ORFs 1b and 2 enabled amplification of the genomic RNA (gRNA) while excluding most subgenomic and defective RNAs. Single RT-PCR products of 204 bp (isolate T36) or 186 bp (other isolates) were obtained with no primer-dimer or non-specific amplifications detected. Melting curve analysis revealed distinct melting temperature peaks (T(m)) for severe and mild isolates. External standard curves using RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) gRNA copies and with very low variation coefficient values. This protocol enabled reliable assessments of CTV accumulation in different tissues and from different citrus species, grown in the greenhouse or under field conditions, and infected with CTV isolates differing in their pathogenicity. CTV accumulation was higher in bark and fruits than in roots or leaves and showed minimal differences among several susceptible citrus species, but it was significantly lower in sour orange. This quantitative detection assay will be a valuable tool for diagnosis and molecular studies on CTV biology.

An element of the tertiary structure of Peach latent mosaic viroid RNA revealed by UV irradiation.

Following UV irradiation, denaturing polyacrylamide gel electrophoresis and Northern blot hybridization revealed a cross-link in Peach latent mosaic viroid (PLMVd) plus-strand RNA. Primer extension and partial alkaline hydrolysis of the UV-irradiated PLMVd plus-strand RNA resulting from the hammerhead-mediated self-cleavage mapped the cross-link at U81 and at the 3'-terminal C289 (or at a very proximal nucleotide). Supporting this notion, in vitro-synthesized PLMVd plus-strand RNAs with short insertions/deletions at their 3' termini failed to cross-link. Because U81 and C289 are conserved in PLMVd variants and because the initiation site of PLMVd minus-strand RNA maps at a short double-stranded motif containing C289, the UV-photo-cross-linkable element of tertiary structure may be functionally significant. A second cross-linked species similar in size and sequence to the monomeric circular PLMVd form, observed in some PLMVd variants, probably derives from UV-induced ligation of the two termini resulting from self-cleavage.

Peach latent mosaic viroid: not so latent.

SUMMARY Taxonomy: Peach latent mosaic viroid (PLMVd) is the type species of the genus Pelamoviroid within the family Avsunviroidae of chloroplastic viroids with hammerhead ribozymes. Physical properties: A small circular RNA of 336-351 nt (differences in size result from the absence or presence of certain insertions) adopting a branched conformation stabilized by a pseudoknot between two kissing loops. This particular conformation is most likely responsible for the insolubility of PLMVd in highly saline conditions (in which other viroids adopting a rod-like conformation are soluble). Both polarity strands are able to form hammerhead structures and to self-cleave during replication as predicted by these ribozymes. Biological properties: Although most infections occur without conspicuous symptoms, certain PLMVd isolates induce leaf mosaics, blotches and in the most extreme cases albinism (peach calico, PC), flower streaking, delays in foliation, flowering and ripening, deformations and decolorations of fruits, which usually present cracked sutures and enlarged roundish stones, bud necrosis, stem pitting and premature ageing of the trees, which also adopt a characteristic growing pattern (open habit). The molecular determinant for PC has been mapped at a 12-14-nt insertion that folds into a hairpin capped by a U-rich loop present only in certain variants. PLMVd is horizontally transmitted by the propagation of infected buds and to a lesser extent by pruning tools and aphids, but not by pollen; the viroid is not vertically transmitted through seed. Interesting features: This provides a suitable system for studying how a minimal non-protein-coding catalytic RNA replicates (subverting a DNA-dependent RNA polymerase to transcribe an RNA template), moves, interferes with the metabolism of its host (inciting specific symptoms and a defensive RNA silencing response) and evolves following a quasi-species model characterized by a complex spectrum of variants.