Highly abundant small interfering RNAs derived from a satellite RNA contribute to symptom attenuation by binding helper virus-encoded RNA silencing suppressors

Research in RF laboratory is currently supported by the Spanish Ministerio de Economıía y Competitividad (grant BFU2014-56812-P)Flores Pedauye, R. (2016). Highly abundant small interfering RNAs derived from a satellite RNA contribute to symptom attenuation by binding helper virus-encoded RNA silencing suppressors. Frontiers in Plant Science. 7(692):1-3. https://doi.org/10.3389/fpls.2016.00692S13769

Apple hammerhead viroid-like RNA is a bona fide viroid: Autonomous replication and structural features support its inclusion as a new member in the genus Pelamoviroid

[EN] Apple hammerhead viroid-like RNA (AHVd RNA) has been reported in different apple cultivars and geographic regions and, considering the presence of hammerhead ribozymes in both polarity strands, suspected to be either a viroid of the family Avsunviroidae or a viroid-like satellite RNA. Here we report that dimeric head-to-tail in vitro transcripts of a 433-nt reference variant of AHVd RNA from cultivar "Pacific Gala" are infectious when mechanically inoculated to apple, thus showing that this RNA is a bona fide viroid for which we have kept the name apple hammerhead viroid (AHVd) until its pathogenicity, if any, is better assessed. By combining thermodynamics-based predictions with co-variation analyses of the natural genetic diversity found in AHVd we have inferred the most likely conformations for both AHVd polarity strands in vivo, with that of the (+) polarity strand being stabilized by a kissing loop-interaction similar to those reported in peach latent mosaic viroid and chrysathemum chlorotic mottle viroid, the two known members of the genus Pelamoviroid (family Avsunviroidae). Therefore, AHVd RNA fulfills the biological and molecular criteria to be allocated to this genus, the members of which, intriguingly, display low global sequence identity but high structural conservation.We wish to express our gratitude to Dr. Marcos de la Pefia for valuable suggestions, to Maria Pedrote for excellent technical assistance, and to Dr. Miguel Cambra for facilitating access to the apple material. This research was partly funded by grant BFU2014-56812-P (to R.F.) from the Ministerio de Economia y Competitividad (MINECO) of Spain (which included a posdoctoral contract for P.S.), and by the Canadian Food Inspection Agency's Plant Research and Strategies Technology Development (TD) Program (to D.J.).Serra Alfonso, P.; Messmer, A.; Sanderson, D.; James, D.; Flores Pedauye, R. (2018). Apple hammerhead viroid-like RNA is a bona fide viroid: Autonomous replication and structural features support its inclusion as a new member in the genus Pelamoviroid. Virus Research. 249:8-15. https://doi.org/10.1016/j.virusres.2018.03.001S81524

Interference between variants of peach latent mosaic viroid reveals novel features of its fitness landscape: implications for detection

[EN] Natural populations of peach latent mosaic viroid (PLMVd) are complex mixtures of variants. During routine testing, TaqMan rtRT-PCR and RNA gel-blot hybridization produced discordant results with some PLMVd isolates. Analysis of the corresponding populations showed that they were exclusively composed of variants (of class II) with a structural domain different from that of the reference and many other variants (of class I) targeted by the TaqMan rtRT-PCR probe. Bioassays in peach revealed that a representative PLMVd variant of class II replicated without symptoms, generated a progeny with low nucleotide diversity, and, intriguingly, outcompeted a representative symptomatic variant of class I when co-inoculated in equimolecular amounts. A number of informative positions associated with the higher fitness of variants of class II have been identified, and novel sets of primers and probes for universal or specific TaqMan rtRT-PCR detection of PLMVd variants have been designed and tested.We thank A. Ahuir for excellent technical assistance and Dr. Francesco Di Serio for suggestions. This work was supported by grant BFU2014-56812-P (to R.F.) from Ministerio de Economia y Competitividad (MINECO) of Spain. P.S. was the recipient of a postdoctoral contract from MINECO and E. Bertolini of an INIA-CCA2011-2016 contract also from MINECO.Serra Alfonso, P.; Bertolini, E.; Martinez, MC.; Cambra, M.; Flores Pedauye, R. (2017). Interference between variants of peach latent mosaic viroid reveals novel features of its fitness landscape: implications for detection. Scientific Reports. 7. https://doi.org/10.1038/srep42825S7Diener, T. O. Discovering viroids - a personal perspective. Nat. Rev. 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Variants of peach latent mosaic viroid inducing peach calico: uneven distribution in infected plants and requirements of the insertion containing the pathogenicity determinant. J. Gen. Virol. 87, 231–240 (2006).Rodio, M. E. et al. A viroid RNA with a specific structural motif inhibits chloroplast development. Plant Cell 19, 3610–3626 (2007).Navarro, B. et al. Small RNAs containing the pathogenic determinant of a chloroplast-replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing. Plant J. 70, 991–1003 (2012).Yazarlou, A., Jafarpour, B., Tarighi, S., Habili, N. & Randles, J. W. New Iranian and Australian peach latent mosaic viroid variants and evidence for rapid sequence evolution. Arch. Virol. 157, 343–347 (2012).Wang, L. P. et al. Virulence determination and molecular features of peach latent mosaic viroid isolates derived from phenotypically different peach leaves: a nucleotide polymorphism in L11 contributes to symptom alteration. Virus Res. 177, 171–178 (2013).Desvignes, J. C. The virus diseases detected in greenhouse and in the field by the peach seedling GF 305 indicator. Acta Hortic. 67, 315–323 (1976).Flores, R. & Llácer, G. Isolation of a viroid-like RNA associated with peach latent mosaic disease. Acta Hortic. 235, 325–332 (1988).Flores, R., Hernández, C., Desvignes, J. C. & Llácer, G. Some properties of the viroid inducing the peach latent mosaic disease. Res. Virol. 141, 109–118 (1990).Ambrós, S., Desvignes, J. C., Llácer, G. & Flores, R. Peach latent mosaic and pear blister canker viroids: detection by molecular hybridization and relationships with specific maladies affecting peach and pear trees. Acta Hortic. 386, 515–521 (1995).Loreti, S., Faggioli, F. & Barba, M. A rapid extraction method to detect peach latent mosaic viroid by molecular hybridization. Acta Hortic. 386, 560–564 (1995).Skrzeczkowski, L. J., Howell, W. E. & Mink, G. I. 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Development of a multiplex TaqMan real-time RT-PCR assay for simultaneous detection of Asian prunus viruses, plum bark necrosis stem pitting associated virus, and peach latent mosaic viroid. Eur. J. Plant Pathol. 137, 797–804 (2013).Zhang, Y. J. et al. A universal oligonucleotide microarray with a minimal number of probes for the detection and identification of viroids at the genus level. PLoS One 8, e64474 (2013).Di Serio, F. et al. Deep sequencing of the small RNAs derived from two symptomatic variants of a chloroplastic viroid: implications for their genesis and for pathogenesis. PLoS One 4, e7539 (2009).Bolduc, F., Hoareau, C., St-Pierre, P. & Perreault, J. P. In-depth sequencing of the siRNAs associated with peach latent mosaic viroid infection. BMC Mol. Biol. 11, 16 (2010).Glouzon, J. P. S., Bolduc, F., Wang, S., Najmanovich, R. J. & Perreault, J. P. Deep-sequencing of the peach latent mosaic viroid reveals new aspects of population heterogeneity. 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Viroid diseases in pome and stone fruit trees and Koch s postulates: a critical assessment

[EN] 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. View Full-TextThis project has received funding from the European Union's Horizon 2020 Research and Innovation Scientific Exchange Program under the Marie Sklodowska-Curie grant agreement No. 734736. This publication reflects only the authors' view. The Agency is not responsible for any use that may be made of the information it contains.Di Serio, F.; Ambros Palaguerri, S.; Sano, T.; Flores Pedauye, R.; Navarro, B. (2018). Viroid diseases in pome and stone fruit trees and Koch s postulates: a critical assessment. Viruses. 10(11). https://doi.org/10.3390/v101106121011Diener, T. O. (1971). Potato spindle tuber «virus». Virology, 45(2), 411-428. doi:10.1016/0042-6822(71)90342-4Flores, R., Minoia, S., Carbonell, A., Gisel, A., Delgado, S., López-Carrasco, A., … Di Serio, F. (2015). Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection. Virus Research, 209, 136-145. doi:10.1016/j.virusres.2015.02.027López-Carrasco, A., & Flores, R. (2016). Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A «naked» rod-like conformation similar but not identical to that observed in vitro. RNA Biology, 14(8), 1046-1054. doi:10.1080/15476286.2016.1223005López-Carrasco, A., & Flores, R. (2017). The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins. Journal of General Virology, 98(7), 1913-1922. doi:10.1099/jgv.0.000846Flores, R., Hernández, C., Alba, A. E. M. de, Daròs, J.-A., & Serio, F. D. (2005). Viroids and Viroid-Host Interactions. Annual Review of Phytopathology, 43(1), 117-139. doi:10.1146/annurev.phyto.43.040204.140243Di Serio, F., Flores, R., Verhoeven, J. T. J., Li, S.-F., Pallás, V., Randles, J. W., … Owens, R. A. (2014). Current status of viroid taxonomy. Archives of Virology, 159(12), 3467-3478. doi:10.1007/s00705-014-2200-6Di Serio, F., Li, S.-F., Matoušek, J., Owens, R. A., Pallás, V., … Randles, J. W. (2018). ICTV Virus Taxonomy Profile: Avsunviroidae. Journal of General Virology, 99(5), 611-612. doi:10.1099/jgv.0.001045Diener, T. O., Smith, D. R., & O’Brien, M. J. (1972). Potato spindle tuber viroid. Virology, 48(3), 844-846. doi:10.1016/0042-6822(72)90166-3Diener, T. O. (1972). Potato spindle tuber viroid. Virology, 50(2), 606-609. doi:10.1016/0042-6822(72)90412-6Semancik, J. S. (1970). Properties of the Infectious Forms of Exocortis Virus of Citrus. Phytopathology, 60(4), 732. doi:10.1094/phyto-60-732Semancik, J. S., Morris, T. J., & Weathers, L. G. (1973). Structure and conformation of low molecular weight pathogenic RNA from exocortis disease. Virology, 53(2), 448-456. doi:10.1016/0042-6822(73)90224-9Bos, L. (1981). Hundred years of Koch’s Postulates and the history of etiology in plant virus research. 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Chrysanthemum chlorotic mottle viroid: Unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes. Proceedings of the National Academy of Sciences, 94(21), 11262-11267. doi:10.1073/pnas.94.21.11262De la Pena, M., Navarro, B., & Flores, R. (1999). Mapping the molecular determinant of pathogenicity in a hammerhead viroid: A tetraloop within the in vivo branched RNA conformation. Proceedings of the National Academy of Sciences, 96(17), 9960-9965. doi:10.1073/pnas.96.17.9960Bellamy, A. R., & Ralph, R. K. (1968). [104] Recovery and purification of nucleic acids by means of cetyltrimethylammonium bromide. Nucleic Acids, Part B, 156-160. doi:10.1016/0076-6879(67)12125-3Codoñer, F. M., Darós, J.-A., Solé, R. V., & Elena, S. F. (2006). The Fittest versus the Flattest: Experimental Confirmation of the Quasispecies Effect with Subviral Pathogens. PLoS Pathogens, 2(12), e136. doi:10.1371/journal.ppat.0020136Hashimoto, J., & Koganezawa, H. (1987). 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NATURAL INFECTION OF WILD AND CULTIVATED PEARS WITH APPLE SCAR SKIN VIROID IN GREECE. Acta Horticulturae, (472), 617-626. doi:10.17660/actahortic.1998.472.82Ambros, S., Desvignes, J. C., Llacer, G., & Flores, R. (1995). Pear blister canker viroid: sequence variability and causal role in pear blister canker disease. Journal of General Virology, 76(10), 2625-2629. doi:10.1099/0022-1317-76-10-2625Sano, T., Hataya, T., Terai, Y., & Shikata, E. (1989). Hop Stunt Viroid Strains from Dapple Fruit Disease of Plum and Peach in Japan. Journal of General Virology, 70(6), 1311-1319. doi:10.1099/0022-1317-70-6-1311Flores, R., Hernández, C., Desvignes, J. C., & Llácer, G. (1990). Some properties of the viroid inducing peach latent mosaic disease. Research in Virology, 141(1), 109-118. doi:10.1016/0923-2516(90)90060-vMalfitano, M., Di Serio, F., Covelli, L., Ragozzino, A., Hernández, C., & Flores, R. (2003). Peach latent mosaic viroid variants inducing peach calico (extreme chlorosis) contain a characteristic insertion that is responsible for this symptomatology. Virology, 313(2), 492-501. doi:10.1016/s0042-6822(03)00315-5Puchta, H., Luckinger, R., Yang, X., Hadidi, A., & S�nger, H. L. (1990). Nucleotide sequence and secondary structure of apple scar skin viroid (ASSVd) from China. Plant Molecular Biology, 14(6), 1065-1067. doi:10.1007/bf00019406KOGANEZAWA, H. (1985). Transmission to apple seedlings of a low molecular weight RNA extracted from apple scar skin diseased trees. Japanese Journal of Phytopathology, 51(2), 176-182. doi:10.3186/jjphytopath.51.176Koganezawa, H. (1986). FURTHER EVIDENCE FOR VIROID ETIOLOGY OF APPLE SCAR SKIN AND DAPPLE APPLE DISEASES. Acta Horticulturae, (193), 29-34. doi:10.17660/actahortic.1986.193.2Yamaguch, A., & Yanase, H. (1976). POSSIBLE RELATIONSHIP BETWEEN THE CAUSAL AGENT OF DAPPLE APPLE AND SCAR SKIN. 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Specific Argonautes Selectively Bind Small RNAs Derived from Potato Spindle Tuber Viroid and Attenuate Viroid Accumulation In Vivo

Research in the laboratory of R. F. is currently funded by grant BFU2011-28443 from the Ministerio de Economia y Competitividad (MINECO, Spain). S.M. has been supported by a fellowship and a pre-doctoral contract from MINECO. Research in the laboratory of B.N. and F.D.S. has been funded by a dedicated grant from the Ministero dell'Economia e Finanze Italiano to the CNR (CISIA; Legge no. 191/2009). Research in the laboratory of J.C.C. was supported by grants from the National Science Foundation (MCB-0956526 and MCB-1231726) and the National Institutes of Health (AI043288)Minoia, S.; Carbonell, A.; Di Serio, F.; Gisel, A.; Carrinton, JC.; Navarro, B.; Flores Pedauye, R. (2014). Specific Argonautes Selectively Bind Small RNAs Derived from Potato Spindle Tuber Viroid and Attenuate Viroid Accumulation In Vivo. Journal of Virology. 88(20):11933-11945. https://doi.org/10.1128/JVI.01404-14S11933119458820Flores, R., Hernández, C., Alba, A. E. M. de, Daròs, J.-A., & Serio, F. D. (2005). 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Symptomatic plant viroid infections in phytopathogenic fungi: a request for a critical reassessment

Serra, P.; Carbonell, A.; Navarro, B.; Gago Zachert, S.; Li, S.; Di Serio, F.; Flores Pedauye, R. (2020). Symptomatic plant viroid infections in phytopathogenic fungi: a request for a critical reassessment. Proceedings of the National Academy of Sciences of the United States of America (Online). 117(19):10126-10128. https://doi.org/10.1073/pnas.1922249117S10126101281171

Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs

[EN] 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 Avsunviroidae 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 Avsunviroidae, 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.We thank Dr. J. A. Daros for critical reading and suggestions, Dr. B. Ding for kindly providing Figures 4 and 5, and Dr. M. de la Pena for kindly providing Figure 6. Research in Ricardo FLores laboratory is presently supported by grant BFU2011-28443 from the Ministerio de Educacion y Ciencia (MEC) of Spain. During this work Pedro Serra has been supported by postdoctoral contracts from the Generalitat Valenciana (APOSTD/2010, program VALi+d) and the MEC (program Juan de la Cierva), and Sofia Minoia by a predoctoral fellowship from the MEC.Flores Pedauye, R.; Serra Alfonso, P.; Minoia, S.; Di Serio, F.; Navarro, B. (2012). Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs. Frontiers in Microbiology. 3:217-1-217-13. https://doi.org/10.3389/fmicb.2012.00217S217-1217-13

e-Book on Closteroviridae

Flores Pedauye, R.; Moreno, P.; Falk, B.; Martelli, GP.; Dawson, WO. (2013). e-Book on Closteroviridae. Frontiers in Microbiology. 4:411-1-411-3. doi:10.3389/fmicb.2013.00411S411-1411-3

Viroid RNA turnover: characterization of the subgenomic RNAs of potato spindle tuber viroid accumulating in infected tissues provides insights into decay pathways operating in vivo

[EN] 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.Ministerio de Economia y Competitividad (MINECO) [BFU2011-28443 to R. F. laboratory]; MINECO [to S.M. and S.D.]. Funding for open access charge: MINECO (BFU2011-28443 to R. F.); Ministero dell'Economia e Finanze Italiano to the CNR (CISIA, Legge 191/2009 to B.N. and F.D.S.).Minoia, S.; Navarro, B.; Delgado Villar, SG.; Di Serio, F.; Flores Pedauye, R. (2015). Viroid RNA turnover: characterization of the subgenomic RNAs of potato spindle tuber viroid accumulating in infected tissues provides insights into decay pathways operating in vivo. Nucleic Acids Research. 43(4):2313-2325. https://doi.org/10.1093/nar/gkv034S2313232543

Cytopathic effects incited by viroid RNAs and putative underlying mechanisms

[EN] 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.Research in Beatriz Navarro and Francesco Di Serio laboratory is supported by a dedicated grant (CISIA) of the Ministero dell'Economia e Finanze Italiano to the CNR (Legge n. 191/2009). Research in Ricardo Flores laboratory is presently supported by grant BFU2011-28443 from the Ministerio de Ciencia e Innovacion of Spain. We apologize for not quoting the original work of many authors due to space limitations.Di Serio, F.; De Stradis, A.; Delgado Villar, SG.; Flores Pedauye, R.; Navarro, B. (2013). Cytopathic effects incited by viroid RNAs and putative underlying mechanisms. Frontiers in Plant Science. 3:288-1-288-7. https://doi.org/10.3389/fpls.2012.00288288-1288-7