Kinetic Modeling of Solketal Synthesis from Glycerol and Acetone Catalyzed by an Iron(III) Complex

In the last few years, the depletion of the fossil sources and their negative effect on the environment has led to find new alternatives; among these, biodiesel is considered one of the most promising for this purpose. Biodiesel can be produced from the transesterification of vegetable oils or animal fats, obtaining glycerol as a by-product. Glycerol can be used in different processes and one of the most interesting is the condensation with acetone to produce solketal. Among its applications, plasticizers, solvents, and pharmaceutical formulations are the most common. In this work, the attention was focused on the reaction between glycerol and acetone to give solketal promoted by an iron(III) complex. The reaction mechanism was hypothesized, and the kinetics was studied in a batch reactor. Finally, the thermodynamic and kinetic parameters were determined with a reliable model investigating the phenomena that occurred in the reaction network. Keywords

Viroids: From Genotype to Phenotype Just Relying on RNA Sequence and Structural Motifs

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.Research in Ricardo FLores laboratory is presently supported by grant BFU2011-28443 from the Ministerio de Educación 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.Peer reviewedPeer Reviewe

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). Nucleotide sequence and secondary structure of apple scar skin viroid. Nucleic Acids Research, 15(17), 7045-7052. doi:10.1093/nar/15.17.7045Zhu, S. F., Hadidi, A., & Hammond, R. W. (1998). AGROINFECTION OF PEAR AND APPLE WITH DAPPLE APPLE VIROID RESULTS IN SYSTEMIC INFECTION. Acta Horticulturae, (472), 613-616. doi:10.17660/actahortic.1998.472.81OSAKI, H., KUDO, A., & OHTSU, Y. (1996). Japanese Pear Fruit Dimple Disease Caused by Apple Scar Skin Viroid (ASSVd). Japanese Journal of Phytopathology, 62(4), 379-385. doi:10.3186/jjphytopath.62.379Ito, T., & Yoshida, K. (1998). REPRODUCTION OF APPLE FRUIT CRINKLE DISEASE SYMPTOMS BY APPLE FRUIT CRINKLE VIROID. Acta Horticulturae, (472), 587-594. doi:10.17660/actahortic.1998.472.78Hadidi, A., & Yang, X. (1990). Detection of pome fruit viroids by enzymatic cDNA amplification. Journal of Virological Methods, 30(3), 261-269. doi:10.1016/0166-0934(90)90068-qKyriakopoulou, P. E., & Hadidi, A. (1998). 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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. Acta Horticulturae, (67), 249-254. doi:10.17660/actahortic.1976.67.31Desvignes, J. C., Grasseau, N., Boyé, R., Cornaggia, D., Aparicio, F., Di Serio, F., & Flores, R. (1999). Biological Properties of Apple Scar Skin Viroid: Isolates, Host Range, Different Sensitivity of Apple Cultivars, Elimination, and Natural Transmission. Plant Disease, 83(8), 768-772. doi:10.1094/pdis.1999.83.8.768Walia, Y., Dhir, S., Bhadoria, S., Hallan, V., & Zaidi, A. A. (2011). Molecular characterization of Apple scar skin viroid from Himalayan wild cherry. Forest Pathology, 42(1), 84-87. doi:10.1111/j.1439-0329.2011.00723.xDi Serio, F., Aparicio, F., Alioto, D., Ragozzino, A., & Flores, R. (1996). Identification and molecular properties of a 306 nucleotide viroid associated with apple dimple fruit disease. Journal of General Virology, 77(11), 2833-2837. doi:10.1099/0022-1317-77-11-2833Di Serio, F., Giunchedi, L., Alioto, D., Ragozzino, A., & Flores, R. (1998). IDENTIFICATION OF APPLE DIMPLE FRUIT VIROID IN DIFFERENT COMMERCIAL VARIETIES OF APPLE GROWN IN ITALY. Acta Horticulturae, (472), 595-602. doi:10.17660/actahortic.1998.472.79Roumi, V., Gazel, M., & Caglayan, K. (2017). First report of Apple dimple fruit viroid in apple trees in Iran. New Disease Reports, 35, 3. doi:10.5197/j.2044-0588.2017.035.003He, Y.-H., Isono, S., Kawaguchi-Ito, Y., Taneda, A., Kondo, K., Iijima, A., … Sano, T. (2010). Characterization of a new Apple dimple fruit viroid variant that causes yellow dimple fruit formation in ‘Fuji’ apple trees. Journal of General Plant Pathology, 76(5), 324-330. doi:10.1007/s10327-010-0258-xChiumenti, M., Torchetti, E. M., Di Serio, F., & Minafra, A. (2014). Identification and characterization of a viroid resembling apple dimple fruit viroid in fig (Ficus carica L.) by next generation sequencing of small RNAs. Virus Research, 188, 54-59. doi:10.1016/j.virusres.2014.03.026ITO, T., KANEMATSU, S., KOGANEZAWA, H., TSUCHIZAKI, T., & YOSHIDA, K. (1993). Detection of a Viroid Associated with Apple Fruit Crinkle Disease. Japanese Journal of Phytopathology, 59(5), 520-527. doi:10.3186/jjphytopath.59.520Sano, T., Yoshida, H., Goshono, M., Monma, T., Kawasaki, H., & Ishizaki, K. (2004). Characterization of a new viroid strain from hops: evidence for viroid speciation by isolation in different host species. Journal of General Plant Pathology, 70(3), 181-187. doi:10.1007/s10327-004-0105-zNakaune, R., & Nakano, M. (2008). Identification of a new Apscaviroid from Japanese persimmon. Archives of Virology, 153(5), 969-972. doi:10.1007/s00705-008-0073-2Hernandez, C., Elena, S. F., Moya, A., & Flores, R. (1992). Pear Blister Canker Viroid is a Member of the Apple Scar Skin Subgroup (apscaviroids) and also has Sequence Homology with Viroids from other Subgroups. Journal of General Virology, 73(10), 2503-2507. doi:10.1099/0022-1317-73-10-2503Lemoine, J. (1986). PROBLEMS REGARDING THE DETECTION OF GRAFT TRANSMITTED PEAR CANKER. Acta Horticulturae, (193), 251-260. doi:10.17660/actahortic.1986.193.43Ambrós, S., Llácer, G., Desvignes, J. C., & Flores, R. (1995). PEACH LATENT MOSAIC AND PEAR BLISTER CANKER VIROIDS: DETECTION BY MOLECULAR HYBRIDIZATION AND RELATIONSHIPS WITH SPECIFIC MALADIES AFFECTING PEACH AND PEAR TREES. Acta Horticulturae, (386), 515-521. doi:10.17660/actahortic.1995.386.74Flores, R., Hernandez, C., Llacer, G., & Desvignes, J. C. (1991). Identification of a new viroid as the putative causal agent of pear blister canker disease. Journal of General Virology, 72(6), 1199-1204. doi:10.1099/0022-1317-72-6-1199Desvignes, J. C., Cornaggia, D., Grasseau, N., Ambrós, S., & Flores, R. (1999). Pear Blister Canker Viroid: Host Range and Improved Bioassay with Two New Pear Indicators, Fieud 37 and Fieud 110. Plant Disease, 83(5), 419-422. doi:10.1094/pdis.1999.83.5.419SASAKI, M., & SHIKATA, E. (1977). On Some Properties of Hop Stunt Disease Agent, a Viroid. Proceedings of the Japan Academy. Ser. B: Physical and Biological Sciences, 53(3), 109-112. doi:10.2183/pjab.53.109Ohno, T., Takamatsu, N., Meshi, T., & Okada, Y. (1983). Hop stunt viroid: molecular cloning and nucleotide sequence of the complete cDNA copy. Nucleic Acids Research, 11(18), 6185-6197. doi:10.1093/nar/11.18.6185Kofalvi, S. A., Pall√°s, V., Marcos, J. F., Candresse, T., & Ca√±izares, M. C. (1997). Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. Journal of General Virology, 78(12), 3177-3186. doi:10.1099/0022-1317-78-12-3177Amari, K., Gomez, G., Myrta, A., Di Terlizzi, B., & Pallás, V. (2001). The molecular characterization of 16 new sequence variants of Hop stunt viroid reveals the existence of invariable regions and a conserved hammerhead-like structure on the viroid molecule The sequences described in this work have been deposited in the EMBL database and received accession numbers AJ297825 to AJ297840. Journal of General Virology, 82(4), 953-962. doi:10.1099/0022-1317-82-4-953SANO, T., HATAYA, T., TERAI, Y., & SHIKATA, E. (1986). Association of a viroid-like RNA from plum dapple disease occurring in Japan. Proceedings of the Japan Academy. Ser. B: Physical and Biological Sciences, 62(3), 98-101. doi:10.2183/pjab.62.98Hernandez, C., & Flores, R. (1992). Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures. Proceedings of the National Academy of Sciences, 89(9), 3711-3715. doi:10.1073/pnas.89.9.3711Ambros, S. (1998). In vitro and in vivo self-cleavage of a viroid RNA with a mutation in the hammerhead catalytic pocket. Nucleic Acids Research, 26(8), 1877-1883. doi:10.1093/nar/26.8.1877Ambrós, S., Hernández, C., & Flores, R. (1999). Rapid generation of genetic heterogeneity in progenies from individual cDNA clones of peach latent mosaic viroid in its natural host The data reported in this paper are in the EMBL nucleotide sequence database and assigned the accession nos AJ241818–AJ241850. Journal of General Virology, 80(8), 2239-2252. doi:10.1099/0022-1317-80-8-2239Fekih Hassen, I., Massart, S., Motard, J., Roussel, S., Parisi, O., Kummert, J., … Jijakli, M. H. (2007). Molecular features of new Peach Latent Mosaic Viroid variants suggest that recombination may have contributed to the evolution of this infectious RNA. Virology, 360(1), 50-57. doi:10.1016/j.virol.2006.10.021DUBÉ, A., BOLDUC, F., BISAILLON, M., & PERREAULT, J.-P. (2011). Mapping studies of the Peach latent mosaic viroid reveal novel structural features. Molecular Plant Pathology, 12(7), 688-701. doi:10.1111/j.1364-3703.2010.00703.xBussière, F., Ouellet, J., Côté, F., Lévesque, D., & Perreault, J. P. (2000). Mapping in Solution Shows the Peach Latent Mosaic Viroid To Possess a New Pseudoknot in a Complex, Branched Secondary Structure. Journal of Virology, 74(6), 2647-2654. doi:10.1128/jvi.74.6.2647-2654.2000FLORES, R., DELGADO, S., RODIO, M.-E., AMBRÓS, S., HERNÁNDEZ, C., & SERIO, F. D. (2006). Peach latent mosaic viroid: not so latent. Molecular Plant Pathology, 7(4), 209-221. doi:10.1111/j.1364-3703.2006.00332.xDesvignes, J. C. (1976). THE VIRUS DISEASES DETECTED IN GREENHOUSE AND IN FIELD BY THE PEACH SEEDLING GF 305 INDICATOR. Acta Horticulturae, (67), 315-323. doi:10.17660/actahortic.1976.67.41DESVIGNES, J. C. (1986). PEACH LATENT MOSAIC AND ITS RELATION TO PEACH MOSAIC AND PEACH YELLOW MOSAIC VIRUS DISEASES. Acta Horticulturae, (193), 51-58. doi:10.17660/actahortic.1986.193.6Flores, R., & Llácer, G. (1989). ISOLATION OF A VIROID-LIKE RNA ASSOCIATED WITH PEACH LATENT MOSAIC DISEASE. Acta Horticulturae, (235), 325-332. doi:10.17660/actahortic.1989.235.47Rodio, M.-E., Delgado, S., Flores, R., & Serio, F. D. (2006). Variants of Peach latent mosaic viroid inducing peach calico: uneven distribution in infected plants and requirements of the insertion containing the pathogenicity determinant. Journal of General Virology, 87(1), 231-240. doi:10.1099/vir.0.81356-0Rodio, M.-E., Delgado, S., De Stradis, A., Gómez, M.-D., Flores, R., & Di Serio, F. (2007). A Viroid RNA with a Specific Structural Motif Inhibits Chloroplast Development. The Plant Cell, 19(11), 3610-3626. doi:10.1105/tpc.106.049775Navarro, B., Gisel, A., Rodio, M. E., Delgado, S., Flores, R., & Di Serio, F. (2012). Small RNAs containing the pathogenic determinant of a chloroplast-replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing. The Plant Journal, 70(6), 991-1003. doi:10.1111/j.1365-313x.2012.04940.xWang, L., He, Y., Kang, Y., Hong, N., Farooq, A. B. U., Wang, G., & Xu, W. (2013). 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 Research, 177(2), 171-178. doi:10.1016/j.virusres.2013.08.005Zhang, Z., Qi, S., Tang, N., Zhang, X., Chen, S., Zhu, P., … Wu, Q. (2014). Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms. PLoS Pathogens, 10(12), e1004553. doi:10.1371/journal.ppat.1004553Serra, P., Messmer, A., Sanderson, D., James, D., & Flores, 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. 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Identification and characterization of privet leaf blotch-associated virus, a novel idaeovirus

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

Discovery and Survey of a New Mandarivirus Associated with Leaf Yellow Mottle Disease of Citrus in Pakistan.

During biological indexing for viruses in citrus trees, in a collection of Symons sweet orange (SSO) (Citrus sinensis L. Osbeck) graft inoculated with bark tissues of citrus trees from the Punjab Province in Pakistan, several SSO trees exhibited leaf symptoms of vein yellowing and mottle. High-throughput sequencing by Illumina of RNA preparation depleted of ribosomal RNAs from one symptomatic tree, followed by BLAST analyses, allowed identification of a novel virus, tentatively named citrus yellow mottle-associated virus (CiYMaV). Genome features of CiYMaV are typical of members of the genus Mandarivirus (family Alphaflexiviridae). Virus particles with elongated flexuous shape and size resembling those of mandariviruses were observed by transmission electron microscopy. The proteins encoded by CiYMaV share high sequence identity, conserved motifs, and phylogenetic relationships with the corresponding proteins encoded by Indian citrus ringspot virus (ICRSV) and citrus yellow vein clearing virus (CYVCV), the two current members of the genus Mandarivirus. Although CYVCV is the virus most closely related to CiYMaV, the two viruses can be serologically and biologically discriminated from each other. A reverse-transcription PCR method designed to specifically detect CiYMaV revealed high prevalence (62%) of this virus in 120 citrus trees from the Punjab Province, Pakistan, where the novel virus was found mainly in mixed infection with CYVCV and citrus tristeza virus. However, a preliminary survey on samples from 200 citrus trees from the Yunnan Province, China failed to detect CiYMaV in this region, suggesting that the molecular, serological, and biological data provided here are timely and can help to prevent the spread of this virus in citrus-producing countries

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

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A neural network (NN) approach is proposed to combine future infrared (IASI-NG) and microwave (MWS) observations to retrieve cloud liquid and ice water path. The methodology is applied to simulated IASI-NG and MWS observations in the period January–October 2019. IASI-NG and MWS observations are simulated globally at synoptic hours (00:00, 06:00, 12:00, 18:00 UTC) and on a regular spatial grid (0.125° × 0.125°) from ECMWF 5-generation reanalysis (ERA5). The state-of-the-art σ-IASI and RTTOV radiative transfer codes are used to simulate IASI-NG and MWS observations, respectively, from the earth's state vector given by ERA5. A principal component analysis of the simulated IASI-NG observations is performed. Accordingly, a NN is developed to retrieve cloud liquid and ice water path from a combination of 24 MWS channels and 30 IASI-NG PCs. Validation indicates that this combination results in liquid and ice water path retrievals with overall accuracy of 1.85 10 −2 kg/m 2 and 1.18 10 −2 kg/m 2 , respectively, and 0.97 correlation with respect to reference values. The root-mean-square error (RMSE) for CLWP results in about 30% of the mean value (5.91 10 −2 kg/m 2 ) and 22% of the variability (1-sigma). Similarly, the RMSE for CIWP results in about 41% of the mean value (2.91 10 −2 kg/m 2 ) and 22% of the variability. Two more NN are developed, retrieving cloud liquid and ice water path from microwave observations only (24 MWS channels) and infrared observations only (30 IASI-NG PCs), demonstrating quantitatively the advantage of using the combination of infrared and microwave observations with respect to either one alone

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Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21–24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations. To overcome this restraint we have examined by deep sequencing (Solexa-Illumina) and computational approaches the vd-sRNAs accumulating in GF-305 peach seedlings infected by two molecular variants of Peach latent mosaic viroid (PLMVd) inciting peach calico (albinism) and peach mosaic. Our results show in both samples multiple PLMVd-sRNAs, with prevalent 21-nt (+) and (−) RNAs presenting a biased distribution of their 5′ nucleotide, and adopting a hotspot profile along the genomic (+) and (−) RNAs. Dicer-like 4 and 2 (DCL4 and DCL2, respectively), which act hierarchically in antiviral defense, likely also mediate the genesis of the 21- and 22-nt PLMVd-sRNAs. More specifically, because PLMVd replicates in plastids wherein RNA silencing has not been reported, DCL4 and DCL2 should dice the PLMVd genomic RNAs during their cytoplasmic movement or the PLMVd-dsRNAs generated by a cytoplasmic RNA-dependent RNA polymerase (RDR), like RDR6, acting in concert with DCL4 processing. Furthermore, given that vd-sRNAs derived from the 12–14-nt insertion containing the pathogenicity determinant of peach calico are underrepresented, it is unlikely that symptoms may result from the accidental targeting of host mRNAs by vd-sRNAs from this determinant guiding the RNA silencing machinery

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