New Insights into the Nucleolar Localization of a Plant RNA Virus-Encoded Protein That Acts in Both RNA Packaging and RNA Silencing Suppression: Involvement of Importins Alpha and Relevance for Viral Infection

[EN] Despite the fact that replication of plus-strand RNA viruses takes place in the cytoplasm of host cells, different proteins encoded by these infectious agents have been shown to localize in the nucleus, with high accumulation at the nucleolus. In most cases, the molecular determinants or biological significance of such subcellular localization remains elusive. Recently, we reported that protein p37 encoded by Pelargonium line pattern virus (family Tombusviridae) acts in both RNA packaging and RNA silencing suppression. Consistently with these functions, p37 was detected in the cytoplasm of plant cells, although it was also present in the nucleus and, particularly, in the nucleolus. Here, we searched for further insights into factors influencing p37 nucleolar localization and into its potential relevance for viral infection. Besides mapping the protein region containing the nucleolar localization signal, we have found that p37 interacts with distinct members of the importin alpha familymain cellular transporters for nucleo-cytoplasmic traffic of proteins-and that these interactions are crucial for nucleolar targeting of p37. Impairment of p37 nucleolar localization through downregulation of importin alpha expression resulted in a reduction of viral accumulation, suggesting that sorting of the protein to the major subnuclear compartment is advantageous for the infection process.This work was supported by Ministerio de Economia y Competitividad (MINECO, Spain) Fundo Europeu de Desenvolvimento Regional (FEDER) (grant BFU2015-70261 to C. Hernandez). M. Perez-Canamas was the recipient of a predoctoral contract from MINECO-FEDER.Perez-Cañamas, M.; Hernandez Fort, C. (2018). New Insights into the Nucleolar Localization of a Plant RNA Virus-Encoded Protein That Acts in Both RNA Packaging and RNA Silencing Suppression: Involvement of Importins Alpha and Relevance for Viral Infection. Molecular Plant-Microbe Interactions. 31(11):1134-1144. https://doi.org/10.1094/MPMI-02-18-0050-RS11341144311

Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus

[EN] Replication of all positive RNA viruses occurs in association with intracellular membranes. In many cases, the mechanism of membrane targeting is unknown and there appears to be no correlation between virus phylogeny and the membrane systems recruited for replication. Pelargonium flower break virus (PFBV, genus Carmovirus, family Tombusviridae) encodes two proteins, p27 and its read-through product p86 (the viral RNA dependent-RNA polymerase), that are essential for replication. Recent reports with other members of the family Tombusviridae have shown that the smaller replicase protein is targeted to specific intracellular membranes and it is assumed to determine the subcellular localization of the replication complex. Using in vivo expression of green fluorescent protein (GFP) fusions in plant and yeast cells, we show here that PFBV p27 localizes in mitochondria. The same localization pattern was found for p86 that contains the p27 sequence at its N-terminus. Cellular fractionation of p27GFP-expressing cells confirmed the confocal microscopy observations and biochemical treatments suggested a tight association of the protein to membranes. Analysis of deletion mutants allowed identification of two regions required for targeting of p27 to mitochondria. These regions mapped toward the N- and C-terminus of the protein, respectively, and could function independently though with distinct efficiency. In an attempt to search for putative cellular factors involved in p27 localization, the subcellular distribution of the protein was checked in a selected series of knockout yeast strains and the outcome of this approach is discussed. (C) 2011 Elsevier B.V. All rights reserved.This research was supported by grant BFU2006-11230 and BFU2009-11699 from MEC and MICINN (Spain), respectively, and by grants ACOM/2006/210 and ACOMP/2009/040 (Generalitat Valenciana, GV) to C.H. S.M.-T. was the recipient of a predoctoral fellowship from GV and of a predoctoral contract from MEC.Martínez Turiño, S.; Hernandez Fort, C. (2012). Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus. Virus Research. 163(2):580-591. https://doi.org/10.1016/j.virusres.2011.12.011S580591163

Epigenetic Changes in Host Ribosomal DNA Promoter Induced by an Asymptomatic Plant Virus Infection

[EN] DNA cytosine methylation is one of the main epigenetic mechanisms in higher eukaryotes and is considered to play a key role in transcriptional gene silencing. In plants, cytosine methylation can occur in all sequence contexts (CG, CHG, and CHH), and its levels are controlled by multiple pathways, including de novo methylation, maintenance methylation, and demethylation. Modulation of DNA methylation represents a potentially robust mechanism to adjust gene expression following exposure to different stresses. However, the potential involvement of epigenetics in plant-virus interactions has been scarcely explored, especially with regard to RNA viruses. Here, we studied the impact of a symptomless viral infection on the epigenetic status of the host genome. We focused our attention on the interaction between Nicotiana benthamiana and Pelargonium line pattern virus (PLPV, family Tombusviridae), and analyzed cytosine methylation in the repetitive genomic element corresponding to ribosomal DNA (rDNA). Through a combination of bisulfite sequencing and RT-qPCR, we obtained data showing that PLPV infection gives rise to a reduction in methylation at CG sites of the rDNA promoter. Such a reduction correlated with an increase and decrease, respectively, in the expression levels of some key demethylases and of MET1, the DNA methyltransferase responsible for the maintenance of CG methylation. Hypomethylation of rDNA promoter was associated with a five-fold augmentation of rRNA precursor levels. The PLPV protein p37, reported as a suppressor of post-transcriptional gene silencing, did not lead to the same effects when expressed alone and, thus, it is unlikely to act as suppressor of transcriptional gene silencing. Collectively, the results suggest that PLPV infection as a whole is able to modulate host transcriptional activity through changes in the cytosine methylation pattern arising from misregulation of methyltransferases/demethylases balance.This work was funded by Ministerio de Economia y Competitividad (MINECO, Spain)-European Regional Development Fund (FEDER) (grants BFU2012-36095 and BFU2015-70261 to C.H) and by the Generalitat Valenciana (GVA, Valencia, Spain) (grant PROMETEO/2019/012 to C.H.). E.H. was the recipient of a contract from MINECO-FEDER and M.P.-C. was the recipient of contracts from MINECO-FEDER and GVA.Pérez-Cañamás, M.; Hevia, E.; Hernandez Fort, C. (2020). Epigenetic Changes in Host Ribosomal DNA Promoter Induced by an Asymptomatic Plant Virus Infection. Biology. 9(5):1-13. https://doi.org/10.3390/biology9050091S11395Wang, A. (2015). Dissecting the Molecular Network of Virus-Plant Interactions: The Complex Roles of Host Factors. 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Nature Reviews Genetics, 15(6), 394-408. doi:10.1038/nrg3683Gong, Z., Morales-Ruiz, T., Ariza, R. R., Roldán-Arjona, T., David, L., & Zhu, J.-K. (2002). ROS1, a Repressor of Transcriptional Gene Silencing in Arabidopsis, Encodes a DNA Glycosylase/Lyase. Cell, 111(6), 803-814. doi:10.1016/s0092-8674(02)01133-9Penterman, J., Zilberman, D., Huh, J. H., Ballinger, T., Henikoff, S., & Fischer, R. L. (2007). DNA demethylation in the Arabidopsis genome. Proceedings of the National Academy of Sciences, 104(16), 6752-6757. doi:10.1073/pnas.0701861104Zhu, J.-K. (2009). Active DNA Demethylation Mediated by DNA Glycosylases. Annual Review of Genetics, 43(1), 143-166. doi:10.1146/annurev-genet-102108-134205Baulcombe, D. C., & Dean, C. (2014). Epigenetic Regulation in Plant Responses to the Environment. Cold Spring Harbor Perspectives in Biology, 6(9), a019471-a019471. doi:10.1101/cshperspect.a019471Ding, B., & Wang, G.-L. (2015). Chromatin versus pathogens: the function of epigenetics in plant immunity. Frontiers in Plant Science, 6. doi:10.3389/fpls.2015.00675Butterbach, P., Verlaan, M. G., Dullemans, A., Lohuis, D., Visser, R. G. F., Bai, Y., & Kormelink, R. (2014). Tomato yellow leaf curl virus resistance by Ty-1 involves increased cytosine methylation of viral genomes and is compromised by cucumber mosaic virus infection. Proceedings of the National Academy of Sciences, 111(35), 12942-12947. doi:10.1073/pnas.1400894111Raja, P., Sanville, B. C., Buchmann, R. C., & Bisaro, D. M. (2008). Viral Genome Methylation as an Epigenetic Defense against Geminiviruses. Journal of Virology, 82(18), 8997-9007. doi:10.1128/jvi.00719-08Yang, L.-P., Fang, Y.-Y., An, C.-P., Dong, L., Zhang, Z.-H., Chen, H., … Guo, H.-S. (2013). C2-mediated decrease in DNA methylation, accumulation of siRNAs, and increase in expression for genes involved in defense pathways in plants infected with beet severe curly top virus. The Plant Journal, 73(6), 910-917. doi:10.1111/tpj.12081Kanazawa, A., Inaba, J., Shimura, H., Otagaki, S., Tsukahara, S., Matsuzawa, A., … Masuta, C. (2010). Virus-mediated efficient induction of epigenetic modifications of endogenous genes with phenotypic changes in plants. The Plant Journal, 65(1), 156-168. doi:10.1111/j.1365-313x.2010.04401.xKon, T., & Yoshikawa, N. (2014). Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing. Frontiers in Microbiology, 5. doi:10.3389/fmicb.2014.00595Otagaki, S., Kawai, M., Masuta, C., & Kanazawa, A. (2011). Size and positional effects of promoter RNA segments on virus-induced RNA-directed DNA methylation and transcriptional gene silencing. Epigenetics, 6(6), 681-691. doi:10.4161/epi.6.6.16214Diezma‐Navas, L., Pérez‐González, A., Artaza, H., Alonso, L., Caro, E., Llave, C., & Ruiz‐Ferrer, V. (2019). Crosstalk between epigenetic silencing and infection by tobacco rattle virus in Arabidopsis. Molecular Plant Pathology, 20(10), 1439-1452. doi:10.1111/mpp.12850Wang, C., Wang, C., Xu, W., Zou, J., Qiu, Y., Kong, J., … Zhu, S. (2018). Epigenetic Changes in the Regulation of Nicotiana tabacum Response to Cucumber Mosaic Virus Infection and Symptom Recovery through Single-Base Resolution Methylomes. Viruses, 10(8), 402. doi:10.3390/v10080402Wang, C., Wang, C., Zou, J., Yang, Y., Li, Z., & Zhu, S. (2019). Epigenetics in the plant–virus interaction. Plant Cell Reports, 38(9), 1031-1038. doi:10.1007/s00299-019-02414-0Scheets, K., Jordan, R., White, K. A., & Hernández, C. (2015). Pelarspovirus, a proposed new genus in the family Tombusviridae. Archives of Virology, 160(9), 2385-2393. doi:10.1007/s00705-015-2500-5Castaño, A., & Hernández, C. (2005). Complete nucleotide sequence and genome organization of Pelargonium line pattern virus and its relationship with the family Tombusviridae. Archives of Virology, 150(5), 949-965. doi:10.1007/s00705-004-0464-yCastaño, A., Ruiz, L., & Hernández, C. (2009). Insights into the translational regulation of biologically active open reading frames of Pelargonium line pattern virus. Virology, 386(2), 417-426. doi:10.1016/j.virol.2009.01.017Pérez-Cañamás, M., & Hernández, C. (2015). Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing. Journal of Biological Chemistry, 290(5), 3106-3120. doi:10.1074/jbc.m114.593707Alonso, M., & Borja, M. (2005). High incidence of Pelargonium line pattern virus infecting asymptomatic Pelargonium spp. in Spain. European Journal of Plant Pathology, 112(2), 95-100. doi:10.1007/s10658-005-0803-1Ivars, P., Alonso, M., Borja, M., & Hernández, C. (2004). Development of a Non-radioactive Dot-blot Hybridisation Assay for the Detection of Pelargonium Flower Break Virus and Pelargonium line Pattern Virus. European Journal of Plant Pathology, 110(3), 275-283. doi:10.1023/b:ejpp.0000019798.87567.22Pérez-Cañamás, M., Blanco-Pérez, M., Forment, J., & Hernández, C. (2017). Nicotiana benthamiana plants asymptomatically infected by Pelargonium line pattern virus show unusually high accumulation of viral small RNAs that is neither associated with DCL induction nor RDR6 activity. Virology, 501, 136-146. doi:10.1016/j.virol.2016.11.018Tucker, S., Vitins, A., & Pikaard, C. S. (2010). Nucleolar dominance and ribosomal RNA gene silencing. Current Opinion in Cell Biology, 22(3), 351-356. doi:10.1016/j.ceb.2010.03.009Blanco-Pérez, M., & Hernández, C. (2016). Evidence supporting a premature termination mechanism for subgenomic RNA transcription in Pelargonium line pattern virus: identification of a critical long-range RNA–RNA interaction and functional variants through mutagenesis. Journal of General Virology, 97(6), 1469-1480. doi:10.1099/jgv.0.000459Pérez-Cañamás, M., & Hernández, C. (2018). New Insights into the Nucleolar Localization of a Plant RNA Virus-Encoded Protein That Acts in Both RNA Packaging and RNA Silencing Suppression: Involvement of Importins Alpha and Relevance for Viral Infection. Molecular Plant-Microbe Interactions®, 31(11), 1134-1144. doi:10.1094/mpmi-02-18-0050-rLi, L.-C., & Dahiya, R. (2002). MethPrimer: designing primers for methylation PCRs. Bioinformatics, 18(11), 1427-1431. doi:10.1093/bioinformatics/18.11.1427Hetzl, J., Foerster, A. M., Raidl, G., & Scheid, O. M. (2007). CyMATE: a new tool for methylation analysis of plant genomic DNA after bisulphite sequencing. The Plant Journal, 51(3), 526-536. doi:10.1111/j.1365-313x.2007.03152.xLiu, D., Shi, L., Han, C., Yu, J., Li, D., & Zhang, Y. (2012). Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR. PLoS ONE, 7(9), e46451. doi:10.1371/journal.pone.0046451McStay, B., & Grummt, I. (2008). The Epigenetics of rRNA Genes: From Molecular to Chromosome Biology. Annual Review of Cell and Developmental Biology, 24(1), 131-157. doi:10.1146/annurev.cellbio.24.110707.175259Pikaard, C. S. (2000). The epigenetics of nucleolar dominance. Trends in Genetics, 16(11), 495-500. doi:10.1016/s0168-9525(00)02113-2Buchmann, R. C., Asad, S., Wolf, J. N., Mohannath, G., & Bisaro, D. M. (2009). Geminivirus AL2 and L2 Proteins Suppress Transcriptional Gene Silencing and Cause Genome-Wide Reductions in Cytosine Methylation. Journal of Virology, 83(10), 5005-5013. doi:10.1128/jvi.01771-08Rodríguez‐Negrete, E., Lozano‐Durán, R., Piedra‐Aguilera, A., Cruzado, L., Bejarano, E. R., & Castillo, A. G. (2013). Geminivirus R ep protein interferes with the plant DNA methylation machinery and suppresses transcriptional gene silencing. New Phytologist, 199(2), 464-475. doi:10.1111/nph.12286Yang, L., Xu, Y., Liu, Y., Meng, D., Jin, T., & Zhou, X. (2016). HC-Pro viral suppressor from tobacco vein banding mosaic virus interferes with DNA methylation and activates the salicylic acid pathway. Virology, 497, 244-250. doi:10.1016/j.virol.2016.07.024Alonso, C., Ramos‐Cruz, D., & Becker, C. (2018). The role of plant epigenetics in biotic interactions. New Phytologist, 221(2), 731-737. doi:10.1111/nph.15408Sáez-Vásquez, J., & Delseny, M. (2019). Ribosome Biogenesis in Plants: From Functional 45S Ribosomal DNA Organization to Ribosome Assembly Factors. The Plant Cell, 31(9), 1945-1967. doi:10.1105/tpc.18.00874Jan, E., Mohr, I., & Walsh, D. (2016). A Cap-to-Tail Guide to mRNA Translation Strategies in Virus-Infected Cells. Annual Review of Virology, 3(1), 283-307. doi:10.1146/annurev-virology-100114-055014Cao, M., Du, P., Wang, X., Yu, Y.-Q., Qiu, Y.-H., Li, W., … Ding, S.-W. (2014). Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs inArabidopsis. Proceedings of the National Academy of Sciences, 111(40), 14613-14618. doi:10.1073/pnas.1407131111Martinez, G., Castellano, M., Tortosa, M., Pallas, V., & Gomez, G. (2013). A pathogenic non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants. Nucleic Acids Research, 42(3), 1553-1562. doi:10.1093/nar/gkt968Csorba, T., Kontra, L., & Burgyán, J. (2015). viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence. Virology, 479-480, 85-103. doi:10.1016/j.virol.2015.02.028Deleris, A., Halter, T., & Navarro, L. (2016). DNA Methylation and Demethylation in Plant Immunity. Annual Review of Phytopathology, 54(1), 579-603. doi:10.1146/annurev-phyto-080615-100308Le, T.-N., Schumann, U., Smith, N. A., Tiwari, S., Au, P. C. K., Zhu, Q.-H., … Wang, M.-B. (2014). DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis. 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Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus

[EN] In plants, RNA silencing functions as a potent antiviral mechanism. Virus-derived double-stranded RNAs (dsRNAs) trigger this mechanism, being cleaved by Dicer-like (DCL) enzymes into virus small RNAs (vsRNAs). These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the Argonaute (AGO) family. Host RNA dependent-RNA polymerases, particularly RDR6, strengthen antiviral silencing by generating more dsRNA templates from RISC-cleavage products that, in turn, are converted into secondary vsRNAs by DCLs. Previous work showed that Pelargonium line pattern virus (PLPV) is a very efficient inducer and target of RNA silencing as PLPV-infected Nicotiana benthamiana plants accumulate extraordinarily high amounts of vsRNAs that, strikingly, are independent of RDR6 activity. Several scenarios may explain these observations including a major contribution of dicing versus slicing for defence against PLPV, as the dicing step would not be affected by the RNA silencing suppressor encoded by the virus, a protein that acts via vsRNA sequestration. Taking advantage of the availability of lines of N. benthamiana with DCL or AGO2 functions impaired, here we have tried to get further insights into the components of the silencing machinery that are involved in anti-PLPV-silencing. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection. Interestingly, AGO2 apparently makes even a higher contribution in the defence against PLPV, extending the number of viruses that are affected by this particular slicer. The data support that both dicing and slicing activities participate in the host race against PLPV.This work was supported by grant BFU2015--70261 from the Ministerio de Economia y Competitividad (MINECO, Spain)--FEDER and PROMETEO/2019/012 from Generalitat Valenciana (GVA) (to C. H). M.P.-C. was the recipient of contracts from MINECO--FEDER and GVA, and E.H. was the recipient of a contract from MINECO--FEDER. K.K. was supported by the grant `Emblematic Action for Research in the Cretan Agrofood sector: Four Institutions, Four References' (AGRO4CRETE -2018S.01300000) held by the General Secretary for Research and Technology of Greece.Pérez-Cañamás, M.; Hevia, E.; Hernandez Fort, C.; Katsarou, K. (2021). Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus. Journal of General Virology. 102(10):1-9. https://doi.org/10.1099/jgv.0.001656191021

Population differentiation and selective constraints in Pelargonium line

[EN] The genomic structure of Pelargonium line pattern virus (PLPV), a tentative member of a proposed new genus within the family Tombusviridae, has been recently determined. However, little is known about the genetic variability and population structure of this pathogen. Here, we have investigated the heterogeneity of PLPV isolates from different origins by sequence analysis of a 1817 nt fragment encompassing the movement (p7 and p9.7) and coat protein genes as well as flanking segments including the complete 3` untranslated region. We have evaluated the selective pressures operating on both viral proteins and RNA genome in order to assess the relative functional and/or structural relevance of different amino acid or nucleotide sites. The results of the study have revealed that distinct protein domains are under different selective constraints and that maintenance of certain primary and/or secondary structures in RNA regulatory sequences might be an important factor limiting viral heterogeneity. We have also performed covariation analyses to uncover potential dependencies among amino acid sites of the same protein or of different proteins. The detection of linked amino acid substitutions has permitted to draw a putative network of intra- and interprotein interactions that are likely required to accomplish the different steps of the infection cycle. Finally, we have obtained phylogenetic trees that support geographical segregation of PLPV sequences. (c) 2010 Elsevier B.V. All rights reserved.We are indebted to Dr. Jan van der Meij (Ball Flora Plant, Chicago) for providing PLPV isolate from USA, and to Dr. Marise Borja (Fundacion Promiva, Madrid) for the Spanish isolates and for valuable comments in the course of this work. We thank to Dr. Selma Gago (IBMCP, Valencia) for critical reading of the manuscript. We are also grateful to Dolores Arocas and Isabella Avellaneda for excellent technical assistance. This research was supported by grants BFU2006-11230 and BFU2009-11699 from Ministerio de Ciencia e Innovacion (MICINN, Spain), ACOM09/040 from Generalitat Valenciana (to C.H.), and by grant BFU2009-06993 (MICINN) (to S.F.E.). A.C. was recipient of predoctoral fellowships from the Generalitat Valenciana and from CSIC-Fundacion Bancaja and L.R. received a postdoctoral contract from the Juan de la Cierva program of MEC.Castaño Sansano, MA.; Ruiz Garcia, ML.; Elena Fito, SF.; Hernandez Fort, C. (2011). Population differentiation and selective constraints in Pelargonium line. Virus Research. 155(1):274-282. doi:10.1016/j.virusres.2010.10.022S274282155

Molecular and biological characterization of an isolate of Tomato mottle mosaic virus (ToMMV) infecting tomato and other experimental hosts in eastern Spain

[EN] Tomato is known to be a natural and experimental reservoir host for many plant viruses. In the last few years a new tobamovirus species, Tomato mottle mosaic virus (ToMMV), has been described infecting tomato and pepper plants in several countries worldwide. Upon observation of symptoms in tomato plants growing in a greenhouse in Valencia, Spain, we aimed to ascertain the etiology of the disease. Using standard molecular techniques, we first detected a positive sense single-stranded RNA virus as the probable causal agent. Next, we amplified and sequenced its full-length genomic RNA which identified the virus as a new ToMMV isolate. Through extensive assays on distinct plant species, we investigated the host range of the Spanish ToMMV isolate. Several plant species were locally and/or systemically infected by the virus, some of which had not been previously reported as ToMMV hosts despite they are commonly used in research greenhouses. Finally, two reliable molecular diagnostic techniques were developed and used to assess the presence of ToMMV. This is the first observation of ToMMV in tomato plants in Europe. We discuss the possibility that, given the high sequence homology between ToMMV and Tomato mosaic virus, the former may have been mistakenly diagnosed as the latter by serological methods.This work was supported by grants BFU2015-70261-P and BFU2015-65037-P (to C.H. and S.F.E., respectively) from Spain Ministry of Economy, Industry and Competitiveness/FEDER.Ambros Palaguerri, S.; Martinez, F.; Ivars, P.; Hernandez Fort, C.; De La Iglesia Jordán, F.; Elena Fito, SF. (2017). Molecular and biological characterization of an isolate of Tomato mottle mosaic virus (ToMMV) infecting tomato and other experimental hosts in eastern Spain. European Journal of Plant Pathology. 149(2):261-268. https://doi.org/10.1007/s10658-017-1180-2S2612681492Fillmer, K., Adkins, S., Pongam, P., & D’Ella, T. (2015). Complete genome sequence of a Tomato mottle mosaic virus isolated from the United States. Genome Announcements, 3(2), e00167–e00115.Hadas, R., Pearlsman, M., Gefen, T., Lachman, O., Hadar, E., Sharabany, G., et al. (2004). Indexing system for Tomato mosaic virus (ToMV) in commercial tomato seed lots. Phytoparasitica, 32(4), 421–424.Lewandowski, D. J., & Dawson, W. O. (1998). Tobamoviruses. In A. Granoff & R. G. Webster (Eds.), Encyclopedia of virology (Vol. 3, 2nd ed., pp. 1780–1783). New York: Academic Press Inc..Li, R., Gao, S., Fel, Z., & Ling, K. (2013). Complete genome sequence of a new Tobamovirus naturally infecting tomatoes in Mexico. Genome Announcements, 1(5), e00794–e00713.Li, Y. Y., Wang, C. L., Xiang, D., Li, R. H., Liu, Y., & Li, F. (2014). First report of Tomato mottle mosaic virus infection of pepper in China. Plant Disease, 98(10), 1447.Martin, D. P., Murrell, B., Golden, M., Khoosal, A., Muhire, B. (2015). RDP4: detection and analysis of recombination patterns in virus genomes. Virus Evolution, 1(1), vev003.Moreira, S. R., Eiras, M., Chaves, A. L. R., Galleti, S. R., & Colariccio, A. (2003). Characterição de uma nova estirpe do Tomato mosaic virus isolada de tomateiro no estado de São Paulo. Fitopatologia Brasileira, 28(6), 602–607.Padmanabhan, C., Zheng, Y., Li, R., Martin, G. B., Fei, Z., & Ling, K. S. (2015). Complete genome sequence of a tomato-infecting Tomato mottle mosaic virus in New York. Genome Announcements, 3(6), e01523–e01515.Pirovano, W., Boetzer, M., Miozzi, L., & Pantaleo, V. (2015). Bioinformatics approaches for viral metagenomics in plants using short RNAs: Model case of study and application to a Cicer arietinum population. Frontiers in Microbiology, 5, 790.Ruiz-Ruiz, S., Moreno, P., Guerri, J., & Ambrós, S. (2006). The complete nucleotide sequence of a severe stem pitting isolate of Citrus tristeza virus from Spain: Comparison with isolates from different origins. Archives of Virology, 151(2), 387398.Salem, N., Mansour, A., Ciuffo, M., Falk, B. W., & Turina, M. (2016). A new tobamovirus infecting tomato crops in Jordan. Archives of Virology, 161(2), 503–506.Soler, S., Prohens, J., López, C., Aramburu, J., Galipienso, L., & Nuez, F. (2010). Viruses infecting tomato in València, Spain: Occurrence, distribution and effect of seed origin. Journal of Phytopathology, 158(11–12), 797–805.Tamura, L., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30(12), 2725–2729.Turina, M., Geraats, B. P. J., & Ciuffo, M. (2016). First report of Tomato mottle mosaic virus in tomato crops in Israel. New Disease Reports, 33, 1.Webster, C. G., Rosskopf, E. N., Lucas, L., Mellinger, H. C., & Adkins, S. (2014). First report of Tomato mottle mosaic virus infecting tomato in the United States. Plant Health Progress. doi: 10.1094/PHP-BR-14-0023

In memoriam of Ricardo Flores: The career, achievements, and legacy of an inspirational plant virologist

[EN] Ricardo Flores (1947-2020) focused his research on the identification, replication, pathogenesis, and evolution of viroids, the minimal non-protein-coding circular RNAs (250-400 nt) able to replicate and incite diseases in plants that are remarkable for being at the lowest step of the biological scale. He and his collaborators initially identified and characterized additional group members, adding six new ones to the family Pospiviroidae, and expanding the Avsunviroidae from one to four members. They showed that members of the second family "encode" ribozymes, a property that, together with others, makes them candidates for being the most primitive replicons that emerged on our planet 3500 million years ago. He also made important contributions regarding how viroids replicate, providing relevant data on the templates, enzymes, and ribozymes that mediate this process and on the mutation rate, which turned out to be the highest reported for any biological entity. More recently, he concentrated on the role that RNA silencing could play on viroid-host interactions, describing details of this process. Ricardo also worked on citrus tristeza virus, a widely different type of subcellular pathogen, and made important contributions on the structure, localization and functions of its unique p23 protein. His research has produced 170 original articles and reviews, according to Web of Science. He encouraged the scientific careers of a large number of researchers, and collaborated with many others, some of whom have recapitulated his scientific legacy in this review and contributed with other chapters in this special issue.This work was supported by the Spanish Agencia Estatal de Investigaci ' on (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), grant number PID2020-115571RB-100. We apologize to colleagues whose work was not cited in this review due to the page limit.Pallás Benet, V.; Hernandez Fort, C.; Marcos, JF.; Daròs, J.; Ambrós, S.; Navarro, B.; Navarro Bohigues, JA.... (2022). In memoriam of Ricardo Flores: The career, achievements, and legacy of an inspirational plant virologist. Virus Research. 312(198718):1-9. https://doi.org/10.1016/j.virusres.2022.1987181931219871

Effectiveness of a strategy that uses educational games to implement clinical practice guidelines among Spanish residents of family and community medicine (e-EDUCAGUIA project):A clinical trial by clusters

This study was funded by the Fondo de Investigaciones Sanitarias FIS Grant Number PI11/0477 ISCIII.-REDISSEC Proyecto RD12/0001/0012 AND FEDER Funding.Background: Clinical practice guidelines (CPGs) have been developed with the aim of helping health professionals, patients, and caregivers make decisions about their health care, using the best available evidence. In many cases, incorporation of these recommendations into clinical practice also implies a need for changes in routine clinical practice. Using educational games as a strategy for implementing recommendations among health professionals has been demonstrated to be effective in some studies; however, evidence is still scarce. The primary objective of this study is to assess the effectiveness of a teaching strategy for the implementation of CPGs using educational games (e-learning EDUCAGUIA) to improve knowledge and skills related to clinical decision-making by residents in family medicine. The primary objective will be evaluated at 1 and 6months after the intervention. The secondary objectives are to identify barriers and facilitators for the use of guidelines by residents of family medicine and to describe the educational strategies used by Spanish teaching units of family and community medicine to encourage implementation of CPGs. Methods/design: We propose a multicenter clinical trial with randomized allocation by clusters of family and community medicine teaching units in Spain. The sample size will be 394 residents (197 in each group), with the teaching units as the randomization unit and the residents comprising the analysis unit. For the intervention, both groups will receive an initial 1-h session on clinical practice guideline use and the usual dissemination strategy by e-mail. The intervention group (e-learning EDUCAGUIA) strategy will consist of educational games with hypothetical clinical scenarios in a virtual environment. The primary outcome will be the score obtained by the residents on evaluation questionnaires for each clinical practice guideline. Other included variables will be the sociodemographic and training variables of the residents and the teaching unit characteristics. The statistical analysis will consist of a descriptive analysis of variables and a baseline comparison of both groups. For the primary outcome analysis, an average score comparison of hypothetical scenario questionnaires between the EDUCAGUIA intervention group and the control group will be performed at 1 and 6months post-intervention, using 95% confidence intervals. A linear multilevel regression will be used to adjust the model. Discussion: The identification of effective teaching strategies will facilitate the incorporation of available knowledge into clinical practice that could eventually improve patient outcomes. The inclusion of information technologies as teaching tools permits greater learning autonomy and allows deeper instructor participation in the monitoring and supervision of residents. The long-term impact of this strategy is unknown; however, because it is aimed at professionals undergoing training and it addresses prevalent health problems, a small effect can be of great relevance. Trial registration: ClinicalTrials.gov: NCT02210442.Publisher PDFPeer reviewe

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

A Viral Suppressor of RNA Silencing May Be Targeting a Plant Defence Pathway Involving Fibrillarin

[EN] To establish productive infections, viruses must be able both to subdue the host metabolism for their own benefit and to counteract host defences. This frequently results in the establishment of viral-host protein-protein interactions that may have either proviral or antiviral functions. The study of such interactions is essential for understanding the virus-host interplay. Plant viruses with RNA genomes are typically translated, replicated, and encapsidated in the cytoplasm of infected cells. Despite this, a significant array of their encoded proteins has been reported to enter the nucleus, often showing high accumulation at subnuclear structures such as the nucleolus and/or Cajal bodies. However, the biological significance of such a distribution pattern is frequently unknown. Here, we explored whether the nucleolar/Cajal body localization of protein p37 of Pelargonium line pattern virus (PLPV, genus Pelarspovirus, family Tombusviridae), might be related to potential interactions with the nucleolar/Cajal body marker proteins, fibrillarin and coilin. The results revealed that p37, which has a dual role as coat protein and as suppressor of RNA silencing, a major antiviral system in plants, is able to associate with these cellular factors. Analysis of (wildtype and/or mutant) PLPV accumulation in plants with up- or downregulated levels of fibrillarin or coilin have suggested that the former might be involved in an as yet unknown antiviral pathway, which may be targeted by p37. The results suggest that the growing number of functions uncovered for fibrillarin can be wider and may prompt future investigations to unveil the plant antiviral responses in which this key nucleolar component may take part.This work was supported by grant BFU2015-70261 from the Ministerio de Economia y Competitividad (MINECO, Spain)-FEDER and PROMETEO/2019/012 from Generalitat Valenciana (GVA) (to C.H). M.P.-C. was the recipient of contracts from MINECO-FEDER and GVA.Perez-Cañamas, M.; Taliansky, M.; Hernandez Fort, C. (2022). A Viral Suppressor of RNA Silencing May Be Targeting a Plant Defence Pathway Involving Fibrillarin. Plants. 11(15):1-14. https://doi.org/10.3390/plants11151903114111