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. Annual Review of Phytopathology, 53(1), 45-66. doi:10.1146/annurev-phyto-080614-120001Garcia‐Ruiz, H. (2019). Host factors against plant viruses. Molecular Plant Pathology, 20(11), 1588-1601. doi:10.1111/mpp.12851Garcia-Ruiz, H. (2018). Susceptibility Genes to Plant Viruses. Viruses, 10(9), 484. doi:10.3390/v10090484Han, G. (2019). Origin and evolution of the plant immune system. New Phytologist, 222(1), 70-83. doi:10.1111/nph.15596García, J. A., & Pallás, V. (2015). Viral factors involved in plant pathogenesis. Current Opinion in Virology, 11, 21-30. doi:10.1016/j.coviro.2015.01.001Whitham, S. A., Yang, C., & Goodin, M. M. (2006). Global Impact: Elucidating Plant Responses to Viral Infection. Molecular Plant-Microbe Interactions®, 19(11), 1207-1215. doi:10.1094/mpmi-19-1207Eichten, S. R., Schmitz, R. J., & Springer, N. M. (2014). Epigenetics: Beyond Chromatin Modifications and Complex Genetic Regulation. Plant Physiology, 165(3), 933-947. doi:10.1104/pp.113.234211Feng, S., Jacobsen, S. E., & Reik, W. (2010). Epigenetic Reprogramming in Plant and Animal Development. Science, 330(6004), 622-627. doi:10.1126/science.1190614Matzke, M. A., Kanno, T., & Matzke, A. J. M. (2015). RNA-Directed DNA Methylation: The Evolution of a Complex Epigenetic Pathway in Flowering Plants. Annual Review of Plant Biology, 66(1), 243-267. doi:10.1146/annurev-arplant-043014-114633Zhang, H., Lang, Z., & Zhu, J.-K. (2018). Dynamics and function of DNA methylation in plants. Nature Reviews Molecular Cell Biology, 19(8), 489-506. doi:10.1038/s41580-018-0016-zMovahedi, A., Sun, W., Zhang, J., Wu, X., Mousavi, M., Mohammadi, K., … Zhuge, Q. (2015). RNA-directed DNA methylation in plants. Plant Cell Reports, 34(11), 1857-1862. doi:10.1007/s00299-015-1839-0Matzke, M. A., & Mosher, R. A. (2014). RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. 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. Genome Biology, 15(9). doi:10.1186/s13059-014-0458-3Yu, A., Lepere, G., Jay, F., Wang, J., Bapaume, L., Wang, Y., … Navarro, L. (2013). Dynamics and biological relevance of DNA demethylation in Arabidopsis antibacterial defense. Proceedings of the National Academy of Sciences, 110(6), 2389-2394. doi:10.1073/pnas.1211757110Palukaitis, P., & García-Arenal, F. (2003). Cucumoviruses. Advances in Virus Research, 241-323. doi:10.1016/s0065-3527(03)62005-1Ratcliff, F., Martin-Hernandez, A. M., & Baulcombe, D. C. (2008). Technical Advance: Tobacco rattle virus as a vector for analysis of gene function by silencing. The Plant Journal, 25(2), 237-245. doi:10.1046/j.0960-7412.2000.00942.

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

Efficient Translation of Pelargonium line pattern virus RNAs Relies on a TED-Like 3 '-Translational Enhancer that Communicates with the Corresponding 5 '-Region through a Long-Distance RNA-RNA Interaction

[EN] Cap-independent translational enhancers (CITEs) have been identified at the 3'-terminal regions of distinct plant positive-strand RNA viruses belonging to families Tombusviridae and Luteoviridae. On the bases of their structural and/or functional requirements, at least six classes of CITEs have been defined whose distribution does not correlate with taxonomy. The so-called TED class has been relatively under-studied and its functionality only confirmed in the case of Satellite tobacco necrosis virus, a parasitic subviral agent. The 3' untranslated region of the monopartite genome of Pelargonium line pattern virus (PLPV), the recommended type member of a tentative new genus (Pelarspovirus) in the family Tombusviridae, was predicted to contain a TED-like CITE. Similar CITEs can be anticipated in some other related viruses though none has been experimentally verified. Here, in the first place, we have performed a reassessment of the structure of the putative PLPV-TED through in silico predictions and in vitro SHAPE analysis with the full-length PLPV genome, which has indicated that the presumed TED element is larger than previously proposed. The extended conformation of the TED is strongly supported by the pattern of natural sequence variation, thus providing comparative structural evidence in support of the structural data obtained by in silico and in vitro approaches. Next, we have obtained experimental evidence demonstrating the in vivo activity of the PLPV-TED in the genomic (g) RNA, and also in the subgenomic (sg) RNA that the virus produces to express 3'-proximal genes. Besides other structural features, the results have highlighted the key role of long-distance kissing-loop interactions between the 3'-CITE and 5'-proximal hairpins for gRNA and sgRNA translation. Bioassays of CITE mutants have confirmed the importance of the identified 5'-3' RNA communication for viral infectivity and, moreover, have underlined the strong evolutionary constraints that may operate on genome stretches with both regulatory and coding functions.This work was supported by grants BFU2009-11699 and BFU2012-36095 from the Ministerio de Investigacion, Ciencia e Innovacion (MICINN, Spain, www.micinn.es) and the Ministerio de Economia y Competitividad (MINECO, Spain, http://www.mineco.gob.es), respectively, and ACOMP/2012/100 from the Generalitat Valenciana (http://www.gva.es) (to C.H.). MBP and LR were the recipients of a predoctoral and postdoctoral (Juan de la Cierva program) contract, respectively, from MICINN, and MPC was the recipient of a predoctoral contract from MINECO.Blanco Pérez, M.; Pérez Cañamás, M.; Ruiz, L.; Hernandez Fort, C. (2016). Efficient Translation of Pelargonium line pattern virus RNAs Relies on a TED-Like 3 '-Translational Enhancer that Communicates with the Corresponding 5 '-Region through a Long-Distance RNA-RNA Interaction. PLoS ONE. 11(4):1-24. https://doi.org/10.1371/journal.pone.0152593S12411

Análisis de la interacción del virus del arabesco del Pelargonium con la ruta de silenciamiento por RNA del huésped

Tesis por compendio[ES] En plantas, el silenciamiento por RNA constituye un potente mecanismo de defensa frente a virus. Los RNA virales de doble cadena activan este tipo de procesos y son digeridos por las enzimas DCL (Dicer-like), cuya acción genera pequeños RNA (sRNA) de entre 20 y 24 nt. Estos sRNA promueven la degradación de RNA de secuencia complementaria a través de un complejo multiproteico conocido como RISC (RNA-induced silencing complex), cuya molécula efectora es una proteína Argonauta (AGO). Con el fin de evadir esta barrera defensiva del huésped, la mayoría de los virus de plantas codifican supresores del silenciamiento por RNA (VSR), cuyos mecanismos de acción son diversos y en muchos casos no se comprenden del todo. Aunque todas las etapas de la ruta de silenciamiento pueden ser inhibidas por los VSR, los sRNA y las proteínas AGO parecen ser las dianas más frecuentes. Se ha postulado que motivos GW/WG podrían ser fundamentales para la actividad de algunos VSR, al intervenir en la interacción con proteínas AGO. En este trabajo se ha pretendido seguir profundizando en el estudio de la respuesta antiviral en plantas y de los mecanismos de acción de los supresores de silenciamiento. El primer objetivo abordado ha sido identificar el VSR codificado por el virus del arabesco del Pelargonium (Pelargonium line pattern virus, PLPV), un miembro del género Pelarspovirus dentro de la amplia familia Tombusviridae. Los resultados han mostrado que la proteína de cubierta del virus (p37) es capaz de inhibir de manera eficiente el silenciamiento inducido por RNA. La generación de un batería de variantes capaces e incapaces de actuar como VSR y/o de empaquetar el RNA viral mediante mutagénesis dirigida de distintos motivos de la proteína, incluido un motivo GW que está conservado en ortólogos, nos han permitido conocer que: (i) tanto la función de supresión del silenciamiento como la función de encapsidación son esenciales para que el PLPV alcance una infección sistémica y (ii) p37, a pesar de contener un motivo GW funcional e interaccionar con distintas AGO, emplea el secuestro de sRNA como estrategia principal para inhibir el silenciamiento. A pesar de que ambas funciones conocidas de p37 deben ser llevadas a cabo esencialmente en el citoplasma, esta proteína se localiza en citoplasma y núcleo, con gran acumulación en nucleolo, por lo que nos planteamos como segundo objetivo en este trabajo profundizar acerca de la localización nucleolar de p37. Además de mapear la región de la proteína que contiene la señal de localización nucleolar (NoLS) en los primeros 45 aminoácidos de la molécula, también hemos observado que p37 interacciona con diferentes miembros de la familia de las importinas ¿, adaptadores moleculares del transporte nucleocitoplasmático, y que esta interacción es esencial para la localización nucleolar de la proteína. Adicionalmente, la anulación de la localización nucleolar de p37 mediante el silenciamiento de importinas ¿ ha correlacionado con una disminución de acumulación del virus, lo que sugiere que dicha localización es ventajosa para la infección viral. Por último, para intentar conocer más datos acerca de las actividades de la ruta de silenciamiento que están implicados en la defensa frente al PLPV, analizamos la infección viral en líneas transgénicas de N. benthamiana con la expresión o actividad distintos componentes de la ruta comprometida. Los resultados han mostrado que DCL4 y, en menor medida, DCL2 afectan a la infección viral y que ambas tienen un efecto aditivo, tal y como se ha descrito en diversas interacciones virus-planta. Adicionalmente, AGO2 se ha revelado como un factor clave en la respuesta frente al PLPV, ampliando el número de virus que están afectados por esta endonucleasa particular. En conjunto, los resultados obtenidos muestran que tanto el procesamiento de dsRNA mediado por enzimas DCL como el corte de RNA mediado AGO, contribuyen a la defen[CA] En plantes, el silenciament per RNA constitueixen un potent mecanisme de defensa davant virus. Els RNA virals de doble cadena activen aquest tipus de processos, i són digerits per els enzims DCL (Dicer-like), donant lloc a xicotets RNA (sRNA) d'entre 20 i 24 nt. Estos sRNA promouen la degradació de RNA de seqüència complementària a través d'un complex multiproteic conegut com RISC (RNA-induced silencing complex), la molècula efectora del qual és una proteïna Argonauta (AGO). Per tal d'evadir aquesta barrera defensiva de l'hoste, la majoria dels virus de plantes codifiquen supressors del silenciament per RNA (VSR), els mecanismes d'acció dels quals són diversos i en molts casos no es comprenen del tot. Encara que totes les etapes de la ruta poden ser inhibides, els sRNA i les proteïnes AGO semblen ser les dianes més freqüents. S'ha postulat que els motius GW/WG podrien ser fonamentals per a l'activitat d'alguns VSR, en intervindre en la interacció amb proteïnes AGO. En aquest treball s'ha pretés continuar aprofundint en l'estudi de la resposta antiviral en plantes i dels mecanismes d'acció dels supressors de silenciament. El primer objectiu abordat ha sigut identificar l'VSR codificat pel virus de l'arabesc del Pelargonium (Pelargonium line pattern virus, PLPV), un membre del gènere Pelarspovirus dins de l'amplia família Tombusviridae. Els resultats han mostrat que la proteïna de coberta del virus (p37) és capaç d'inhibir de manera eficient el silenciament induït per RNA. La generació d'una bateria de variants capaces i incapaces d'actuar com VSR i/o d'empaquetar l'RNA viral mitjançant la mutagènesi dirigida de diferents motius de la proteïna, inclòs un motiu GW que està conservat en ortòlegs, ens ha permés conèixer que: (i) tant la funció de supressió del silenciament com la funció d'encapsidació són essencials per a que el PLPV aconseguisca una infecció sistèmica i (ii) p37, malgrat contindre un motiu GW funcional i interaccionar amb diferents AGO, empra el segrest de sRNA com a estratègia principal per a inhibir el silenciament. Malgrat que ambdues funcions conegudes de p37 han de ser dutes a terme essencialment en el citoplasma, esta proteïna localitza en citoplasma i nucli, amb gran acumulació en nuclèol, per la qual cosa ens hem plantejat com a segon objectiu en este treball aprofundir sobre la localització nucleolar de p37. Ademés de mapejar la senyal de localització nucleolar (NoLS) en els primers 45 aminoàcids de la molècula, també hem observat que p37 interacciona amb diferents membres de la família de les importines ¿, i que aquesta interacció és essencial per a la localització nucleolar de la proteïna. A més, l'anul¿lació de la localització nucleolar de p37 mitjançant el silenciament d'importines ¿ s'ha correlacionat amb una disminució d'acumulació del virus, la qual cosa suggereix que aquesta localització és avantatjosa per a la infecció viral. Finalment, per a intentar conéixer més dades sobre les activitats de la ruta de silenciament que estan implicats en la defensa front al PLPV, hem analitzat la infecció viral en línies transgèniques de N. benthamiana amb l'expressió o activitat diferents components de la ruta compromesa. Els resultats han mostrat que DCL4 i, en menor mesura, DCL2 afecten la infecció viral i que ambdues tenen un efecte additiu, tal com s'ha descrit en diverses interacciones virus-planta. Addicionalment, AGO2 s'ha revelat com un factor clau en la resposta front al PLPV, ampliant el nombre de virus que estan afectats per aquesta endonucleasa particular. En conjunt, els resultats obtinguts mostren que tant el processament de dsRNA mediat per enzims DCL com el tall d'RNA mediat AGO, contribueixen a la defensa de N. benthamiana front al PLPV.[EN] n plants, RNA silencing functions as a potent antiviral mechanism. Viral-derived double-stranded RNAs trigger this type of processes, being digested by DCL (Dicer-like) enzymes into virus-derived small RNAs (vsRNAs) of 20-24 nt. 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. To counteract this host defence response, most plant viruses encode suppressors of RNA silencing (VSRs), whose mechanisms of action are diverse and often not well understood. Though virtually all stages of the antiviral silencing pathway can be blocked by VSRs, sRNAs and AGO proteins seem to be the most common targets. It has been postulated that GW/WG motifs could be fundamental for the activity of some VSRs by directing interaction with AGOs. In this work, we have pursued to get further insights into the antiviral silencing in plants and the mechanisms of action of silencing suppressors. The first objective has been to identify the VSR encoded by Pelargonium line pattern virus (PLPV), a member of the genus Pelarspovirus within family Tombusviridae. The results have shown that the viral coat protein (p37) is able to efficiently inhibit RNA silencing. Generation of suppressor-competent and incompetent molecules and uncoupling of the RSS and particle assembly capacities through site-directed mutagenesis of some p37 sequence traits, including a conserved GW motif, allowed us to know that: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic PLPV infection and, (ii) p37, even though it has a functional GW motif and interacts with different AGOs, inhibits silencing most likely through vsRNA sequestration. Despite both p37 functions have to be executed essentially in the cytoplasm, this protein localizes in cytoplasm and nucleus, with high accumulation at the nucleolus, so the second objective of this work has been to gain further insights into the nucleolar localization of p37. Besides mapping the protein region containing the nucleolar localization signal (NoLS) in the first 45 amino acids of the molecule, we have found that p37 interacts with distinct members of the importin ¿ family, main cellular transporters for nucleo-cytoplasmic traffic of proteins, and that these interactions are crucial for nucleolar targeting of p37. In addition, impairment of p37 nucleolar localization through down-regulation of importin ¿ expression has been correlated with a reduction of viral accumulation, suggesting that sorting of the protein to the major subnuclear compartment is advantageous for the infection process. Finally, in order to obtain information on which activities of RNA silencing pathway are involved in the defense against PLPV, we analyzed the viral infection in N. benthamiana transgenic lines with the functions of distinct components of the pathway impaired. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection and that they have additive effects, in agreement with that observed in other plant-virus interactions. Additionally, AGO2 was found to be a key factor in the defense against PLPV, extending the number of viruses that are affected by this particular slicer. Altogether, the results supported that both dicing and slicing activities participate in the defense of N. benthamiana against PLPV.Pérez Cañamás, M. (2019). Análisis de la interacción del virus del arabesco del Pelargonium con la ruta de silenciamiento por RNA del huésped [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/122297TESISCompendi

Análisis de las interacciones moleculares del supresor del silenciamiento por RNA del virus del arabesco del Pelargonium

[EN] RNA silencing processes play an essential role in plant responses against exogenous nucleic acids such as transgenes, transposons or viruses. Viral-derived double-stranded RNAs (dsRNAs) trigger this type of processes as they are recognized by RNase III-type enzymes, called Dicer-like (DCL), and digested into small RNAs (sRNAs) of 20-24 nt. Then, one strand of those sRNAs is incorporated into an RNA-induced silencing complex (RISC) whose main component is an Argonaute (AGO) protein.. Within the activated RISC, the sRNA act as guide to bring the RISC complex into contact with complementary RNA and thereby cause their degradation. To counteract this host defense barrier, most plant viruses encode suppressors of RNA silencing (VSR). Such VSRs are structurally diverse and their mechanisms of action may differ and are often not well understood. Though virtually all stages of the antiviral silencing pathway can be inhibited by VSRs, sRNAs and AGO proteins seem to be the most common targets. Recently, GW/WG motifs present at the sequence of some VSRs have been proposed to dictate their suppressor activity by directing interaction with AGOs. In this work, we have studied the VSR encoded by Pelargonium line pattern virus (PLPV), a member of the family Tombusviridae that causes frequent infections in geranium. Previous experiments have shown that the viral coat protein of PLPV (CP or p37) acts as a VSR. In order to get insights into the mode of action of this suppressor, different protein motifs have been modified by site directed mutagenesis, including a GW motif conserved in homologous proteins, generating suppressor-competent and incompetent molecules and uncoupling the VSR and particle assembly capacities. The engineered mutants have been used, on the one hand, to assess the importance of the silencing suppression and the encapsidation functions of p37 for viral infection and, on the other hand, to analyze different molecular interactions of p37 (sRNAs binding, dimerization, subcellular localization, association with AGO proteins) in an attempt to establish possible correlations between such interactions and the suppression activity. Two main conclusions can be drawn from this work: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic PLPV infection and, (ii) p37 inhibits silencing most likely through sequestration of sRNAs, preventing the activation of RISC.[ES] Los procesos de silenciamiento por RNA constituyen uno de los principales mecanismos de respuesta de las plantas frente a ácidos nucleicos exógenos, como transgenes, transposones o virus. Este mecanismo es activado por RNAs virales de doble cadena (double stranded RNAs, dsRNAs), producidos en el curso de la infección, que son reconocidos por RNasas III de tipo Dicer, cuya acción genera pequeños RNAs (small RNAs, sRNAs) de entre 20 y 24 nt. A continuación, una de las cadenas de esos sRNAs es incorporada a un complejo multiproteico conocido como RISC (RNA-induced silencing complex), cuyo componente principal es un endonucleasa de la familia de las Argonautas (AGO). Una vez activado, RISC es dirigido por el sRNA asociado hasta RNAs de secuencia complementaria provocando su degradación. Con el fin de evadir esta respuesta defensiva del huésped, los virus codifican proteínas conocidas como supresores del silenciamiento por RNA (viral RNA silencing suppressors, VSRs). Los VSRs son muy diversos en cuanto a tamaño y secuencia, y las bases moleculares de su actividad no se conocen con precisión en muchos casos. Aunque todas las etapas de la ruta de silenciamiento pueden ser inhibidas por los VSR, los sRNAs y las proteínas AGO parecen ser las dianas más frecuentes. Recientemente se ha propuesto que los motivos GW/WG presentes en la secuencia de algunos VSR son los que dictaminan su función supresora por interacción directa con las AGOs. Este trabajo se ha centrado en el estudio del VSR codificado por el virus del arabesco del Pelargonium (Pelargonium line pattern virus, PLPV), un miembro de la familia Tombusviridae que causa infecciones frecuentes en geranio. Experimentos anteriores habían permitido identificar a la proteína de cubierta viral (CP o p37) como el VSR del PLPV. Para tratar de obtener datos acerca del modo de acción de este supresor, se han modificado mediante mutagénesis dirigida distintos motivos de la proteína, incluido un motivo GW que está conservado en proteínas homólogas, y se ha obtenido una batería de moléculas capaces e incapaces de actuar como VSR y/o de empaquetar el RNA viral. Esta batería de moléculas ha permitido, por una parte, evaluar la importancia de las funciones de supresión del silenciamiento y de encapsidación de p37 para la infección viral y, por otra, analizar distintas interacciones moleculares de p37 (unión a sRNAs, dimerización, localización subcelular, asociación con proteínas AGO) y establecer posibles correlaciones entre las mismas y la actividad supresora. Dos conclusiones principales se pueden extraer de este trabajo: (i) tanto la función de supresión del silenciamiento como la función de encapsidación de p37 son necesarias para la infección sistémica del PLPV y, (ii) p37 inhibe el silenciamiento, muy probablemente, a a través del secuestro de sRNAs, impidiendo la activación de RISC.Pérez Cañamás, M. (2014). Análisis de las interacciones moleculares del supresor del silenciamiento por RNA del virus del arabesco del Pelargonium. http://hdl.handle.net/10251/53784Archivo delegad

Effect of mutations in the TED-CITE and/or in a hairpin within p27 ORF on translation of reporter-based genomic PLPV transcripts.

<p>(A) Putative secondary structure of the 5´-proximal 125 nt of PLPV according Mfold predictions. A potential long-range interaction between the apical loop of a hairpin (gHP3) predicted within p27 ORF and the apical loop of the 3´-CITE is shown. Nucleotides that can putatively pair are connected by dotted lines. (B) Translation efficiency of transcripts bearing mutations that disrupted or reconstituted the potential kissing-loop interaction between apical loops of gHP3 and the CITE. All mutants were generated from parental construct gFF and assayed in <i>N</i>. <i>benthamiana</i> protoplasts as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.g005" target="_blank">Fig 5</a> legend. Only the sequences of the loops involved in the potential base-pairing are shown. The engineered nucleotide substitutions are on a gray background and putative G:U base pairs are in italics. (C) Translation efficiency of transcripts bearing mutations in the CITE outside of its apical loop. In B and C, levels of translation as a percentage of that of gFF construct are given. Results are from three experiments with standard deviations. Other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.g005" target="_blank">Fig 5</a>.</p

Examination of the structure of the 3´-terminal region of PLPV through <i>in silico</i>, <i>in vitro</i> and comparative structural analyses.

<p>(A) Secondary structure of the 3´-terminal region of PLPV (bottom). The represented structure embraces the 3´-terminal sequence of the p37 gene and the entire 3´ UTR (arrow), contains several hairpins (HP1 to HP4) and corresponds to the optimal one according to Mfold predictions using the entire PLPV gRNA. Nucleotides likely involved in an interaction that presumably acts as repressor of minus-strand synthesis are within grey circles. Underlined nucleotides are predicted to base-pair with a segment of the gRNA out of the 3´-terminal region. HP4, that might be a CITE of the TED class, shows a SHAPE-derived flexibility profile (autoradiographs at the top) consistent with the Mfold predictions. In autoradiographs of SHAPE analysis, the lanes have been labelled as: N, for the NMIA treated RNA, D, for the DMSO treated RNA, G, C and A, for sequencing ladders. Residues in the putative TED and flanking region with high and medium reactivity to NMIA are denoted with red and green colors, respectively, whereas those with low or no reactivity to the reagent are in black. Bulges (B) and loops (L) of HP4 have been numbered on the secondary structure representation to facilitate comparison with SHAPE profile. The apical loop and a lateral bulge of the upper part of HP4 have been labelled as AL and BG, respectively. The region of HP4 previously proposed as putative TED-like CITE (15) is indicated by a square bracket with discontinuous line at the left side of the structure. (B) SHAPE profiles across a portion of the 3´-terminal region of the PLPV wild-type (wt) gRNA and a mutated (mut) gRNA carrying nucleotide replacements (in italics) in the apical loop of HP4. Strong NMIA reactivity of the apical loop in the mutant is observed when compared with the wt molecule. This result is in agreement with the involvement of the loop in RNA-RNA interaction as previously proposed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.ref015" target="_blank">15</a>]. The lanes have been labelled as in panel A and the sequencing ladders have been performed on the wt template. (C) Distribution of natural sequence heterogeneity along HP4. Twenty natural PLPV variants [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.ref043" target="_blank">43</a>] were included in the analysis. Nucleotide replacements are in blue and the number of variants with a given mutation is shown in parentheses. Co-variations and mutations resulting in conversion of canonical Watson-Crick to wobble base pairs, or vice versa, in stems are boxed. Only two minority mutations affecting residue U3723 (marked by an asterisk), could lead to slight destabilization of a stem.</p

Schematic representation of the PLPV genome.

<p>PLPV gRNA contains five ORFs (represented by grey boxes) flanked by 5´- and 3´- UTRs (striped boxes). The viral RNA dependent-RNA polymerase, p87, is the ribosomal read-through (RT) product of p27, an auxiliary replication protein. PLPV sgRNA is tri-cistronic and serves as mRNA for expression of two small movement proteins, p7 and p9.7, and of protein p37, which functions as coat protein and as the suppressor of RNA silencing.</p

Assessment of infectivity of PLPV mutants.

<p>Uncapped transcripts corresponding to the gRNA of wt and CITE-related mutants of PLPV were inoculated onto <i>N</i>. <i>benthamiana</i> plants. Local leaves were collected at 15 days post-inoculation and viral accumulation was determined by Northern blot analysis. Bioassayed mutants are indicated above the lanes of the autoradiograph. A mock inoculated sample (lane M) was also included. Positions of PLPV gRNA and sgRNA are indicated at the right. Ethidium bromide staining of ribosomal RNAs is shown below the autoradiograph as loading control.</p

Proposed structures of STNV-TED and of TED-like CITEs predicted in several viruses.

<p>Virus acronyms: <i>Satellite tobacco necrosis virus</i> (STNV), <i>Calibrachoa mottle virus</i> (CbMV), <i>Pelargonium chlorotic ring pattern virus</i> (PCRPV), <i>Elderberry latent virus</i> (ELV), <i>Pelargonium ring spot virus</i> (PelRSV), <i>Rosa rugosa leaf distortion virus</i> (RrLDV) and <i>Pelargonium line pattern virus</i> (PLPV). CbMV is a member of genus <i>Carmovirus</i> and PCRPV, ELV, PelRSV, RrLDV and PLPV are recommended to be included in the tentative new genus <i>Pelarspovirus</i>, all within family <i>Tombusviridae</i>. The region corresponding to the previously proposed STNV-TED as well as those corresponding to the previously proposed TED-like CITEs of CbMV, PCRPV and PLPV [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.ref015" target="_blank">15</a>] are shown on a grey background.</p