4 research outputs found
Estudio de la Compartimentalización subcelular en Viroides miembros de la familia Avsunviroidae
[ES] EL objetivo del trabajo fue determinar el movimiento subcelular de los viroides ASBVd y PLMVd. Para
ello se crearon unas construcciones dónde la secuencia del viroide actuase como extremo 5'UTR del
gen de la GFP. Gracias a la fuorescencia emitida por esta molécula, fue posible determia la localización
subcelular del RNA del viroide[EN] The objective of the study was to determine the subcellular movement ASBVd and PLMVd viroids. For
this, some viroid sequences were created where the secuence of the viroid could act as a as 5'UTR end
GFP gene. Thanks to flourescence emitted by this molecule, it was possible to determinate the
subcelular localization of the viroid RNA.Castellano Pérez, M. (2013). Estudio de la Compartimentalización subcelular en Viroides miembros de la familia Avsunviroidae. http://hdl.handle.net/10251/35384Archivo delegad
ESTUDIO DE LAS ALTERACIONES EN EL PATRON DE METILACIÓN DEL DNA DEL HUÉSPED INDUCIDAS POR UN VIROIDE NUCLEAR
Tesis por compendioIn the first chapter, we determined that cucumber plants infected with hop stunt viroid (HSVd) accumulated high levels of sRNAs derived of ribosomal RNA (rb-sRNAs).Moreover, this effect was correlated with an increase of the transcription of the ribosomal RNAs precursors (rRNAs) due to a decrease in DNA methylation in its promoter region, revealing that certain ribosomal genes (usually silenced) reactivated its transcriptional activity during the infection. In the following chapter and in order to determine whether this process could be a common phenomenon to other plant-pathogen systems, we analyzed N.benthamiana transgenic plants that expressed, in a constitutive way, the viroid dimeric sequence. It was observed that the accumulation of the sRNAs in transgenic plants was similar to the one seen in infected cucumbers, promoting the imbalance of the rb-sRNAs accumulation. By bysulfited DNA sequencing we proved that this phenomenon turned to be linked with the loss of cytosine methylation in a symmetrical context. As with that observed in cucumber, this phenomenon was correlated with an increase of the transcription of these hypomethylated DNA regions. These data supported the idea that the HSVd was able to interfere with the regulation mechanisms in the host epigenetic level (methylation), suggesting that this phenomenon could happen generally in other viroid-host systems. In the third chapter, by immunoprecipitation essays, it was possible to determine that both in cucumberas in N. benthamiana plants, the HSVd formed an in vivo stable complex with the HISTONE DEACETYLASE 6 (HDA6), a key component in the process of methylation of diverse repetitive DNAs. These results suggested that the interaction HSVd-HDA6 would generate a functional deficit of HDA6 that might be responsible of the epigenetic alterations observed in the host during the infection. This hypothesis was consistent with the observation that the transient overexpression of HDA6 in infected plants reverted to the hypomethylation status of the rDNA. Unexpectedly, we observed that the HDA6 overexpression induced a significant reduction in the accumulation levels of the viroid in the infected plants. Also, the viroid accumulation in the infected cells increased when we transiently silenced the HDA6 expression, demonstrating the existence of an antagonistic relationship between the HDA6 concentration and the viroid. A hypothesis that allows explaining the information obtained and correlating them with an increase of biological viroid efficacy in the host is described in detail in the discussion of this chapter. Once determined that, in vegetative host tissue, the HSVd induces alterations in the epigenetic map of the promotor areas of rDNA, in the last chapter of this thesis we analyzed whether or not reproductive host tissue showed similar alterations during the infection. We analyzed pollen grains of infected cucumber plants. The structural analysis of these reproductive cells indicated that the HSVd accumulation induced a nuclear chromatin decodensation, responsible of the rRNAs transcription in the nucleus of the generative cell. This alteration was correlated with a significant demethylation of the ribosomal DNAs and transposable elements. By RT-PCR analysis it was possible to determine that this methylation pattern alteration correlated with a significant increase of transcriptional activity. This observation revealed that the HSVd infection also induced alterations in the transcriptional regulation mechanisms in the host reproductive tissue. This result allowed speculating with the possibility that these epigenetic modifications could happen in the next generation plants, awarding to the viroid an advantage for host adaptation.En el primer capítulo determinamos que plantas de pepino infectadas con el viroide del enanismo del lúpulo (HSVd) acumulaban altos niveles de sRNAs derivados del RNA ribosomal. Además, este efecto se correlacionó con un aumento de la transcripción de los precursores de los RNAs ribosomales debido a una disminución de la metilación del DNA en su región promotora poniendo de manifiesto que ciertos genes ribosomales (normalmente silenciados) reactivaban su actividad transcripcional durante la infección. En el siguiente capítulo y con el objetivo de determinar si este proceso podía ser un fenómeno común a otros sistemas planta-patógeno, analizamos plantas de N.benthamiana transgénicas que expresaban de forma constitutiva la secuencia dimérica del viroide. Se observó cómo la acumulación de los sRNAs en plantas transgénicas era similar a la observada en los pepinos infectados, promoviendo el desequilibrio de la acumulación de rb-sRNAs. Mediante secuenciación de DNA bisulfitado demostramos que este fenómeno volvía a estar ligado con la pérdida de metilación de citosinas en un contexto simétrico. Al igual que en pepino este fenómeno correlacionaba con un aumento de la transcripción de estas zonas de DNA hipometiladas. En el tercer capítulo y mediante ensayos de immuno-precipitación, fue posible determinar que tanto en pepino como en N. benthaminana el HSVd formaba complejos estables in vivo con la proteína HISTONA DEACETILASA 6 (HDA6), un componente clave del proceso de metilación de diversos DNAs repetitivos, entre los que se encuentra el DNA ribosomal. Estos resultados sugerían que esta interacción HSVd-HDA6 generaría un déficit funcional de HDA6 que podría ser responsable de las alteraciones epigenéticas observadas en el huésped durante la infección. Esta hipótesis fue consistente con la observación de que la sobreexpresión transitoria de HDA6 en plantas infectadas revirtió el estado de hipometilación del rDNA inducido por el viroide. Inesperadamente, observamos que la sobreexpresión de HDA6 inducía una significativa reducción en los niveles de acumulación del viroide en la planta infectada. Además, la acumulación del viroide en las células infectadas aumentó al silenciar de forma transitoria la expresión de HDA6 evidenciando la existencia de una relación antagónica entre la concentración de HDA6 y la del viroide. Una vez determinado que, en tejidos vegetativos del huésped, el HSVd induce alteraciones en el mapa epigenético de las zonas promotoras del rDNA, en el último capítulo de esta tesis analizamos si tejidos reproductivos del huésped mostraban alteraciones similares durante la infección. Para ello se analizaron granos de polen de flores provenientes de plantas de pepino infectadas por el HSVd. El análisis estructural de estas células reproductivas indico que la acumulación de HSVd inducía la descondensación de la cromatina nucleolar responsable de la transcripción de los rRNAs en el núcleo generativo. Esta alteración correlacionó con una significativa desmetilación de DNAs ribosomales y los asociados a Elementos Transponibles. Mediante análisis de qRT-PCR fue posible determinar que esta alteración en los patrones de metilación se correspondía con un significativo aumento de su actividad transcripcional lo que permite afirmar que al igual que lo observado en hoja, la infección por HSVd induce alteraciones a nivel de los mecanismos de regulación transcripcional también en tejidos reproductivos del huésped. Esta observación permite especular con la posibilidad de que estas modificaciones epigenéticas podrían pasar a la siguiente generación de plantas, confiriendo de esta manera al viroide una ventaja en la adaptación al huésped.En el primer capítol determinem que plantes de cogombre infectades amb el viroide del nanisme del llúpol (HSVd) acumulaven alts nivells d'sRNA derivats de l'RNA ribosòmic (rb-sRNA). A més, aquest efecte es va correlacionar amb un augment de la transcripció dels precursors dels RNA ribosòmics (rRNA) a causa d'una disminució de la metilació del DNA en la seua regió promotora, i va posar de manifest que certs gens ribosòmics (normalment silenciats) reactivaven la seua activitat transcripcional durant la infecció. En el següent capítol i amb l'objectiu de determinar si aquest procés podia ser un fenomen comú a altres sistemes planta-patogen, analitzem plantes de N. benthamiana transgèniques que expressaven de forma constitutiva la seqüència dimèrica del viroide. Es va observar como l'acumulació dels sRNA en plantes transgèniques era similar a l'observada en els cogombres infectats, promovent el desequilibri de l'acumulació d'rb-sRNA. Mitjançant la seqüenciació del DNA bisulfitat vam demostrar que aquest fenomen tornava a estar lligat a la pèrdua de metilació de citosines en un context simètric. De la mateixa forma que en el cogombre, aquest fenomen es correlacionava amb un augment de la transcripció d'aquestes zones de DNA hipometilades. En el tercer capítol, mitjançant assajos d'immunoprecipitació, va ser possible determinar que tant en cogombre com en N. benthaminana, l'HSVd formava complexos estables in vivo amb la proteïna HISTONA DEACETILASA 6 (HDA6), un component clau del procés de metilació de diversos DNA repetitius, entre els quals es troba el DNA ribosòmic. Aquests resultats suggerien que aquesta interacció HSVd-HDA6 generaria un dèficit funcional d'HDA6 que podria ser responsable de les alteracions epigenètiques observades en l'hoste durant la infecció. Aquesta hipòtesi va ser consistent amb l'observació que la sobreexpressió transitòria d'HDA6 en plantes infectades revertia l'estat d'hipometilació de l'rDNA induït pel viroide. Inesperadament, observem que la sobreexpressió d'HDA6 induïa una significativa reducció en els nivells d'acumulació del viroide en la planta infectada. A més, l'acumulació del viroide en les cèl·lules infectades va augmentar en silenciar de forma transitòria l'expressió d'HDA6, evidenciant l'existència d'una relació antagònica entre la concentració d'HDA6 i la del viroide. Una vegada determinat que, en teixits vegetatius de l'hoste, l'HSVd indueix alteracions en el mapa epigenètic de les zones promotores de l'rDNA, en l'últim capítol d'aquesta tesi analitzem si teixits reproductius de l'hoste mostraven alteracions similars durant la infecció. Amb aquesta finalitat, es van analitzar grans de pol·len de flors provinents de plantes de cogombre infectades per l'HSVd . L'anàlisi estructural d'aquestes cèl·lules reproductives va indicar que l'acumulació d'HSVd induïa la descondensació de la cromatina nucleolar responsable de la transcripció dels rRNA en el nucli generatiu. Aquesta alteració es va correlacionar amb una significativa desmetilació de DNA ribosòmics i els associats a elements transposables (TE). Mitjançant anàlisi de qRT-PCR va ser possible determinar que aquesta alteració en els patrons de metilació es corresponia amb un significatiu augment de la seua activitat transcripcional, la qual cosa va permetre afirmar que, igual que l'observat en la fulla, la infecció per HSVd induïa, també en els teixits reproductius de l'hoste, alteracions dels mecanismes de regulació transcripcional. Aquesta observació va permetre especular amb la possibilitat que aquestes modificacions epigenètiques pogueren passar a la següent generació de plantes, conferint d'aquesta manera al viroide un avantatge d'adaptació a l'hoste.Castellano Pérez, M. (2017). ESTUDIO DE LAS ALTERACIONES EN EL PATRON DE METILACIÓN DEL DNA DEL HUÉSPED INDUCIDAS POR UN VIROIDE NUCLEAR [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/77992TESISCompendi
Bacillus subtilis IAB/BS03 as a potential biological control agent
[EN] We describe the efficacy of Bacillus subtilis strain IAB/BS03 in reducing disease incidence of B. subtilis IAB/BS03 as a foliar treatment against Botrytis cinerea and Pseudomonas syringae on greenhouse-grown tomato (Solanum lycopersicon) plants. We also tested the effect of foliar treatments on lettuce (Lactuca sativa) against lettuce downy mildew caused by Bremia lactucae in multiple trials under different field conditions. All the assays indicated that B. subtilis IAB/BS03 reduced disease. To ascertain the mechanism of action, the induction of pathogenesis-related (PR) proteins, the accumulation of salicylic acid and the activation of peroxidase caused by foliar or root treatments with B. subtilis IAB/BS03 were studied in tomato. A salicylic acid-independent induction of the antifungal protein PR1 was observed after treatment with B. subtilis IAB/BS03, with the strongest induction due to root treatment compared with foliar application. A metabolic analysis of B. subtilis IAB/BS03 culture broth using Ultra Performance Liquid Chromatography coupled with ultraviolet and mass spectrometric detection determined surfactin and iturin A isomers. These compounds have been described as antifungal and antibiotic lipopeptides. The results indicated that B. subtilis IAB/BS03 could be effectively used as a biocontrol agent.This work was funded by IAB S. L. (Investigaciones y Aplicaciones Biotecnologicas, S. L.), and by grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness. Mayte Castellano was the recipient of a research grant also funded by IAB S. L. The authors would like to thank Cristina Torres (IBMCP, UPV-CSIC) for her excellent technical assistance.Hinarejos, E.; Castellano Pérez, M.; Rodrigo Bravo, I.; Belles Albert, JM.; Conejero Tomás, V.; López-Gresa, MP.; Lisón, P. (2016). Bacillus subtilis IAB/BS03 as a potential biological control agent. European Journal of Plant Pathology. 146(3):597-608. https://doi.org/10.1007/s10658-016-0945-3S5976081463Abbott, W. S. (1925). A method for computing the effectiveness of an insecticide. Journal Economic Entomology, 18, 265–267.Chen, H., Wang, L., Su, C.X., Gong, G. H., Wang, P., Yu, Z. L. (2008). Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis. Letters in Applied Microbiology. 47, 180–186.Cho, S. J., Lee, S. K., Cha, B. J., Kim, Y. H., & Shin, K. S. (2003). Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin a from Bacillus subtilis strain KS03. FEMS Microbiology Letters, 223, 47–51.Choudhary, D. K., & Johri, B. N. (2009). Interactions of Bacillus spp. and plants with special reference to induced systemic resistance (ISR). Microbiology Research, 164, 493–513.Coego, A., Ramírez, V., Ellul, P., Mayda, E., & Vera, P. (2005). The H2O2-regulated Ep5C gene encodes a peroxidase required for bacterial speck susceptibility in tomato. The Plant Journal, 42, 283–293.Conrath, U., Pieterse, C. M. J., & Mauch-Mani, B. (2002). Priming in plant-pathogen interactions. Trends in Plant Science, 7, 210–216.Fleming, A. J., Mandel, T., Roth, I., & Kuhlemier, C. (1993). The patterns of gene expression in the tomato shoot apical meristem. The Plant Cell, 5, 297–309.Fravel, D. R. (2005). Commercialization and implementation of biocontrol. Annual Review of Phytopathology, 43, 337–359.Hammerschmidt, R., Nuckles, E. M., & Kuc, J. (1982). Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiological Plant Pathology, 20, 73–76.Kawagoe, Y., Shiraishi, S., Kondo, H., Yamamoto, S., Aoki, Y., & Suzuki, S. (2015). Cyclic lipopeptide iturin a structure-dependently induces defense response in Arabidopsis plants by activating SA and JA signaling pathways. Biochemical and Biophysical Research Communications, 460, 1015–1020.Kloepper, J. W., Ryu, C. M., & Zhang, S. A. (2004). Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology, 94, 1259–1266.Liu, H.-X., Li, S.-M., Luo, Y.-M., Luo, L.-X., Li, J.-Q., & Guo, J.-H. (2014). Biological control of Ralstonia wilt, Phytophthora blight, Meloidogyne root-knot on bell pepper by the combination of Bacillus subtilis AR12, Bacillus subtilis SM21 and Chryseobacterium sp. R89. European Journal of Plant Pathology, 139, 107–116.Mohammadi, M., & Kazemi, H. (2002). Changes in peroxidase and polyphenol oxidase activities in susceptible and resistant wheat heads inoculated with Fusarium graminearum and induced resistance. Plant Science, 162, 491–498.Niderman, T., Genetet, I., Bruyere, T., Gees, R., Stintzi, A., Legrand, M., et al. (1995). Pathogenesis-related PR-1 proteins are antifungal - isolation and characterization of 3 14-Kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans. Plant Physiology, 108, 17–27.Ohno, A., Ano, T., & Shoda, M. (1995). Effect of temperature on production of lipopeptide antibiotics, iturin a and surfactin by a dual producer, Bacillus subtilis Rb14, in solid-state fermentation. Journal of Fermentation and Bioengineering, 80, 517–519.Ongena, M., & Jacques, P. (2008). Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology, 16, 115–125.Pérez-García, A., Romero, D., & De Vicente, A. (2011). Plant protection and growth stimulation by microorganisms: biotechnological applications of bacilli in agriculture. Current Opinion in Biotechnology, 22, 187–193.Phister, T. G., O’Sullivan, D. J., & McKay, L. L. (2004). Identification of bacilysin, chlorotetaine, and iturin a produced by Bacillus sp strain CS93 isolated from pozol, a Mexican fermented maize dough. Applied Environmental Microbiology, 70, 631–634.Pieterse, C. M. J., vanWees, S. C. M., Hoffland, E., Van Pelt, J. A., & Van Loon, L. C. (1996). Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression. The Plant Cell, 8, 1225–1237.Pryor, S. W., Gibson, D. M., Krasnoff, S. B., & Walker, L. P. (2006). Identification of antifungal compounds in a biological control product using a microplate inhibition bioassay. Transactions of the ASAE, 49, 1643–1649.Robert-Seilaniantz, A., Navarro, L., Bari, R., & Jones, J. D. (2007). Pathological hormone imbalances. Current Opinion in Plant Biology, 10, 372–379.Rudrappa, T., Biedrzycki, M. L., Kunjeti, S. G., Donofrio, N. M., Czymmek, K. J., Paul W, P., et al. (2010). The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana. Communicative and Integrative Biology, 3, 130–138.Summermatter, K., Sticher, L., & Métraux, J. P. (1995). Systemic responses in Arabidopsis thaliana infected and challenged with Pseudomonas syringae pv syringae. Plant Physiology, 108, 1379–1385.Tang, J. S., Zhao, F., Gao, H., Dai, Y., Yao, Z. H., Hong, K., et al. (2010). Characterization and online detection of surfactin isomers based on HPLC-MSn analyses and their inhibitory effects on the overproduction of nitric oxide and the release of TNF-α and IL-6 in LPS induced macrophages. Marine Drugs, 8, 2605–2618.Tornero, P., Gadea, J., Conejero, V., & Vera, P. (1997). Two PR-1 genes from tomato are differentially regulated and reveal a novel mode of expression for a pathogenesis-related gene during the hypersensitive response and development. Molecular Plant-Microbe Interactions, 10, 624–634.Tsavkelova, E. A., Klimova, S. Y., Cherdyntseva, T. A., & Netrusov, A. I. (2006). Microbial producers of plant growth stimulators and their practical use: a review. Applied Biochemistry and Microbiology, 42, 117–126.Van Loon, L. C. (2007). Plant responses to plant growth-promoting rhizobacteria. European Journal of Plant Pathology, 119, 243–254.Verhagen, B. W. M., Glazebrook, J., Zhu, T., Chang, H. S., Van Loon, L. C., & Pieterse, C. M. J. (2004). The transcriptome of rhizobacteria-induced systemic resistance in Arabidopsis. Molecular Plant-Microbe Interactions, 17, 895–908.Wulff, B. B. H., Horvath, D. M., & Ward, E. R. (2011). Improving immunity in crops: new tactics in an old game. Current Opinion in Plant Biology, 14, 468–476.Yáñez-Mendizábal, V., Zeriouh, H., Viñas, I., Torres, R., Usall, J., de Vicente, A., et al. (2012). Biological control of peach brown rot (Monilinia spp.) by Bacillus subtilis CPA-8 is based on production of fengycin-like lipopeptides. European Journal of Plant Pathology, 132, 609–619.Zacarés, L., López-Gresa, M. P., Fayos, J., Primo, J., Bellés, J. M., & Conejero, V. (2007). Induction of p-coumaroyldopamine and feruloyldopamine, two novel metabolites, in tomato by the bacterial pathogen Pseudomonas syringae. Molecular Plant-Microbe Interactions, 20, 1439–1448.Zadoks, J. C., Chang, T. T., & Konzak, C. F. (1974). A decimal code for the growth stages of cereals. Weed Research, 14, 415–421.Zeriouh, H., Romero, D., García-Gutiérrez, L., Cazorla, F. M., De Vicente, A., & Pérez-García, A. (2011). The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits. Molecular Plant-Microbe Interactions, 24, 1540–1552
A non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants
[EN] Viroids are plant-pathogenic non-coding RNAs able to interfere with as yet poorly known host-regulatory pathways and to cause alterations recognized as diseases. The way in which these RNAs coerce the host to express symptoms remains to be totally deciphered. In recent years, diverse studies have proposed a close interplay between viroid-induced pathogenesis and RNA silencing, supporting the belief that viroid-derived small RNAs mediate the post-transcriptional cleavage of endogenous mRNAs by acting as elicitors of symptoms expression. Although the evidence supporting the role of viroid-derived small RNAs in pathogenesis is robust, the possibility that this phenomenon can be a more complex process, also involving viroid-induced alterations in plant gene expression at transcriptional levels, has been considered. Here we show that plants infected with the 'Hop stunt viroid' accumulate high levels of sRNAs derived from ribosomal transcripts. This effect was correlated with an increase in the transcription of ribosomal RNA (rRNA) precursors during infection. We observed that the transcriptional reactivation of rRNA genes correlates with a modification of DNA methylation in their promoter region and revealed that some rRNA genes are demethylated and transcriptionally reactivated during infection. This study reports a previously unknown mechanism associated with viroid (or any other pathogenic RNA) infection in plants providing new insights into aspects of host alterations induced by the viroid infectious cycle.The Spanish granting agency Direccion General de Investigacion Cientifica [BIO2011-25018 to V.P.] and from the Prometeo program [2011/003] from the Generalitat Valenciana. GM is the recipient of a Marie Curie IOF fellowship. Funding for open access charge: Direccion General de Investigacion Cientifica [BIO2011-25018].Martínez Arias, GE.; Castellano Pérez, M.; Tortosa-Viqueira, M.; Pallás Benet, V.; Gomez, GG. (2014). A non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants. Nucleic Acids Research. 42(3):1553-1562. https://doi.org/10.1093/nar/gkt968S15531562423Ding, B. (2009). The Biology of Viroid-Host Interactions. Annual Review of Phytopathology, 47(1), 105-131. doi:10.1146/annurev-phyto-080508-081927Ding, B. (2010). Viroids: self-replicating, mobile, and fast-evolving noncoding regulatory RNAs. Wiley Interdisciplinary Reviews: RNA, 1(3), 362-375. doi:10.1002/wrna.22Navarro, B., Gisel, A., Rodio, M.-E., Delgado, S., Flores, R., & Di Serio, F. (2012). Viroids: How to infect a host and cause disease without encoding proteins. Biochimie, 94(7), 1474-1480. doi:10.1016/j.biochi.2012.02.020Zhao, Y., Hammond, R. W., & Owens, R. A. (2001). Use of a vector based on Potato virus X in a whole plant assay to demonstrate nuclear targeting of Potato spindle tuber viroid. Journal of General Virology, 82(6), 1491-1497. doi:10.1099/0022-1317-82-6-1491Gómez, G., & Pallas, V. (2012). Studies on Subcellular Compartmentalization of Plant Pathogenic Noncoding RNAs Give New Insights into the Intracellular RNA-Traffic Mechanisms. Plant Physiology, 159(2), 558-564. doi:10.1104/pp.112.195214Navarro, J.-A., Vera, A., & Flores, R. (2000). A Chloroplastic RNA Polymerase Resistant to Tagetitoxin Is Involved in Replication of Avocado Sunblotch Viroid. Virology, 268(1), 218-225. doi:10.1006/viro.1999.0161Nohales, M.-A., Flores, R., & Daros, J.-A. (2012). Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase. Proceedings of the National Academy of Sciences, 109(34), 13805-13810. doi:10.1073/pnas.1206187109Nohales, M.-A., Molina-Serrano, D., Flores, R., & Daros, J.-A. (2012). Involvement of the Chloroplastic Isoform of tRNA Ligase in the Replication of Viroids Belonging to the Family Avsunviroidae. Journal of Virology, 86(15), 8269-8276. doi:10.1128/jvi.00629-12Zhong, X., & Ding, B. (2008). Distinct RNA motifs mediate systemic RNA trafficking. Plant Signaling & Behavior, 3(1), 58-59. doi:10.4161/psb.3.1.4890Gomez, G., & Pallas, V. (2004). A Long-Distance Translocatable Phloem Protein from Cucumber Forms a Ribonucleoprotein Complex In Vivo with Hop Stunt Viroid RNA. Journal of Virology, 78(18), 10104-10110. doi:10.1128/jvi.78.18.10104-10110.2004Gómez, G., & Pallás, V. (2001). Identification of an In Vitro Ribonucleoprotein Complex Between a Viroid RNA and a Phloem Protein from Cucumber Plants. Molecular Plant-Microbe Interactions, 14(7), 910-913. doi:10.1094/mpmi.2001.14.7.910Owens, R. A., Blackburn, M., & Ding, B. (2001). Possible Involvement of the Phloem Lectin in Long-Distance Viroid Movement. Molecular Plant-Microbe Interactions, 14(7), 905-909. doi:10.1094/mpmi.2001.14.7.905Gómez, G., & Pallás, V. (2013). Viroids: a light in the darkness of thelncRNA-directed regulatory networks in plants. New Phytologist, 198(1), 10-15. doi:10.1111/nph.12196Diener, T. O. (1981). Are viroids escaped introns? Proceedings of the National Academy of Sciences, 78(8), 5014-5015. doi:10.1073/pnas.78.8.5014Haas, B., Klanner, A., Ramm, K., & Sänger, H. L. (1988). The 7S RNA from tomato leaf tissue resembles a signal recognition particle RNA and exhibits a remarkable sequence complementarity to viroids. The EMBO Journal, 7(13), 4063-4074. doi:10.1002/j.1460-2075.1988.tb03300.xVisvader, J. E., & Symons, R. H. (1985). Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity. Nucleic Acids Research, 13(8), 2907-2920. doi:10.1093/nar/13.8.2907Schnölzer, M., Haas, B., Ramm, K., Hofmann, H., & Sänger, H. L. (1985). Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV). The EMBO Journal, 4(9), 2181-2190. doi:10.1002/j.1460-2075.1985.tb03913.xPapaefthimiou, I. (2001). Replicating potato spindle tuber viroid RNA is accompanied by short RNA fragments that are characteristic of post-transcriptional gene silencing. Nucleic Acids Research, 29(11), 2395-2400. doi:10.1093/nar/29.11.2395Wang, M.-B., Bian, X.-Y., Wu, L.-M., Liu, L.-X., Smith, N. A., Isenegger, D., … Waterhouse, P. M. (2004). On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites. Proceedings of the National Academy of Sciences, 101(9), 3275-3280. doi:10.1073/pnas.0400104101Gómez, G., Martínez, G., & Pallás, V. (2009). Interplay between viroid-induced pathogenesis and RNA silencing pathways. Trends in Plant Science, 14(5), 264-269. doi:10.1016/j.tplants.2009.03.002Gómez, G., Martínez, G., & Pallás, V. (2008). Viroid-Induced Symptoms in Nicotiana benthamiana Plants Are Dependent on RDR6 Activity. Plant Physiology, 148(1), 414-423. doi:10.1104/pp.108.120808Navarro, 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.xDowen, R. H., Pelizzola, M., Schmitz, R. J., Lister, R., Dowen, J. M., Nery, J. R., … Ecker, J. R. (2012). Widespread dynamic DNA methylation in response to biotic stress. Proceedings of the National Academy of Sciences, 109(32), E2183-E2191. doi:10.1073/pnas.1209329109Law, J. A., & Jacobsen, S. E. (2010). Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nature Reviews Genetics, 11(3), 204-220. doi:10.1038/nrg2719Matzke, M., Kanno, T., Daxinger, L., Huettel, B., & Matzke, A. J. (2009). RNA-mediated chromatin-based silencing in plants. Current Opinion in Cell Biology, 21(3), 367-376. doi:10.1016/j.ceb.2009.01.025Zhang, X., Henderson, I. R., Lu, C., Green, P. J., & Jacobsen, S. E. (2007). Role of RNA polymerase IV in plant small RNA metabolism. Proceedings of the National Academy of Sciences, 104(11), 4536-4541. doi:10.1073/pnas.0611456104Haag, J. R., & Pikaard, C. S. (2011). Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nature Reviews Molecular Cell Biology, 12(8), 483-492. doi:10.1038/nrm3152Gong, 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)01133-9Nuthikattu, S., McCue, A. D., Panda, K., Fultz, D., DeFraia, C., Thomas, E. N., & Slotkin, R. K. (2013). The Initiation of Epigenetic Silencing of Active Transposable Elements Is Triggered by RDR6 and 21-22 Nucleotide Small Interfering RNAs. Plant Physiology, 162(1), 116-131. doi:10.1104/pp.113.216481Wassenegger, M., Heimes, S., Riedel, L., & Sänger, H. L. (1994). RNA-directed de novo methylation of genomic sequences in plants. Cell, 76(3), 567-576. doi:10.1016/0092-8674(94)90119-8Itaya, A., Matsuda, Y., Gonzales, R. A., Nelson, R. S., & Ding, B. (2002). Potato spindle tuber viroidStrains of Different Pathogenicity Induces and Suppresses Expression of Common and Unique Genes in Infected Tomato. Molecular Plant-Microbe Interactions, 15(10), 990-999. doi:10.1094/mpmi.2002.15.10.990Owens, R. A., Tech, K. B., Shao, J. Y., Sano, T., & Baker, C. J. (2012). Global Analysis of Tomato Gene Expression DuringPotato spindle tuber viroidInfection Reveals a Complex Array of Changes Affecting Hormone Signaling. Molecular Plant-Microbe Interactions, 25(4), 582-598. doi:10.1094/mpmi-09-11-0258Tessitori, M., Maria, G., Capasso, C., Catara, G., Rizza, S., De Luca, V., … Carginale, V. (2007). Differential display analysis of gene expression in Etrog citron leaves infected by Citrus viroid III. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1769(4), 228-235. doi:10.1016/j.bbaexp.2007.03.004Herranz, M. C., Niehl, A., Rosales, M., Fiore, N., Zamorano, A., Granell, A., & Pallas, V. (2013). A remarkable synergistic effect at the transcriptomic level in peach fruits doubly infected by prunus necrotic ringspot virus and peach latent mosaic viroid. Virology Journal, 10(1). doi:10.1186/1743-422x-10-164Lisón, P., Tárraga, S., López-Gresa, P., Saurí, A., Torres, C., Campos, L., … Rodrigo, I. (2013). A noncoding plant pathogen provokes both transcriptional and posttranscriptional alterations in tomato. PROTEOMICS, 13(5), 833-844. doi:10.1002/pmic.201200286GÓMEZ, G., & PALLÁS, V. (2006). Hop stunt viroid is processed and translocated in transgenic Nicotiana benthamiana plants. Molecular Plant Pathology, 7(6), 511-517. doi:10.1111/j.1364-3703.2006.00356.xMARTINEZ, G., DONAIRE, L., LLAVE, C., PALLAS, V., & GOMEZ, G. (2010). High-throughput sequencing ofHop stunt viroid-derived small RNAs from cucumber leaves and phloem. Molecular Plant Pathology, 11(3), 347-359. doi:10.1111/j.1364-3703.2009.00608.xPallas, V., Navarro, A., & Flores, R. (1987). Isolation of a Viroid-like RNA from Hop Different from Hop Stunt Viroid. Journal of General Virology, 68(12), 3201-3205. doi:10.1099/0022-1317-68-12-3201Gómez, G., & Pallás, V. (2007). Mature monomeric forms of Hop stunt viroid resist RNA silencing in transgenic plants. The Plant Journal, 51(6), 1041-1049. doi:10.1111/j.1365-313x.2007.03203.xDellaporta, S. L., Wood, J., & Hicks, J. B. (1983). A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter, 1(4), 19-21. doi:10.1007/bf02712670Martínez, G., Forment, J., Llave, C., Pallás, V., & Gómez, G. (2011). High-Throughput Sequencing, Characterization and Detection of New and Conserved Cucumber miRNAs. PLoS ONE, 6(5), e19523. doi:10.1371/journal.pone.0019523Long, E. O., & Dawid, I. B. (1980). Repeated Genes in Eukaryotes. Annual Review of Biochemistry, 49(1), 727-764. doi:10.1146/annurev.bi.49.070180.003455Henras, A. K., Soudet, J., Gérus, M., Lebaron, S., Caizergues-Ferrer, M., Mougin, A., & Henry, Y. (2008). The post-transcriptional steps of eukaryotic ribosome biogenesis. Cellular and Molecular Life Sciences, 65(15), 2334-2359. doi:10.1007/s00018-008-8027-0Pontvianne, F., Blevins, T., Chandrasekhara, C., Feng, W., Stroud, H., Jacobsen, S. E., … Pikaard, C. S. (2012). Histone methyltransferases regulating rRNA gene dose and dosage control in Arabidopsis. Genes & Development, 26(9), 945-957. doi:10.1101/gad.182865.111Preuss, S., & Pikaard, C. S. (2007). rRNA gene silencing and nucleolar dominance: Insights into a chromosome-scale epigenetic on/off switch. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1769(5-6), 383-392. doi:10.1016/j.bbaexp.2007.02.005Earley, K. W., Pontvianne, F., Wierzbicki, A. T., Blevins, T., Tucker, S., Costa-Nunes, P., … Pikaard, C. S. (2010). Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation. Genes & Development, 24(11), 1119-1132. doi:10.1101/gad.1914110Tucker, 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.2010.03.009Layat, E., Cotterell, S., Vaillant, I., Yukawa, Y., Tutois, S., & Tourmente, S. (2012). Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development. The Plant Journal, 71(1), 35-44. doi:10.1111/j.1365-313x.2012.04948.xRodio, 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.106.049775Yu, 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.1211757110Sha, A. H., Lin, X. H., Huang, J. B., & Zhang, D. P. (2005). Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis. Molecular Genetics and Genomics, 273(6), 484-490. doi:10.1007/s00438-005-1148-3Boyko, A., Kathiria, P., Zemp, F. J., Yao, Y., Pogribny, I., & Kovalchuk, I. (2007). Transgenerational changes in the genome stability and methylation in pathogen-infected plants. Nucleic Acids Research, 35(5), 1714-1725. doi:10.1093/nar/gkm029Rodríguez-Negrete, E., Lozano-Durán, R., Piedra-Aguilera, A., Cruzado, L., Bejarano, E. R., & Castillo, A. G. (2013). Geminivirus Rep protein interferes with the plant DNA methylation machinery and suppresses transcriptional gene silencing. New Phytologist, 199(2), 464-475. doi:10.1111/nph.12286Vaillant, I., Tutois, S., Cuvillier, C., Schubert, I., & Tourmente, S. (2007). Regulation of Arabidopsis thaliana 5S rRNA Genes. Plant and Cell Physiology, 48(5), 745-752. doi:10.1093/pcp/pcm043MÜHLBACH, H.-P., & SÄNGER, H. L. (1979). Viroid replication is inhibited by α-amanitin. Nature, 278(5700), 185-188. doi:10.1038/278185a0Li, L.-C., & Dahiya, R. (2002). MethPrimer: designing primers for methylation PCRs. Bioinformatics, 18(11), 1427-1431. doi:10.1093/bioinformatics/18.11.142