Citrus exocortis viroid causes ribosomal stress in tomato plants

[EN] Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.Spanish Ministry of Science, Innovation and Universities [BIO2009-11818, BIO2015-70483-R to A.F.]; Spanish Ministry of Science, Innovation and Universities [BFU2009-11958]; Generalitat Valenciana (Valencia, Spain) [AICO/2017/048]; Natural Sciences and Engineering Research Council of Canada [155219-17 to J.-P.P.]; The RNA group is supported by a grant from the Universite de Sherbrooke; J.-P.P. holds the Research Chair of the Universite de Sherbrooke in RNA Structure and Genomics, and is a member of the Centre de Recherche du CHUS; B.B.-P. was a recipient of a VALi+d postdoctoral contract of the Generalitat Valenciana [APOSTD/2017/039]; Schleiff group is funded through the Deutsche Forschungsgemeinschaft [SFB 902]. Funding for open access charge: Spanish Ministry of Science, Innovation and Universities.Cottilli, P.; Belda-Palazón, B.; Adkar-Purushothama, CR.; Perreault, J.; Schleiff, E.; Rodrigo Bravo, I.; Ferrando Monleón, AR.... (2019). Citrus exocortis viroid causes ribosomal stress in tomato plants. Nucleic Acids Research. 47(16):8649-8661. https://doi.org/10.1093/nar/gkz679864986614716Di Serio, F., & Flores, R. (2008). Viroids: Molecular implements for dissecting RNA trafficking in plants. RNA Biology, 5(3), 128-131. doi:10.4161/rna.5.3.6638Flores, R., Owens, R. A., & Taylor, J. (2016). Pathogenesis by subviral agents: viroids and hepatitis delta virus. Current Opinion in Virology, 17, 87-94. doi:10.1016/j.coviro.2016.01.022Di Serio, F., Flores, R., Verhoeven, J. T. J., Li, S.-F., Pallás, V., Randles, J. W., … Owens, R. A. (2014). Current status of viroid taxonomy. Archives of Virology, 159(12), 3467-3478. doi:10.1007/s00705-014-2200-6Vogt, U., Pélissier, T., Pütz, A., Razvi, F., Fischer, R., & Wassenegger, M. (2004). Viroid-induced RNA silencing of GFP-viroid fusion transgenes does not induce extensive spreading of methylation or transitive silencing. The Plant Journal, 38(1), 107-118. doi:10.1111/j.1365-313x.2004.02029.xMartínez de Alba, A. E., Flores, R., & Hernández, C. (2002). Two Chloroplastic Viroids Induce the Accumulation of Small RNAs Associated with Posttranscriptional Gene Silencing. Journal of Virology, 76(24), 13094-13096. doi:10.1128/jvi.76.24.13094-13096.2002Markarian, N., Li, H. W., Ding, S. W., & Semancik, J. S. (2004). RNA silencing as related to viroid induced symptom expression. Archives of Virology, 149(2), 397-406. doi:10.1007/s00705-003-0215-5Carbonell, A., Martínez de Alba, Á.-E., Flores, R., & Gago, S. (2008). Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families. Virology, 371(1), 44-53. doi:10.1016/j.virol.2007.09.031St-Pierre, P., Hassen, I. F., Thompson, D., & Perreault, J. P. (2009). Characterization of the siRNAs associated with peach latent mosaic viroid infection. Virology, 383(2), 178-182. doi:10.1016/j.virol.2008.11.008MARTINEZ, 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.xIvanova, D., Milev, I., Vachev, T., Baev, V., Yahubyan, G., Minkov, G., & Gozmanova, M. (2014). Small RNA analysis of Potato Spindle Tuber Viroid infected Phelipanche ramosa. Plant Physiology and Biochemistry, 74, 276-282. doi:10.1016/j.plaphy.2013.11.019Islam, W., Noman, A., Qasim, M., & Wang, L. (2018). Plant Responses to Pathogen Attack: Small RNAs in Focus. International Journal of Molecular Sciences, 19(2), 515. doi:10.3390/ijms19020515Minoia, S., Carbonell, A., Di Serio, F., Gisel, A., Carrington, J. C., Navarro, B., & Flores, R. (2014). Specific Argonautes Selectively Bind Small RNAs Derived from Potato Spindle Tuber Viroid and Attenuate Viroid Accumulation In Vivo. Journal of Virology, 88(20), 11933-11945. doi:10.1128/jvi.01404-14Katsarou, K., Mavrothalassiti, E., Dermauw, W., Van Leeuwen, T., & Kalantidis, K. (2016). Combined Activity of DCL2 and DCL3 Is Crucial in the Defense against Potato Spindle Tuber Viroid. PLOS Pathogens, 12(10), e1005936. doi:10.1371/journal.ppat.1005936Dadami, E., Boutla, A., Vrettos, N., Tzortzakaki, S., Karakasilioti, I., & Kalantidis, K. (2013). DICER-LIKE 4 But Not DICER-LIKE 2 May Have a Positive Effect on Potato Spindle Tuber Viroid Accumulation in Nicotiana benthamiana. Molecular Plant, 6(1), 232-234. doi:10.1093/mp/sss118Navarro, 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.xEamens, A. L., Smith, N. A., Dennis, E. S., Wassenegger, M., & Wang, M.-B. (2014). In Nicotiana species, an artificial microRNA corresponding to the virulence modulating region of Potato spindle tuber viroid directs RNA silencing of a soluble inorganic pyrophosphatase gene and the development of abnormal phenotypes. Virology, 450-451, 266-277. doi:10.1016/j.virol.2013.12.019Adkar-Purushothama, C. R., Brosseau, C., Giguère, T., Sano, T., Moffett, P., & Perreault, J.-P. (2015). Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants. The Plant Cell, 27(8), 2178-2194. doi:10.1105/tpc.15.00523Adkar-Purushothama, C. R., Iyer, P. S., & Perreault, J.-P. (2017). Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants. Scientific Reports, 7(1). doi:10.1038/s41598-017-08823-zCarbonell, A., & Daròs, J.-A. (2017). Artificial microRNAs and synthetictrans-acting small interfering RNAs interfere with viroid infection. Molecular Plant Pathology, 18(5), 746-753. doi:10.1111/mpp.12529Lisón, 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.201200286Dubé, A., Bisaillon, M., & Perreault, J.-P. (2009). Identification of Proteins from Prunus persica That Interact with Peach Latent Mosaic Viroid. Journal of Virology, 83(23), 12057-12067. doi:10.1128/jvi.01151-09Eiras, M., Nohales, M. A., Kitajima, E. W., Flores, R., & Daròs, J. A. (2010). Ribosomal protein L5 and transcription factor IIIA from Arabidopsis thaliana bind in vitro specifically Potato spindle tuber viroid RNA. Archives of Virology, 156(3), 529-533. doi:10.1007/s00705-010-0867-xMartinez, 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/gkt968Castellano, M., Martinez, G., Marques, M. C., Moreno-Romero, J., Köhler, C., Pallas, V., & Gomez, G. (2016). Changes in the DNA methylation pattern of the host male gametophyte of viroid-infected cucumber plants. Journal of Experimental Botany, 67(19), 5857-5868. doi:10.1093/jxb/erw353Mills, E. W., & Green, R. (2017). Ribosomopathies: There’s strength in numbers. Science, 358(6363), eaan2755. doi:10.1126/science.aan2755Mayer, C., & Grummt, I. (2005). Cellular Stress and Nucleolar Function. Cell Cycle, 4(8), 1036-1038. doi:10.4161/cc.4.8.1925Boulon, S., Westman, B. J., Hutten, S., Boisvert, F.-M., & Lamond, A. I. (2010). The Nucleolus under Stress. Molecular Cell, 40(2), 216-227. doi:10.1016/j.molcel.2010.09.024Ohbayashi, I., & Sugiyama, M. (2018). Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses. Frontiers in Plant Science, 8. doi:10.3389/fpls.2017.02247Weis, B. L., Kovacevic, J., Missbach, S., & Schleiff, E. (2015). Plant-Specific Features of Ribosome Biogenesis. Trends in Plant Science, 20(11), 729-740. doi:10.1016/j.tplants.2015.07.003Waltz, F., Nguyen, T.-T., Arrivé, M., Bochler, A., Chicher, J., Hammann, P., … Giegé, P. (2019). Small is big in Arabidopsis mitochondrial ribosome. Nature Plants, 5(1), 106-117. doi:10.1038/s41477-018-0339-yOhbayashi, I., Lin, C.-Y., Shinohara, N., Matsumura, Y., Machida, Y., Horiguchi, G., … Sugiyama, M. (2017). Evidence for a Role of ANAC082 as a Ribosomal Stress Response Mediator Leading to Growth Defects and Developmental Alterations in Arabidopsis. The Plant Cell, 29(10), 2644-2660. doi:10.1105/tpc.17.00255Kressler, D., Hurt, E., & Baßler, J. (2017). A Puzzle of Life: Crafting Ribosomal Subunits. Trends in Biochemical Sciences, 42(8), 640-654. doi:10.1016/j.tibs.2017.05.005Rorbach, J., Aibara, S., & Amunts, A. (2017). Ribosome origami. Nature Structural & Molecular Biology, 24(11), 879-881. doi:10.1038/nsmb.3497Ahmed, T., Yin, Z., & Bhushan, S. (2016). Cryo-EM structure of the large subunit of the spinach chloroplast ribosome. Scientific Reports, 6(1). doi:10.1038/srep35793Henras, 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/s00018-008-8027-0Baßler, J., & Hurt, E. (2019). Eukaryotic Ribosome Assembly. Annual Review of Biochemistry, 88(1), 281-306. doi:10.1146/annurev-biochem-013118-110817Hang, R., Wang, Z., Deng, X., Liu, C., Yan, B., Yang, C., … Cao, X. (2018). Ribosomal RNA Biogenesis and Its Response to Chilling Stress in Oryza sativa. Plant Physiology, 177(1), 381-397. doi:10.1104/pp.17.01714Palm, D., Streit, D., Shanmugam, T., Weis, B. L., Ruprecht, M., Simm, S., & Schleiff, E. (2018). Plant-specific ribosome biogenesis factors in Arabidopsis thaliana with essential function in rRNA processing. Nucleic Acids Research, 47(4), 1880-1895. doi:10.1093/nar/gky1261Tomecki, R., Sikorski, P. J., & Zakrzewska-Placzek, M. (2017). Comparison of preribosomal RNA processing pathways in yeast, plant and human cells - focus on coordinated action of endo- and exoribonucleases. FEBS Letters, 591(13), 1801-1850. doi:10.1002/1873-3468.12682Perry, K. L., & Palukaitis, P. (1990). Transcription of tomato ribosomal DNA and the organization of the intergenic spacer. Molecular and General Genetics MGG, 221(1), 102-112. doi:10.1007/bf00280374Echevarría-Zomeño, S., Yángüez, E., Fernández-Bautista, N., Castro-Sanz, A., Ferrando, A., & Castellano, M. (2013). Regulation of Translation Initiation under Biotic and Abiotic Stresses. International Journal of Molecular Sciences, 14(3), 4670-4683. doi:10.3390/ijms14034670Wang, Z., Ying, T., Bao, B., & Huang, X. (2005). Characteristics of fruit ripening in tomato mutantepi. Journal of Zhejiang University SCIENCE, 6B(6), 502-507. doi:10.1631/jzus.2005.b0502Bellés, J. M., Carbonell, J., & Conejero, V. (1991). Polyamines in Plants Infected by Citrus Exocortis Viroid or Treated with Silver Ions and Ethephon. Plant Physiology, 96(4), 1053-1059. doi:10.1104/pp.96.4.1053Adkar-Purushothama, C. R., & Perreault, J.-P. (2018). Alterations of the viroid regions that interact with the host defense genes attenuate viroid infection in host plant. RNA Biology, 15(7), 955-966. doi:10.1080/15476286.2018.1462653Rivera, M. C., Maguire, B., & Lake, J. A. (2015). Isolation of Ribosomes and Polysomes. Cold Spring Harbor Protocols, 2015(3), pdb.prot081331. doi:10.1101/pdb.prot081331Hsu, P. Y., Calviello, L., Wu, H.-Y. L., Li, F.-W., Rothfels, C. J., Ohler, U., & Benfey, P. N. (2016). Super-resolution ribosome profiling reveals unannotated translation events in Arabidopsis. Proceedings of the National Academy of Sciences, 113(45), E7126-E7135. doi:10.1073/pnas.1614788113Mustroph, A., Juntawong, P., & Bailey-Serres, J. (2009). Isolation of Plant Polysomal mRNA by Differential Centrifugation and Ribosome Immunopurification Methods. Methods in Molecular Biology™, 109-126. doi:10.1007/978-1-60327-563-7_6López-Gresa, M. P., Lisón, P., Yenush, L., Conejero, V., Rodrigo, I., & Bellés, J. M. (2016). Salicylic Acid Is Involved in the Basal Resistance of Tomato Plants to Citrus Exocortis Viroid and Tomato Spotted Wilt Virus. PLOS ONE, 11(11), e0166938. doi:10.1371/journal.pone.0166938Missbach, S., Weis, B. L., Martin, R., Simm, S., Bohnsack, M. T., & Schleiff, E. (2013). 40S Ribosome Biogenesis Co-Factors Are Essential for Gametophyte and Embryo Development. PLoS ONE, 8(1), e54084. doi:10.1371/journal.pone.0054084Verhoeven, J. th. j., Jansen, C. C. C., Willemen, T. M., Kox, L. F. F., Owens, R. A., & Roenhorst, J. W. (2004). Natural infections of tomato by Citrus exocortis viroid, Columnea latent viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid. European Journal of Plant Pathology, 110(8), 823-831. doi:10.1007/s10658-004-2493-5Campos, L., Granell, P., Tárraga, S., López-Gresa, P., Conejero, V., Bellés, J. M., … Lisón, P. (2014). Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens. Plant Physiology and Biochemistry, 77, 35-43. doi:10.1016/j.plaphy.2014.01.016Kalantidis, K., Denti, M. A., Tzortzakaki, S., Marinou, E., Tabler, M., & Tsagris, M. (2007). Virp1 Is a Host Protein with a Major Role in Potato Spindle Tuber Viroid Infection in Nicotiana Plants. Journal of Virology, 81(23), 12872-12880. doi:10.1128/jvi.00974-07Barry, C. S., Fox, E. A., Yen, H., Lee, S., Ying, T., Grierson, D., & Giovannoni, J. J. (2001). Analysis of the Ethylene Response in theepinastic Mutant of Tomato. Plant Physiology, 127(1), 58-66. doi:10.1104/pp.127.1.58Diener, T. O. (2003). Discovering viroids — a personal perspective. Nature Reviews Microbiology, 1(1), 75-80. doi:10.1038/nrmicro736Ding, B., & Itaya, A. (2007). Viroid: A Useful Model for Studying the Basic Principles of Infection and RNA Biology. Molecular Plant-Microbe Interactions®, 20(1), 7-20. doi:10.1094/mpmi-20-0007Jakab, G., Kiss, T., & Solymosy, F. (1986). Viroid pathogenicity and pre-rRNA processing: A model amenable to experimental testing. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 868(4), 190-197. doi:10.1016/0167-4781(86)90054-0Meduski, C. J., & Velten, J. (1990). PSTV sequence similarity to large rRNA. Plant Molecular Biology, 14(4), 625-627. doi:10.1007/bf00027509Kiss, T., Pósfai, J., & Solymosy, F. (1983). Sequence homology between potato spindle tuber viroid and U3B snRNA. FEBS Letters, 163(2), 217-220. doi:10.1016/0014-5793(83)80822-9Hughes, J. M., & Ares, M. (1991). Depletion of U3 small nucleolar RNA inhibits cleavage in the 5′ external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA. The EMBO Journal, 10(13), 4231-4239. doi:10.1002/j.1460-2075.1991.tb05001.xSharma, K., & Tollervey, D. (1999). Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing. Molecular and Cellular Biology, 19(9), 6012-6019. doi:10.1128/mcb.19.9.6012Dragon, F., Gallagher, J. E. G., Compagnone-Post, P. A., Mitchell, B. M., Porwancher, K. A., Wehner, K. A., … Baserga, S. J. (2002). A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature, 417(6892), 967-970. doi:10.1038/nature00769Dutca, L. M., Gallagher, J. E. G., & Baserga, S. J. (2011). The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing. Nucleic Acids Research, 39(12), 5164-5180. doi:10.1093/nar/gkr044Qi, Y., & Ding, B. (2003). Differential Subnuclear Localization of RNA Strands of Opposite Polarity Derived from an Autonomously Replicating Viroid[W]. The Plant Cell, 15(11), 2566-2577. doi:10.1105/tpc.016576Idol, R. A., Robledo, S., Du, H.-Y., Crimmins, D. L., Wilson, D. B., Ladenson, J. H., … Mason, P. J. (2007). Cells depleted for RPS19, a protein associated with Diamond Blackfan Anemia, show defects in 18S ribosomal RNA synthesis and small ribosomal subunit production. Blood Cells, Molecules, and Diseases, 39(1), 35-43. doi:10.1016/j.bcmd.2007.02.001Cho, H. K., Ahn, C. S., Lee, H.-S., Kim, J.-K., & Pai, H.-S. (2013). Pescadillo plays an essential role in plant cell growth and survival by modulating ribosome biogenesis. The Plant Journal, 76(3), 393-405. doi:10.1111/tpj.12302Weis, B. L., Missbach, S., Marzi, J., Bohnsack, M. T., & Schleiff, E. (2014). The 60S associated ribosome biogenesis factor LSG1-2 is required for 40S maturation inArabidopsis thaliana. The Plant Journal, 80(6), 1043-1056. doi:10.1111/tpj.12703Kojima, K., Tamura, J., Chiba, H., Fukada, K., Tsukaya, H., & Horiguchi, G. (2018). Two Nucleolar Proteins, GDP1 and OLI2, Function As Ribosome Biogenesis Factors and Are Preferentially Involved in Promotion of Leaf Cell Proliferation without Strongly Affecting Leaf Adaxial–Abaxial Patterning in Arabidopsis thaliana. Frontiers in Plant Science, 8. doi:10.3389/fpls.2017.02240Maekawa, S., Ishida, T., & Yanagisawa, S. (2017). Reduced Expression of APUM24, Encoding a Novel rRNA Processing Factor, Induces Sugar-Dependent Nucleolar Stress and Altered Sugar Responses in Arabidopsis thaliana. The Plant Cell, 30(1), 209-227. doi:10.1105/tpc.17.00778Bonfiglioli, R. G., McFadden, G. I., & Symons, R. H. (1994). In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus. The Plant Journal, 6(1), 99-103. doi:10.1046/j.1365-313x.1994.6010099.xHill, J. M., Zhao, Y., Bhattacharjee, S., & Lukiw, W. J. (2014). miRNAs and viroids utilize common strategies in genetic signal transfer. Frontiers in Molecular Neuroscience, 7. doi:10.3389/fnmol.2014.00010Li, S., Liu, L., Zhuang, X., Yu, Y., Liu, X., Cui, X., … Chen, X. (2013). MicroRNAs Inhibit the Translation of Target mRNAs on the Endoplasmic Reticulum in Arabidopsis. Cell, 153(3), 562-574. doi:10.1016/j.cell.2013.04.005Fukaya, T., Iwakawa, H., & Tomari, Y. (2014). MicroRNAs Block Assembly of eIF4F Translation Initiation Complex in Drosophila. Molecular Cell, 56(1), 67-78. doi:10.1016/j.molcel.2014.09.004Lanet, E., Delannoy, E., Sormani, R., Floris, M., Brodersen, P., Crété, P., … Robaglia, C. (2009). Biochemical Evidence for Translational Repression by Arabidopsis MicroRNAs. The Plant Cell, 21(6), 1762-1768. doi:10.1105/tpc.108.063412Iwakawa, H., & Tomari, Y. (2013). Molecular Insights into microRNA-Mediated Translational Repression in Plants. Molecular Cell, 52(4), 591-601. doi:10.1016/j.molcel.2013.10.033Reis, R. S., Hart-Smith, G., Eamens, A. L., Wilkins, M. R., & Waterhouse, P. M. (2015). Gene regulation by translational inhibition is determined by Dicer partnering proteins. Nature Plants, 1(3). doi:10.1038/nplants.2014.27Flores, R., Navarro, B., Kovalskaya, N., Hammond, R. W., & Di Serio, F. (2017). Engineering resistance against viroids. Current Opinion in Virology, 26, 1-7. doi:10.1016/j.coviro.2017.07.00