Emergence and phylodynamics of Citrus tristeza virus in Sicily, Italy

[EN] Citrus tristeza virus (CTV) outbreaks were detected in Sicily island, Italy for the first time in 2002. To gain insight into the evolutionary forces driving the emergence and phylogeography of these CTV populations, we determined and analyzed the nucleotide sequences of the p20 gene from 108 CTV isolates collected from 2002 to 2009. Bayesian phylogenetic analysis revealed that mild and severe CTV isolates belonging to five different clades (lineages) were introduced in Sicily in 2002. Phylogeographic analysis showed that four lineages co-circulated in the main citrus growing area located in Eastern Sicily. However, only one lineage (composed of mild isolates) spread to distant areas of Sicily and was detected after 2007. No correlation was found between genetic variation and citrus host, indicating that citrus cultivars did not exert differential selective pressures on the virus. The genetic variation of CTV was not structured according to geographical location or sampling time, likely due to the multiple introduction events and a complex migration pattern with intense co- and recirculation of different lineages in the same area. The phylogenetic structure, statistical tests of neutrality and comparison of synonymous and nonsynonymous substitution rates suggest that weak negative selection and genetic drift following a rapid expansion may be the main causes of the CTV variability observed today in Sicily. Nonetheless, three adjacent amino acids at the p20 N-terminal region were found to be under positive selection, likely resulting from adaptation events.A.W. and S.F.E. were supported by grant BFU2012-30805 from the Spanish Secretaria de Estado de Investigacion, Desarrollo e Innovacion and by a grant 22371 from the John Templeton Foundation. The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the John Templeton Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Davino, S.; Willemsen, A.; Panno. Stefano; Davino, M.; Catara, A.; Elena Fito, SF.; Rubio, L. (2013). Emergence and phylodynamics of Citrus tristeza virus in Sicily, Italy. PLoS ONE. 8:66700-66700. doi:10.1371/journal.pone.0066700S66700667008Domingo, E., & Holland, J. J. (1997). RNA VIRUS MUTATIONS AND FITNESS FOR SURVIVAL. Annual Review of Microbiology, 51(1), 151-178. doi:10.1146/annurev.micro.51.1.151Grenfell, B. T. (2004). Unifying the Epidemiological and Evolutionary Dynamics of Pathogens. Science, 303(5656), 327-332. doi:10.1126/science.1090727Moya, A., Holmes, E. C., & González-Candelas, F. (2004). The population genetics and evolutionary epidemiology of RNA viruses. Nature Reviews Microbiology, 2(4), 279-288. doi:10.1038/nrmicro863Gray, R. R., Tatem, A. J., Lamers, S., Hou, W., Laeyendecker, O., Serwadda, D., … Salemi, M. (2009). Spatial phylodynamics of HIV-1 epidemic emergence in east Africa. AIDS, 23(14), F9-F17. doi:10.1097/qad.0b013e32832faf61Holmes, E. C. (2008). Evolutionary History and Phylogeography of Human Viruses. Annual Review of Microbiology, 62(1), 307-328. doi:10.1146/annurev.micro.62.081307.162912Pybus, O. G., Suchard, M. A., Lemey, P., Bernardin, F. J., Rambaut, A., Crawford, F. W., … Delwart, E. L. (2012). Unifying the spatial epidemiology and molecular evolution of emerging epidemics. Proceedings of the National Academy of Sciences, 109(37), 15066-15071. doi:10.1073/pnas.1206598109Talbi, C., Lemey, P., Suchard, M. A., Abdelatif, E., Elharrak, M., Jalal, N., … Bourhy, H. (2010). Phylodynamics and Human-Mediated Dispersal of a Zoonotic Virus. PLoS Pathogens, 6(10), e1001166. doi:10.1371/journal.ppat.1001166Vijaykrishna, D., Bahl, J., Riley, S., Duan, L., Zhang, J. X., Chen, H., … Guan, Y. (2008). Evolutionary Dynamics and Emergence of Panzootic H5N1 Influenza Viruses. PLoS Pathogens, 4(9), e1000161. doi:10.1371/journal.ppat.1000161Gómez, P., Sempere, R. N., Aranda, M. A., & Elena, S. F. (2012). Phylodynamics of Pepino mosaic virus in Spain. European Journal of Plant Pathology, 134(3), 445-449. doi:10.1007/s10658-012-0019-0Lefeuvre, P., Martin, D. P., Harkins, G., Lemey, P., Gray, A. J. A., Meredith, S., … Heydarnejad, J. (2010). The Spread of Tomato Yellow Leaf Curl Virus from the Middle East to the World. PLoS Pathogens, 6(10), e1001164. doi:10.1371/journal.ppat.1001164TOMITAKA, Y., & OHSHIMA, K. (2006). A phylogeographical study of the Turnip mosaic virus population in East Asia reveals an ‘emergent’ lineage in Japan. Molecular Ecology, 15(14), 4437-4457. doi:10.1111/j.1365-294x.2006.03094.xWu, B., Blanchard-Letort, A., Liu, Y., Zhou, G., Wang, X., & Elena, S. F. (2011). Dynamics of Molecular Evolution and Phylogeography of Barley yellow dwarf virus-PAV. PLoS ONE, 6(2), e16896. doi:10.1371/journal.pone.0016896MORENO, P., AMBRÓS, S., ALBIACH-MARTÍ, M. R., GUERRI, J., & PEÑA, L. (2008). Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Molecular Plant Pathology, 9(2), 251-268. doi:10.1111/j.1364-3703.2007.00455.xTatineni, S., Robertson, C. J., Garnsey, S. M., & Dawson, W. O. (2011). A plant virus evolved by acquiring multiple nonconserved genes to extend its host range. Proceedings of the National Academy of Sciences, 108(42), 17366-17371. doi:10.1073/pnas.1113227108Folimonova, S. Y. (2012). Superinfection Exclusion Is an Active Virus-Controlled Function That Requires a Specific Viral Protein. Journal of Virology, 86(10), 5554-5561. doi:10.1128/jvi.00310-12Bar-Joseph, M., Marcus, R., & Lee, R. F. (1989). The Continuous Challenge of Citrus Tristeza Virus Control. Annual Review of Phytopathology, 27(1), 291-316. doi:10.1146/annurev.py.27.090189.001451Davino, S., Rubio, L., & Davino, M. (2005). Molecular analysis suggests that recent Citrus tristeza virus outbreaks in Italy were originated by at least two independent introductions. European Journal of Plant Pathology, 111(3), 289-293. doi:10.1007/s10658-003-2815-zAlbiach-Marti, M. R., Mawassi, M., Gowda, S., Satyanarayana, T., Hilf, M. E., Shanker, S., … Dawson, W. O. (2000). Sequences of Citrus Tristeza Virus Separated in Time and Space Are Essentially Identical. Journal of Virology, 74(15), 6856-6865. doi:10.1128/jvi.74.15.6856-6865.2000Rubio, L., Ayllon, M. A., Kong, P., Fernandez, A., Polek, M., Guerri, J., … Falk, B. W. (2001). Genetic Variation of Citrus Tristeza Virus Isolates from California and Spain: Evidence for Mixed Infections and Recombination. Journal of Virology, 75(17), 8054-8062. doi:10.1128/jvi.75.17.8054-8062.2001Silva, G., Marques, N., & Nolasco, G. (2011). The evolutionary rate of citrus tristeza virus ranks among the rates of the slowest RNA viruses. Journal of General Virology, 93(2), 419-429. doi:10.1099/vir.0.036574-0Mawassi, M., Mietkiewska, E., Gofman, R., Yang, G., & Bar-Joseph, M. (1996). Unusual Sequence Relationships Between Two Isolates of Citrus Tristeza Virus. Journal of General Virology, 77(9), 2359-2364. doi:10.1099/0022-1317-77-9-2359Vives, M. C., Dawson, W. O., Flores, R., L√≥pez, C., Albiach-Mart√≠, M. R., Rubio, L., … Moreno, P. (1999). The complete genome sequence of the major component of a mild citrus tristeza virus isolate. Journal of General Virology, 80(3), 811-816. doi:10.1099/0022-1317-80-3-811Martín, S., Elena, S. F., Guerri, J., Moreno, P., Sambade, A., Rubio, L., … Vives, M. C. (2009). Contribution of recombination and selection to molecular evolution of Citrus tristeza virus. Journal of General Virology, 90(6), 1527-1538. doi:10.1099/vir.0.008193-0Vives, M. C., Rubio, L., Sambade, A., Mirkov, T. E., Moreno, P., & Guerri, J. (2005). Evidence of multiple recombination events between two RNA sequence variants within a Citrus tristeza virus isolate. Virology, 331(2), 232-237. doi:10.1016/j.virol.2004.10.037D’Urso, F., Sambade, A., Moya, A., Guerri, J., & Moreno, P. (2003). Variation of haplotype distributions of two genomic regions of Citrus tristeza virus populations from eastern Spain. Molecular Ecology, 12(2), 517-526. doi:10.1046/j.1365-294x.2000.01747.xSambade, A., Rubio, L., Garnsey, S. M., Costa, N., Muller, G. W., Peyrou, M., … Moreno, P. (2002). Comparison of viral RNA populations of pathogenically distinct isolates of Citrus tristeza virus : application to monitoring cross-protection. Plant Pathology, 51(3), 257-265. doi:10.1046/j.1365-3059.2002.00720.xReed, J. C., Kasschau, K. D., Prokhnevsky, A. I., Gopinath, K., Pogue, G. P., Carrington, J. C., & Dolja, V. V. (2003). Suppressor of RNA silencing encoded by Beet yellows virus. Virology, 306(2), 203-209. doi:10.1016/s0042-6822(02)00051-xFolimonova, S. Y., Robertson, C. J., Shilts, T., Folimonov, A. S., Hilf, M. E., Garnsey, S. M., & Dawson, W. O. (2009). Infection with Strains of Citrus Tristeza Virus Does Not Exclude Superinfection by Other Strains of the Virus. Journal of Virology, 84(3), 1314-1325. doi:10.1128/jvi.02075-09Kong, P., Rubio, L., Polek, M., & Falk, B. W. (2000). Virus Genes, 21(3), 139-145. doi:10.1023/a:1008198311398Powell, C. A., Pelosi, R. R., Rundell, P. A., & Cohen, M. (2003). Breakdown of Cross-Protection of Grapefruit from Decline-Inducing Isolates of Citrus tristeza virus Following Introduction of the Brown Citrus Aphid. Plant Disease, 87(9), 1116-1118. doi:10.1094/pdis.2003.87.9.1116Roistacher C, Dodds J. (1993) Failure of 100 mild Citrus tristeza virus isolates from california to cross protect against a challenge by severe sweet orange stem pitting isolates. Proc 12th Conf IOCV: 100–107.Ayllón, M. A., Rubio, L., Sentandreu, V., Moya, A., Guerri, J., & Moreno, P. (2006). Variations in Two Gene Sequences of Citrus Tristeza Virus after Host Passage. Virus Genes, 32(2), 119-128. doi:10.1007/s11262-005-6866-4Ayllón, M. A., Rubio, L., Moya, A., Guerri, J., & Moreno, P. (1999). The Haplotype Distribution of Two Genes of Citrus Tristeza Virus Is Altered after Host Change or Aphid Transmission. Virology, 255(1), 32-39. doi:10.1006/viro.1998.9566Sentandreu, V., Castro, J. A., Ayllón, M. A., Rubio, L., Guerri, J., González-Candelas, F., … Moya, A. (2005). Evolutionary analysis of genetic variation observed in citrus tristeza virus (CTV) after host passage. Archives of Virology, 151(5), 875-894. doi:10.1007/s00705-005-0683-xMatos, L. A., Hilf, M. E., Cayetano, X. A., Feliz, A. O., Harper, S. J., & Folimonova, S. Y. (2013). Dramatic Change in Citrus tristeza virus Populations in the Dominican Republic. Plant Disease, 97(3), 339-345. doi:10.1094/pdis-05-12-0421-reDavino, S., Davino, M., Sambade, A., Guardo, M., & Caruso, A. (2003). The First Citrus tristeza virus Outbreak Found in a Relevant Citrus Producing Area of Sicily, Italy. Plant Disease, 87(3), 314-314. doi:10.1094/pdis.2003.87.3.314aRUBIO, L., AYLLONl, M. A., GUERRI, J., PAPPU, H., NIBLETT, C., & MORENO, P. (1996). Differentiation of citrus tristeza closterovirus (CTV) isolates by single-strand conformation polymorphism analysis of the coat protein gene. Annals of Applied Biology, 129(3), 479-489. doi:10.1111/j.1744-7348.1996.tb05770.xLarkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., … Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23(21), 2947-2948. doi:10.1093/bioinformatics/btm404Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28(10), 2731-2739. doi:10.1093/molbev/msr121Kosakovsky Pond, S. L., Posada, D., Gravenor, M. B., Woelk, C. H., & Frost, S. D. W. (2006). GARD: a genetic algorithm for recombination detection. Bioinformatics, 22(24), 3096-3098. doi:10.1093/bioinformatics/btl474Martin, D. P., Lemey, P., Lott, M., Moulton, V., Posada, D., & Lefeuvre, P. (2010). RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics, 26(19), 2462-2463. doi:10.1093/bioinformatics/btq467Librado, P., & Rozas, J. (2009). DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25(11), 1451-1452. doi:10.1093/bioinformatics/btp187Kimura M. (1985) The neutral theory of molecular evolution. Cambridge Univ Pr.Weir, B. S., & Cockerham, C. C. (1984). Estimating F-Statistics for the Analysis of Population Structure. Evolution, 38(6), 1358. doi:10.2307/2408641Pond, S. L. K., & Frost, S. D. W. (2005). Datamonkey: rapid detection of selective pressure on individual sites of codon alignments. Bioinformatics, 21(10), 2531-2533. doi:10.1093/bioinformatics/bti320Drummond, A. J., & Rambaut, A. (2007). BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7(1), 214. doi:10.1186/1471-2148-7-214Bielejec, F., Rambaut, A., Suchard, M. A., & Lemey, P. (2011). SPREAD: spatial phylogenetic reconstruction of evolutionary dynamics. Bioinformatics, 27(20), 2910-2912. doi:10.1093/bioinformatics/btr481Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22(21), 2688-2690. doi:10.1093/bioinformatics/btl446Ott M, Zola J, Stamatakis A, Aluru S. (2007) Large-scale maximum likelihood-based phylogenetic analysis on the IBM BlueGene/L. Proceedings of the 19th ACM/IEEE conference on Supercomputing. Article No. 4.Shimodaira, H., & Hasegawa, M. (1999). Multiple Comparisons of Log-Likelihoods with Applications to Phylogenetic Inference. Molecular Biology and Evolution, 16(8), 1114-1116. doi:10.1093/oxfordjournals.molbev.a026201Soria-Carrasco, V., Talavera, G., Igea, J., & Castresana, J. (2007). The K tree score: quantification of differences in the relative branch length and topology of phylogenetic trees. Bioinformatics, 23(21), 2954-2956. doi:10.1093/bioinformatics/btm466Puigbo, P., Garcia-Vallve, S., & McInerney, J. O. (2007). TOPD/FMTS: a new software to compare phylogenetic trees. Bioinformatics, 23(12), 1556-1558. doi:10.1093/bioinformatics/btm13

Evolutionary analysis of Citrus tristeza virus outbreaks in Calabria, Italy: two rapidly spreading and independent introductions of mild and severe isolates

The evolution of citrus tristeza virus (CTV) from outbreaks occurred in Calabria, Italy, was compared with that of CTV outbreaks reported previously in another two proximal Italian regions, Sicily and Apulia. Examination of four genomic regions (genes p20, p25 and p23, and one fragment of open reading frame 1) showed two recombination events, and phylogenetic analysis disclosed two divergent CTV groups in Calabria: one formed by severe and the other by mild isolates. This analysis, together with others involving population genetic parameters, revealed a low migration rate of CTV between the three Italian regions, as well as significant differences in selective pressures, epidemiology and demography, all affecting the genetic structure of CTV populations.This research was carried out in the frame of the national project "Armonizzazione della diagnosi e valutazione del rischio di patogeni da quarantena e nocivi ai vegetali e ai prodotti vegetali (ARNADIA-ARON)", supported by the Italian Ministry of Agriculture. A. Fontana has been the recipient of a postdoctoral fellowship from "Progetto cofinanziato POR Calabria FSE 2007/2013-Obiettivo Operativo M2", and D. E. Debreczeni the recipient of a FPU predoctoral fellowship from the Spanish Ministry of Education and Science.Fontana, A.; Debreczeni, DE.; Albanese, G.; Davino, S.; Flores Pedauye, R.; Rubio, L. (2014). Evolutionary analysis of Citrus tristeza virus outbreaks in Calabria, Italy: two rapidly spreading and independent introductions of mild and severe isolates. European Journal of Plant Pathology. 140(3):607-613. doi:10.1007/s10658-014-0489-3S6076131403Abou Kubaa, R., D’Onghia, A. M., Djelouah, K., Savino, V., & Saponari, M. (2012). Characterization of Citrus tristeza virus isolates recovered in Syria and Apulia (southern Italy) using different molecular tools. Phytopathologia Mediterranea, 51, 496–504.Acosta-Leal, R., Duffy, S., Xiong, Z., Hammond, R., & Elena, S. F. (2011). Advances in plant virus evolution: translating evolutionary insights into better disease management. Phytopathology, 101, 1136–1148.Albanese, G., Schimio, R., Fontana, A., Ferretti, L., Palmeri, V., Campolo, O., et al. (2010). Assessment of Citrus tristeza virus (CTV) incidence in Calabria, southern Italy: results of a three-year survey. Phytopathologia Mediterranea, 49, 27–34.Albiach-Martí, M. R., Mawassi, M., Gowda, S., Satyanarayana, T., Hilf, M. E., Shanker, S., et al. (2000). Sequences of Citrus tristeza virus separated in time and space are essentially identical. Journal of Virology, 74, 6856–6865.Bar-Joseph, M., Marcus, R., & Lee, R. F. (1989). The continuous challenge of Citrus tristeza virus control. Annual Review of Phytopathology, 27, 291–316.Davino, S., Davino, M., Sambade, A., Guardo, M., & Caruso, A. (2003). The first Citrus tristeza virus outbreak found in a relevant citrus producing area of Sicily, Italy. Plant Disease, 87, 314–314.Davino, S., Rubio, L., & Davino, M. (2005). Molecular analysis suggests that recent Citrus tristeza virus outbreaks in Italy were originated by at least two independent introductions. European Journal of Plant Pathology, 111, 289–293.Davino, S., Willemsen, A., Panno, S., Davino, M., Catara, A., Elena, S. F., et al. (2013). Emergence and phylodynamics of Citrus tristeza virus in Sicily, Italy. PLoS ONE, 8, e66700.Domingo, E., & Holland, J. (1997). RNA virus mutations and fitness for survival. Annual Review of Microbiology, 51, 151–178.Efron, B., Halloran, E., & Holmes, S. (1996). Bootstrap confidence levels for phylogenetic trees. Proceedings of the National Academy of Sciences of the United States of America, 93, 7085–7090.Febres, V., Ashoulin, L., Mawassi, M., Frank, A., Bar-Joseph, M., Manjunath, K., et al. (1996). The p27 protein is present at one end of Citrus tristeza virus particles. Phytopathology, 86, 1331–1335.Ferretti, L., Fontana, A., Sciarroni, R., Schimio, R., Loconsole, G., Albanese, G., et al. (2014). Molecular and biological evidence for a severe seedling yellows strain of Citrus tristeza virus spreading in Calabria (Southern Italy). Phytopathologia Mediterranea, 53, 3–13.Fu, Y. X., & Li, W. H. (1993). Maximum likelihood estimation of population parameters. Genetics, 134, 261–1270.Harper, S. J. (2013). Citrus tristeza virus: evolution of complex and varied genotypic groups. Frontiers in Microbiology, 4, 93.Hilf, M., & Garnsey, S. (2000). Characterization and classification of Citrus tristeza virus isolates by amplification of multiple molecular markers. Proceedings of the Fourteenth Conference of the International Organization of Citrus Virologists, 14, 18–27.Hudson, R. R. (2000). A new statistic for detecting genetic differentiation. Genetics, 155, 2011–2014.Hudson, R. R., Boos, D. D., & Kaplan, N. L. (1992). A statistical test for detecting geographic subdivision. Molecular Biology and Evolution, 9, 138–151.Karasev, A., Boyko, V., Gowda, S., Nikolaeva, O., Hilf, M., Koonin, E., et al. (1995). Complete sequence of the Citrus tristeza virus RNA genome. Virology, 208, 511–520.Kimura, M. (1985). The neutral theory of molecular evolution. Cambridge: Cambridge University Press.Kosakovsky Pond, S. L., Posada, D., Gravenor, M. B., Woelk, C. H., & Frost, S. D. (2006). GARD: a genetic algorithm for recombination detection. Bioinformatics, 22, 3096–3098.Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947–2948.Librado, P., & Rozas, J. (2009). DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451–1452.Lu, R., Folimonov, A., Shintaku, M., Li, W., Falk, B. W., Dawson, W. O., et al. (2004). Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome. Proceedings of the National Academy of Sciences of the United States of America, 101, 15742–15747.Martín, S., Sambade, A., Rubio, L., Vives, M. C., Moya, P., Guerri, J., et al. (2009). Contribution of recombination and selection to molecular evolution of Citrus tristeza virus. Journal of General Virology, 90, 1527–1538.Martín, D. P., Lemey, P., Lott, M., Moulton, V., Posada, D., & Lefeuvre, P. (2010). RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics, 26, 2462–2463.Moreno, P., Ambrós, S., Albiach-Martí, M. R., Guerri, J., & Peña, L. (2008). Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Molecular Plant Pathology, 9, 251–268.Moya, A., Holmes, E. C., & González-Candelas, F. (2004). The population genetics and evolutionary epidemiology of RNA viruses. Nature Reviews Microbiology, 2, 279–288.Nagy, P. D. (2008). Recombination in plant RNA viruses. In M. J. Roossinck (Ed.), Plant virus evolution (pp. 133–164). Berlin: Springer.Nei, M., & Kumar, S. (2000). Molecular evolution and phylogenetics. USA: Oxford University Press.Niblett, C. L., Genc, H., Cevik, B., Halbert, S., Brown, L., Nolasco, G., et al. (2000). Progress on strain differentiation and its application to the epidemiology of Citrus Tristeza Virus. Virus Research, 71, 97–106.Pappu, H. R., Pappu, S. S., Manjunath, K. L., Lee, R. F., & Niblett, C. L. (1993). Molecular characterization of a structural epitope that is largely conserved among severe isolates of a plant virus. Proceedings of the National Academy of Sciences of the United States of America, 90, 3641–3644.Rubio, L., Ayllón, M. A., Kong, P., Fernández, A., Polek, M. L., Guerri, J., et al. (2001). Genetic variation of Citrus tristeza virus isolates from California and Spain: evidence for mixed infections and recombination. Journal of Virology, 75, 8054–8062.Rubio, L., Guerri, J., & Moreno, P. (2013). Genetic variability and evolutionary dynamics of viruses of the family Closteroviridae. Frontiers in Microbiology, 4, 151.Ruiz-Ruiz, S., Soler, N., Sánchez-Navarro, J., Fagoaga, C., López, C., Navarro, L., et al. (2013). Citrus tristeza virus p23: determinants for nucleolar localization and their influence on suppression of RNA silencing and pathogenesis. Molecular Plant-Microbe Interactions, 26, 306–318.Sambade, A., López, C., Rubio, L., Flores, R., Guerri, J., & Moreno, P. (2003). Polymorphism of a specific region in gene p23 of Citrus tristeza virus allows discrimination between mild and severe isolates. Archives of Virology, 148, 2325–2340.Silva, G., Marques, N., & Nolasco, G. (2012). The evolutionary rate of Citrus tristeza virus ranks among the rates of the slowest RNA viruses. Journal of General Virology, 93, 419–429.Tajima, F. (1989). Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics, 123, 585–595.Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739.Vives, M. C., Rubio, L., López, C., Navas-Castillo, J., Albiach-Martí, M. R., Dawson, W. O., et al. (1999). The complete genome sequence of the major component of a mild Citrus tristeza virus isolate. Journal of General Virology, 80, 811–816.Vives, M. C., Rubio, L., Sambade, A., Mirkov, T. E., Moreno, P., & Guerri, J. (2005). Evidence of multiple recombination events between two RNA sequence variants within a Citrus tristeza virus isolate. Virology, 331, 232–237.Weir, B. S., & Cockerham, C. C. (1984). Estimating F-statistics for the analysis of population structure. Evolution, 38, 1358–1370

Citrus tristeza virus: Host RNA Silencing and Virus Counteraction

[EN] To dissect the host RNA silencing response incited by citrus tristeza virus (CTV, genus Closterovirus), a (+) ssRNA of similar to 19300 nt, and the counter reaction deployed by the virus via its three RNA silencing suppressors (RSS), the small RNAs (sRNAs) of three virus-host combinations were deep sequenced. The subsequent analysis indicated that CTV sRNAs (1) constitute more than half of the total sRNAs in the susceptible Mexican lime and sweet orange, while only 3.5% in the restrictive sour orange; (2) are mostly of 21-22 nt, with those of (+) sense predominating slightly; and (3) derive from all the CTV genome, as evidenced by its entire recomposition from viral sRNA contigs but adopt an asymmetric pattern with a hotspot mapping at the 3'-terminal similar to 2500 nt. The citrus homologues of Arabidopsis Dicer-like (DCL) 4 and 2 most likely generate the 21 and 22 nt CTV sRNAs, respectively, by dicing the gRNA and the 3' co-terminal sgRNAs and, particularly, their double-stranded forms accumulating in infected cells. The plant sRNA profile, very similar and dominated by the 24 nt sRNAs in the three mock-inoculated controls, displayed a major reduction of the 24 nt sRNAs in Mexican lime and sweet orange, but not in sour orange. CTV infection also influences the levels of certain microRNAs. The high accumulation of CTV sRNAs in two of the citrus hosts examined suggests that it is not their synthesis, but their function, the target of the RSS encoded by CTV: p25 (intercellular), p23 (intracellular) and p20 (both). The two latter might block the loading of CTV sRNAs into the RNA silencing complex or interfere with it through alternative mechanisms. Of the three CTV RSS, p23 is the one that has been more thoroughly studied. It is a multifunctional RNA-binding protein with a putative Zn finger domain and basic motifs that (1) has no homologues in other closteroviruses, (2) accumulates in the nucleolus and plasmodesmata, (3) regulates the asymmetric balance of CTV (+) and (-) RNA strands, and (4) induces CTV syndromes and stimulates systemic infection in certain citrus species when expressed as a transgene ectopically or in phloem-associated cells.This research was supported by a grant (Prometeo/2008/121) from the Generalitat Valenciana, Spain, and by a grant (AGL2009-08052) from the Ministerio de Ciencia e Innovacio¿nFondo Europeo de Desarrollo Regional. S. Ruiz-Ruiz was additionally supported by a postdoctoral contract from the Generalitat Valenciana (APOSTD/2012/020, Program VALi+d).Ruiz-Ruiz, S.; Navarro, B.; Peña Garcia, L.; Navarro, L.; Moreno, P.; Di Serio, F.; Flores Pedauye, R. (2019). Citrus tristeza virus: Host RNA Silencing and Virus Counteraction. Methods in Molecular Biology. 2015:195-207. https://doi.org/10.1007/978-1-4939-9558-5_14S195207201