Plant Molecular Biology

, Volume 60, Issue 2, pp 153–165 | Cite as

Post-Transcriptional Gene Silencing of the p23 Silencing Suppressor of Citrus tristeza virus Confers Resistance to the Virus in Transgenic Mexican Lime

  • Carmen Fagoaga
  • Carmelo López
  • Alfonso Hermoso de Mendoza
  • Pedro Moreno
  • Luis Navarro
  • Ricardo Flores
  • Leandro Peña


Previously, we have shown that most Mexican limes (Citrus aurantifolia (Christ.) Swing.) expressing the p23 gene of Citrus tristeza virus (CTV) exhibit aberrations resembling viral leaf symptoms. Here we report that five independent transgenic lines having normal phenotype displayed characteristics typical of post-transcriptional gene silencing (PTGS): multiple copies of the transgene, low levels of the corresponding mRNA, methylation of the silenced transgene, and accumulation of p23-specific small interfering RNAs (siRNAs). When graft- or aphid-inoculated with CTV, some propagations of these silenced lines were immune: they neither expressed symptoms nor accumulated virions and viral RNA as estimated by DAS-ELISA and Northern blot hybridization, respectively. Other propagations were moderately resistant because they became infected later and showed attenuated symptoms compared to controls. The susceptible propagations, in addition to symptom expression and elevated virus titer, accumulated p23-specific siRNAs at levels significantly higher than immune or non-inoculated propagations, and showed transgene demethylation. This variable response among clonal transformants indicates that factors other than the genetic background of the transgenic plants play a key role in PTGS-mediated resistance.


CTV methylation pathogenicity determinant RNA silencing small interfering RNAs virus resistance 


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  1. Ayllón, M.A., Gowda, S., Satyanarayana, T., Karasev, A.V., Adkins, S., Mawassi, M., Guerri, J., Moreno, P., Dawson, W.O. 2003Effects of modification of the transcription initiation site context on citrus tristeza virus subgenomic RNA synthesisJ. Virol.7792329243PubMedGoogle Scholar
  2. Bar-Joseph, M., Marcus, R., Lee, R.F. 1989The continuous challenge of citrus tristeza virus controlAnnu. Rev. Phytopathol.27291316CrossRefGoogle Scholar
  3. Ballester-Olmos, J.F., Pina, J.A., Carbonell, E., Moreno, P., Hermoso de Mendoza, A., Cambra, M., Navarro, L. 1993Biological diversity of citrus tristeza virus (CTV) isolates in SpainPlant Pathol.42219229Google Scholar
  4. Baulcombe, D. 2004RNA silencing in plantsNature431356363PubMedCrossRefGoogle Scholar
  5. Cambra, M., Garnsey, S.M., Permar, T.A., Henderson, C., Gumpf, D., Vela, C. 1990Detection of citrus tristeza virus (CTV) with a mixture of monoclonal antibodiesPhytopathology801034Google Scholar
  6. Carpenter, C.D., Simon, A.E. 1998Preparation of RNAMethods Mol. Biol.828589PubMedGoogle Scholar
  7. Chiu, W., Niwa, Y., Zeng, W., Hirano, T. 1996Engineered GFP as a vital reporter in plantsCurr. Biol6325330PubMedCrossRefGoogle Scholar
  8. Costa, A.S., Müller, G.W. 1980Tristeza control by cross protection: a US–Brazil cooperative successPlant Dis.64538541Google Scholar
  9. Covey, S.N., AlKaff, N.S., Langara, A., Turner, D.S. 1997Plants combat infection by gene silencingNature385781782CrossRefGoogle Scholar
  10. Dellaporta, S.L., Wood, J., Hicks, J.B. 1983A plant DNA minipreparation: version IIPlant Mol. Biol. Rep.41921Google Scholar
  11. Domínguez, A., Hermoso de Mendoza, A., Guerri, J., Cambra, M., Navarro, L., Moreno, P., Peña, L. 2002Pathogen-derived resistance to citrus tristeza virus (CTV) in transgenic Mexican lime (Citrus aurantifolia (Christ.) Swing.) plants expressing its p25 coat protein geneMol. Breed.10110Google Scholar
  12. English, J.J., Baulcombe, D.C. 1997The influence of small changes in transgene transcription on homology-dependent virus resistance and gene silencingPlant J.1213111318Google Scholar
  13. Fagoaga, C., López, C., Moreno, P., Navarro, L., Flores, R., Peña, L. 2005Viral-like symptoms induced by the ectopic expression of the p23 gene of citrus tristeza virus are citrus-specific and do not correlate with the pathogenicity of the virus strainMol. Plant Microbe Interact.18435445PubMedGoogle Scholar
  14. Flores, R. 1986Detection of citrus exocortis viroid in crude extracts by dot-blot hybridization: conditions for reducing spurious hybridization results and for enhancing the sensitivity of the techniqueJ. Virol. Meth.13161169Google Scholar
  15. Ghorbel, R., López, C., Fagoaga, C., Moreno, P., Navarro, L., Flores, R., Peña, L. 2001Transgenic citrus plants expressing the citrus tristeza virus p23 protein exhibit viral-like symptomsMol. Plant Pathol.22736CrossRefGoogle Scholar
  16. Gowda, S., Satyanarayana, T., Davis, C.L., Navas-Castillo, J., Albiach-Martí, M.R., Mawassi, M., Valkov, N., Bar-Joseph, M., Moreno, P., Dawson, W.O. 2000The p20 gene product of citrus tristeza virus accumulates in the amorphous inclusion bodiesVirology274246254PubMedCrossRefGoogle Scholar
  17. Guo, H.S., Ding, S.W. 2002A viral protein inhibits the long range signalling activity of the gene silencing signalEMBO J.21398407PubMedCrossRefGoogle Scholar
  18. Hamilton, A.J., Baulcombe, D.C. 1999A species of small antisense RNA in posttranscriptional gene silencing in plantsScience286950952PubMedCrossRefGoogle Scholar
  19. Hammond, S.M., Bernstein, E., Beach, D., Hannon, G.J. 2000An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cellsNature404293296PubMedGoogle Scholar
  20. Hermoso de Mendoza, A., Ballester-Olmos, J.F. and Pina, J.A. 1984. Transmission of citrus tristeza virus by aphids in Spain. In: S.M. Garnsey, L.W. Timmer and J.A. Dodds (Eds.), Tristeza and Related Diseases. Proceedings of the 9th Conference of the International Organization of Citrus Virologist, 9–13 May 1983, Argentina, pp. 23–27. International Organization of Citrus Virologists (IOCV), Riverside, CA, USAGoogle Scholar
  21. Hilf, M.E., Karasev, A.V., Pappu, H.R., Gumpf, D.J., Niblett, C.L., Garnsey, S.M. 1995Characterization of citrus tristeza virus subgenomic RNAs in infected tissueVirology208576582PubMedCrossRefGoogle Scholar
  22. Kalantidis, K., Psaradakis, S., Tabler, M., Tsagris, M. 2002The occurrence of CMV-specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virusMol. Plant Microbe Interact.15826833PubMedGoogle Scholar
  23. Karasev, A.V., Boyko, V.P., Gowda, S., Nikolaeva, O.V., Hilf, M.E., Koonin, E.V., Niblett, C.L., Cline, K., Gumpf, D.J., Lee, R.F., Garnsey, S.M., Lewandowsky, D.J., Dawson, W.O. 1995Complete sequence of the citrus tristeza virus RNA genomeVirology208511520PubMedCrossRefGoogle Scholar
  24. Lindbo, J.A., Silva, R. L., Proebsting, W.M., Dougherty, W.G. 1993Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistancePlant Cell517491759PubMedCrossRefGoogle Scholar
  25. López, C., Navas-Castillo, J., Gowda, S., Moreno, P., Flores, R. 2000The 23 kDa protein coded by the 3′-terminal gene of citrus tristeza virus is an RNA-binding proteinVirology269462470PubMedGoogle Scholar
  26. Lu, R., Folimonov, A., Shintaku, M., Li, W.-X., Falk, B.W., Dawson, W.O., Ding, S.W. 2004Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genomeProc. Natl. Acad. Sci. USA1011574215747PubMedGoogle Scholar
  27. Mallory, A.C., Ely, L., Smith, T.H., Marathe, R., Anandalakshmi, R., Fagard, M., Vaucheret, H., Pruss, G., Bowman, L., Vance, V.B. 2001HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signalPlant Cell13571583PubMedCrossRefGoogle Scholar
  28. Martínez de Alba, A.E., Flores, R., Hernández, C. 2002Two chloroplastic viroids induce the accumulation of small RNAs associated with posttranscriptional gene silencingJ. Virol.761309413096PubMedGoogle Scholar
  29. Pang, S.-Z., Jan, F.-J., Carney, K., Stout, J., Tricoli, D.M., Quemada, H.D., Gonsalves, D. 1996Post-transcriptional transgene silencing and consequent tospovirus resistance in transgenic lettuce are affected by transgene dosage and plant developmentPlant J.9899909CrossRefGoogle Scholar
  30. Pappu, H.R., Karasev, A.V., Anderson, E.J., Pappu, S.S., Hilf, M.E., Febres, V.J., Eckloff, R.M.G., Mccaffery, M., Boyko, V., Gowda, S., Dolja, V.V., Koonin, E.V., Gumpf, D.J., Cline, K.C., Garmsey, S.M., Dawson, W.O., Lee, R.F., Niblett, C.L. 1994Nucleotide sequence and organization of eight 3′ open reading frames of the citrus tristeza closterovirus genomeVirology1993546PubMedCrossRefGoogle Scholar
  31. Ratcliff, F., Harrison, B.D., Baulcombe, D.C. 1997A similarity between viral defense and gene silencing in plantsScience27615581560CrossRefGoogle Scholar
  32. Ratcliff, F., MacFarlane, S., Baulcombe, D.C. 1999Gene silencing without DNA. RNA-mediated cross-protection between virusesPlant Cell1112071216PubMedCrossRefGoogle Scholar
  33. Roistacher, C.N. and Moreno, P. 1991. The worldwide threat from destructive isolates of citrus tristeza virus – a review. In: R.H. Brlansky, R.F. Lee and L.W. Timmer (Eds.), Tristeza. Proceedings of the 11th Conference of the International Organization of Citrus Virologist, 6–10 November 1989, Orlando, USA, pp. 7–19. International Organization of Citrus Virologists (IOCV), Riverside, CA, U.S.AGoogle Scholar
  34. Satyanarayana, T., Gowda, S., Mawassi, M., Albiach-Martí, M.R., Ayllón, M.A., Robertson, C., Garnsey, S.M., Dawson, W.O. 2000Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assemblyVirology278253265PubMedCrossRefGoogle Scholar
  35. Satyanarayana, T., Gowda, S., Ayllón, M.A., Albiach-Martí, M.R., Rabindran, S., Dawson, W.O. 2002The p23 protein of citrus tristeza virus controls asymmetrical RNA accumulationJ. Virol.76473483PubMedCrossRefGoogle Scholar
  36. Satyanarayana, T., Gowda, S., Ayllón, M.A., Dawson, W.O. 2004Closterovirus bipolar virion: evidence for initiation of assembly by minor coat protein and its restriction to the genomic RNA 5′ regionProc. Natl. Acad. Sci. USA101799804PubMedCrossRefGoogle Scholar
  37. Scorza, R., Callahan, A., Levy, L., Damsteegt, V., Webb, K., Ravelonandro, M. 2001Post-transcriptional gene silencing in plum pox virus resistant transgenic European plum containing the plum pox potyvirus coat protein geneTransgenic Res.10201209PubMedCrossRefGoogle Scholar
  38. Smith, H.A., Swaney, S.L., Parks, T.D., Wernsman, E.A., Dougherty, W.G. 1994Transgenic plant virus resistance mediated by untranslatable sense RNAs: expression, regulation, and fate of nonessential RNAsPlant Cell614411453PubMedCrossRefGoogle Scholar
  39. Szittya, G., Silhavy, D., Molnár, A., Havelda, Z., Lovas, A., Lakatos, L., Bánfalvi, Z., Burgyán, J. 2003Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generationEMBO J.22633640PubMedCrossRefGoogle Scholar
  40. Tennant, P., Fermin, G., Fitch, M.M., Manshardt, R.M., Slightom, J.L., Gonsalves, D. 2001Papaya ringspot virus resistance of transgenic rainbow and SunUp is affected by gene dosage, plant development, and coat protein homologyEur. J. Plant Pathol.107645653CrossRefGoogle Scholar
  41. Vuuren, S.P., Collins, R.P., Graça, J.V. 1993Evaluation of citrus tristeza virus isolates for cross protection of grapefruit in South AfricaPlant Dis.772428Google Scholar
  42. Xu, J.P., Schubert, J., Altpeter, F. 2001Dissection of RNA-mediated ryegrass mosaic virus resistance in fertile transgenic perennial ryegrass (Lolium perenne L.)Plant J.26265274PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • Carmen Fagoaga
    • 1
  • Carmelo López
    • 2
  • Alfonso Hermoso de Mendoza
    • 1
  • Pedro Moreno
    • 1
  • Luis Navarro
    • 1
  • Ricardo Flores
    • 2
  • Leandro Peña
    • 1
  1. 1.Instituto Valenciano de Investigaciones Agrarias (IVIA)ValenciaSpain
  2. 2.Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC) Universidad Politécnica de ValenciaValenciaSpain

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