European Journal of Plant Pathology

, Volume 139, Issue 1, pp 151–159 | Cite as

Reaction of transgenic Citrus sinensis plants to Citrus tristeza virus infection by Toxoptera citricida

  • Fabiana R. Muniz
  • Amancio Souza
  • Ricardo Harakava
  • Francisco de Assis Alves Mourão Filho
  • Dagmar R. Stach-Machado
  • Jorge A. M. Rezende
  • Vicente J. Febres
  • Gloria A. Moore
  • Beatriz M. J. MendesEmail author


Transgenic Citrus sinensis ‘Hamlin’ and ‘Valencia’ plants containing Citrus tristeza virus (CTV)-derived sequences were propagated and inoculated with CTV. For propagation, selected buds from transgenic and non-transgenic control plants were grafted onto C. aurantium and C. limonia rootstock plants. CTV inoculation was performed via viruliferous aphids (Toxoptera citricida), and viral detection post-inoculation was performed through DASI-ELISA or RT-qPCR. After four inoculations, none of the transgenic lines tested showed complete resistance. However, viral multiplication was undetectable in some of the propagated clones. These resistant clones mainly came from transgenic ‘Valencia’ sweet orange plants grafted onto C. limonia rootstock containing the pCTV-CS gene construct. Although the tested viral inoculation method represents natural field infection conditions, the results did not differ significantly from those previously reported when the same transgenic lines were bud-graft inoculated. This finding indicates that the difficulties in producing CTV-resistant transgenic citrus lines may be unrelated to the inoculation method. Transgene expression level was quantified by RT-qPCR analysis and it was not possible to relate transgene mRNA level with resistance to the pathogen.


Genetic engineering Pathogen derived resistance Vector inoculation 



The authors acknowledge Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Fundo de Defesa da Citricultura (Fundecitrus) for research financial support. FRM acknowledges Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for doctoral fellowship. FAAMF, JAMR, RH, and BMJM acknowledge CNPq for research fellowship.

Supplementary material

10658_2013_373_Fig3_ESM.jpg (5.1 mb)

(JPEG 5235 kb)

10658_2013_373_Fig4_ESM.jpg (5.1 mb)

(JPEG 5200 kb)

10658_2013_373_Fig5_ESM.jpg (4.8 mb)

(JPEG 4930 kb)

10658_2013_373_Fig6_ESM.jpg (4.8 mb)

(JPEG 4868 kb)


  1. Ananthakrishnan, G., Orbović, V., Pasquali, G., Ćalović, M., & Grosser, J. W. (2007). Transfer of citrus tristeza virus (CTV)-derived resistance candidate sequences to four grapefruit cultivars through Agrobacterium-mediated genetic transformation. In Vitro Cellular and Developmental Biology - Plant, 43, 593–601.CrossRefGoogle Scholar
  2. Batuman, O., Mawassi, M., & Bar-Joseph, M. (2006). Transgenes consisting of a dsRNA of an RNAi suppressor plus the 3′ UTR provide resistance to Citrus tristeza virus sequences in Nicotiana benthamiana but not in citrus. Virus Genes, 33, 319–327.PubMedGoogle Scholar
  3. Boscariol, R. L., Monteiro, M., Takahashi, E. K., Chabregas, S. M., Vieira, M. L. C., Vieira, L. G. E., Pereira, L. F. P. P., Mourão Filho, F. A. A., Cardoso, S. C., Christiano, R. S. C., Bergamin Filho, A., Barbosa, J. M., Azevedo, F. A., & Mendes, B. M. J. (2006). Attacin A gene from Tricloplusia ni reduces susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis cv. ‘Hamlin’. Journal of the American Society for Horticultural Science, 131, 530–536.Google Scholar
  4. Cardoso, S. C., Barbosa-Mendes, J. M., Boscariol-Camargo, R. L., Christiano, R. S. C., Bergamin Filho, A., Vieira, M. L. C., Mendes, B. M. J., & Mourão Filho, F. A. A. (2010). Transgenic sweet orange (Citrus sinensis L. Osbeck) expressing the attacin A gene for resistance to Xanthomonas citri subsp. citri. Plant Molecular Biology Reporter, 28, 185–192.CrossRefGoogle Scholar
  5. Cervera, M., Esteban, O., Gil, M., Gorris, M. T., Martínez, M. C., Peña, L., & Cambra, M. (2010). Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance. Transgenic Research, 19, 1001–1015.PubMedCrossRefGoogle Scholar
  6. Collinge, D. B., Lund, O. S., & Thordal-Christensen, H. (2008). What are the prospects for genetically engineered, disease resistant plants? European Journal of Plant Pathology, 121, 217–231.CrossRefGoogle Scholar
  7. Domínguez, A., Mendoza, A. H., Guerri, J., Cambra, M., Navarro, L., Moreno, P., & Peña, L. (2002). Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic mexican lime (Citrus aurantifolia (Christ.) Swing.) plants expressing its p25 coat protein gene. Molecular Breeding, 10, 1–10.CrossRefGoogle Scholar
  8. Dutt, M., & Grosser, J. W. (2009). Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus. Plant Cell, Tissue and Organ Culture, 98, 331–340.CrossRefGoogle Scholar
  9. Fagoaga, C., López, C., Mendoza, A. H., Moreno, P., Navarro, L., Flores, R., & Peña, L. (2006). Post-transcriptional gene silencing of the p23 silencing suppressor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime. Plant Molecular Biology, 60, 153–165.PubMedCrossRefGoogle Scholar
  10. Febres, V. J., Niblett, C. L., Lee, R. F., & Moore, G. A. (2003). Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes. Plant Cell Reports, 21, 421–428.PubMedGoogle Scholar
  11. Febres, V. J., Lee, R. F., & Moore, G. A. (2008). Transgenic resistance to Citrus tristeza virus in grapefruit. Plant Cell Reports, 27, 93–104.PubMedCrossRefGoogle Scholar
  12. Gambino, G., & Gribaudo, I. (2012). Genetic transformation of fruit trees: current status and remaining challenges. Transgenic Research, 21, 1163–1181.PubMedCrossRefGoogle Scholar
  13. Garnsey, S. M. (1999). Systemic diseases. In L. W. Timmer & L. W. Duncan (Eds.), Citrus health management (pp. 95–106). St. Paul: APS Press.Google Scholar
  14. Ghorbel, R., López, C., Fagoaga, C., Moreno, P., Navarro, L., Flores, R., & Peña, L. (2001). Transgenic citrus plants expressing the citrus tristeza virus p23 protein exhibit viral-like symptoms. Molecular Plant Pathology, 2, 27–36.PubMedCrossRefGoogle Scholar
  15. Gonsalves, D. (1998). Control of papaya ringspot virus in papaya: a case study. Annual Review of Phytopathology, 36, 415–437.PubMedCrossRefGoogle Scholar
  16. Gutiérrez-E, M. A., Luth, D., & Moore, G. A. (1997). Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of citrus tristeza virus. Plant Cell Reports, 16, 745–753.CrossRefGoogle Scholar
  17. Kobayashi, S., & Uchimiya, H. (1989). Expression and integration of a foreign gene in orange (Citrus sinensis Osb.) protoplasts by direct DNA transfer. Japanese Journal of Genetics, 64, 91–97.CrossRefGoogle Scholar
  18. Kubista, M., Andrade, J. M., Bengtsson, M., Forootan, A., Jonák, J., Lind, K., Sindelka, R., Sjöback, R., Sjögreen, B., Strömbom, L., Ståhlberg, A., & Zoric, N. (2006). The real-time polymerase chain reaction. Molecular Aspects of Medicine, 27, 95–125.PubMedCrossRefGoogle Scholar
  19. Marshall, A. (2010). 2nd-generation GM traits progress. Nature Biotechnology, 28, 306.PubMedCrossRefGoogle Scholar
  20. Mendes, B. M. J., Boscariol, R. L., Mourão Filho, F. A. A., & Almeida, W. A. B. (2002). Agrobacterium-mediated genetic transformation of ‘Hamlin’ sweet orange. Pesquisa Agropecuária Brasileira, 37, 955–961.CrossRefGoogle Scholar
  21. Mendes, B. M. J., Cardoso, S. C., Boscariol-Camargo, R. L., Cruz, R. B., Mourão Filho, F. A. A., & Bergamin Filho, A. (2010). Reduction in susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis expressing the rice Xa21 gene. Plant Pathology, 59, 68–75.CrossRefGoogle Scholar
  22. Monteiro-Hara, A. C. B. A., Jadão, A. S., Mendes, B. M. J., Rezende, J. A. M., Trevisan, F., Mello, A. P. O. A., Vieira, M. L. C., Meletti, L. M. M., & Piedade, S. M. S. (2011). Genetic transformation of passionflower and evaluation of R1 and R2 generations for resistance to Cowpea aphid borne mosaic virus. Plant Disease, 95, 1021–1025.CrossRefGoogle Scholar
  23. Moreno, P., Ambrós, S., Albiach-Mart, 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.PubMedCrossRefGoogle Scholar
  24. Muniz, F. R., Souza, A. J., Stipp, L. C. L., Schinor, E. H., Freitas Junior, W., Harakava, R., Stach-Machado, D. R., Rezende, J. A. M., Mourão Filho, F. A. A., & Mendes, B. M. J. (2012). Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV)-derived sequences and reaction of transgenic lines to CTV infection. Biologia Plantarum, 56, 162–166.CrossRefGoogle Scholar
  25. Peña, L., Perez, R. M., Cervera, M., Juarez, J. A., & Navarro, L. (2004). Early events in Agrobacterium-mediated genetic transformation of citrus explants. Annals of Botany, 94, 67–74.PubMedCrossRefGoogle Scholar
  26. Ramakers, C., Ruijter, J. M., Deprez, R. H. L., & Moorman, A. F. M. (2003). Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neuroscience Letters, 339, 62–66.PubMedCrossRefGoogle Scholar
  27. Soler, N., Plomer, M., Fagoaga, C., Moreno, P., Navarro, L., Flores, R., & Peña, L. (2012). Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus. Plant Biotechnology Journal, 10, 597–608.PubMedCrossRefGoogle Scholar
  28. Waterhouse, P. M., Wang, M. B., & Lough, T. (2001). Gene silencing as an adaptative defence against viruses. Nature, 411, 834–842.PubMedCrossRefGoogle Scholar
  29. Zanek, M. C., Reyes, C. A., Cervera, M., Peña, E. J., Velázquez, K., Costa, N., Plata, M. I., Grau, O., Peña, L., & Garcia, M. L. (2008). Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus. Plant Cell Reports, 27, 57–66.PubMedCrossRefGoogle Scholar

Copyright information

© KNPV 2014

Authors and Affiliations

  • Fabiana R. Muniz
    • 1
  • Amancio Souza
    • 1
  • Ricardo Harakava
    • 2
  • Francisco de Assis Alves Mourão Filho
    • 1
  • Dagmar R. Stach-Machado
    • 3
  • Jorge A. M. Rezende
    • 1
  • Vicente J. Febres
    • 4
  • Gloria A. Moore
    • 4
  • Beatriz M. J. Mendes
    • 5
    Email author
  1. 1.Escola Superior de Agricultura “Luiz de Queiroz”Universidade de São PauloPiracicabaBrazil
  2. 2.Instituto Biológico de São PauloSão PauloBrazil
  3. 3.Instituto de BiologiaUniversidade Estadual de CampinasCampinasBrazil
  4. 4.University of FloridaGainesvilleUSA
  5. 5.Centro de Energia Nuclear na AgriculturaUniversidade de São PauloPiracicabaBrazil

Personalised recommendations