European Journal of Plant Pathology

, Volume 149, Issue 4, pp 865–873 | Cite as

Enhanced resistance to citrus canker in transgenic sweet orange expressing the sarcotoxin IA gene

  • Adilson K. Kobayashi
  • Luiz Gonzaga E. Vieira
  • João Carlos Bespalhok Filho
  • Rui Pereira LeiteJr
  • Luiz Filipe P. Pereira
  • Hugo Bruno C. Molinari
  • Viviani V. MarquesEmail author


Citrus canker, caused by the bacterial pathogen Xanthomonas citri subp. Citri (Xcc), is a serious disease reported in most citrus-producing areas around the world. Although different levels of field resistance to citrus canker have been reported in sweet oranges, they are usually not sufficient to provide adequate control of the disease. Ectopic over-expression of antibacterial genes is one of the potential strategies to increase plant resistance to bacterial diseases. Previous in vitro results showed that sarcotoxin IA, an antimicrobial peptide isolated from the flesh fly (Sarcophaga peregrina), can be efficient to control different plant pathogenic bacteria, including Xcc. Transgenic “Pera” sweet orange (Citrus sinensis [L.] Osbeck) plants constitutively expressing the sarcotoxin IA peptide fused to the PR1a signal peptide from Nicotiana tabacum for secretion in the intercellular space were obtained by Agrobacterium-mediated transformation using thin sections of mature explants. Citrus canker resistance evaluation in leaves of transgenic and non-transgenic plants was performed through inoculations with Xcc by infiltration and spraying. The Xcc population was up to 2 log unit lower in leaves of the transgenic plants compared to those of non-transgenic controls. Incidence of canker lesions was significantly higher in non-transformed controls (>10 lesions/cm2) than in the transgenic plants (<5 lesions/cm2) after injection infiltration or spraying with Xcc inoculum. Accumulation of sarcotoxin IA peptide in sweet orange tissue did not cause any deleterious effects on the growth and development of the transgenic plants, indicating this approach is suitable to provide resistance to citrus canker.


Citrus canker Antimicrobial peptides Agrobacterium tumefaciens Mature tissue transformation; bacterial disease resistance 



The authors gratefully thanks to Dr. Yuko Ohashi (Plant-Microbe Interactions Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan) for providing the pST10 plasmid. We also thank Suely A. Kudo and Luciana Meneguin for technical assistance. We thank Dr. Nelson A. Wulff, Dr. Leandro Peña and Dr. James Graham for critical reading of this manuscript. This work received financial support from CNPq, Fundação Araucária and Fundo de Defesa da Citricultura – FUNDECITRUS. L. G. E. Vieira and L. F. P. Pereira are CNPq research fellows.

Supplementary material

10658_2017_1234_MOESM1_ESM.docx (206 kb)
Figure 1 (DOCX 205 kb)
10658_2017_1234_MOESM2_ESM.docx (21 kb)
Table 1 (DOCX 21 kb)


  1. Behlau, F., Belasque Jr., J., Bergamin, A. F., Graham, J. H., Leite Jr., R. P., & Gottwald, T. R. (2008). Coppersprays and windbreaks for control of citrus canker on young orange trees in southern Brazil. Crop Protection, 27(3–5), 807–813.CrossRefGoogle Scholar
  2. Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.CrossRefPubMedGoogle Scholar
  3. Bronson, C. H., & Gaskalla, R. (2007). Comprehensive report on citrus canker in Florida. Division of Plant Industry: Florida Department of Agriculture and Consumer Services.Google Scholar
  4. Cardoso, S. C., Barbosa-Mendes, J. M., Boscariol-Camargo, R. L., Christiano, R. S. C., Filho, A. B., 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(2), 185–192.CrossRefGoogle Scholar
  5. Carvalho, S. A., Nunes, W. M. C., Belasque Jr., J., Machado, M. A., Croce-Filho, J., Bock, C. H., & Abdo, Z. (2015). Comparison of resistance to asiatic citrus canker among different genotypes of citrus in a long-term canker-resistance field screening experiment in Brazil. Plant Disease, 99(2), 207–218.CrossRefGoogle Scholar
  6. Cervera, M., Juárez, J., Navarro, A., Pina, J. A., Durán-Vila, N., Navarro, L., & Peña, L. (1998). Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage. Transgenic Research, 7(1), 51–59.CrossRefGoogle Scholar
  7. Da Silva, A. C. R., Ferro, J. A., Reinach, F. C., Farah, C. S., Furlan, L. R., Quaggio, R. B., et al. (2002). Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature, 417, 459–463.Google Scholar
  8. Delaporta, S. L., Wood, J., & Hicks, J. B. (1983). A plant minipreparation: Version II. Plant Molecular Biology Reporter, 4, 19–21.CrossRefGoogle Scholar
  9. Düring, K., Porsch, P., Fladung, M., Lõrz, H. (1993). Transgenic potato plants resistant to the phytopathogenic bacterium Erwinia carotovora. The Plant Journal, 3, 587–598.Google Scholar
  10. Dutt, M., Barthe, G., Irey, M., & Grosser, J. (2015). Transgenic citrus expressing an Arabidopsis NPR1 Gene exhibit enhanced resistance against Huanglongbing (HLB; citrus greening). PloS One, 10(9), e0137134. doi: 10.1371/journal.pone.0137134.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Furman, N., Kobayashi, K., Zanek, M. C., Calcagno, J., Garcia, M. L., & Mentaberry, A. (2013). Transgenic sweet orange plants expressing a dermaseptin coding sequence show reduced symptoms of citrus canker disease. Journal of Biotechnology, 167, 412–419.CrossRefPubMedGoogle Scholar
  12. Gmitter Jr., F. G., Grosser, J. W., & Moore, G. A. (1992). Citrus. In F. A. Hammerschlag & R. E. Litz (Eds.), Biotechnology of perennial crops (pp. 335–369). CAB International: Cambridge.Google Scholar
  13. Gottwald, T. R., Graham, J. H., Civerolo, E. L., Barrett, H. C., & Hearn, C. J. (1993). Differential host range reaction of citrus and citrus relatives to citrus canker and citrus bacterial spot determined by leaf mesophyll susceptibility. Plant Disease, 77, 1004–1009.CrossRefGoogle Scholar
  14. Gottwald, T. R., Graham, J. H., & Schubert, T. S. (2002). Citrus canker: The pathogen and its impact. Plant Health Progress: Resource document Accessed 14 Oct 2016.Google Scholar
  15. Gottwald, T., Graham, J., Bock, C., Bonn, G., Civerolo, E., Irey, M., et al. (2009). The epidemiological significance of post-packinghouse survival of Xanthomonas citri ssp. citri for dissemination of Asiatic citrus canker via infected fruit. Crop Protection, 28, 508–524.CrossRefGoogle Scholar
  16. Graham, J. H., Gottwald, T. R., Cubero, J., & Achor, D. S. (2004). Xanthomonas axonopodis pv. citri: Factors affecting successful eradication of citrus canker. Molecular Plant Pathology, 5(1), 1–15.CrossRefPubMedGoogle Scholar
  17. He, Y. R., Chen, S. C., Peng, A. H., Zou, X. P., Xu, L. Z., Lei, T. G., Liu, X. F., & Yao, L. X. (2011). Production and evaluation of transgenic sweet orange (Citrus sinensis L. Osbeck) containing bivalent antibacterial peptide genes (Shiva A and Cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary buds. Scientia Horticulturae, 128, 99–107.CrossRefGoogle Scholar
  18. Hao, G., Stover, E., & Gupta, G. (2016). Overexpression of a modified plant thionin enhances disease resistance to citrus canker and Huanglongbing (HLB). Frontiers in Plant Science. doi: 10.3389/fpls.2016.01078.Google Scholar
  19. Jaymes, J. M., Nagpala, P., Destéfano-Beltrán, L., Huang, J. H., Kim, J., Denny, T., & Cetiner, S. (1993). Expression of a cecropin B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas solanacearum. Plant Science, 98, 43–53.CrossRefGoogle Scholar
  20. Kanai, A., & Natori, S. (1989). Cloning of gene cluster for sarcotoxin I, antibacterial proteins of Sarcophaga peregrina. FEBS Letters, 258(2), 199–202. doi: 10.1016/0014-5793(89)81652-7.
  21. Kobayashi, A. K., Bespalhok, J. C., Pereira, L. F. P., & Vieira, L. G. E. (2003). Plant regeneration of sweet orange (Citrus sinensis) from thin sections of mature stem segments. Plant Cell Tissue and Organ Culture, 74(1), 99–102.CrossRefGoogle Scholar
  22. Leite, Jr R.P., (1990). Cancro cítrico: prevenção e controle no Paraná. Instituto Agronômico do Paraná, Londrina, p. 51 (IAPAR. Circular, 61).Google Scholar
  23. Lloyd, G.B, & McCown, B.H. (1980). Commercially feasible micropropagation of mountain laurel (Kalmia latifolia) by use of shoot tip culture. Proceedings, international plant propagators, 30: 421-437.Google Scholar
  24. Mitsuhara, I., Matsuura, H., Ohshima, M., Kaku, H., Nakajima, Y., Murai, N., Natori, S., & Ohashi, Y. (2000). Induced expression of sarcotoxin IA enhanced host resistance against both resistance against both bacterial and fungal pathogens in transgenic tobacco. Molecular Plant Microbe Interaction, 13, 860–868.CrossRefGoogle Scholar
  25. Mitsuhara, I., Nakajima, Y., Natori, S., Mitsuoka, T., & Ohashi, Y. (2001). In vitro growth inhibition of human intestinal bacteria by sarcotoxin IA, an insect bactericidal peptide. Biotechnology Letters, 23, 569–573.CrossRefGoogle Scholar
  26. Mourgues, F., Brisset, M., & Chevreau, E. (1998). Strategies to improve plant resistance to bacterial diseases through genetic engineering. Trends in Biotechnology, 16, 203–210.CrossRefPubMedGoogle Scholar
  27. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473–479.CrossRefGoogle Scholar
  28. Nakajima, Y., Qu, X.M. & Natori, S. (1987). Interaction between liposomes and sarcotoxin IA, a potential antibacterial protein of Sarcophaga peregrina (flesh fly). Journal Biological Chemistry, 262, 1665–1669.Google Scholar
  29. Navarro, L. (1992). Citrus shoot-tip grafting in vitro. In Y. P. S. Bajaj (Ed.), Biotechnology in agriculture and forestry (Vol. 18, pp. 328–338). New York: Spring Verlag, Berlin.Google Scholar
  30. Ohshima, M., Mitsuhara, I., Okamoto, M., Sawano, S., Nishiyama, K., Kaku, H., Natori, S., & Ohashi, Y. (1999). Enhanced resistance to bacterial diseases of transgenic tobacco plants overexpressing sarcotoxin IA, a bactericidal peptide of insect. Journal of Biochemistry, 125, 431–435.CrossRefPubMedGoogle Scholar
  31. Okamoto, M., Mitsuhara, I., Ohshima, M., Natori, S., & Ohashi, Y. (1998). Enhanced expression of an antimicrobial peptide sarcotoxin IA by GUS fusion in transgenic tobacco plants. Plant Cell Physiology, 39, 57–63.CrossRefPubMedGoogle Scholar
  32. Rodríguez, A., Cervera, M., Peris, J. E., & Peña, L. (2008). The same treatment for transgenic shoot regeneration elicits the opposite effect in mature explants from two closely related sweet orange (Citrus sinensis (L.) Osb.) genotypes. Plant Cell, Tissue and Organ Culture, 93(1), 97–106.CrossRefGoogle Scholar
  33. Sambrock, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.Google Scholar
  34. Sharma, A., Sharma, R., Imamura, M., Yamakawa, M., & Machii, H. (2000). Transgenic expression of cecropin B, an antibacterial peptide from Bombyx mori, confers enhanced resistance to bacterial leaf blight in rice. FEBS Letters, 484, 7–11.CrossRefPubMedGoogle Scholar
  35. Stall, R. E., Marcó, G. M., & Echenique, B. I. C. (1982). Importance of mesophyll in mature- leaf resistance to cancrosis of citrus. Phytopathology, 72, 1097–1100.CrossRefGoogle Scholar
  36. Viloria, Z., Drouillard, D. L., Graham, J. H., & Grosser, J. W. (2004). Screening triploid hybrids of ‘Lakeland’ limequat for resistance to citrus canker. Plant Disease, 88, 1056–1060.CrossRefGoogle Scholar
  37. Yamada, K., Nakajima, Y., & Natori, S. (1990). Production of recombinant sarcotoxin IA in Bombyx mori cells. Biochemistry Jornal, 272, 633–636.CrossRefGoogle Scholar
  38. Zhang, X., Francis, M. I., Dawson, W. O., Graham, J. H., Orbović, V., Triplett, E. W., & Mou, Z. (2010). Over-expression of the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker. European Journal of Plant Pathology, 128, 91–100.CrossRefGoogle Scholar
  39. Wally, O., & Punja, Z. K. (2010). Genetic engineering for increasing fungal and bacterial disease resistance in crop plants. GM Crops, 1, 199–206.CrossRefPubMedGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2017

Authors and Affiliations

  • Adilson K. Kobayashi
    • 1
    • 2
  • Luiz Gonzaga E. Vieira
    • 1
    • 3
  • João Carlos Bespalhok Filho
    • 1
    • 4
  • Rui Pereira LeiteJr
    • 1
  • Luiz Filipe P. Pereira
    • 5
  • Hugo Bruno C. Molinari
    • 1
    • 2
  • Viviani V. Marques
    • 6
    • 7
    Email author
  1. 1.Instituto Agronômico do Paraná - IAPARLondrinaBrazil
  2. 2.Genetics and Biotechnology Laboratory, Embrapa AgroenergyBrasíliaBrazil
  3. 3.Biotechnology Laboratory, UNOESTEPresidente PrudenteBrazil
  4. 4.Universidade Federal do ParanáCuritibaBrazil
  5. 5.Embrapa Café, Biothecnology Laboratory – IAPARLondrinaBrazil
  6. 6.Fundecitrus – Fundo de Defesa da CitriculturaAraraquaraBrazil
  7. 7.Biothecnology LaboratoryFundo de Defesa da Citricultura – FUNDECITRUSAraraquaraBrazil

Personalised recommendations