European Journal of Plant Pathology

, Volume 139, Issue 2, pp 241–244 | Cite as

Methods for Cercospora coffeicola protoplast isolation and genetic transformation with the green fluorescent protein

  • André Gomes Coelho Souza
  • Sonia Herrero
  • Luiz Antonio Maffia
  • Margaret Elizabeth Daub


Cercospora coffeicola is the causal agent of brown eye spot on coffee leaves. Although the disease has significant importance, few molecular studies have been done with C. coffeicola. Here we report a protocol for isolating protoplasts as well as development of a genetic transformation system using Green Fluorescent Protein. High yields of protoplasts (≈108/ml) were obtained from mycelial cultures from five isolates of C. coffeicola. One isolate was transformed with a vector encoding hygromycin resistance and Green Fluorescent Protein. Out of 43 hygromycin-resistant transformants obtained, Green Fluorescent Protein was highly expressed in one (2.3 %).


Brown eye spot Coffea arabica L. Leaf spot 



Brown eye spot





Support for this research was provided by CNPq/NCSU/UFV/FAPEMIG. We thank Aydin Beseli, Andrew Mealin, Shante Bryant, and Jeff Gillikin for assistance with this work.


  1. Balint-Kurti, P., May, G. D., & Churchill, A. C. L. (2001). Development of a transformation system for Mycosphaerella pathogens of banana: a tool for the study of host/pathogen interaction. FEMS Microbiology Letters, 195, 9–15.PubMedCrossRefGoogle Scholar
  2. Chung, K. R., Ehrenshaft, M., & Daub, M. E. (2002). Functional expression and cellular localization of cercosporin-resistance proteins fused with the GFP in Cercospora nicotianae. Current Genetics, 41, 159–167.PubMedCrossRefGoogle Scholar
  3. Daub, M. E., & Chung, K. R. (2009). Photoactivated perylenequinone toxins in plant pathogenesis. In H. B. Deising (Ed.), The Mycota–Plant Relationships V (pp. 201–219). Berlin Heidelberg: Springer Verlag.Google Scholar
  4. Dell’Acqua, R., Mantovani, E. S., Braghini, M. T., Oliveira, C. M. G., Harakava, R., Robaina, A. S., et al. (2011). Variabilidade in vitro, in vivo e molecular de isolados de Cercospora coffeicola. Tropical Plant Pathology, 36, 313–326.Google Scholar
  5. Ehrenshaft, M., Jenns, A. E., & Daub, M. E. (1995). Targeted gene disruption of carotenoid biosynthesis in Cercospora-nicotianae reveals no role for carotenoids in photosensitizer resistance. Molecular Plant-Microbe Interactions, 8, 569–575.CrossRefGoogle Scholar
  6. Lorang, J. M., Tuori, R. P., Martinez, J. P., Sawyer, T. L., Redman, R. S., Rollins, J. A., et al. (2001). Green fluorescent protein is lighting up fungal biology. Applied and Environmental Microbiology, 67, 1987–1994.PubMedCentralPubMedCrossRefGoogle Scholar
  7. Portal, O., Acosta-Suárez, M., Ocaña, B., Schäfer, W., Jiménez, E., & Höfte, M. (2012). A green fluorescent protein-transformed Mycosphaerella fijiensis strain shows increased aggressiveness on banana. Australasian Plant Pathology, 41, 645–647.CrossRefGoogle Scholar
  8. Shim, W. B., & Dunkle, L. D. (2003). CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis, regulates cercosporin biosynthesis, fungal development, and pathogenesis. Molecular Plant-Microbe Interactions, 16, 760–768.PubMedCrossRefGoogle Scholar
  9. Souza, A. G. C., Rodrigues, F. A., Maffia, L. A., & Mizubuti, E. S. G. (2011). Infection process of Cercospora coffeicola on coffee leaf. Journal of Phytopathology, 159, 6–11.CrossRefGoogle Scholar
  10. Souza, A. G. C., Maffia, L. A., & Mizubuti, E. S. G. (2012). Cultural and aggressiveness variability of Cercospora coffeicola. Journal of Phytopathology, 160, 540–546.CrossRefGoogle Scholar
  11. Walker, D., Hunt, H., & Hager, G. L. (1999). Using inducible vectors to study intracellular trafficking of GFP-tagged steroid/nuclear receptors in living cells. Methods, 19, 386–393.PubMedCrossRefGoogle Scholar

Copyright information

© KNPV 2013

Authors and Affiliations

  • André Gomes Coelho Souza
    • 1
  • Sonia Herrero
    • 2
  • Luiz Antonio Maffia
    • 1
  • Margaret Elizabeth Daub
    • 2
  1. 1.Departamento de FitopatologiaUniversidade Federal de ViçosaViçosaBrasil
  2. 2.Department of Plant BiologyNorth Carolina State UniversityRaleighUSA

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