, Volume 224, Issue 4, pp 740–749 | Cite as

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

  • Siela N. Maximova
  • Jean-Philippe Marelli
  • Ann Young
  • Sharon Pishak
  • Joseph A. Verica
  • Mark J. Guiltinan
Original Article


Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.


Fungal pathogen resistance Theobroma cacao Chitinase Transgenic Colletotrichum gloeosporioides 



We would like to thank Gabriela Antunez de Mayolo Wilmking for her contribution to the construction of vector pGAM00.0511 and Sara Milillo and Amanda Thompson for their technical assistance with performing and data analysis of the chitinase protein activity assay.


  1. Arnold AE, Mejia LC, Kyllo D, Rojas EI, Maynard Z, Robbins N, Herre EA (2003) Fungal endophytes limit pathogen damage in a tropical tree. Proc Natl Acad Sci USA 100:15649–15654CrossRefPubMedGoogle Scholar
  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (2001) Current protocols in molecular biology. Wiley, New YorkGoogle Scholar
  3. Bennett AB (2003) Out of the Amazon: Theobroma cacao enters the genomic era. Trends Plant Sci 8:561–563CrossRefPubMedGoogle Scholar
  4. Bolar J, Norelli J, Harman G, Brown S, Aldwinckle H (2001) Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants. Transgenic Res 10:533–543CrossRefPubMedGoogle Scholar
  5. Broglie K, Chet I, Holliday M, Cressman R, Biddle P, Knowlton S, Mauvais CJ, Broglie R (1991) Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254:1194–1197PubMedCrossRefGoogle Scholar
  6. Carstens M, Vivier MA, Pretorius IS (2003) The Saccharomyces cerevisiae chitinase, encoded by the CTS1–2 gene, confers antifungal activity against Botrytis cinerea to transgenic tobacco. Transgenic Res 12:497–508CrossRefPubMedGoogle Scholar
  7. Cedeño L, Carrero C (2003) Antracnosis del cacao. Universidad de los Andes
  8. Coe SD, Coe MD (1996) The true history of chocolate. Thames and Hudson, New YorkGoogle Scholar
  9. Collinge D, Kragh K, Mikkelsen J, Nielsen K, Rasmussen U, Vad K (1993) Plant chitinases. Plant J 3:31–40CrossRefPubMedGoogle Scholar
  10. De-Block M, Herrerra-Estrella L, Van Montagu M, Shell J, Zambryski P (1984) Expression of foreign genes in regenerated plants and their progeny. EMBO J 3:1681–1689PubMedGoogle Scholar
  11. Dufresne M, Perfect S, Pellier AL, Bailey JA, Langin T (2000) A GAL4-like protein is involved in the switch between biotrophic and necrotrophic phases of the infection process of Colletotrichum lindemuthianum on common bean. Plant Cell 12:1579–1589CrossRefPubMedGoogle Scholar
  12. Dumas B, Centis S, Sarrazin N, Esquerré-Tugayé M-T (1999) Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection. Appl Environ Microbiol 65:1769–1771PubMedGoogle Scholar
  13. Emch M (2003) The human ecology of Mayan cacao farming in Belize. Hum Ecol 31:111–132CrossRefGoogle Scholar
  14. Freeman S, Rodriguez RJ (1993) Genetic conversion of a fungal plant pathogen to a nonpathogenic, endophytic mutualist. Science 260:75–78PubMedCrossRefGoogle Scholar
  15. Jach G, Gornhardt B, Mundy J, Logemann J, Pinsdorf E, Leah R, Schell J, Maas C (1995) Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J 8:97–109CrossRefPubMedGoogle Scholar
  16. Kellmann JW, Kleinow T, Engelhardt K, Philipp C, Wegener D, Schell J, Schreier PH (1996) Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants. Plant Mol Biol 30:351–358CrossRefPubMedGoogle Scholar
  17. Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J 11:945–957CrossRefPubMedGoogle Scholar
  18. Kramer KJ, Muthukrishnan S (1997) Insect chitinases: molecular biology and potential use as biopesticides. Insect Biochem Mol Biol 27:887–900CrossRefPubMedGoogle Scholar
  19. Li Z, Traore A, Maximova S, Guiltinan MJ (1998) Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron. In Vitro Cell Dev Biol Plant 34:293–299CrossRefGoogle Scholar
  20. Linthorst HJ, van Loon LC, van Rossum CM, Mayer A, Bol JF, van Roekel JS, Meulenhoff EJ, Cornelissen BJ (1990) Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco. Mol Plant Microbe Interact 3:252–258PubMedGoogle Scholar
  21. Liu M, Sun ZX, Zhu J, Xu T, Harman GE, Lorito M (2004) Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride. J Zhejiang Univ Sci 5:133–136PubMedCrossRefGoogle Scholar
  22. Lorito M, Woo SL, Fernandez IG, Colucci G, Harman GE, Pintor-Toro JA, Filipone E, Muccifora S, Lawrence CB, Zoina A, Tuzun S, Scala F (1998) Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proc Natl Acad Sci USA 95:7860–7865CrossRefPubMedGoogle Scholar
  23. Marchant R, Davey MR, Lucas JA, Lamb CJ, Dixon RA, Power JB (1998) Expression of a chitinase transgene in rose (Rosa hybrida L) reduces development of blackspot disease (Diplocarpon rosae Wolf). Mol Breed 4:187–194CrossRefGoogle Scholar
  24. Matzke AJ, Matzke MA (1998) Position effects and epigenetic silencing of plant transgenes. Curr Opin Plant Biol 1:142–148CrossRefPubMedGoogle Scholar
  25. Maximova SN, Alemanno L, Young A, Ferriere N, Traore A, Guiltinan M (2002) Efficiency, genotypic variability, and cellular origin of primary and secondary somatic embryogenesis of Theobroma cacao L. In Vitro Cell Dev Biol Plant 38:252–259CrossRefGoogle Scholar
  26. Maximova S, Miller C, Antunez de Mayolo G, Pishak S, Young A, Guiltinan MJ (2003) Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression. Plant Cell Rep 21:872–883PubMedGoogle Scholar
  27. Maximova SN, Young A, Pishak S, Miller C, Traore A, Guiltinan MJ (2005) Integrated system for propagation of Theobroma cacao L. In: Jain SM, Gupta PK (eds) Protocol for somatic embryogenesis in woody plants. Springer, Berlin Heidelberg New York, pp 209–229CrossRefGoogle Scholar
  28. Mendgen K, Hahn M (2002) Plant infection and the establishment of fungal biotrophy. Trends Plant Sci 7:352–356CrossRefPubMedGoogle Scholar
  29. Michiels A, Van den Ende W, Tucker M, Van Riet L, Van Laere A (2003) Extraction of high-quality genomic DNA from latex-containing plants. Anal Biochem 315:85–89CrossRefPubMedGoogle Scholar
  30. Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohashi Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49–59PubMedGoogle Scholar
  31. Mohanan RC, Kaveriappa KM, Nambiar KKN (1989) Epidemiological studies of Colletotrichum gloeosporioides disease of cocoa. Ann Appl Biol 114:15–22CrossRefGoogle Scholar
  32. Mora A, Earl E (2001) Resistance to Alternaria brassicola in transgenic broccoli expressing a Trichoderma harzianum endochitinase gene. Mol Breed 8:1–9CrossRefGoogle Scholar
  33. Motamayor JC, Risterucci AM, Lopez PA, Ortiz CF, Moreno A, Lanaud C (2002) Cacao domestication I: the origin of the cacao cultivated by the Mayas. Heredity 89:380–386CrossRefPubMedGoogle Scholar
  34. Neuhaus J-M (1999) Plant chitinases (PR-3, PR-4, PR-8, PR-11). In: Datta SK, Muthukrishnan S (eds) Pathogenesis-related proteins in plants. CRC Press, New York, pp 77–107Google Scholar
  35. O’Connell RJ, Perfect SE, Hughes HB, Carzaniga R, Bailey JA, Green JR (2000) Dissecting the cell biology of Colletotrichum infection processes. In: Prusky D, Freeman S, Dickman M (eds) Host specificity, pathology, and host–pathogen interaction of Colletotrichum. American Phytopathology Society Press, St Paul, pp 57–77Google Scholar
  36. O’Connell R, Herbert C, Sreenivasaprasad S, Khatib M, Esquerre-Tugaye M-T, Dumas B (2004) A novel Arabidopsis–Colletotrichum pathosystem for the molecular dissection of plant–fungal interactions. Mol Plant Microbe Interact 17(3):272–282PubMedCrossRefGoogle Scholar
  37. Patil VR, Widholm JM (1997) Possible correlation between increased vigor and chitinase activity expression in tobacco. J Exp Bot 48:1943–1950CrossRefGoogle Scholar
  38. Perfect SE, Hugues HB, O’Connel RJ, Green JR (1999) Colletotrichum: a model genus for studies on pathology and fungal–plant interactions. Fungal Genet Biol 27:186–198CrossRefPubMedGoogle Scholar
  39. Piasentin F, Klare-Repnik L (2004) Biodiversity conservation and cocoa agroforests. Gro Cocoa 5:7–8. Google Scholar
  40. Punja ZK, Raharjo SHT (1996) Response of transgenic cucumber and carrot plants expressing different chitinase enzymes to inoculation with fungal pathogens. Plant Dis 80:999–1005CrossRefGoogle Scholar
  41. Raharjo SHT, Hernandez MO, Zhang YY, Punja ZK (1996) Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens. Plant Cell Rep 15:591–596CrossRefGoogle Scholar
  42. Snyder T (1994) Isolation and characterization of a genomic chitinase clone form Theobroma cacao L. PhD thesis, Intercollege Program in Plant Physiology, The Pennsylvania State UniversityGoogle Scholar
  43. Snyder-Leiby TE, Furtek DB (1995) A genomic clone (accession no. U30324) from Theobroma cacao L. with high similarity to plant class I endochitinase sequences. Plant Physiol 109:338Google Scholar
  44. Tabaeizadeh Z (1997) Transgenic tomato plants expressing L. chilense chitinase gene demonstrate resistance to Verticillium dahliae. Plant Physiol 114:299Google Scholar
  45. Vierheilig H, Alt M, Lange J, Gut-Rella M, Wiemken A, Boller T (1995) Colonization of transgenic tobacco constitutively expressing pathogenesis-related proteins by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. Appl Environ Microbiol 61:3031–3034PubMedGoogle Scholar
  46. Wood GAR, Lass RA (1987) Cocoa. Longman Scientific & Technical, copublished by Wiley, New YorkGoogle Scholar
  47. Yamamoto T, Iketani H, Ieki H, Nishizawa Y, Notsuka K, Hibi T, Hayashi T, Matsuta N (2000) Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Rep 19:639–646CrossRefGoogle Scholar
  48. Zeng Y, Yang T (2002) RNA isolation from highly viscous samples rich in polyphenols and polysaccharides. Plant Mol Biol Rep 20:417a–417eCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Siela N. Maximova
    • 1
  • Jean-Philippe Marelli
    • 2
  • Ann Young
    • 1
  • Sharon Pishak
    • 1
  • Joseph A. Verica
    • 1
  • Mark J. Guiltinan
    • 1
  1. 1.The Department of HorticultureThe Pennsylvania State UniversityChesterUSA
  2. 2.The Department of Plant PathologyThe Pennsylvania State UniversityChesterUSA

Personalised recommendations