Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides
- 494 Downloads
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.
KeywordsFungal 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.
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (2001) Current protocols in molecular biology. Wiley, New YorkGoogle Scholar
- Cedeño L, Carrero C (2003) Antracnosis del cacao. Universidad de los Andes http://www.ulauniversidad.com.ve/vnews/display.v/ART/2003/06/30/3f009da56c4c4?in_archive=1
- Coe SD, Coe MD (1996) The true history of chocolate. Thames and Hudson, New YorkGoogle Scholar
- 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
- 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
- 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
- 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
- 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
- Tabaeizadeh Z (1997) Transgenic tomato plants expressing L. chilense chitinase gene demonstrate resistance to Verticillium dahliae. Plant Physiol 114:299Google Scholar
- Wood GAR, Lass RA (1987) Cocoa. Longman Scientific & Technical, copublished by Wiley, New YorkGoogle Scholar