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JXB Advance Access published online on April 29, 2005
Journal of Experimental Botany, doi:10.1093/jxb/eri165
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© The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received October 27, 2004
Accepted March 16, 2005

RESEARCH PAPER

Pathogen-induced expression of a cecropin A-melittin antimicrobial peptide gene confers antifungal resistance in transgenic tobacco

Dmytro P. Yevtushenko 1, Rafael Romero 1, Benjamin S. Forward 1 *, Robert E. Hancock 2, William W. Kay 1, and Santosh Misra 1*

1 Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, V8W 3P6 Canada
2 Department of Microbiology, University of British Columbia, Vancouver, British Columbia, V6T 1W5 Canada

* To whom correspondence should be addressed.
Santosh Misra, E-mail: smisra{at}uvic.ca


  Abstract

Expression of defensive genes from a promoter that is specifically activated in response to pathogen invasion is highly desirable for engineering disease-resistant plants. A plant transformation vector was constructed with transcriptional fusion between the pathogen-responsive win3.12T promoter from poplar and the gene encoding the novel cecropin A-melittin hybrid peptide (CEMA) with strong antimicrobial activity. This promoter-transgene combination was evaluated in transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) for enhanced plant resistance against a highly virulent pathogenic fungus Fusarium solani. Transgene expression in leaves was strongly increased after fungal infection or mechanical wounding, and the accumulation of CEMA transcripts was found to be systemic and positively correlated with the number of transgene insertions. A simple and efficient in vitro regeneration bioassay for preliminary screening of transgenic lines against pathogenic fungi was developed. CEMA had strong antifungal activity in vitro, inhibiting conidia germination at concentrations that were non-toxic to tobacco protoplasts. Most importantly, the expression level of the CEMA peptide in vivo, regulated by the win3.12T promoter, was sufficient to confer resistance against F. solani in transgenic tobacco. The antifungal resistance of plants with high CEMA expression was strong and reproducible. In addition, leaf tissue extracts from transgenic plants significantly reduced the number of fungal colonies arising from germinated conidia. Accumulation of CEMA peptide in transgenic tobacco had no deleterious effect on plant growth and development. This is the first report showing the application of a heterologous pathogen-inducible promoter to direct the expression of an antimicrobial peptide in plants, and the feasibility of this approach to provide disease resistance in tobacco and, possibly, other crops.

Keywords: Antifungal resistance; cecropin A-melittin antimicrobial peptide; Fusarium solani; inducible response; in vitro regeneration bioassay; poplar; tobacco; win3.12T promoter.

*Present address: The Huntsman Marine Science Centre, 1 Lower Campus Road, St Andrews, New Brunswick, E5B 2L7 Canada.


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