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Applied and Environmental Microbiology, March 2008, p. 1339-1349, Vol. 74, No. 5
0099-2240/08/$08.00+0 doi:10.1128/AEM.02126-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Detection of Plant-Modulated Alterations in Antifungal Gene Expression in Pseudomonas fluorescens CHA0 on Roots by Flow Cytometry
Patrice de Werra,1,
Eric Baehler,2,,
Aurélie Huser,1,
Christoph Keel,2* and
Monika Maurhofer1*
Institute of Integrative Biology, Plant Pathology, Swiss Federal Institute of Technology, CH-8092 Zurich,1 Department of Fundamental Microbiology, University of Lausanne, CH-1015 Lausanne, Switzerland2
Received 17 September 2007/ Accepted 19 December 2007
The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.
* Corresponding author. Mailing address for Monika Maurhofer: Plant Pathology, Institute of Integrative Biology, Swiss Federal Institute of Technology, Universitätstrasse 2, CH-8092 Zürich, Switzerland. Phone: 41 44 632 3868. Fax: 41 44 632 1572. E-mail: monika.maurhofer{at}agrl.ethz.ch. Mailing address for Christoph Keel: Department of Fundamental Microbiology, Biophore Building, University of Lausanne, CH-1015 Lausanne, Switzerland. Phone: 41 21 692 5636. Fax: 41 21 692 5605. E-mail: christoph.keel{at}unil.ch
Published ahead of print on 28 December 2007.
P.D.W. and E.B. contributed equally to this study.
Present address: Département Fédéral de l'Intérieur DFI, Office Fédéral de la Santé Publique, Unité de Direction Santé Publique, CH-3011 Bern, Switzerland.
Present address: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Köln, Germany.
Applied and Environmental Microbiology, March 2008, p. 1339-1349, Vol. 74, No. 5
0099-2240/08/$08.00+0 doi:10.1128/AEM.02126-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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