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Ha-DEF1, a sunflower defensin, induces cell death in Orobanche parasitic plants

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Abstract

Plant defensins are small basic peptides of 5–10 kDa and most of them exhibit antifungal activity. In a sunflower resistant to broomrape, among the three defensin encoding cDNA identified, SF18, SD2 and HaDef1, only HaDef1 presented a preferential root expression pattern and was induced upon infection by the root parasitic plant Orobanche cumana. The amino acid sequence deduced from HaDef1 coding sequence was composed of an endoplasmic reticulum signal sequence of 28 amino acids, a standard defensin domain of 50 amino-acid residues and an unusual C-terminal domain of 30 amino acids with a net positive charge. A 5.8 kDa recombinant mature Ha-DEF1 corresponding to the defensin domain was produced in Escherichia coli and was purified by means of a two-step chromatography procedure, Immobilized Metal Affinity Chromatography (IMAC) and Ion Exchange Chromatography. Investigation of in vitro antifungal activity of Ha-DEF1 showed a strong inhibition on Saccharomyces cerevisiae growth linked to a membrane permeabilization, and a morphogenetic activity on Alternaria brassicicola germ tube development, as already reported for some other plant defensins. Bioassays also revealed that Ha-DEF1 rapidly induced browning symptoms at the radicle apex of Orobanche seedlings but not of another parasitic plant, Striga hermonthica, nor of Arabidopsis thaliana. FDA vital staining showed that these browning areas corresponded to dead cells. These results demonstrate for the first time a lethal effect of defensins on plant cells. The potent mode of action of defensin in Orobanche cell death and the possible involvement in sunflower resistance are discussed.

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Abbreviations

Dm-AMP1:

Dahlia merckii antimicrobial peptide1

ES-MS:

Electro-spray mass spectrometer

FDA:

Fluorescein diacetate

Ha-DEF1:

Helianthus annuus defensin protein1

IMAC:

Immobilized metal affinity chromatography

Rs-AFP2:

Raphanus sativus antifungal peptide2

RT-PCR:

Reverse transcriptase-polymerase chain reaction

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Acknowledgments

This work was supported by a grant from the Ministry of Research and Technology of France and partly by the Ouest-genopole and INRA. We thank S. Delgrange, S. Rezé and A. Lelion for technical assistance.

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Authors and Affiliations

  1. LBPV, EA1157, Faculté des Sciences et Techniques, Université de Nantes, Nantes Atlantique Universités, 2, rue de la Houssinière, Bât. 8, Nantes, 44000, France

    Axel de Zélicourt, Séverine Thoiron, Philippe Simier & Philippe Delavault

  2. Laboratoire de Stress, Défenses et Reproduction des Plantes, UFR Sciences, Université de Reims, BP 1039, Reims Cedex 2, 51687, France

    Patricia Letousey

  3. Université d’Angers, UMR PaVé A77, 2 Bd Lavoisier, Angers Cedex, 49045, France

    Claire Campion & Philippe Simoneau

  4. Unité de Recherche Biopolymères Interactions Assemblages, INRA, Nantes Cedex, 44316, France

    Khalil Elmorjani & Didier Marion

Authors
  1. Axel de Zélicourt
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  2. Patricia Letousey
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  3. Séverine Thoiron
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  4. Claire Campion
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  5. Philippe Simoneau
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  6. Khalil Elmorjani
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  7. Didier Marion
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  8. Philippe Simier
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  9. Philippe Delavault
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Correspondence to Philippe Delavault.

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de Zélicourt, A., Letousey, P., Thoiron, S. et al. Ha-DEF1, a sunflower defensin, induces cell death in Orobanche parasitic plants. Planta 226, 591–600 (2007). https://doi.org/10.1007/s00425-007-0507-1

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  • DOI: https://doi.org/10.1007/s00425-007-0507-1

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