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Thanatin confers partial resistance against aflatoxigenic fungi in maize (Zea mays)

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Abstract

Aflatoxin-producing fungi can contaminate plants and plant-derived products with carcinogenic secondary metabolites that present a risk to human and animal health. In this study, we investigated the effect of antimicrobial peptides on the major aflatoxigenic fungi Aspergillus flavus and A. parasiticus. In vitro assays with different chemically-synthesized peptides demonstrated that the broad-spectrum peptide thanatin from the spined soldier bug (Podisus maculiventris) had the greatest potential to eliminate aflatoxigenic fungi. The minimal inhibitory concentrations of thanatin against A. flavus and A. parasiticus were 3.13 and 12.5 µM, respectively. A thanatin cDNA was subsequently cloned in a plant expression vector under the control of the ubiquitin-1 promoter allowing the recombinant peptide to be directed to the apoplast in transgenic maize plants. Successful integration of the thanatin expression cassette was confirmed by PCR and expression was demonstrated by semi-quantitative RT-PCR in transgenic maize kernels. Infection assays with maize kernels from T1 transgenic plants showed up to three-fold greater resistance against Aspergillus spp. infections compared to non-transgenic kernels. We demonstrated for the first time that heterologous expression of the antimicrobial peptide thanatin inhibits the growth of Aspergillus spp. in transgenic maize plants offering a solution to protect crops from aflatoxin-producing fungi and the resulting aflatoxin contamination in the field and under storage conditions.

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Acknowledgments

This work was supported by DAAD Grant No. FKZ 50739422 and FKZ 54364828. The authors acknowledge Katey Warnberg and Dr. Kan Wang (pTF Iowa State University, Ames, USA) for providing the maize transformation vector pTF101.1gw1 and for the initial maize transformation, Prof. Dr. Andreas Vilcinskas (Justus-Liebig University, Giessen, Germany) for providing the thanatin sequence, Elke Stein (Justus-Liebig University, Giessen, Germany) for initial AMP testing, Dr. Thomas Rademacher (Fraunhofer IME, Aachen, Germany) for providing the vectors pTRAkc and pTRAux, and Ibrahim Al-Amedi (Fraunhofer IME) for cultivating the maize plants used in this investigation. We thank Dr. Richard M. Twyman for editorial assistance.

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  1. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany

    Max Schubert, Marcel Houdelet, Rainer Fischer, Stefan Schillberg & Greta Nölke

  2. Institute of Phytopathology and Applied Zoology, Justus-Liebig University, Heinrich-Buff-Ring 26-32, 35392, Giessen, Germany

    Karl-Heinz Kogel & Stefan Schillberg

  3. Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

    Rainer Fischer

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  1. Max Schubert
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Correspondence to Greta Nölke.

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Schubert, M., Houdelet, M., Kogel, KH. et al. Thanatin confers partial resistance against aflatoxigenic fungi in maize (Zea mays). Transgenic Res 24, 885–895 (2015). https://doi.org/10.1007/s11248-015-9888-2

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