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Expression of a polyphemusin variant in transgenic tobacco confers resistance against plant pathogenic bacteria, fungi and a virus

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Abstract

Antimicrobial cationic peptides provide a promising means of engineering plant resistance to a range of plant pathogens, including viruses. PV5 is a synthetic structural variant of polyphemusin, a cationic peptide derived from the horseshoe crab-Limulus polyphemus. PV5 has been shown to be benign toward eukaryotic membranes but with enhanced antimicrobial activity against animal pathogens. In this work, the cytotoxicity of PV5 toward tobacco protoplasts and leaf discs was assessed using TTC (2,3,5-triphenyltetrazolium chloride) and Evans blue colorimetric assays. PV5 showed no measurable cytotoxic effects even at levels as high as 60 μg. As a possible approach to enhancing plant resistance, a gene encoding PV5 was fused to the signal sequence encoding the C-terminus portion of the BiP protein from Pseudotsuga menziesii, under the control of 2 × 35S CaMV promoter. When introduced into Nicotiana tabacum var Xanthi gene integration and expression was confirmed by both Southern and northern analyses. When transgenic plants were subsequently challenged with bacterial and fungal phytopathogens enhanced resistance was observed. Moreover, transgenic plants also displayed antiviral properties against Tobacco Mosaic Virus making PV5 an excellent candidate for conferring unusually broad spectrum resistance to plants and the first anti-plant virus antimicrobial peptide.

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Abbreviations

CAP:

Cationic antimicrobial peptides

dH2O:

Distilled water

HIV:

Human immunodeficiency virus

MS:

Murashige–Skoog

TMV:

Tobacco mosaic virus

TTC:

2,3,5-Triphenyltetrazolium chloride

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Acknowledgements

We are thankful to Dr. D. Yevtushenko for the support provided for protoplast isolation. This work was funded by grants to Santosh Misra from The Canadian Bacterial Diseases Network (CBDN) and the Advanced Food and Biomaterials Network (AFMnet), National Centers of Excellence.

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Authors and Affiliations

  1. Department of Biochemistry and Microbiology and Centre for Forest Biology, University of Victoria, Victoria, BC, Canada, V8W 3P6

    Apurva Bhargava, Milan Osusky, Benjamin S. Forward, William W. Kay & Santosh Misra

  2. Department of Microbiology, University of British Columbia, Vancouver, BC, Canada

    Robert E. Hancock

Authors
  1. Apurva Bhargava
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  2. Milan Osusky
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  3. Benjamin S. Forward
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  4. Robert E. Hancock
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  5. William W. Kay
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  6. Santosh Misra
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Correspondence to Santosh Misra.

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Bhargava, A., Osusky, M., Forward, B.S. et al. Expression of a polyphemusin variant in transgenic tobacco confers resistance against plant pathogenic bacteria, fungi and a virus. Plant Cell Tiss Organ Cult 88, 301–312 (2007). https://doi.org/10.1007/s11240-007-9204-9

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  • DOI: https://doi.org/10.1007/s11240-007-9204-9

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