Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens

Nature Biotechnology volume 18, pages 1162–1166 (2000)Cite this article

Abstract

Here we describe a strategy for engineering transgenic plants with broad-spectrum resistance to bacterial and fungal phytopathogens. We expressed a synthetic gene encoding a N terminus-modified, cecropin–melittin cationic peptide chimera (MsrA1), with broad-spectrum antimicrobial activity. The synthetic gene was introduced into two potato (Solanum tuberosum L.) cultivars, Desiree and Russet Burbank, stable incorporation was confirmed by PCR and DNA sequencing, and expression confirmed by reverse transcription (RT)-PCR and recovery of the biologically active peptide. The morphology and yield of transgenic Desiree plants and tubers was unaffected. Highly stringent challenges with bacterial or fungal phytopathogens demonstrated powerful resistance. Tubers retained their resistance to infectious challenge for more than a year, and did not appear to be harmful when fed to mice. Expression of msrA1 in the cultivar Russet Burbank caused a striking lesion-mimic phenotype during leaf and tuber development, indicating its utility may be cultivar specific. Given the ubiquity of antimicrobial cationic peptides as well as their inherent capacity for recombinant and combinatorial variants, this approach may potentially be used to engineer a range of disease-resistant plants.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The structure and expression constructs for MsrA1.
Figure 2: msrA1 gene integration and mRNA expression.
Figure 3: Morphological characteristics of transgenic potato plants and tubers.
Figure 4: Transgenic potato challenged with the fungal pathogen Phytophthora cactorum.
Figure 5: Transgenic potato challenged with the fungal pathogen Fusarium solani.
Figure 6: Transgenic potatoes resistant to Erwinia carotovora.

Similar content being viewed by others

References

  1. Baker, B., Zambryski, P., Staskawicz, B. & Dinesh-Kumar, S.P. Signaling in plant–microbe interactions. Science 267, 726–733 (1997).

    Article  Google Scholar 

  2. Mourgues, F., Brisset, M-N. & Chevreau, E. Strategies to improve plant resistance to bacterial diseases through genetic engineering. Trends Biotechnol. 16, 203–210 (1998).

    Article  CAS  Google Scholar 

  3. Hancock, R.E.W. & Lehrer, R. Cationic peptides: a new source of antibiotics. Trends Biotechnol. 16, 82–88 (1998).

    Article  CAS  Google Scholar 

  4. Cao, H., Li, X. & Dong, X. Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proc. Natl. Acad. Sci. USA 95, 6531–6536 (1999).

    Article  Google Scholar 

  5. Heo, W.D. et al. Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses. Proc. Natl. Acad. Sci. USA 96, 766–771 (1999).

    Article  CAS  Google Scholar 

  6. Hancock, R.E.W., Falla, T. & Brown, M. Cationic bactericidal peptides. Adv. Microbiol. Physiol. 37, 135–175 (1995).

    Article  CAS  Google Scholar 

  7. Nicholas, P. & Mor, A. Peptides as weapons against microorganisms in the chemical defense system of vertebrates. Annu. Rev. Microbiol. 49, 277–304 (1995).

    Article  Google Scholar 

  8. Zasloff, M. Antibiotic peptides as mediators of innate immunity. Curr. Opin. Immunol. 4, 3–7 (1992).

    Article  CAS  Google Scholar 

  9. Cociancich, S. et al. Novel inducible antibacterial peptides from a hemipteran insect, the sap-sucking bug Pyrrhocoris apterus. Biochem. J. 300, 567–575 (1994).

    Article  CAS  Google Scholar 

  10. Piers, K.L. & Hancock, R.E.W. The interaction of a recombinant cecropin/melittin hybrid peptide with the outer membrane of Pseudomonas aeruginosa. Mol. Microbiol. 12, 951–958 (1994).

    Article  CAS  Google Scholar 

  11. Jaynes, J.M. et al. Expression of a cecropin B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas solanacearum. Plant Sci. 89, 43–53 (1993).

    Article  CAS  Google Scholar 

  12. Mitra, A. & Zhang, Z. Expression of human lactoferrin cDNA in tobacco cells produces antibacterial protein(s). Plant Physiol. 106, 977–981 (1994).

    Article  CAS  Google Scholar 

  13. Florack, D.E.A, Dirkse, W.G., Visser, B., Heidekamp, F. & Stiekema, W. Expression of biologically active hordothionin in tobacco. Effects of pre- and prosequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting. Plant Mol. Biol. 24, 83–96 (1994).

    Article  CAS  Google Scholar 

  14. Carmona, M.J., Molina, A., Fernandez, J.A., Lopez-Fando, J.J. & Garcia-Olmedo, F. Expression of the alpha-thionin from barley in tobacco confers enhanced resistance to bacterial pathogens. Plant J. 3, 457–462 (1993).

    Article  CAS  Google Scholar 

  15. Allefs, S.J.H.M., De Jong, E.R., Florack, D.E.A., Hoogendoorn, C. & Stiekema, W.J. Erwinia soft rot resistance of potato cultivars expressing antimicrobial peptide tachyplesin I. Molecular Breeding 2, 97–105 (1996).

    Article  CAS  Google Scholar 

  16. Allefs, S.J.H.M., Florack, D.E.A., Hoogendoorn, C. & Stiekema W.J. Erwinia soft rot resistance of potato cultivars transformed with a gene construct coding for antimicrobial peptide cecropin B is not altered. Potato J. 72, 437–445 (1995).

    Article  CAS  Google Scholar 

  17. Florack, D.E.A. et al. Expression of giant silkmoth cecropin B genes in tobacco. Transgenic Res. 4, 132–141 (1995).

    Article  CAS  Google Scholar 

  18. During, K. Genetic engineering for resistance to bacteria in transgenic plants by introduction of foreign genes. Molecular Breeding 2, 297–305 (1996).

    Article  Google Scholar 

  19. Epple, P., Apel, K. & Bohlmann, H. Overexpression of an endogenous thionin enhances resistance of Arabidopsis against Fusarium oxysporum. Plant Cell 9, 509–520 (1997).

    Article  CAS  Google Scholar 

  20. Bohman, H.G. & Hultmark, D. Cell-free immunity in insects. Annu. Rev. Microbiol. 41, 103–126 (1987).

    Article  Google Scholar 

  21. van Hofsten, P. et al. Molecular cloning, cDNA sequencing, and chemical synthesis of cecropin B from Hyalophora cecropia. Proc.Natl. Acad. Sci. USA 82, 2240–2243 (1985).

    Article  CAS  Google Scholar 

  22. Hultmark, D., Engstrom, A., Bennich, H., Kapur, R. & Boman, H.G. Insect immunity: isolation and structure of cecropin D and four minor antimicrobial components from cecropia pupae. Eur. J. Biochem. 127, 207–217 (1982).

    Article  CAS  Google Scholar 

  23. Jaynes, J.M., Xanthopoulos, K.G., Destefano-Beltran, L. & Dodds, J.H. Increasing bacterial disease resistance utilizing antibacterial genes from insects. BioEssays 6, 263–270 (1987).

    Article  CAS  Google Scholar 

  24. Nordeen, R.G., Sinden, S.L., Jaynes, J.M. & Owens, L.D. Activity of cecropin SB 37 against protoplast from several plant species and their bacterial pathogens. Plant Sci. 82, 101–107 (1992).

    Article  CAS  Google Scholar 

  25. Habermann, E. Bee and wasp venoms. Science 177, 314–322 (1972).

    Article  CAS  Google Scholar 

  26. Tosteson, M.T., Holmes, S.J., Razin, M. & Tosteson, D.C. Melittin lysis of red cells. J. Membr. Biol. 87, 35–44 (1985).

    Article  CAS  Google Scholar 

  27. Hancock, R.E.W., Brown, M.H. & Piers, K. Cationic peptides and method of preparation. US Patent application serial No. 07/913, 492, filed August 21, 1992.

  28. Datla, R.S.S. et al. Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence. Plant Sci. 94, 139–149 (1993).

    Article  CAS  Google Scholar 

  29. Ross, H. Potato breeding—problems and perspectives: advances in plant breeding. J. Plant Breeding (Supp.) 13, 132 (1986).

    Google Scholar 

  30. Cavallarin L., Andreu, D. & San Segundo, B. Cecropin A-derived peptides are potent inhibitors of fungal pathogens. Mol. Plant Microbe Interact. 11, 218–227 (1998).

    Article  CAS  Google Scholar 

  31. Powell, W.A., Catranis, C.M. & Maynard, C.A. Synthetic antimicrobial peptide design. Mol. Plant Microbe Interact. 8, 792–794 (1995).

    Article  CAS  Google Scholar 

  32. Mittler, R., Shulaev, V. & Lam, E. Coordinated activation of programmed cell death and defense mechanisms in transgenic tobacco plants expressing a bacterial proton pump. Plant Cell 7, 29–42 (1995).

    Article  CAS  Google Scholar 

  33. Dangl, J.L., Dietrich, R.A. & Richberg, M.H. Death don't have no mercy: cell death programs in plant–microbe interactions. Plant Cell 8, 1793–1807 (1996).

    Article  CAS  Google Scholar 

  34. Mittler, R. & Lam, E. Sacrifice in the face of foes: pathogen-induced programmed cell death in plants. Trends Microbiol. 4, 10–15 (1996).

    Article  CAS  Google Scholar 

  35. Abad, M.S. et al. Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin. Mol. Plant Microbe Interact. 10, 635–645 (1997).

    Article  CAS  Google Scholar 

  36. Dempsey, D.A., Silva, H. & Klessig, D.F. Engineering disease and pest resistance in plants. Trends Microbiol. 6, 54–61 (1997).

    Article  Google Scholar 

  37. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning: a laboratory manual, Edn. 2. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1989).

    Google Scholar 

  38. Holsters, M. et al. Transfection and transformation of A. tumefaciens. Mol. Gen. Genet. 163, 181–187 (1978).

    Article  CAS  Google Scholar 

  39. De Block, M. Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor. Appl. Genet. 76, 767–774 (1988).

    Article  CAS  Google Scholar 

  40. Wagner, D.B. et al. Chloroplast DNA polymorphisms in lodgepole and jack pines and their hybrids. Proc. Natl. Acad. Sci. USA 84, 2097–2100 (1987).

    Article  CAS  Google Scholar 

  41. Verwoerd, T.C., Dekker, B.M.M. & Hoekema, A. A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res. 17, 2362 (1989).

  42. Schägger, H. & von Jagow, G. Tricine–sodium dodecyl sulphate–polyacrylamide gelelectrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379 (1987).

    Article  Google Scholar 

  43. Friedrich, C., Scott, M., Karunaratne, N., Yan, H. & Hancock, R.E.W. NaCl resistant α-helical cationic antimicrobial peptides. Antimicrob. Agents Chemother. 43, 1542–1548 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by the Natural Sciences and Engineering Research Council (NSERC) Strategic grant to S.M. and W.W.K. We thank N. Vettakorrumkankav, L. Sun, X. Yu, and T. Stevenson for expert technical assistance, and Dr. Zamir Punja (Simon Fraser University, Burnaby, BC, Canada) for providing fungal phytopathogens.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Microbiology, University of Victoria, Victoria, V8W 3P6, BC, Canada

    Milan Osusky, Guoqing Zhou, Lubica Osuska, William W. Kay & Santosh Misra

  2. Department of Microbiology, University of British Columbia, Vancouver, V6T 1W5, BC, Canada

    Robert E. Hancock

Authors
  1. Milan Osusky
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guoqing Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lubica Osuska
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert E. Hancock
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William W. Kay
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Santosh Misra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to William W. Kay or Santosh Misra.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Osusky, M., Zhou, G., Osuska, L. et al. Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nat Biotechnol 18, 1162–1166 (2000). https://doi.org/10.1038/81145

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/81145

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing