Skip to main content

Transgenic Brassica juncea Plants Expressing MsrA1, a Synthetic Cationic Antimicrobial Peptide, Exhibit Resistance to Fungal Phytopathogens

Molecular Biotechnology volume 56, pages 535–545 (2014)Cite this article

Abstract

Cationic antimicrobial peptides (CAPs) have shown potential against broad spectrum of phytopathogens. Synthetic versions with desirable properties have been modeled on these natural peptides. MsrA1 is a synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties. We generated transgenic Brassica juncea plants expressing the msrA1 gene aimed at conferring fungal resistance. Five independent transgenic lines were evaluated for resistance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devastating pathogens of B. juncea crops. In vitro assays showed inhibition by MsrA1 of Alternaria hyphae growth by 44–62 %. As assessed by the number and size of lesions and time taken for complete leaf necrosis, the Alternaria infection was delayed and restricted in the transgenic plants with the protection varying from 69 to 85 % in different transgenic lines. In case of S. sclerotiorum infection, the lesions were more severe and spread profusely in untransformed control compared with transgenic plants. The sclerotia formed in the stem of untransformed control plants were significantly more in number and larger in size than those present in the transgenic plants where disease protection of 56–71.5 % was obtained. We discuss the potential of engineering broad spectrum biotic stress tolerance by transgenic expression of CAPs in crop plants.

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

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Food and Agriculture Organization (2010) The state of food insecurity in the world. http://www.fao.org/docrep/013/i1683e/i1683e.pdf.

  2. Agrios, G. N. (2005). Plant Pathology. London: Academic Press.

    Google Scholar 

  3. Ali, G. S., & Reddy, A. S. N. (2000). Inhibition of fungal and bacterial plant pathogens by synthetic peptides: In vitro growth inhibition, interaction between peptides and inhibition of disease progression. Molecular Plant–Microbe Interactions, 13, 847–859.

    CAS  Article  Google Scholar 

  4. Brown, K. L., & Hancock, R. E. W. (2006). Cationic host defense (antimicrobial) peptides. Current Opinion in Immunology, 18, 24–30.

    CAS  Article  Google Scholar 

  5. Hancock, R. E. W., & Lehrer, R. (1998). Cationic peptides: A new source of antibiotics. Trends in Biotechnology, 16, 82–88.

    CAS  Article  Google Scholar 

  6. Yount, N. Y., & Yeaman, M. R. (2005). Immunocontinuum: Perspectives in antimicrobial peptide mechanisms of action and resistance. Protein and Peptide Letters, 12, 49–67.

    CAS  Article  Google Scholar 

  7. Hancock, R. E. W. (2005). Mechanisms of action of newer antibiotics for Gram-positive pathogens. Lancet Infectious Diseases, 5, 209–218.

    CAS  Article  Google Scholar 

  8. Yevtushenko, D. P., Romero, R., Forward, B. S., Hancock, R. E., Kay, W. W., & Misra, S. (2005). Pathogen-induced expression of a cecropin A melittin antimicrobial peptide gene confers antifungal resistance in transgenic tobacco. Journal of Experimental Botany, 56, 1685–1695.

    CAS  Article  Google Scholar 

  9. Koo, J. C., Chun, H. J., Park, H. C., Kim, M. C., Koo, Y. D., Koo, S. C., et al. (2002). Over-expression of a seed specific hevein-like antimicrobial peptide from Pharbitis nil enhances resistance to a fungal pathogen in transgenic tobacco plants. Plant Molecular Biology, 50, 441–452.

    CAS  Article  Google Scholar 

  10. Jaynes, J. M., Nagpala, P., Destefanobeltran, L., Huang, J. H., Kim, J. H., Denny, T., et al. (2002). Expression of a cecropin-B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas solanacearum. Plant Science, 89, 43–53.

    Article  Google Scholar 

  11. Huang, Y., Nordeen, R. O., Di, M., Owens, L. D., & McBeath, J. H. (1997). Expression of an engineered cecropin gene cassette in transgenic tobacco plants confers resistance to Pseudomonas syringae pv. tabaci. Phytopathology, 87, 494–499.

    CAS  Article  Google Scholar 

  12. Sharma, A., Sharma, R., Imamura, M., Yamakawa, M., & Machii, H. (2000). Transgenic expression of cecropin B, an antibacterial peptide from Bombyx mori, confers enhanced resistance to bacterial leaf blight in rice. FEBS Letters, 484, 7–11.

    CAS  Article  Google Scholar 

  13. Osusky, M., Zhou, G., Osuska, L., Hancock, R. E., Kay, W. W., & Misra, S. (2000). Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nature Biotechnology, 18, 1162–1166.

    CAS  Article  Google Scholar 

  14. Gao, A. G., Hakimi, S. M., Mittanck, C. A., Wu, Y., Woerner, B. M., Stark, D. M., et al. (2000). Fungal pathogen protection in potato by expression of a plant defensin peptide. Nature Biotechnology, 18, 1307–1310.

    CAS  Article  Google Scholar 

  15. Chakrabarti, A., Ganapathi, T. R., Mukherjee, P. K., & Bapat, V. A. (2003). MSI-99, a magainin analogue, imparts enhanced disease resistance in transgenic tobacco and banana. Planta, 216, 587–596.

    CAS  Google Scholar 

  16. Alan, A. R., Blowers, A., & Earle, E. D. (2004). Expression of a magainin-type antimicrobial peptide gene (MSI-99) in tomato enhances resistance to bacterial speck disease. Plant Cell Reports, 22, 388–396.

    CAS  Article  Google Scholar 

  17. Mentag, R., Luckevich, M., Morency, M. J., & Se′guin, A. (2003). Bacterial disease resistance of transgenic hybrid poplar expressing the synthetic antimicrobial peptide D4E1. Tree Physiology, 23, 405–411.

    CAS  Article  Google Scholar 

  18. Vidal, J. R., Kikkert, J. R., Malnoy, M. A., Wallace, P. G., Barnard, J., & Reisch, B. I. (2006). Evaluation of transgenic ‘Chardonnay’ (Vitis vinifera) containing magainin genes for resistance to crown gall and powdery mildew. Transgenic Research, 15, 69–82.

    CAS  Article  Google Scholar 

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

  20. Niknam, S. R., & Turner, D. W. (1999). A single drought event, at different stages of development, has different effects on the final yield of Brassica napus cv. Monty and B. juncea line 397-23-2-3-3. In G. Shea, (Ed.), Oilseed Crop Updates (pp. 14–15). Northam: Agriculture Western Australia.

    Google Scholar 

  21. Wright, P. R., Morgan, J. M., & Jessop, R. S. (1996). Comparative adaptation of canola (Brassica napus) and Indian mustard (B. juncea) to soil water deficits: Plant water relations and growth. Field Crop Research, 49, 51–64.

    Article  Google Scholar 

  22. Shekhawat, K., Rathore, S. S., Premi, O. P., Kandpal, B. K., & Chauhan, J. S. (2012). Advances in agronomic management of Indian mustard (Brassica juncea (L.) Czernj. Cosson): An overview. International Journal Of Agronomy, 2012, 1–14.

    Article  Google Scholar 

  23. Mondal, K. K., Bhattacharya, R. C., Koundal, K. R., & Chatterjee, S. C. (2007). Transgenic Indian mustard (Brassica juncea) expressing tomato glucanase leads to arrested growth of Alternaria brassicae. Plant Cell Reports, 26, 247–252.

    CAS  Article  Google Scholar 

  24. Ghasolia, R. P., Shivpuri, A., & Bhargava, A. K. (2004). Sclerotinia rot of Indian mustard (Brassica juncea) in Rajasthan. Indian Phytopathology, 57, 76–79.

    Google Scholar 

  25. Höfgen, R., & Willmitzer, L. (1988). Storage of competent cells for Agrobacterium transformation. Nucleic Acids Research, 16, 9877.

    Article  Google Scholar 

  26. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiologia Plantarum, 15, 473–497.

    CAS  Article  Google Scholar 

  27. Yusuf, M. A., & Sarin, N. B. (2007). Antioxidant value addition in human diets: genetic transformation of Brassica juncea with gamma-TMT gene for increased alpha-tocopherol content. Transgenic Research, 16, 109–113.

    CAS  Article  Google Scholar 

  28. Murray, H. G., & Thompson, W. F. (1980). Rapid isolation of high molecular weight DNA. Nucleic Acids Research, 8, 4321–4325.

    CAS  Article  Google Scholar 

  29. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning. A laboratory manual. New York: Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

    Google Scholar 

  30. Sharma, N., Rahman, M. H., Strelkov, S., Thiagarajah, M., Bansal, V. K., & Kav, N. N. V. (2007). Proteome-level changes in two Brassica napus lines exhibiting differential responses to the fungal pathogen Alternaria brassicae. Plant Science, 172, 95–110.

    CAS  Article  Google Scholar 

  31. Jensen, B. D., Finckh, M. R., Munk, L., & Hauser, T. P. (2008). Susceptibility of wild carrot (Daucus carota ssp. carota) to Sclerotinia sclerotiorum. European Journal of Plant Pathology, 122, 359–367.

    Article  Google Scholar 

  32. Grover, A., & Gowthaman, R. (2003). Strategies for development of fungus-resistant transgenic plants. Current Science, 84, 330–340.

    Google Scholar 

  33. Ghasolia, R. P., & Shivpuri, A. (2007). Management of Sclerotinia rot of Indian mustard through cultural practices. Journal of Mycology and Plant Pathology, 37, 244–247.

    Google Scholar 

  34. Kang, I. S., & Chahal, S. S. (2000). Prevalence and incidence of white rot of mustard incited by Sclerotinia sclerotiorum in Punjab. Plant Disease Research, 15, 232–233.

    Google Scholar 

  35. Sharma, S., Yadav, J. L., & Sharma, G. R. (2001). Effect of various agronomic practices on the incidence of white rot of Indian mustard caused by Sclerotinia sclerotiorum. Journal of Mycology and Plant Pathology, 31, 83–84.

    Google Scholar 

  36. Finnegan, J., & McElroy, D. (1994). Transgene inactivation: Plants fight back. Biotechnology, 12, 883–888.

    Article  Google Scholar 

  37. Day, C. D., Lee, E., Kobayashi, J., Holappa, L. D., Albert, H., & Ow, D. W. (2000). Transgene integration into the same chromosome location can produce alleles that express at a predictable level, or alleles that are differentially silenced. Genes & Development, 14, 2869–2880.

    CAS  Article  Google Scholar 

Download references

Acknowledgments

This work was funded by Grant (No. BT/PR9616/AGR/02/458/2007) from the Department of Biotechnology, India to N.B.S. A.R. and D.K. were recipients of Senior Research Fellowship from the Council of Science and Industrial Research, India. M.A.Y. is a U.G.C. Dr. D.S. Kothari Postdoctoral Fellow. The authors thank Dr. Pratibha Sharma, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, for her suggestions in fungal infection experiments.

Author information

Author notes

  1. Anjana Rustagi

    Present address: Department of Botany, Ramjas College, University of Delhi, Delhi, 110007, India

Affiliations

  1. School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

    Anjana Rustagi, Deepak Kumar, Shashi Shekhar, Mohd Aslam Yusuf & Neera Bhalla Sarin

  2. Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W3P6, Canada

    Santosh Misra

Authors
  1. Anjana Rustagi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Deepak Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shashi Shekhar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mohd Aslam Yusuf
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Santosh Misra
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Neera Bhalla Sarin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Neera Bhalla Sarin.

Additional information

Anjana Rustagi and Deepak Kumar contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rustagi, A., Kumar, D., Shekhar, S. et al. Transgenic Brassica juncea Plants Expressing MsrA1, a Synthetic Cationic Antimicrobial Peptide, Exhibit Resistance to Fungal Phytopathogens. Mol Biotechnol 56, 535–545 (2014). https://doi.org/10.1007/s12033-013-9727-8

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-013-9727-8

Keywords

  • Biotic stress
  • Brassica juncea
  • Cationic antimicrobial peptides
  • MsrA1
  • Transgenic plants