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A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus)

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Abstract

Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it’s in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.

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Acknowledgments

This research was supported by funding from Agriculture Funding Consortium, the Alberta Canola Producers Commission (ACPC), Alberta Crop Industry Development Fund (ACIDF) and the Canola Council of Canada which is gratefully acknowledged. We are also thankful to MBSU and the microscopy unit, as well as Department of Agricultural, Food and Nutritional Science of University of Alberta for providing research facilities. We would also like to thank A. Kamran for help with the statistical analysis of the data.

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Authors and Affiliations

  1. Department of Agricultural, Food and Nutritional Science, University of Alberta, 4-10 Agriculture/Forestry Centre, Edmonton, AB, T6G 2P5, Canada

    Shiv S. Verma, Muhammad H. Rahman & Nat N. V. Kav

  2. Department of Plant Pathology, North Dakota State University, Fargo, ND, 58108, USA

    William R. Yajima

  3. Plant Biotechnology, Alberta Innovates—Technology Futures, Vegreville, AB, T9C 1T4, Canada

    Saleh Shah

  4. Pacific Forestry Centre, Victoria, BC, V8Z 1M5, Canada

    Jun-Jun Liu & Abul K. M. Ekramoddoullah

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  1. Shiv S. Verma
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Correspondence to Nat N. V. Kav.

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11103_2012_9895_MOESM1_ESM.tif

T-DNA of plant transformation vector pPKYLX-71. PmAMP1 cDNA driven by constitutive promoter CAMV35S and pea rbcS30 polyA terminator and neomycin phosphotransferase (nptII) coding gene between nos promoter and nos terminator are shown within the T-DNA; LB, left T-DNA border; RB, right T-DNA border (TIFF 462 kb)

11103_2012_9895_MOESM2_ESM.tif

Immunohistochemical analysis of Alternaria brassicae (a), Leptosphaeria maculans (b) and Sclerotinia sclerotiorum (c) showing the immunolocalization of PmAMP1. The insets show the transverse section of fungal mycelia. One representative control (A. brassicae without primary antibody) is shown (d) out of all six negative controls of 3 species with either primary or secondary antibody. The arrows indicate the cell walls of the fungal hyphae, which are not detected by the antibody (TIFF 8,714 kb)

11103_2012_9895_MOESM3_ESM.tif

Predicted three dimensional structure of PmAMP1 employing the tool available on the ExPASY-Pyhre2 server package. The putative structure shows the organization of the protein into β-strands which are furthered ordered into motifs similar to previously reported structure of the MiAMP1 (TIFF 2,475 kb)

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Verma, S.S., Yajima, W.R., Rahman, M.H. et al. A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus). Plant Mol Biol 79, 61–74 (2012). https://doi.org/10.1007/s11103-012-9895-0

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