Plant Molecular Biology

, Volume 100, Issue 4–5, pp 481–494 | Cite as

Enhancing disease resistance in poplar through modification of its natural defense pathway

  • Dmytro P. YevtushenkoEmail author
  • Santosh Misra


Key message

Modification of the poplar defense pathway through pathogen-induced expression of an amphibian host defense peptide modulates plant innate immunity and confers robust and reliable resistance against a major poplar pathogen, Septoria musiva.


Host defense peptides (HDPs), also known as cationic antimicrobial peptides, represent a diverse group of small membrane-active molecules that are part of the innate defense system of their hosts against pathogen invasion. Here we describe a strategy for development of poplar plants with enhanced HDP production and resistance to the commercially significant fungal pathogen Septoria musiva. The naturally occurring linear amphipathic α-helical HDP dermaseptin B1, which has 31 residues and originated from the skin secretion of arboreal frogs, was N-terminally modified (MsrA2) and evaluated in vitro for antifungal activity and phytotoxicity. The MsrA2 peptide inhibited germination of S. musiva conidia at physiologically relevant low micromolar concentrations that were non-toxic to poplar protoplasts. The nucleotide sequence of MsrA2, optimized for expression in plants, was introduced into the commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6) via Agrobacterium-mediated transformation. Transgene expression was regulated by the pathogen-inducible poplar promoter win3.12T, a part of the poplar innate defense system. Most importantly, the induced accumulation of MsrA2 peptide in poplar leaves was sufficient to confer resistance against S. musiva. The antifungal resistance of plants with high MsrA2 expression and MsrA2 accumulation was strong and reproducible, and without deleterious effects on plant growth and development. These results provide an insight into development of new technologies for engineering durable disease resistance against major pathogens of poplar and other plants.


Host defense peptides MsrA2 win3.12T poplar promoter Populus nigra L. × P. maximowiczii A. Henry Septoria musiva Disease resistance 



We thank Dr. Arun Goyal (University of Minnesota-Duluth, MN, USA) for providing plants of hybrid poplar P. nigra L. × P. maximowiczii A. Henry, genotype NM6; Dr. Brenda Callan (Pacific Forestry Centre, Victoria, BC, Canada) for providing the in vitro culture of Septoria musiva; Dr. C. Peter Constabel (University of Victoria, BC, Canada) for providing the fungus Melampsora medusae; Dr. Bob Chow (University of Victoria, BC, Canada) for helping with microscopy imaging; and Dr. Barbara Hawkins (University of Victoria, BC, Canada) for her kind support of this project.

Authors contribution

D.Y. and S.M. designed the study, discussed and interpreted the results. DY conducted the experiments and wrote the manuscript. Both authors contributed to the final manuscript.


This study was funded by grants from the National Centre of Excellence, and the Advanced Foods and Materials Network to S.M.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2019_874_MOESM1_ESM.pdf (990 kb)
Online Resource 1 Resistance of NM6 leaves to M. medusae infection (PDF 990 kb)


  1. Ali GS, Reddy ASN (2000) Inhibition of fungal and bacterial plant pathogens by synthetic peptides: in vitro growth inhibition, interaction between peptides and inhibition of disease progression. Mol Plant Microbe Interact 13:847–859CrossRefPubMedGoogle Scholar
  2. Allefs SJHM, Florack DEA, Hoogendoorn C, Stiekema WJ (1995) Erwinia soft rot resistance of potato cultivars transformed with a gene construct coding for antimicrobial peptide cecropin B is not altered. Am Potato J 72:437–445CrossRefGoogle Scholar
  3. Balatinecz JJ, Kretschmann DE (2001) Properties and utilization of poplar wood. In: Dickmann DI et al (eds) Poplar culture in North America. NRC Research Press, Ottawa, pp 277–290Google Scholar
  4. Bent AF, Mackey D (2007) Elicitors, effectors, and R genes: the new paradigm and a lifetime supply of questions. Annu Rev Phytopathol 45:399–436CrossRefPubMedGoogle Scholar
  5. Burdsall HH Jr, Dorworth EB (1994) Preserving cultures of wood-decaying Basidiomycotina using sterile distilled water in cryovials. Mycologia 86(2):275–280CrossRefGoogle Scholar
  6. Callan BE, Leal I, Foord B, Dennis JJ, van Oosten C (2007) Septoria musiva isolated from cankered stems in hybrid poplar stool beds, Fraser Valley, British Columbia. Pac Northwest Fungi 2(7):1–9Google Scholar
  7. Cavallarin L, Andreu D, San Segundo S (1998) Cecropin A-derived peptides are potent inhibitors of fungal plant pathogens. Mol Plant Microbe Interact 11:218–227CrossRefPubMedGoogle Scholar
  8. Feau N, Mottet M-J, Périnet P, Hamelin RC, Bernier L (2010) Recent advances related to poplar leaf spot and canker caused by Septoria musiva. Can J Plant Pathol 32(2):122–134CrossRefGoogle Scholar
  9. Florack D, Allefs S, Bollen R, Bosch D, Visser B, Stiekema W (1995) Expression of giant silkmoth cecropin B genes in tobacco. Transgenic Res 4:132–141CrossRefPubMedGoogle Scholar
  10. Gao A, Hakimi SM, Mittanck CA, Wu Y, Woerner BM, Stark DM, Shah DM, Liang J, Rommens CMT (2000) Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat Biotechnol 18:1307–1310CrossRefPubMedGoogle Scholar
  11. Govrin EM, Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Curr Biol 10:751–757CrossRefPubMedGoogle Scholar
  12. Häggman H, Raybould A, Borem A et al (2013) Genetically engineered trees for plantation forests: key considerations for environmental risk assessment. Plant Biotechnol J 11(7):785–798CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hancock REW, Lehrer R (1998) Cationic peptides: a new source of antibiotics. Trends Biotechnol 16:82–88CrossRefGoogle Scholar
  14. Haney EF, Hancock REW (2013) Peptide design for antimicrobial and immunomodulatory applications. Biopolymers 100(6):572–583CrossRefPubMedPubMedCentralGoogle Scholar
  15. Harfouche A, Meilan R, Kirst M, Morgante M, Boerjan W, Sabatti M, Scarascia Mugnozza G (2012) Accelerating the domestication of forest trees in a changing world. Trends Plant Sci 17:64–72CrossRefPubMedGoogle Scholar
  16. Hightower R, Baden C, Penzes E, Dunsmuir P (1994) The expression of cecropin peptide in transgenic tobacco does not confer resistance to Pseudomonas syringae pv. tabaci. Plant Cell Rep 13:295–299CrossRefPubMedGoogle Scholar
  17. Hollick JB, Gordon MP (1993) A poplar tree proteinase inhibitor-like gene promoter is responsive to wounding in transgenic tobacco. Plant Mol Biol 22:561–572CrossRefPubMedGoogle Scholar
  18. Hollick JB, Gordon MP (1995) Transgenic analysis of a hybrid poplar wound-inducible promoter reveals developmental patterns of expression similar to that of storage protein genes. Plant Physiol 109:73–85CrossRefPubMedPubMedCentralGoogle Scholar
  19. Huang Y, Liu H, Jia Z, Fang Q, Luo K (2012) Combined expression of antimicrobial genes (Bbchit1 and LJAMP2) in transgenic poplar enhances resistance to fungal pathogens. Tree Physiol 32:1313–1320CrossRefPubMedGoogle Scholar
  20. Jacobi V, Plourde A, Charest PJ, Hamelin RC (2000) In vitro toxicity of natural and designed peptides to tree pathogens and pollen. Can J Bot 78:455–461Google Scholar
  21. JrRS Zalesny, Wiese AH, Bauer EO, Riemenschneider DE (2006) Sapflow of hybrid poplar (Populus nigra L. × P. maximowiczii A. Henry ‘NM6’) during phytoremediation of landfill leachate. Biomass Bioenergy 30:784–793CrossRefGoogle Scholar
  22. Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126:105–110CrossRefPubMedGoogle Scholar
  23. Labrecque M, Teodorescu TI (2005) Field performance and biomass production of 12 willow and poplar clones in short-rotation coppice in southern Quebec (Canada). Biomass Bioenergy 29:1–9CrossRefGoogle Scholar
  24. Larson PI, Isebrands JG (1971) The plastochron index as applied to developmental studies of cottonwood. Can J For Res 1:1–11CrossRefGoogle Scholar
  25. Liang H, Maynard CA, Allen RD, Powell WA (2001) Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene. Plant Mol Biol 45:619–629CrossRefPubMedGoogle Scholar
  26. Liang H, Catranis CM, Maynard CA, Powell WA (2002) Enhanced resistance to the poplar fungal pathogen, Septoria musiva, in hybrid poplar clones transformed with genes encoding antimicrobial peptides. Biotechnol Lett 24:383–389CrossRefGoogle Scholar
  27. Liang H, Staton M, Xu Y, Xu T, LeBoldus J (2014) Comparative expression analysis of resistant and susceptible Populus clones inoculated with Septoria musiva. Plant Sci 223:69–78CrossRefPubMedGoogle Scholar
  28. Lo MH, Abrahamson LP, White EH, Manion PD (1995) Early measures of basal area and canker disease predict growth potential of some hybrid poplar clones. Can J Res 25:1113–1118CrossRefGoogle Scholar
  29. Marcos JF, Muñoz A, Párez-Payá E, Misra S, López-García B (2008) Identification and rational design of novel antimicrobial peptides for plant protection. Annu Rev Phytopathol 46:273–301CrossRefPubMedGoogle Scholar
  30. Marmiroli MF, Pietrini E, Maestri M, Zacchini N, Marmiroli A, Massacci A (2011) Growth, physiological and molecular traits in Salicaceae trees investigated for phytoremediation of heavy metals and organics. Tree Physiol 31:1319–1334CrossRefPubMedGoogle Scholar
  31. Matzke MA, Matzke AJM (1995) How and why do plants inactivate homologous (trans)genes? Plant Physiol 107:679–685CrossRefPubMedPubMedCentralGoogle Scholar
  32. McDonald B (2010) How can we achieve durable disease resistance in agricultural ecosystems? New Phytol 185:3–5CrossRefPubMedGoogle Scholar
  33. McPhee JB, Hancock RE (2005) Function and therapeutic potential of host defence peptides. J Pept Sci 11(11):677–687CrossRefPubMedGoogle Scholar
  34. Medgyesy P, Menczel L, Maliga P (1980) The use of cytoplasmic streptomycin resistance: chloroplast transfer from Nicotiana tabacum into Nicotiana sylvestris, and isolation of their somatic hybrids. Mol Gen Genet 179:693–698CrossRefGoogle Scholar
  35. Minocha SC (2000) Optimization of the expression of a transgene in plants. In: Mohan Jain S, Minocha SC (eds) Molecular biology of woody plants. Kluwer, Dordrecht, pp 1–30Google Scholar
  36. Miranda M, Ralph SG, Mellway R, White R, Heath MC, Bohlmann J, Constable CP (2007) The transcriptional response of hybrid poplar (Populus trichocarpa × P. deltoides) to infection by Melampsora medusae leaf rust involves induction of flavonoid pathway genes leading to the accumulation of proanthocyanidins. Mol Plant Microbe Interact 20:816–831CrossRefPubMedGoogle Scholar
  37. Mookherjee N, Chow LNY, Hancock REW (2012) Immunomodulatory cationic peptide therapeutics: a new paradigm in infection and immunity. In: Rajasekaran K, Cary JW, Jaynes JM, Montesinos E (eds) Small wonders: peptides for disease control. ACS Symposium Series 1095, Oxford University Press, Washington, pp 1–19Google Scholar
  38. Mor A, Amiche M, Nicolas P (1994) Structure, synthesis, and activity of dermaseptin b, a novel vertebrate defensive peptide from frog skin: relationship with adenoregulin. Biochemistry 33:6642–6650CrossRefPubMedGoogle Scholar
  39. Mundt CC (2014) Durable resistance: a key to sustainable management of pathogens and pests. Infect Genet Evol 27:446–455CrossRefPubMedGoogle Scholar
  40. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  41. Nicolas P, Amri CEI (2009) The dermaseptin superfamily: a gene-based combinatorial library of antimicrobial peptides. Biomembranes (BBA) 1788(8):1537–1550CrossRefGoogle Scholar
  42. Noël A, Levasseur C, Le VQ, Séguin A (2005) Enhanced resistance to fungal pathogens in forest trees by genetic transformation of black spruce and hybrid poplar with a Trichoderma harzianum endochitinase gene. Physiol Mol Plant Pathol 67:92–99CrossRefGoogle Scholar
  43. Ostry ME, McRoberts RE, Ward KT, Resendez R (1988) Screening hybrid poplars in vitro for resistance to leaf spot caused by Septoria musiva. Plant Dis 72:497–499CrossRefGoogle Scholar
  44. Osusky M, Zhou G, Osuska L, Hancock RE, Kay WW, Misra S (2000) Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nat Biotechnol 18:1162–1166CrossRefPubMedGoogle Scholar
  45. Osusky M, Osuska L, Kay WW, Misra S (2005) Genetic modification of potato against microbial diseases: in vitro and in planta activity of a dermaseptin B1 derivative, MsrA2. Theor Appl Genet 111:711–722CrossRefPubMedGoogle Scholar
  46. Park YG, Son SH (1992) In vitro shoot regeneration from leaf mesophyll protoplasts of hybrid poplar (Populus nigra × P. maximowiczii). Plant Cell Rep 11:2–6CrossRefPubMedGoogle Scholar
  47. Rajasekaran K, Cary JW, Chlan CA, Jaynes JM, Bhatnagar D (2012) Strategies for controlling plant diseases and mycotoxin contamination using antimicrobial synthetic peptides. In: Rajasekaran K, Cary JW, Jaynes JM, Montesinos E (eds) Small wonders: peptides for disease control. ACS Symposium Series 1095. Oxford University Press, Washington, pp 295–316Google Scholar
  48. Roedl A (2010) Production and energetic utilization of wood from short rotation coppice—a life cycle assessment. Int J Life Cycle Assess 15:567–578CrossRefGoogle Scholar
  49. Rommens CM (2004) All-native DNA transformation: a new approach to plant genetic engineering. Trends Plant Sci 9 (9):457–464CrossRefPubMedGoogle Scholar
  50. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  51. Schnetkamp PPM, Kaupp UB, Junge W (1981) Interfacial potentials at the disk membranes of isolated intact cattle rod outer segments as a function of the occupation state of the intradiskal cation-exchange binding sites. Biomembranes (BBA) 642(2):213–230CrossRefGoogle Scholar
  52. Stanosz JC, Stanosz GR (2002) A medium to enhance identification of Septoria musiva from poplar cankers. For Path 32:145–152CrossRefGoogle Scholar
  53. Strauss S, Ma C, Ault K, Klocko AL (2016) Lessons from two decades of field trials with genetically modified trees in the USA: biology and regulatory c compliance. In: Vettori C et al (eds) Biosafety of forest transgenic trees. Springer, Dordrecht, pp 101–124CrossRefGoogle Scholar
  54. Yevtushenko DP, Misra S (2007) Comparison of pathogen-induced expression and efficacy of two amphibian antimicrobial peptides, MsrA2 and temporin A, for engineering wide-spectrum disease resistance in tobacco. Plant Biotechnol J 5:720–734CrossRefPubMedGoogle Scholar
  55. Yevtushenko DP, Misra S (2010) Efficient method for Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry. Plant Cell Rep 29:211–221CrossRefPubMedGoogle Scholar
  56. Yevtushenko DP, Misra S (2012) Transgenic expression of antimicrobial peptides in plants: Strategies for enhanced disease resistance, improved productivity, and production of therapeutics. In: Rajasekaran K, Cary JW, Jaynes JM, Montesinos E (eds) Small wonders: peptides for disease control. ACS Symposium Series 1095. Oxford University Press, Washington, pp 445–458Google Scholar
  57. Yevtushenko DP, SidorovVA Romero R, Kay WW, Misra S (2004) Wound-inducible promoter from poplar is responsive to fungal infection in transgenic potato. Plant Sci 167:715–724CrossRefGoogle Scholar
  58. Yevtushenko DP, Romero R, Forward BS, Hancock RE, Kay WW, Misra S (2005) Pathogen-induced expression of a cecropin A-melittin antimicrobial peptide gene confers antifungal resistance in transgenic tobacco. J Exp Bot 56:1685–1695CrossRefPubMedGoogle Scholar
  59. Yi JY, Seo HW, Yang MS, Robb EJ, Nazar RN, Lee SW (2004) Plant defense gene promoter enhances the reliability of shiva-1 gene-induced resistance to soft rot disease in potato. Planta 220:165–171CrossRefPubMedGoogle Scholar
  60. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Biological SciencesUniversity of LethbridgeLethbridgeCanada
  2. 2.Department of Biochemistry & Microbiology, Centre for Forest BiologyUniversity of VictoriaVictoriaCanada

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