Journal of Plant Research

, Volume 125, Issue 1, pp 115–124 | Cite as

Defense gene expression is potentiated in transgenic barley expressing antifungal peptide metchnikowin throughout powdery mildew challenge

  • Mohammad Rahnamaeian
  • Andreas Vilcinskas
Regular Paper


Transgenesis of antimicrobial peptides (AMPs) from different origins has emerged as an option for improvement of crop disease resistance since proof-of-concept for their activities against microbial phytopathogens is provided, persistently. Nevertheless, a more systematic approach based on knowledge of AMPs modes of action including elucidation of their cellular targets and possible impact on immune system considerably improves and diversifies the armory against harmful plant diseases. In present study, the impact of Metchnikowin (Mtk) expression in barley in terms of modulating different immune pathways was investigated. Monitoring of transcript abundance of different genes involved in key immune pathways of SAR, ISR, and redox milieu during interaction of Mtk barley with biotrophic Blumeria graminis f. sp. hordei (Bgh) demonstrated that several immune responses are modulated in Mtk transgenic plants. Present findings substantiate the higher activation of SAR pathway as well as ISR pathway in transgenic plants. Regarding susceptibility factors, nonetheless MLO gene is expressed more in Mtk plants and should lead to an increased cellular accessibility to Bgh, its impact is presumably overwhelmed by other mechanism(s) so that the plants show more resistance when challenging with Bgh. On the other hand, no obvious difference was observed between expression level of Bax inhibitor-1 (BI-1) in transgenic and wild type plants, which could be an indicative of its neutrality in resistance/susceptibility of transgenic plants to Bgh. The provided evidence on the involved pathways in Mtk-induced resistance improves our knowledge concerning impacts of AMPs expressed in diverse plant species on immune system of relevant transgenic plants.


Antimicrobial peptides Barley SAR ISR Metchnikowin 



The authors thank the research grant from Shahid Bahonar University of Kerman (Iran) as well as Ministry for Science and Art of the state of Hesse (Germany) for funding both the LOEWE research focus “Insect Biotechnology” and the Fraunhofer group Bioresources.

Supplementary material

10265_2011_420_MOESM1_ESM.doc (137 kb)
Supplementary material 1 (DOC 150 kb)
10265_2011_420_MOESM2_ESM.doc (42 kb)
Supplementary material 2 (DOC 47 kb)


  1. Aerts AM, Francois I, Meert EMK, Li Q, Cammue BPA, Thevissen K (2007a) The antifungal activity of RsAFP2, a plant defensin from Raphanus sativus, involves the induction of reactive oxygen species in Candida albicans. J Mol Microbiol Biotechnol 13:243–247PubMedCrossRefGoogle Scholar
  2. Aerts AM, Thevissen K, Bresseleers SM, Sels J, Wouters P, Cammue BPA, Francois I (2007b) Arabidopsis thaliana plants expressing human beta-defensin-2 are more resistant to fungal attack: functional homology between plant and human defensins. Plant Cell Rep 26:1391–1398PubMedCrossRefGoogle Scholar
  3. Almasia NI, Bazzini AA, Hopp HE, Vazquez-Rovere C (2008) Overexpression of snakin-1 gene enhances resistance to Rhizoctonia solani and Erwinia carotovora in transgenic potato plants. Mol Plant Pathol 9:329–338PubMedCrossRefGoogle Scholar
  4. Altincicek B, Lindner M, Linder D, Preissner K, Vilcinskas A (2007) Microbial metalloproteinases mediate sensing of invading pathogens and activate innate immune responses in the lepidopteran model host Galleria mellonella. Infect Immun 75:175–183PubMedCrossRefGoogle Scholar
  5. Atsumi G, Kagaya U, Kitazawa H, Nakahara KS, Uyeda I (2009) Activation of the salicylic acid signaling pathway enhances clover yellow vein virus virulence in susceptible pea cultivars. Mol Plant Microbe Interact 22(2):166–175PubMedCrossRefGoogle Scholar
  6. Ausubel FM (2005) Are innate immune signaling pathways in plants and animals conserved? Nat Immunol 6:973–979PubMedCrossRefGoogle Scholar
  7. Birch PR, Armstrong M, Bos J, Boevink P, Gilroy EM, Taylor RM, Wawra S, Pritchard L, Conti L, Ewan R, Whisson SC, van West P, Sadanandom A, Kamoun S (2009) Towards understanding the virulence functions of RXLR effectors of the oomycete plant pathogen Phytophthora infestans. J Exp Bot 60:1133–1140PubMedCrossRefGoogle Scholar
  8. Bowdish DM, Davidson DJ, Hancock RE (2005) A re-evaluation of the role of host defence peptides in mammalian immunity. Curr Protein Peptide Sci 6:35–51CrossRefGoogle Scholar
  9. Brogden K (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238–250PubMedCrossRefGoogle Scholar
  10. Campo S, Manrique S, García-Martínez J, San Segundo B (2008) Production of cecropin A in transgenic rice plants has an impact on host gene expression. Plant Biotechnol J 6:585–608PubMedCrossRefGoogle Scholar
  11. Cary JW, Rajasekaran K, Jaynes JM, Cleveland TE (2000) Transgenic expression of a gene encoding a synthetic antimicrobial peptide results in inhibition of fungal growth in vitro and in planta. Plant Sci 154:171–181PubMedCrossRefGoogle Scholar
  12. Chakrabarti A, Ganapathi TR, Mukherjee PK, Bapat VA (2003) MSI-99, a magainin analogue, imparts enhanced disease resistance in transgenic tobacco and banana. Planta 216:587–596PubMedGoogle Scholar
  13. Coca M, Bortolotti C, Rufat M, Penas G, Eritja R, Tharreau D, del Pozo AM, Messeguer J, San Segundo B (2004) Transgenic rice plants expressing the antifungal AFP protein from Aspergillus giganteus show enhanced resistance to the rice blast fungus Magnaporthe grisea. Plant Mol Biol 54:245–259PubMedCrossRefGoogle Scholar
  14. Coca M, Penas G, Gomez J, Campo S, Bortolotti C, Messeguer J, San Segundo B (2006) Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice. Planta 223:392–406PubMedCrossRefGoogle Scholar
  15. Consonni C, Humphry ME, Hartmann HA, Livaja M, Durner J, Westphal L, Vogel J, Lipka V, Kemmerling B, Schulze-Lefert P, Somerville SC, Panstruga R (2006) Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nat Genet 38:716–720PubMedCrossRefGoogle Scholar
  16. DeGray G, Rajasekaran K, Smith F, Sanford J, Daniell H (2001) Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol 127:852–862PubMedCrossRefGoogle Scholar
  17. Devoto A, Hartmann HA, Piffanelli P, Elliott C, Simmons C, Taramino G, Goh CS, Cohen FE, Emerson BC, Schulze-Lefert P, Panstruga R (2003) Molecular phylogeny and evolution of the plant-specific seven-transmembrane MLO family. J Mol Evol 56:77–88PubMedCrossRefGoogle Scholar
  18. Distefano G, La Malfa S, Vitale A, Lorito M, Deng Z, Gentile A (2008) Defence-related gene expression in transgenic lemon plants producing an antimicrobial Trichoderma harzianum endochitinase during fungal infection. Transgenic Res 17:873–879PubMedCrossRefGoogle Scholar
  19. Dong X (2004) NPR1, all things considered. Curr Opin Plant Biol 7:547–552PubMedCrossRefGoogle Scholar
  20. Du T, Wang Y, Hu QX, Chen J, Liu S, Huang WJ, Lin ML (2005) Transgenic Paulownia expressing shiva-1 gene has increased resistance to paulownia witches’ broom disease. J Integ Plant Biol 47:1500–1506CrossRefGoogle Scholar
  21. Gao AG, Hakimi SM, Mittanck CA, Wu Y, Woerner BM, Stark DM, Shah DM, Liang JH, Rommens CMT (2000) Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat Biotechnol 18:1307–1310PubMedCrossRefGoogle Scholar
  22. Gillespie J, Bailey A, Cobb B, Vilcinskas A (2000) Fungal elicitors of insect immune responses. Arch Insect Biochem Physiol 44:49–68PubMedCrossRefGoogle Scholar
  23. Hammond-Kosack KE, Jones JDG (1996) Resistance gene-dependent plant defence responses. Plant Cell 8:1773–1791PubMedCrossRefGoogle Scholar
  24. Hancock R (2001) Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 1:156–164PubMedCrossRefGoogle Scholar
  25. Harfouche AL, Rugini E, Mencarelli F, Botondi R, Muleo R (2008) Salicylic acid induces H2O2 production and endochitinase gene expression but not ethylene biosynthesis in Castanea sativa in vitro model system. J Plant Physiol 165(7):734–744PubMedCrossRefGoogle Scholar
  26. He P, Shan L, Sheen J (2007) Elicitation and suppression of microbe-associated molecular pattern-triggered immunity in plant–microbe interactions. Cell Microbiol 9(6):1385–1396PubMedCrossRefGoogle Scholar
  27. Huang Y, Nordeen RO, Di M, Owens LD, McBeth JH (1997) Expression of an engineered cecropin gene cassette in transgenic tobacco plants confers disease resistance to Pseudomonas syringae pv. tabaci. Phytopathology 87:494–499PubMedCrossRefGoogle Scholar
  28. Huckelhoven R, Dechert C, Trujillo M, Kogel KH (2001) Differential expression of putative cell death regulator genes in near-isogenic, resistant and susceptible barley lines during interaction with the powdery mildew fungus. Plant Mol Biol 47:739–748PubMedCrossRefGoogle Scholar
  29. Imamura T, Yasuda M, Kusano H, Nakashita H, Ohno Y, Kamakura T, Taguchi S, Shimada H (2010) Acquired resistance to the rice blast in transgenic rice accumulating the antimicrobial peptide thanatin. Transgenic Res 19:415–424PubMedCrossRefGoogle Scholar
  30. Johrde A, Schweizer P (2008) A class III peroxidase specifically expressed in pathogen-attacked barley epidermis contributes to basal resistance. Mol Plant Pathol 9:687–696PubMedCrossRefGoogle Scholar
  31. Kanzaki H, Nirasawa S, Saitoh H, Ito M, Nishihara M, Terauchi R, Nakamura I (2002) Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in transgenic rice. Theor Appl Genet 105:809–814PubMedCrossRefGoogle Scholar
  32. Kato M, Hayakawa Y, Hyodo Y, Yano M (2000) Wound-induced ethylene synthesis and expression and formation of 1-aminocyclopropane-1-carboxylate (ACC) synthase, ACC oxidase, phenylalanine ammonia-lyase and peroxidase in wounded mesocarp tissue of Cucurbita maxima. Plant Cell Physiol 41:440–447PubMedGoogle Scholar
  33. Koo JC, Chun HJ, Park HC, Kim MC, Koo YD, Koo SC, Ok HM, Park SJ, Lee SH, Yun DJ, Lim CO, Bahk JD, Lee SY, Cho MJ (2002) Over-expression of a seed specific hevein-like antimicrobial peptide from Pharbitis nilenhances resistance to a fungal pathogen in transgenic tobacco plants. Plant Mol Biol 50:441–452PubMedCrossRefGoogle Scholar
  34. Koornneef A, Leon-Reyes A, Ritsema T, Verhage A, Den Otter FC, Van Loon LC, Pieterse CM (2008) Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation. Plant Physiol 147(3):1358–1368PubMedCrossRefGoogle Scholar
  35. Langen G, Imani J, Altincicek B, Kieseritzky G, Kogel KH, Vilcinskas A (2006) Transgenic expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco. Biol Chem 387:549–557PubMedCrossRefGoogle Scholar
  36. Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9:963–967PubMedCrossRefGoogle Scholar
  37. Lazzaro BP, Clark AG (2003) Molecular population genetics of inducible antibacterial peptide genes in Drosophila melanogaster. Mol Biol Evol 20:914–923PubMedCrossRefGoogle Scholar
  38. Leon J, Lawton MA, Raskin L (1995) Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco. Plant Physiol 108:1673–1678PubMedGoogle Scholar
  39. Levashina EA, Ohresser S, Bulet P, Reichhart JM, Hetru C, Hoffmann JA (1995) Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties. Eur J Biochem 233:694–700PubMedCrossRefGoogle Scholar
  40. Levine A, Tenhaken R, Dixon R, Lamb C (1994) H202 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79(9):583–593PubMedCrossRefGoogle Scholar
  41. Li Q, Lawrence CB, Xing HY, Babbit RA, Bass WT, Maiti IB, Everett NP (2001) Enhanced disease resistance conferred by expression of an antimicrobial magainin analog in transgenic tobacco. Planta 212:635–639PubMedCrossRefGoogle Scholar
  42. Li HP, Zhang JB, Shi RP, Huang T, Fischer R, Liao YC (2008) Engineering Fusarium head blight resistance in wheat by expression of a fusion protein containing a Fusarium-specific antibody and an antifungal peptide. Mol Plant Microbe Interact 21:1242–1248PubMedCrossRefGoogle Scholar
  43. Liu Q, Ingersoll J, Owens L, Salih S, Meng R, Hammerschlag F (2001) Response of transgenic Royal Gala apple (Malus domestica Borkh.) shoots carrying a modified cecropin MB39 gene, to Erwinia amylovora. Plant Cell Rep 20:306–312CrossRefGoogle Scholar
  44. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−DDC(T)) method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  45. Miyashita S, Shirako Y (2008) Chromosomal integration of a binding domain: bait gene into yeast enhances detection in the two-hybrid system. J Microbiol Methods 73:179–184PubMedCrossRefGoogle Scholar
  46. Moreno AB, Penas G, Rufat M, Bravo JM, Estopa M, Messeguer J, San Segundo B (2005) Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice. Mol Plant Microbe Interact 18:960–972PubMedCrossRefGoogle Scholar
  47. Nürnberger T, Brunner F, Kemmerling B, Piater L (2004) Innate immunity in plants and animals: striking similarities and obvious differences. Immunol Rev 198:249–266PubMedCrossRefGoogle Scholar
  48. Oard SV, Enright FM (2006) Expression of the antimicrobial peptides in plants to control phytopathogenic bacteria and fungi. Plant Cell Rep 25:561–572PubMedCrossRefGoogle Scholar
  49. Osusky M, Zhou GQ, Osuska L, Hancock RE, Kay WW, Misra S (2000) Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nat Biotechnol 18:1162–1166PubMedCrossRefGoogle Scholar
  50. Osusky M, Osuska L, Hancock RE, Kay WW, Misra S (2004) Transgenic potatoes expressing a novel cationic peptide are resistant to late blight and pink rot. Transgenic Res 13:181–190PubMedCrossRefGoogle Scholar
  51. Osusky M, Osuska L, Kay W, 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 111:711–722PubMedCrossRefGoogle Scholar
  52. Panstruga R (2003) Establishing compatibility between plants and obligate biotrophic pathogens. Curr Opin Plant Biol 6:320–326PubMedCrossRefGoogle Scholar
  53. Park CH, Kang YH, Chun HJ, Koo JC, Cheong YH, Kim CY, Kim MC, Chung WS, Kim JC, Yoo JH, Koo YD, Koo SC, Lim CO, Lee SY, Cho MJ (2002) Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage. Plant Mol Biol 50:57–68CrossRefGoogle Scholar
  54. Patkar RN, Chattoo BB (2006) Transgenic indica rice expressing ns-LTP-Like protein shows enhanced resistance to both fungal and bacterial pathogens. Mol Breed 17:159–171CrossRefGoogle Scholar
  55. Quilis J, Peñas G, Messeguer J, Brugidou C, San Segundo B (2008) The Arabidopsis AtNPR1 inversely modulates defense responses against fungal, bacterial, or viral pathogens while conferring hypersensitivity to abiotic stresses in transgenic rice. Mol Plant Microbe Interact 21(9):1215–1231PubMedCrossRefGoogle Scholar
  56. Rahnamaeian M, Langen G, Imani J, Khalifa W, Altincicek B, von Wettstein D, Kogel KH, Vilcinskas A (2009) Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens. J Exp Bot 60:4105–4114PubMedCrossRefGoogle Scholar
  57. Rajasekaran K, Cary JW, Jaynes JM, Cleveland TE (2005) Disease resistance conferred by the expression of a gene encoding a synthetic peptide in transgenic cotton (Gossypium hirsutum L.) plants. Plant Biotechnol J 3:545–554PubMedCrossRefGoogle Scholar
  58. Reiss E, Horstmann C (2001) Drechslera teres-infected barley (Hordeum vulgare L.) leaves accumulate eight isoforms of thaumatin-like proteins. Physiol Mol Plant Pathol 58:183–188CrossRefGoogle Scholar
  59. Scalla R, Roulet A (2002) Cloning and characterization of a glutathione S-transferase induced by a herbicide safener in barley (Hordeum vulgare). Physiol Plant 116:336–344CrossRefGoogle Scholar
  60. Schulze-Lefert P, Panstruga R (2003) Establishment of biotrophy by parasitic fungi and reprogramming of host cells for disease resistance. Ann Rev Phytopathol 41:641–647CrossRefGoogle Scholar
  61. Schweizer P, Pokorny J, Abderhalden O, Dudler R (1999) A transient assay system for the functional assessment of defense-related genes in wheat. Mol Plant Microbe Interact 12:647–654CrossRefGoogle Scholar
  62. Steiner-Lange S, Fischer A, Boettcher A, Rouhara I, Liedgens H, Schmelzer E, Knogge W (2003) Differential defense reactions in leaf tissues of barley in response to infection by Rhynchosporium secalis and to treatment with a fungal avirulence gene product. Mol Plant Microbe Interact 16:893–902PubMedCrossRefGoogle Scholar
  63. Stevens C, Titarenko E, Hargreaves JA, Gurr SJ (1996) Defence-related gene activation during an incompatible interaction between Stagonospora (Septoria) nodorum and barley (Hordeum vulgare L.) coleoptile cells. Plant Mol Biol 31:741–749PubMedCrossRefGoogle Scholar
  64. Swathi Anuradha T, Divya K, Jami SK, Kirti PB (2008) Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens. Plant Cell Rep 27:1777–1786PubMedCrossRefGoogle Scholar
  65. Terras FRG, Eggermont K, Kovaleva V, Raikhel NV, Osborn RW, Kester A, Rees SB, Torrekens S, Leuven FV, Vanderleyden J, Cammue BPA, Broekaert WF (1995) Small cysteine-rich antifungal proteins from radish: their role in host defense. Plant Cell 7:573–588PubMedCrossRefGoogle Scholar
  66. Turrini A, Sbrana C, Pitto L, Ruffini Castiglione M, Giorgetti L, Briganti R, Bracci T, Evangelista M, Nuti MP, Giovannetti M (2004) The antifungal Dm-AMP1 protein from Dahlia merckii expressed in Solanum melongena is released in root exudates and differentially affects pathogenic fungi and mycorrhizal symbiosis. New Phytol 163:393–403CrossRefGoogle Scholar
  67. Tuzun S (2001) The relationship between pathogen-induced systemic resistance (ISR) and multigenic (horizontal) resistance in plants. Eur J Plant Pathol 107:85–93CrossRefGoogle Scholar
  68. Uquillas C, Letelier I, Blanco F, Jordana X, Holuigue L (2004) NPR1-independent activation of immediate early salicylic acid-responsive genes in Arabidopsis. Mol Plant Microbe Interact 17(1):34–42PubMedCrossRefGoogle Scholar
  69. van der Weerden NL, Lay FT, Anderson MA (2008) The plant defensin, NaD1, enters the cytoplasm of Fusarium oxysporum hyphae. J Biol Chem 283:14445–14452PubMedCrossRefGoogle Scholar
  70. van Hulten M, Pelser M, van Loon LC, Pieterse CM, Ton J (2006) Costs and benefits of priming for defense in Arabidopsis. Proc Nat Acad Sci USA 103:5602–5607PubMedCrossRefGoogle Scholar
  71. Van Loon LC, Van Strien EA (1999) The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant Pathol 55:85–97CrossRefGoogle Scholar
  72. Verpoorte R (2000) Secondary metabolism. Kluwer Academic Publishers, DordrechtGoogle Scholar
  73. Vidal JR, Kikkert JR, Malnoy MA, Wallace PG, Barnard J, Reisch BI (2006) Evaluation of transgenic ‘chardonnay’ (Vitis vinifera) containing magainin genes for resistance to crown gall and powdery mildew. Transgenic Res 15:69–82PubMedCrossRefGoogle Scholar
  74. Wang Y, Nowak G, Culley D, Hadwiger LA, Fristensky B (1999) Constitutive expression of pea defense gene DRR206 confers resistance to blackleg (Leptosphaeria maculans) disease in transgenic canola (Brassica napus). Mol Plant Microbe Interact 12:410–418CrossRefGoogle Scholar
  75. Wiberg A (1974) Genetical studies of spontaneous sources of resistance to powdery mildew in barley. Hereditas 77:89–148PubMedCrossRefGoogle Scholar
  76. Xing H, Lawrence CB, Chambers O, Davies HM, Everett NP, Li QQ (2006) Increased pathogen resistance and yield in transgenic plants expressing combinations of the modified antimicrobial peptides based on indolicidin and magainin. Planta 223:1024–1032PubMedCrossRefGoogle Scholar
  77. Yang D, Biragyn A, Hoover DM, Lubkowski J, Oppenheim JJ (2004) Multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neurotoxin in host defense. Ann Rev Immunol 22:181–215CrossRefGoogle Scholar
  78. 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 56:1685–1695PubMedCrossRefGoogle Scholar
  79. Zhu YJ, Agbayani R, Moor PH (2007) Ectopic expression of Dahlia merckii defensin DmAMP1 improves papaya resistance to Phytophthora palmivora by reducing pathogen vigor. Planta 226:87–97PubMedCrossRefGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer 2011

Authors and Affiliations

  1. 1.Department of Plant Biotechnology, College of AgricultureShahid Bahonar UniversityKermanIran
  2. 2.Institute of Phytopathology and Applied Zoology, Research Centre for BioSystems, Land Use and Nutrition (IFZ)Justus Liebig UniversityGiessenGermany

Personalised recommendations