, Volume 223, Issue 3, pp 392–406 | Cite as

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice

  • María Coca
  • Gisela Peñas
  • Jorge Gómez
  • Sonia Campo
  • Cristina Bortolotti
  • Joaquima Messeguer
  • Blanca San Segundo
Original Article


Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthegrisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.


Antifungal Cecropin A Oryza Rice blast fungus Transgenic rice 



endoplasmic reticulum


intercellular fluid


pathogenesis related



María Coca is a researcher from the Ministerio de Educación y Ciencia (Ramón y Cajal). Gisela Peñas is a recipient of a predoctoral fellowship from the Generalitat de Catalunya. We thank Dr U. Schaffrath for providing us with the PR1a rice cDNA probe. We are grateful to A.B. Moreno and M. Rufat for their collaboration in parts of this work and to P. Fontanet for taking care of the greenhouse plants. We also acknowledge Dr. R. Eritja for synthesis of oligonucleotides and Dr. D. Tharreau for providing us with the M. grisea PR9 isolate. This research was supported by the European Commission (QLRT-CT99-1484, EURICE) and by the Ministerio de Ciencia y Tecnologia (BIO2003-04936-C02). We also thank the “Centre de Referència en Biotecnología” (CeRBa) for substantial support.


  1. Alberola J, Rodrigued A, Francino O, Roura X, Rivas L, Andreu D (2004) Safety and efficacy of antimicrobial peptides against naturally acquired leishmaniasis. Antimicrob Agents Chemother 48:641–643CrossRefPubMedGoogle Scholar
  2. Allefs S, 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 72:437–445Google Scholar
  3. Bohlmann H (1999) The role of thionins in the resistance of plants. In: Datta SK, Muthudrishan S (eds) Pathogenesis-related proteins in plants. CRC Press, New York, pp 207–234Google Scholar
  4. Boman HG (1995) Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 13:61–92CrossRefPubMedGoogle Scholar
  5. Boman HG, Steiner H (1981) Humoral immunity in Cecropia pupae. Curr Topics Microbiol Immunol 94:75–91Google Scholar
  6. Bradford M (1976) A rapid and sensitive method for the quentification of microgram quantities utilizing the principle of protein-dye binding. Ann Biochem 72:248–254CrossRefGoogle Scholar
  7. Broekaert WF, Terras FRG, Cammue BP, Osborn RW (1995) Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Physiol 108:1353–1358PubMedCrossRefGoogle Scholar
  8. Broglie K, Chet I, Holliday M, Cressman R, Riddle P, Knowlton S, Mauvais CJ, Broglie R (1991) Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254:1194–1197PubMedCrossRefGoogle Scholar
  9. Cao H, Li X, Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proc Natl Acad Sci USA 95:6532–6536Google Scholar
  10. Castro MS, Fontes W (2005) Plant defense and antimicrobial peptides. Protein and Peptide Lett 12:13–18Google Scholar
  11. Cavallarin L, Andreu D, San Segundo B (1998) Cecropin A-derived peptides are potent inhibitors of fungal plant pathogens. Mol Plant-Microbe Interact 11:218–227PubMedCrossRefGoogle Scholar
  12. Christensen AH, Scharrock RA, Quail PJ (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689CrossRefPubMedGoogle Scholar
  13. Christensen AH, Quail PH (1996) Ubiquitin promoter based vectors for high level expression of selectable and/or screenable marker genes in monocotyledoneus plants. Transgenic Res 5:216–218CrossRefGoogle Scholar
  14. Christensen B, Fink J, Merrifield RB, Mauzerall D (1988) Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes. Proc Natl Acad Sci USA 85:5072–5076PubMedCrossRefGoogle Scholar
  15. Coca M, Bortolotti C, Rufat M, Peñas G, Eritja R, Tharreau D, Martinez del Pozo A, 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 54:245–259CrossRefPubMedGoogle Scholar
  16. Cornelissen BJC, Horowitz J, van Kan JAL, Goldberg RB, Bol JF (1987) Structure of tobacco genes encoding pathogenesis-related proteins from the PR-1 group. Nucl Acid Res 15:6799–6811CrossRefGoogle Scholar
  17. Datta S, Muthukrisnan S, Datta SK (1999) Expression and function of PR proteins in transgenic plants. In: Datta SK, Muthudrishan S (eds) Pathogenesis-related proteins in plants. CRC Press, New York, pp 261–277Google Scholar
  18. 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
  19. 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 13:295–299CrossRefGoogle Scholar
  20. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218CrossRefGoogle Scholar
  21. Huang Y, Nordeen RO, Di M, Owens LD, McBeath JH (1997) Expression of an engineered cecropin gene cassette in transgenic tobacco plants confers disease resistance to Pseudomonas syringae pv. tabaci. Phytopathology 87:494–499CrossRefPubMedGoogle Scholar
  22. Iwai H, Nakajima Y, Natori S, Arata Y, Shimada I (1993) Solution conformation of an antibacterial peptide, sarcotoxin IA, as determined by 1H-NMR. Eur J Biochem 217:639–644CrossRefPubMedGoogle Scholar
  23. Jach G, Gornhardt B, Mundy J, Logemann J, Pinsdorf E, Leah R, Schell J, Maas C (1995) Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J 8:97–103CrossRefPubMedGoogle Scholar
  24. Jaynes JM, Burton CA, Barr SB, Jeffers GW, Julian GR, White KL, Enright FM, Klei TR, Laine RA (1988) In vitro cytocidal effect of novel lytic peptides on Plasmodium falciparum and Trypanosoma cruzi. FASEB J 2:2878–2883PubMedGoogle Scholar
  25. Jaynes JM, Nagpala P, Destéfano-Beltrán L, Huang J-H, Kim J, Denny T, Cetiner S (1993) Expression of a cecropin B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas solanacearum. Plant Sci 89:43–53CrossRefGoogle Scholar
  26. Liu Q, Feng Y, Zhao X, Dong H, Xue Q (2004) Synonymous codon usage bias in Oryza sativa. Plant Sci 167:101–105CrossRefGoogle Scholar
  27. Logeman J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Ann Biochem 163:16–20CrossRefGoogle Scholar
  28. Lorito M, Woo SL, García-Fernandez I, Colucci G, Harman GE, Pintor-Toro JA, Filippone E, Muccifora S, Lawrence CB, Zoina A, Tuzun S, Scala F (1998) Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proc Natl Acad Sci USA 95:7860–7865CrossRefPubMedGoogle Scholar
  29. Mittler R, Shulaev V, Lam E (1995) Coordinated activation of programmed cell death and defense mechanisms in transgenic tobacco platns expressing a bacterial proton pump. Plant Cell 7:29–42CrossRefPubMedGoogle Scholar
  30. Mills D, Hammerschlag FA (1993) Effect of cecropin B on peach pathogens, protoplasts, and cells. Plant Sci 93:143–150CrossRefGoogle Scholar
  31. Mills D, Hammerschlag FA, Nordeen RO, Owens LD (1994) Evidence for the breakdown of cecropin B by proteinases in the intercellular fluid of peach leaves. Plant Sci 104:17–22CrossRefGoogle Scholar
  32. Mitsuhara I, Matsufuru H, Ohshima M, Kaku H, Nakajima Y, Murai N, Natori S, Ohashi Y (2000) Induced expression of sarcotoxin IA enhanced host resistance against both bacterial and fungal pathogens in transgenic tobacco. Mol Plant-Microbe Interact 13:860–868PubMedCrossRefGoogle Scholar
  33. Molina A, Ahl Goy P, Fraile A, Sánchez-Monje R, García-Olmedo F (1993) Inhibition of bacterial and fungal plant pathogens by thionins types I and II. Plant Sci 92:169–177CrossRefGoogle Scholar
  34. Moreno AB, Martinez del Pozo A, Borja M, San Segundo B (2003) Activity of the antifungal protein from Aspergillus giganteus against Botrytis cinerea. Phytopathology 93:1344–1353CrossRefPubMedGoogle Scholar
  35. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucl Acid Res 8:4321–4325CrossRefGoogle Scholar
  36. Nordeen RO, Sinden SL, Jaynes JM, Owens LD (1992) Activity of cecropin SB37 against protoplasts from several plant species and their bacterial pathogens. Plant Sci 82:101–107CrossRefGoogle Scholar
  37. Osusky M, Zhou G, Osuska L, Hancock E, Kay WW, Misra S (2000) Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nature Biotechnol 18:1162–1166CrossRefGoogle Scholar
  38. 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 13:181–190CrossRefPubMedGoogle Scholar
  39. Ou SH (1985) Rice Diseases, second edition, Commonwealth Mycological Institute, Kew, EnglandGoogle Scholar
  40. Owens LD, Heutte TM (1997) A single amino acid substitution in the antimicrobial defense protein cecropin B is associated with diminished degradation by leaf intercellular fluid. Mol Plant-Microbe Interact 10:525–528PubMedCrossRefGoogle Scholar
  41. Pons MJ, Marfà V, Melé E, Messeguer J (2000) Regeneration and genetic transformation of Spanish rice cultivars using mature embryos. Euphytica 114:117–122CrossRefGoogle Scholar
  42. Prodromou Ch, Pearl LH (1992) Recursive PCR: a novel technique for total gene synthesis. Protein Eng 5:827–829PubMedCrossRefGoogle Scholar
  43. Rao AG (1995) Antimicrobial peptides. Mol Plant-Microbe Interact 8:6–13PubMedGoogle Scholar
  44. Reed WA, Elzer PH, Enright FM, Jaynes JM, Morrey JD, White KL (1997) Interleukin 2 promoter/enhancer controlled expression of a synthetic cecropin-class lytic peptide in transgenic mice and subsequent resistance to Brucella abortus. Transgenic Res 6:337–347CrossRefPubMedGoogle Scholar
  45. Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Ann Biochem 166:368–379CrossRefGoogle Scholar
  46. 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 Lett 484:7–11CrossRefPubMedGoogle Scholar
  47. Shai Y (1999) Mechanism of the binding, insertion and desestabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochem Biophys Acta 1462:55–70PubMedCrossRefGoogle Scholar
  48. Silvestro L, Axelsen PH (2000) Membrane-induced folding of cecropin A. Biophys J 79:1465–1477PubMedCrossRefGoogle Scholar
  49. Steiner H, Hultmark D, Engström A, Bennich H, Boman HG (1981) Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292:246–248CrossRefPubMedGoogle Scholar
  50. Steiner H, Andreu D, Merrifield RB (1988) Binding and action of cecropin and cecropin analogs: antibacterial peptides from insects. Biochim Biophys Acta 939:260–266PubMedCrossRefGoogle Scholar
  51. Vila L, Lacadena V, Fontanet P, Martinez del Pozo A, San Segundo B (2001) A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens. Mol Plant-Microbe Interact 14:1327–1331PubMedCrossRefGoogle Scholar
  52. Zhu Q, Maher EA, Masoud S, Dixon RA, Lamb CJ (1994) Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Bio/Technology 12:807–812CrossRefGoogle Scholar
  53. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • María Coca
    • 1
    • 2
  • Gisela Peñas
    • 1
    • 3
  • Jorge Gómez
    • 1
    • 2
  • Sonia Campo
    • 1
    • 2
  • Cristina Bortolotti
    • 1
    • 2
  • Joaquima Messeguer
    • 1
    • 3
  • Blanca San Segundo
    • 1
    • 2
  1. 1.Laboratorio de Genética Molecular VegetalConsorcio CSIC-IRTABarcelonaSpain
  2. 2.Departamento de Genética MolecularInstituto de Biología Molecular de Barcelona, CSICBarcelonaSpain
  3. 3.Departamento de Genética VegetalIRTA Centro de CabrilsBarcelonaSpain

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