Transgenic Research

, Volume 24, Issue 5, pp 885–895 | Cite as

Thanatin confers partial resistance against aflatoxigenic fungi in maize (Zea mays)

  • Max Schubert
  • Marcel Houdelet
  • Karl-Heinz Kogel
  • Rainer Fischer
  • Stefan Schillberg
  • Greta Nölke
Original Paper


Aflatoxin-producing fungi can contaminate plants and plant-derived products with carcinogenic secondary metabolites that present a risk to human and animal health. In this study, we investigated the effect of antimicrobial peptides on the major aflatoxigenic fungi Aspergillus flavus and A. parasiticus. In vitro assays with different chemically-synthesized peptides demonstrated that the broad-spectrum peptide thanatin from the spined soldier bug (Podisus maculiventris) had the greatest potential to eliminate aflatoxigenic fungi. The minimal inhibitory concentrations of thanatin against A. flavus and A. parasiticus were 3.13 and 12.5 µM, respectively. A thanatin cDNA was subsequently cloned in a plant expression vector under the control of the ubiquitin-1 promoter allowing the recombinant peptide to be directed to the apoplast in transgenic maize plants. Successful integration of the thanatin expression cassette was confirmed by PCR and expression was demonstrated by semi-quantitative RT-PCR in transgenic maize kernels. Infection assays with maize kernels from T1 transgenic plants showed up to three-fold greater resistance against Aspergillus spp. infections compared to non-transgenic kernels. We demonstrated for the first time that heterologous expression of the antimicrobial peptide thanatin inhibits the growth of Aspergillus spp. in transgenic maize plants offering a solution to protect crops from aflatoxin-producing fungi and the resulting aflatoxin contamination in the field and under storage conditions.


Aflatoxin Aspergillus flavus Aspergillus parasiticus Corn Transgenic plants 



This work was supported by DAAD Grant No. FKZ 50739422 and FKZ 54364828. The authors acknowledge Katey Warnberg and Dr. Kan Wang (pTF Iowa State University, Ames, USA) for providing the maize transformation vector pTF101.1gw1 and for the initial maize transformation, Prof. Dr. Andreas Vilcinskas (Justus-Liebig University, Giessen, Germany) for providing the thanatin sequence, Elke Stein (Justus-Liebig University, Giessen, Germany) for initial AMP testing, Dr. Thomas Rademacher (Fraunhofer IME, Aachen, Germany) for providing the vectors pTRAkc and pTRAux, and Ibrahim Al-Amedi (Fraunhofer IME) for cultivating the maize plants used in this investigation. We thank Dr. Richard M. Twyman for editorial assistance.


  1. Abbas H, Mascagni H Jr, Bruns H, Shier W (2012) Effect of planting density, irrigation regimes, and maize hybrids with varying ear size on yield, and aflatoxin and fumonisin contamination levels. Am J Plant Sci 3:1341–1354. doi: 10.4236/ajps.2012.310162 CrossRefGoogle Scholar
  2. Allen A, Islamovic E, Kaur J, Gold S, Shah D, Smith TJ (2011) Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut. Plant Biotechnol J 9:857–864. doi: 10.1111/j.1467-7652.2011.00590.x CrossRefPubMedGoogle Scholar
  3. Armstrong CL, Green CE, Phillips RL (1991) Development and availability of germplasm with high Type II culture formation response. Maize Genet Coop Newsl 65:92–93Google Scholar
  4. Attílio LB, Mourão Filho FdAA, Harakava R, da Silva TL, Miyata LY, Stipp LCL, Mendes BMJ (2013) Genetic transformation of sweet oranges with the D4E1 gene driven by the AtPP2 promoter. Pesq Agropec Bras 48:741–747. doi: 10.1590/S0100-204X2013000700006 CrossRefGoogle Scholar
  5. Azziz-Baumgartner E et al (2005) Case–control study of an acute aflatoxicosis outbreak, Kenya, 2004. Environ Health Perspect 113:1779–1783PubMedCentralCrossRefPubMedGoogle Scholar
  6. Blount WP (1961) Turkey “X” disease. Turkeys 52:55–58Google Scholar
  7. Brown RL, Cleveland TE, Payne GA, Woloshuk CP, Campbell KW, White DG (1995) Determination of resistance to aflatoxin production in maize kernels and detection of fungal colonization using an Aspergillus flavus transformant expressing Escherichia coli glucuronidase. Phytopathology 85:983–989. doi: 10.1094/Phyto-85-98 CrossRefGoogle Scholar
  8. Brown RL, Chen ZY, Cleveland TE, Menkir A, Fakhoury A (2009) Identification of maize breeding markers through investigations of proteins associated with aflatoxin-resistance. In: Mycotoxin prevention and control in agriculture, vol 1031. American Chemical Society, pp 157–165. doi: 10.1021/bk-2009-1031.ch011
  9. Brown RL, Menkir A, Chen ZY, Bhatnagar D, Yu J, Yao H, Cleveland TE (2013) Breeding aflatoxin-resistant maize lines using recent advances in technologies—a review. Food Addit Contam 30:1382–1391. doi: 10.1080/19440049.2013.812808 CrossRefGoogle Scholar
  10. Bulet P, Hetru C, Dimarcq JL, Hoffmann D (1999) Antimicrobial peptides in insects; structure and function. Dev Comp Immunol 23:329–344CrossRefPubMedGoogle Scholar
  11. Campbell KW, Hamblin AM, White DG (1997) Inheritance of resistance to aflatoxin production in the cross between corn inbreds B73 and LB31. Phytopathology 87:1144–1147. doi: 10.1094/PHYTO.1997.87.11.1144 CrossRefPubMedGoogle Scholar
  12. 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 154:171–181CrossRefPubMedGoogle Scholar
  13. Cary JW, Rajasekaran K, Brown RL, Luo M, Chen ZY, Bhatnagar D (2011) Developing resistance to aflatoxin in maize and cottonseed. Toxins 3:678–696. doi: 10.3390/toxins3060678 PubMedCentralCrossRefPubMedGoogle Scholar
  14. Chakrabarti A, Ganapathi TR, Mukherjee PK, Bapat VA (2003) MSI-99, a magainin analogue, imparts enhanced disease resistance in transgenic tobacco and banana. Planta 216:587–596. doi: 10.1007/s00425-002-0918-y PubMedGoogle Scholar
  15. Chikwamba RK, Scott MP, Mejia LB, Mason HS, Wang K (2003) Localization of a bacterial protein in starch granules of transgenic maize kernels. Proc Natl Acad Sci U S A 100:11127–11132. doi: 10.1073/pnas.1836901100 PubMedCentralCrossRefPubMedGoogle Scholar
  16. Cleveland TE, Dowd PF, Desjardins AE, Bhatnagar D, Cotty PJ (2003) United States Department of Agriculture—Agricultural Research Service research on pre-harvest prevention of mycotoxins and mycotoxigenic fungi in US crops. Pest Manag Sci 59:629–642CrossRefPubMedGoogle Scholar
  17. De Lucca AJ et al (1998) Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1. Can J Microbiol 44:514–520CrossRefPubMedGoogle Scholar
  18. 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 127:852–862. doi: 10.1104/pp.010233 PubMedCentralCrossRefPubMedGoogle Scholar
  19. Diener UL, Cole RJ, Sanders TH, Payne GA, Lee LS, Klich MA (1987) Epidemiology of aflatoxin formation by Aspergillus flavus. Annu Rev Phytopathol 25:249–270CrossRefGoogle Scholar
  20. Dorner JW (2008) Management and prevention of mycotoxins in peanuts. Food Addit Contam 25:203–208. doi: 10.1080/02652030701658357 CrossRefGoogle Scholar
  21. Dorner JW, Cole RJ, Wicklow DT (1999) Aflatoxin reduction in corn through field application of competitive fungi. J Food Prot 62:650–656PubMedGoogle Scholar
  22. Drakakaki G et al (2005) Endosperm-specific co-expression of recombinant soybean ferritin and aspergillus phytase in maize results in significant increases in the levels of bioavailable iron. Plant Mol Biol 59:869–880. doi: 10.1007/s11103-005-1537-3 CrossRefPubMedGoogle Scholar
  23. Ehrlich KC (2014) Non-aflatoxigenic to prevent aflatoxin contamination in crops: advantages and limitations. Front Microbiol 5:50. doi: 10.3389/fmicb.2014.00050 PubMedCentralPubMedGoogle Scholar
  24. Fehlbaum P, Bulet P, Michaut L, Lagueux M, Broekaert WF, Hetru C, Hoffmann JA (1994) Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. J Biol Chem 269:33159–33163PubMedGoogle Scholar
  25. Fehlbaum P et al (1996) Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides. Proc Natl Acad Sci USA 93:1221–1225PubMedCentralCrossRefPubMedGoogle Scholar
  26. Fountain JC, Scully BT, Ni X, Kemerait RC, Lee RD, Chen ZY, Guo B (2014) Environmental influences on maize- interactions and aflatoxin production. Front Microbiol 5:40. doi: 10.3389/fmicb.2014.00040 PubMedCentralCrossRefPubMedGoogle Scholar
  27. Frame BR et al (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129:13–22. doi: 10.1104/pp.000653 PubMedCentralCrossRefPubMedGoogle Scholar
  28. Ganapathi TR, Ghosh SB, Laxmi NHS, Bapat VA (2007) Expression of an antimicrobial peptide (MSI-99) confers enhanced resistance to Aspergillus niger in transgenic potato. Indian J Biotechnol 6:63–67Google Scholar
  29. Gong Y, Egal S, Hounsa A, Turner P, Hall A, Cardwell K, Wild C (2003) Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning. Int J Epidemiol 32:556–562. doi: 10.1093/ije/dyg109 CrossRefPubMedGoogle Scholar
  30. Heathcote JG, Hibbert JR (1978) Aflatoxins: chemical and biological aspects. Elsevier Scientific Publishing Company, AmsterdamGoogle Scholar
  31. Hedayati MT, Pasqualotto AC, Warn PA, Bowyer P, Denning DW (2007) Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 153:1677–1692. doi: 10.1099/mic.0.2007/007641-0 CrossRefPubMedGoogle Scholar
  32. Hou Z et al (2013) R-thanatin inhibits growth and biofilm formation of methicillin-resistant Staphylococcus epidermidis in vivo and in vitro. Antimicrob Agents Chemother 57:5045–5052. doi: 10.1128/aac.00504-13 PubMedCentralCrossRefPubMedGoogle Scholar
  33. Imamura T et al (2010) Acquired resistance to the rice blast in transgenic rice accumulating the antimicrobial peptide thanatin. Transgenic Res 19:415–424. doi: 10.1007/s11248-009-9320-x CrossRefPubMedGoogle Scholar
  34. Johnson ET, Berhow MA, Dowd PF (2007) Expression of a maize Myb transcription factor driven by a putative silk-specific promoter significantly enhances resistance to Helicoverpa zea in transgenic maize. J Agric Food Chem 55:2998–3003. doi: 10.1021/jf0633600 CrossRefPubMedGoogle Scholar
  35. Jung YJ, Kang KK (2014) Application of antimicrobial peptides for disease control in plants. Plant Breed Biotechnol 2:1–13CrossRefGoogle Scholar
  36. Koch A, Khalifa W, Langen G, Vilcinskas A, Kogel K-H, Imani J (2012) The antimicrobial peptide thanatin reduces fungal infections in Arabidopsis. J Phytopathol 160:606–610. doi: 10.1111/j.1439-0434.2012.01946.x CrossRefGoogle Scholar
  37. Lawyer C, Pai S, Watabe M, Bakir H, Eagleton L, Watabe K (1996) Effects of synthetic form of tracheal antimicrobial peptide on respiratory pathogens. J Antimicrob Chemother 37:599–604CrossRefPubMedGoogle Scholar
  38. 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 233:694–700CrossRefPubMedGoogle Scholar
  39. Liu Y, Wu F (2010) Global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment. Environ Health Perspect 118:818–824. doi: 10.1289/ehp.0901388 PubMedCentralCrossRefPubMedGoogle Scholar
  40. Makovitzki A, Viterbo A, Brotman Y, Chet I, Shai Y (2007) Inhibition of fungal and bacterial plant pathogens in vitro and in planta with ultrashort cationic lipopeptides. Appl Environ Microbiol 73:6629–6636. doi: 10.1128/aem.01334-07 PubMedCentralCrossRefPubMedGoogle Scholar
  41. Mentag R, Luckevich M, Morency MJ, Seguin A (2003) Bacterial disease resistance of transgenic hybrid poplar expressing the synthetic antimicrobial peptide D4E1. Tree Physiol 23:405–411CrossRefPubMedGoogle Scholar
  42. Mideros SX, Windham GL, Williams WP, Nelson RJ (2009) Aspergillus flavus biomass in maize estimated by quantitative real-time polymerase chain reaction is strongly correlated with aflatoxin concentration. Plant Dis 93:1163–1170. doi: 10.1094/pdis-93-11-1163 CrossRefGoogle Scholar
  43. Mireia B et al (2014) Production of cecropin A antimicrobial peptide in rice seed endosperm. BMC Plant Biol 14:102CrossRefGoogle Scholar
  44. Mohammadi H (2011) A review of aflatoxin M1, milk, and milk products. In: G-G (ed) Aflatoxins—biochemistry and molecular biology. InTech. doi: 10.5772/24353
  45. Montesinos E (2007) Antimicrobial peptides and plant disease control. FEMS Microbiol Lett 270:1–11. doi: 10.1111/j.1574-6968.2007.00683.x CrossRefPubMedGoogle Scholar
  46. Newman SJ, Smith JR, Stenske KA, Newman LB, Dunlap JR, Imerman PM, Kirk CA (2007) Aflatoxicosis in nine dogs after exposure to contaminated commercial dog food. J Vet Diagn Invest 19:168–175CrossRefPubMedGoogle Scholar
  47. Ngindu A et al (1982) Outbreak of acute hepatitis caused by aflatoxin poisoning in Kenya. The Lancet 319:1346–1348CrossRefGoogle Scholar
  48. Paz M, Shou H, Guo Z, Zhang Z, Banerjee A, Wang K (2004) Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant. Euphytica 136:167–179. doi: 10.1023/B:EUPH.0000030669.75809.dc CrossRefGoogle Scholar
  49. Probst C, Njapau H, Cotty PJ (2007) Outbreak of an acute aflatoxicosis in Kenya in 2004: identification of the causal agent. Appl Environ Microbiol 73:2762–2764. doi: 10.1128/aem.02370-06 PubMedCentralCrossRefPubMedGoogle Scholar
  50. Rahnamaeian M et al (2009) Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens. J Exp Bot 60:4105–4114. doi: 10.1093/jxb/erp240 PubMedCentralCrossRefPubMedGoogle Scholar
  51. 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 3:545–554. doi: 10.1111/j.1467-7652.2005.00145.x CrossRefPubMedGoogle Scholar
  52. Rasche S, Martin A, Holzem A, Fischer R, Schinkel H, Schillberg S (2011) One-step protein purification: use of a novel epitope tag for highly efficient detection and purification of recombinant proteins. Biotechnol J 5:1–6. doi: 10.2174/1874070701105010001 Google Scholar
  53. Robens J, Cardwell KF (2003) The costs of mycotoxin management to the USA: management of aflatoxins in the United States. Toxin Rev 22:139–152CrossRefGoogle Scholar
  54. Robert É, Fillion M, Otis F, Voyer N, Auger M (2014) Understanding how the antimicrobial peptide thanatin interacts with the lipid bilayer of cell walls using model membranes. Biophys J 106:85a. doi: 10.1016/j.bpj.2013.11.546 CrossRefGoogle Scholar
  55. Scheidegger KA, Payne GA (2003) Unlocking the secrets behind secondary metabolism: a review of Aspergillus flavus from pathogenicity to functional genomics. Toxin Rev 22:423–459. doi: 10.1081/txr-120024100 CrossRefGoogle Scholar
  56. Schmidt C (2013) Breaking the mold: new strategies for fighting aflatoxins. Environ Health Perspect. doi: 10.1289/ehp.121-A270 Google Scholar
  57. Schubert M, Agdour S, Fischer R, Olbrich Y, Schinkel H, Schillberg S (2010) A monoclonal antibody that specifically binds chitosan in vitro and in situ on fungal cell walls. J Microbiol Biotechnol 20:1179–1184CrossRefPubMedGoogle Scholar
  58. Schuhmann B, Seitz V, Vilcinskas A, Podsiadlowski L (2003) Cloning and expression of gallerimycin, an antifungal peptide expressed in immune response of greater wax moth larvae, Galleria mellonella. Arch Insect Biochem 53:125–133. doi: 10.1002/arch.10091 CrossRefGoogle Scholar
  59. Schumann U, Smith N, Kazan K, Ayliffe M, Wang M-B (2013) Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum. Silence 4:3PubMedCentralCrossRefPubMedGoogle Scholar
  60. Shahryari F, Safarnejad MR, Shams-Bakhsh M, Schillberg S, Nölke G (2013) Generation and expression in plants of a single-chain variable fragment antibody against the immunodominant membrane protein of Candidatus phytoplasma aurantifolia. J Microbiol Biotechnol 23:1047–1054CrossRefPubMedGoogle Scholar
  61. Vaquero C et al (1999) Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves. Proc Natl Acad Sci USA 96:11128–11133PubMedCentralCrossRefPubMedGoogle Scholar
  62. Villers P (2014) Aflatoxins and safe storage. Front Microbiol. doi: 10.3389/fmicb.2014.00158 PubMedCentralPubMedGoogle Scholar
  63. Warburton ML, Williams WP (2014) Aflatoxin resistance in maize: what have we learned lately? Adv Bot 2014:10. doi: 10.1155/2014/352831 Google Scholar
  64. Wild CP (2007) Aflatoxin exposure in developing countries: the critical interface of agriculture and health. Food Nutr Bull 28:372S–380SGoogle Scholar
  65. Williams JH, Phillips TD, Jolly PE, Stiles JK, Jolly CM, Aggarwal D (2004) Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am J Clin Nutr 80:1106–1122PubMedGoogle Scholar
  66. Williams W, Krakowsky MD, Windham GL, Balint-Kurti P, Hawkins LK, Henry W (2008) Identifiying maize germplasm with resistance to aflatoxin accumulation. Toxin Rev 27:319–345. doi: 10.1080/15569540802399838 CrossRefGoogle Scholar
  67. Williams WP, Ozkan S, Ankala A, Windham GL (2011) Ear rot, aflatoxin accumulation, and fungal biomass in maize after inoculation with Aspergillus flavus. Field Crops Res 120:196–200. doi: 10.1016/j.fcr.2010.10.002 CrossRefGoogle Scholar
  68. Williams WP, Krakowsky MD, Scully BT, Brown RL, Menkir A, Warburton ML, Windham GL (2014) Identifying and developing maize germplasm with resistance to accumulation of aflatoxins. World Mycotoxin J. doi: 10.3920/WMJ2014.1751 Google Scholar
  69. Wu G et al (2010) Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin. Biochem Biophys Res Commun 395:31–35. doi: 10.1016/j.bbrc.2010.03.107 CrossRefPubMedGoogle Scholar
  70. Wu G, Li X, Fan X, Wu H, Wang S, Shen Z, Xi T (2011) The activity of antimicrobial peptide S-thanatin is independent on multidrug-resistant spectrum of bacteria. Peptides 32:1139–1145. doi: 10.1016/j.peptides.2011.03.019 CrossRefPubMedGoogle Scholar
  71. Wu G, Deng X, Wu P, Shen Z, Xu H (2012) Subacute toxicity of antimicrobial peptide S-thanatin in ICR mice. Peptides 36:109–113. doi: 10.1016/j.peptides.2012.04.005 CrossRefPubMedGoogle Scholar
  72. Wu T, Tang D, Chen W, Huang H, Wang R, Chen Y (2013) Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria. Gene 527:235–242. doi: 10.1016/j.gene.2013.06.037 CrossRefPubMedGoogle Scholar
  73. Zasloff M (1987) Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci USA 84:5449–5453PubMedCentralCrossRefPubMedGoogle Scholar
  74. Zeitler B, Herrera Diaz A, Dangel A, Thellmann M, Meyer H, Sattler M, Lindermayr C (2013) De-novo design of antimicrobial peptides for plant protection. PLoS One 8:e71687. doi: 10.1371/journal.pone.0071687 PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Max Schubert
    • 1
  • Marcel Houdelet
    • 1
  • Karl-Heinz Kogel
    • 2
  • Rainer Fischer
    • 1
    • 3
  • Stefan Schillberg
    • 1
    • 2
  • Greta Nölke
    • 1
  1. 1.Fraunhofer Institute for Molecular Biology and Applied Ecology IMEAachenGermany
  2. 2.Institute of Phytopathology and Applied ZoologyJustus-Liebig UniversityGiessenGermany
  3. 3.Institute for Molecular BiotechnologyRWTH Aachen UniversityAachenGermany

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