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Arabidopsis thaliana plants expressing human beta-defensin-2 are more resistant to fungal attack: functional homology between plant and human defensins

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Abstract

Human beta-defensin-2 (hBD-2) is a small antimicrobial peptide with potent activity against different Gram-negative bacteria and fungal/yeast species. Since human beta-defensins and plant defensins share structural homology, we set out to analyse whether there also exists a functional homology between these defensins of different eukaryotic kingdoms. To this end, we constructed a plant transformation vector harbouring the hBD-2 coding sequence, which we transformed to Arabidopsis thaliana plants, giving rise to A. thaliana plants indeed expressing hBD-2. Furthermore, we could demonstrate that this heterologously produced hBD-2 possesses antifungal activity in vitro. Finally, we could show that hBD-2 expressing A. thaliana plants are more resistant against the broad-spectrum fungal pathogen Botrytis cinerea as compared to untransformed A. thaliana plants, and that this resistance is correlated with the level of active hBD-2 produced in these transgenic plants. Hence, we demonstrated a functional homology, next to the already known structural homology, between defensins originating from different eukaryotic kingdoms. To our knowledge, this is the first time that this is specifically demonstrated for plant and mammalian defensins.

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Abbreviations

AMP:

Antimicrobial protein

CRP:

Cross-reactive protein

Dm-AMP:

Dahlia merckii antimicrobial protein

hBD-2:

Human beta-defensin-2

IC50 :

Concentration causing 50% inhibition of fungal growth

MAR:

Matrix attachment region

PTGS:

Post-transcriptional gene silencing

RsAFP:

Raphanus sativus antifungal protein

References

  • Aerts AM, François IEJA, Meert EMK, Li Q, Cammue BPA, Thevissen K (2006) The antifungal activity of RsAFP2, a plant defensin from Raphanus sativus, involves the induction of reactive oxygen species in Candida albicans. FEBS Lett 580:1903–1907

    Article  PubMed  CAS  Google Scholar 

  • Banzet N, Latorse MP, Bulet P, François E, Derpierre C, Dubald (2002) Expression of insect cystein-rich antifungal peptides in transgenic tobacco enhances resistance to a fungal disease. Plant Sci 162:995–1006

    Article  CAS  Google Scholar 

  • Boman HG (1995) Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 13:61–92

    Article  PubMed  CAS  Google Scholar 

  • Bode M, Stobe P, Thiede B, Schuphan I, Schmidt B (2004) Biotransformation of atrazine in transgenic tobacco cell culture expressing human P450. Pest Manag Sci 60:49–58

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Broekaert WF, Terras FR, Cammue BPA, Vanderleyden J (1990) An automated quantitative assay for fungal growth. FEMS Microbiol Lett 69:55–60

    Article  CAS  Google Scholar 

  • Brouwer M, Lievens B, Van Hemelrijck W, Van den Ackerveken G, Cammue BP, Thomma BP (2003) Quantification of disease progression of several microbial pathogens on Arabidopsis thaliana using real-time fluorescence PCR. FEMS Microbiol Lett 228:241–248

    Article  PubMed  CAS  Google Scholar 

  • Butaye KM, Goderis IJ, Wouters PF, Pues JM, Delaure SL, Broekaert WF, Depicker A, Cammue BP, De Bolle MF (2004) Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions. Plant J 39:440–449

    Article  PubMed  CAS  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  PubMed  CAS  Google Scholar 

  • Colilla FJ, Rocher A, Mendez E (1990) Gamma-Purothionins: amino acid sequence of two polypeptides of a new family of thionins from wheat endosperm. FEBS Lett 270:191–194

    Article  PubMed  CAS  Google Scholar 

  • De Bolle MF, Butaye KM, Coucke WJ, Goderis IJ, Wouters PF, van Boxel N, Broekaert WF, Cammue BP (2003) Analysis of the influence of promoter elements and a matrix attachment region on the inter-individual variation of transgene expression in populations of Arabidopsis thaliana. Plant Sci 165:169–179

    Article  Google Scholar 

  • Fant F, Vranken W, Broekaert W, Borremans F (1998) Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1HNMR. J Mol Biol 279:257–270

    Article  PubMed  CAS  Google Scholar 

  • Francois IE, De Bolle MF, Dwyer G, Goderis IJ, Woutors PF, Verhaert PD, Proost P, Schaaper WM, Cammue BP, Broekaert WF (2002a) Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins. Plant Physiol 128:1346–1358

    Article  CAS  Google Scholar 

  • Francois IEJA, Dwyer GI, De Bolle MFC, Goderis IJWM, Van Hemelrijck W, Proost P, Wouters P, Broekaert WF, Cammue BPA (2002b) Processing in transgenic Arabidopsis thaliana plants of polyproteins with linker peptide variants derived from the impatiens balsamina antimicrobial polyprotein precursor. Plant Physiol Biochem 40:871–879

    Article  CAS  Google Scholar 

  • Francois IE, Van Hemelrijck W, Aerts AM, Wouters PF, Proost P, Broekaert WF, Cammue BP (2004) Processing in Arabidopsis thaliana of a heterologous polyprotein resulting in defferential targeting of the individual plant defensins. Plant Sci 166:113–121

    Article  CAS  Google Scholar 

  • Gao AG, Hakimi SM, Mittanck CA, Wu Y, Woerner BM, Stark DM, Shah DM, Liang J, Rommens CM (2000) Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat Biotechnol 18:1307–1310

    Article  PubMed  CAS  Google Scholar 

  • Geyer BC, Muralidharan M, Cherni I, Doran J, Fletcher SP, Evron T, Soreq H, Mor TS (2005) Purification of transgenic plant-derived recombinant human acetylcholinesterase-R. Chem Biol Interact 157–158:331–334

    Article  PubMed  Google Scholar 

  • Goderis IJ, De Bolle MF, François IE, Wouters PF, Broekaert WF, Cammue BP (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol 50:17–27

    Article  PubMed  CAS  Google Scholar 

  • Harder J, Bartels J, Christophers E, Schröder JM (1997) A peptide antibiotic from human skin. Nature 387:861

    Article  PubMed  CAS  Google Scholar 

  • Hondred D, Walker JM, Mathews DE, Vierstra RD (1999) Use of ubiquitin fusions to augment protein expression in transgenic plants. Plant Physiol 119:713–724

    Article  PubMed  CAS  Google Scholar 

  • Hoover DM, Rajashankar KR, Blumenthal R, Puri A, Oppenheim JJ, Chertov O, Lubkowwski J (2000) The structure of human β-defensin-2 shows evidence of higher order oligomerization. J Biol Chem 275:32911–32918

    Article  PubMed  CAS  Google Scholar 

  • Hoover DM, Chertov O, Lubkowski J (2001) The structure of human b-defensin-1. New insights into structural properties of b-defensins. J Biol Chem 276:39021–39026

    Article  PubMed  CAS  Google Scholar 

  • 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 105:809–814

    Article  PubMed  CAS  Google Scholar 

  • Kwon SY, Jo SH, Lee OS, Choi SM, Kwak SS, Lee HS (2003) Transgenic ginseng cell lines that produce high levels of a human lactoferrin. Planta Med 69:1005–1008

    Article  PubMed  CAS  Google Scholar 

  • Lamberty M, Caille A, Landon C, Tassin-Moindrot S, Hetru C, Bulet P, Vovelle F (2001) Solution structures of the antifungal heliomicin and a selected variant with both antibacterial and antifungal activities. Biochemistry 40:11995–12003

    Article  PubMed  CAS  Google Scholar 

  • 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 387:549–557

    Article  PubMed  CAS  Google Scholar 

  • Lay FT, Anderson MA (2005) Defensins—components of the innate immune system in plants. Curr Protein Pept Sci 6:85–101

    Article  PubMed  CAS  Google Scholar 

  • Lehrer RI, Lichtenstein AK, Ganz T (1993) Defensins: antimicrobial and cytotoxic peptides of mammalian cells. Annu Rev Immunol 11:105–128

    Article  PubMed  CAS  Google Scholar 

  • Li X, Gasic K, Cammue B, Broekaert W, Korban SS (2003) Transgenic rose lines harboring an antimicrobial protein gene, Ace-AMP1, demonstrate enhanced resistance to powdery mildew (Sphaerotheca pannosa). Planta 218:226–232

    Article  PubMed  CAS  Google Scholar 

  • Matsunaga T, Rahman A (1998) What brought the adaptive immune system to vertebrates?—the jaw hypothesis and the seahorse. Immunol Rev 166:177–186

    Article  PubMed  CAS  Google Scholar 

  • Mendez E, Moreno A, Colilla F, Pelaez F, Limas GG, Mendez R, Soriano F, Salinas M, de Haro C (1990) Primary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, gamma-hordothionin, from barley endosperm. Eur J Biochem 194:533–539

    Article  PubMed  CAS  Google Scholar 

  • Mourrain P, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, Jouette D, Lacombe AM, Nikic S, Picault N, Remoue K, Sanial M, Vo TA, Vaucheret H (2000) Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101:533–542

    Article  PubMed  CAS  Google Scholar 

  • Mygind PH, Fischer RL, Schnorr KM, Hansen MT, Sonksen CP, Ludvigsen S, Raventos D, Buskov S, Christensen B, De Maria L, Taboureau O, Yaver D, Elvig-Jorgensen SG, Sorensen MV, Christensen BE, Kjaerulff S, Frimodt-Moller N, Lehrer RI, Zasloff M, Kristensen HH (2005) Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature 437:975–980

    Article  PubMed  CAS  Google Scholar 

  • Okamoto M, Mitsuhara I, Ohshima M, Natori S, Ohashi Y (1998) Enhanced expression of an antimicrobial peptide sarcotoxin IA by GUS fusion in transgenic tobacco plants. Plant Cell Physiol 39:57–63

    PubMed  CAS  Google Scholar 

  • Osborn RW, De Samblanx GW, Thevissen K, Goderis I, Torrekens S, Van Leuven F, Attenborough S, Rees SB, Broekaert WF (1995) Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Lett 368:257–262

    Article  PubMed  CAS  Google Scholar 

  • Panahi M, Alli Z, Cheng X, Belbaraka L, Belgoudi J, Sardana R, Phipps J, Altosaar I (2004) Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants. Transgenic Res 13:245–259

    Article  PubMed  CAS  Google Scholar 

  • Park HC, 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 50:59–69

    Article  PubMed  CAS  Google Scholar 

  • Penninckx IA, Eggermont K, Terras FR, Thomma BP, De Samblanx GW, Buchala A, Metraux JP, Manners JM, Broekaert WF (1996) Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. Plant Cell 8:2309–2323

    Article  PubMed  CAS  Google Scholar 

  • Sawai MV, Jia HP, Liu L, Aseyev V, Wiencek JM, McCray PB Jr, Ganz T, Kearney WR, Tack BF (2001) The NMR structure of human b-defensin-2 reveals a novel alpha-helical segment. Biochemistry 40:3810–3816

    Article  PubMed  CAS  Google Scholar 

  • Selsted ME, Szklarek D, Lehrer RI (1984) Purification and antibacterial activity of antimicrobial peptides of rabbit granulocytes. Infect Immun 45:150–154

    PubMed  CAS  Google Scholar 

  • Selsted ME, Tang YQ, Morris WL, McGuire PA, Novotny MJ, Smith W, Henschen AH, Cullor JS (1993) Purification, primary structures, and antibacterial activities of b-defensins, a new family of antimicrobial peptides from bovine neutrophils. J Biol Chem 268:6641–6648

    PubMed  CAS  Google Scholar 

  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    Article  PubMed  CAS  Google Scholar 

  • Takaichi M, Oeda K (2000) Transgenic carrots with enhanced resistance against two major pathogens, Erysiphe heraclei and Alternaria dauci. Plant Sci 153:135–144

    Article  PubMed  CAS  Google Scholar 

  • Tang YQ, Yuan J, Osapay G, Osapay K, Tran D, Miller CJ, Ouellette AJ, Selsted ME (1999) A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins. Science 286:498–502

    Article  PubMed  CAS  Google Scholar 

  • Terras FR, Schoofs HM, De Bolle MF, Van Leuven F, Rees SB, Vanderleyden J, Cammue BP, Broekaert WF (1992) Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 267:15301–15309

    PubMed  CAS  Google Scholar 

  • Terras FR, Eggermont K, Kovaleva V, Raikhel NV, Osborn RW, Kester A, Rees SB, Torrekens S, Van Leuven F, Vanderleyden J (1995) Small cysteine-rich antifungal proteins from radish: their role in host defense. Plant Cell 7:573–588

    Article  PubMed  CAS  Google Scholar 

  • Thevissen K, Ghazi A, De Samblanx GW, Brownlee C, Osborn RW, Broekaert WF (1996) Fungal membrane responses induced by plant defensins and thionins. J Biol Chem 271:15018–15025

    Article  PubMed  CAS  Google Scholar 

  • Thevissen K, Terras FR, Broekaert WF (1999) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl Environ Microbiol 65:5451–5458

    PubMed  CAS  Google Scholar 

  • Thevissen K, Osborn RW, Acland DP, Broekaert WF (2000a) Specific binding sites for an antifungal plant defensin from Dahlia (Dahlia merckii) on fungal cells are required for antifungal activity. Mol Plant Microbe Interact 13:54–61

    Article  CAS  Google Scholar 

  • Thevissen K, Cammue BP, Lemaire K, Winderickx J, Dickson RC, Lester RL, Ferket KK, Van Even F, Parret AH, Broekaert WF (2000b) A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of Saccharomyces cerevisiae to an antifungal plant defensin from dahlia (Dahlia merckii). Proc Natl Acad Sci USA 97:9531–9536

    Article  CAS  Google Scholar 

  • Thevissen K, Ferket K, François IEJA, Cammue BPA (2003) Interactions of antifungal plant defensins with fungal membrane components. Peptides 24:1705–1712

    Article  PubMed  CAS  Google Scholar 

  • Thevissen K, Warnecke D, Francois IEJA, Leipelt M, Heinz E, Ott C, Thomma BPHJ, Ferket KKA, Cammue BPA (2004) Fungal glucosylceramides constitute the binding site for plant and insect defensins. J Biol Chem 279:3900–3905

    Article  PubMed  CAS  Google Scholar 

  • Thomma BPHJ, Eggermont K, Penninckx IAMA, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF (1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc Natl Acad Sci USA 95:15107–15111

    Article  PubMed  CAS  Google Scholar 

  • Thomma BPHJ, Eggermont K, Tierens KF, Broekaert WF (1999) Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea. Plant Physiol 121:1093–1102

    Article  PubMed  CAS  Google Scholar 

  • Thomma BPHJ, Cammue BPA, Thevissen K (2002) Plant defensins. Planta 216:193–202

    Article  PubMed  CAS  Google Scholar 

  • Tierens FMJ, Thomma BPHJ, Brouwer M, Schmidt J, Kirstner K, Porzel A, Mauch Mani B, Cammue BPA, Broekaert WF (2001) Study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of Arabidopsis thaliana to microbial pathogens. Plant Physiol 125:1688–1699

    Article  PubMed  CAS  Google Scholar 

  • Trabi M, Schirra HJ, Craik DJ (2001) Three-dimensional structure of RTD-1, a cyclic antimicrobial defensin from Rhesus macaque leukocytes. Biochemistry 40:4211–4221

    Article  PubMed  CAS  Google Scholar 

  • Wang YP, 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 12:410–418

    Article  CAS  Google Scholar 

  • Yang D, Biragyn A, Hoover DM, Lubkowski J, Oppenheim JJ (2004) Multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neurotoxin in host defense. Annu Rev Immunol 22:181–215

    Article  PubMed  Google Scholar 

  • Zambryski P, Joos H, Genetello C, Leemans J, Van Montagu M, Schell J (1983) Ti plasmid vectors for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J 2:2143–2150

    PubMed  CAS  Google Scholar 

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Acknowledgments

The authors wish to thank Dr. Hervé Vaucheret (INRA, Versailles, France) for providing seeds of the sgs2-mutant and Prof. Cassiman (K. U. Leuven, Belgium) for the kind gift of the pUC18 vector containing the hBD-2 coding sequence. This work was supported by grants from the Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen to B. C (G028804N and G.0405.05). A. A. acknowledges the receipt of a pre-doctoral grant of the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (IWT-Vlaanderen). K. T. acknowledges the receipt of a post-doctoral fellowship of the Industrial Research Fund (IOF) of the Katholieke Universiteit Leuven. I.F. acknowledges the receipt of a post-doctoral grant of the IWT-Vlaanderen. The authors wish to thank Dr. Stijn Delauré for his assistance with the infection assays and statistical analysis.

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  1. Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001, Heverlee, Belgium

    An M. Aerts, Karin Thevissen, Sara M. Bresseleers, Jan Sels, Piet Wouters, Bruno P. A. Cammue & Isabelle E. J. A. François

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  1. An M. Aerts
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  2. Karin Thevissen
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  3. Sara M. Bresseleers
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  4. Jan Sels
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  5. Piet Wouters
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  6. Bruno P. A. Cammue
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  7. Isabelle E. J. A. François
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Correspondence to Bruno P. A. Cammue.

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Communicated by E. Prinsen.

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Aerts, A.M., Thevissen, K., Bresseleers, S.M. et al. 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, 1391–1398 (2007). https://doi.org/10.1007/s00299-007-0329-4

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