Advertisement

Plant Cell Reports

, Volume 22, Issue 6, pp 388–396 | Cite as

Expression of a magainin-type antimicrobial peptide gene (MSI-99) in tomato enhances resistance to bacterial speck disease

Genetic Transformation and Hybridization

Abstract

MSI-99 is a synthetic analog of magainin II (MII), a small cationic peptide highly inhibitory to a wide spectrum of microbial organisms. Tomato plants were transformed to express a gene encoding the MSI-99 peptide and tested for possible enhancement of resistance to important pathogens of this crop. Thirty-six tomato transformants carrying an MSI-99 expression vector designed to target the peptide into extracellular spaces were obtained by Agrobacterium tumefaciens-mediated transformation. Expression of MSI-99 caused no obvious cytotoxic effects in these plants. In the tests with Pseudomonas syringae pv. tomato (bacterial speck pathogen) at 105 CFU/ml, several MSI-99-expressing lines developed significantly fewer disease symptoms than controls. However, MSI-99-expressing lines were not significantly different from controls in their responses to the fungal pathogen Alternaria solani (early blight) and the oomycete pathogen Phytophthora infestans (late blight). These findings are in accordance with our previous in vitro inhibition tests, which showed that the MSI-99 peptide is more inhibitory against bacteria than against fungi and oomycetes. Additional in vitro inhibition assays showed that MSI-99 loses its antimicrobial activity in the total or extracellular fluids from leaflets of non-transformed tomato plants; however, P. syringae pv. tomato could not multiply in the extracellular fluid from an MSI-99-expressing line. Our results suggest that expression strategies providing continuous high expression of MSI-99 will be necessary to achieve significant enhancement of plant disease resistance.

Keywords

Disease resistance Cationic peptides Extracellular targeting Lycopersicon esculentum 

Abbreviations

AMP

Antimicrobial peptide

CFU

Colony forming unit

ECF

Extracellular fluid

gus

β-glucuronidase gene

nptII

Neomycin phosphotransferase II

SP

Signal peptide

TF

Total fluid

Notes

Acknowledgements

We gratefully acknowledge the Turkish Ministry of National Education and the Department of Plant Breeding, Cornell University for providing financial support to Ali Alan. The research was also supported by the Cornell University Agricultural Experiment Station Project 149-422 from CSREES/USDA and by the Cornell University Center for Biotechnology. We thank Sanford Scientific for providing the pSAN147 construct. Special thanks to Chris Smart and Hillary Mayton for providing P. infestans cultures and for their assistance in the interpretation of the results obtained from assays with this organism. We also thank Kerrie Seberg for her help with in vitro bioassays.

References

  1. Alan AR (2001) Utilization of lytic peptide and avirulence genes for developing plants with broad spectrum disease resistance. PhD thesis, Cornell University, Ithaca, N.Y.Google Scholar
  2. Alan AR, Earle ED (1999) Enhancing resistance with a lytic peptide gene to plant pathogens by transformation. Phytopathology 89:S2Google Scholar
  3. Alan AR, Earle ED (2002) Sensitivity of bacterial and fungal plant pathogens to the lytic peptides, MSI-99, Magainin II, and Cecropin B. Mol Plant Microbe Interact 15:701–708PubMedGoogle Scholar
  4. 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 72:437–445Google Scholar
  5. Allefs SJHM, De Jong ER, Florack DEA, Hoogendoorn C, Stiekema WJ (1996) Erwinia soft rot resistance of potato cultivars expressing antimicrobial peptide tachyplesin I. Mol Breed 2:97–105Google Scholar
  6. Andreu D, Ubach J, Boman A, Wahlin B, Wade D, Merrifield RB, Boman HG (1992) Shortened cecropin A-melittin hybrids: significant size reduction retains potent antibiotic activity. FEBS Lett 296:190–194CrossRefPubMedGoogle Scholar
  7. Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982) Nucleotide sequence and exact location of the neomycin phosphotransferase gene from transposon Tn5. Gene 19:327–336PubMedGoogle Scholar
  8. Bernatzky R, Tanksley SD (1986) The detection of single or low copy sequences in tomato on Southern blots. Plant Mol Biol Rep 4:37–41Google Scholar
  9. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  10. Broekaert WF, Cammue BPA, De Bolle MFC, Thevissen K, De Samblanx GW, Osborn, RW (1997) Antimicrobial peptides from plants. Crit Rev Plant Sci 16:297–323Google 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 154:171–181CrossRefPubMedGoogle Scholar
  12. Cavallarin L, Andeu D, San Segundo B (1998) Cecropin A-derived peptides are potent inhibitors of fungal plant pathogens. Mol Plant Microbe Interact 11:218–227PubMedGoogle Scholar
  13. 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
  14. Chen HC, Brown J H, Morell JL, Huang CM (1988) Synthetic magainin analogues with improved antimicrobial activity. FEBS Lett 236:462–466CrossRefPubMedGoogle Scholar
  15. Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991–1995PubMedGoogle 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 127:852–862CrossRefPubMedGoogle Scholar
  17. Everett NP (1994) Design of antifungal peptides for agricultural applications. In: Hedin PA, Menn JJ, Hollingworth RM (eds) Natural and engineered pest management agents. ACS Symp Ser 551, Am Chem Soc, Washington, D.C., pp 278–291Google 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 4:132–141PubMedGoogle Scholar
  19. Frary A, Earle ED (1996) An examination of factors affecting the efficiency of Agrobacterium-mediated transformation of tomato. Plant Cell Rep 16:235–240Google Scholar
  20. Hamilton CM, Frary A, Lewis C, Tanksley SD (1995) Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc Natl Acad Sci USA 93:9975–9979CrossRefGoogle Scholar
  21. Hammond-Kosack KE (1992) Preparation and analysis of intercellular fluid. In: Gurr SJ, McPherson MJ, Bowles DJ (eds) Molecular plant pathology. A practical approach, vol 2. Oxford University Press, New York, pp 15–21Google Scholar
  22. 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 13:295–299Google Scholar
  23. Huang Y, Nordeen RO, Di M, Owens LD, McBeath JH (1997) Expression of an engineered cecropin gene cassette in transgenic tobacco plants confers resistance to Pseudomonas syringae pv. tabaci. Phytopathology 87:494–499Google Scholar
  24. Jaynes JM, Nagpala P, Destefano-Beltran L, Huang JH, Kim JH, Denney 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 89:43–53Google Scholar
  25. Legard DE, Lee TY, Fry WE (1995) Aggressive specialization in Phytophthora infestans: aggressiveness on tomato. Phytopathology 85:1362–1367Google Scholar
  26. Li Q, Lawrence CB, Xing H-Y, Babbit RA, Bass WT, Maiti IB, Everett NP (2001) Enhanced disease resistance conferred by expression of an antimicrobial magainin analog in transgenic tobacco. Planta 212:635–639CrossRefPubMedGoogle Scholar
  27. Liang H, Catranis CM, Maynard CE, 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
  28. 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
  29. Maloy WL, MacDonald D, Brasseur M (1990) Design of broad spectrum antibiotic and host defense peptides based on magainin and related peptides. In: Giralt ED, Andreu D (eds) Proc 21st Eur Peptide Symp. ESCOM Science Publ, Leiden, pp 731–745Google Scholar
  30. Mayton H, Forbes GA, Mizubuti ESG, Fray WE (2001) The roles of three fungicides in the epidemiology of potato late blight. Plant Dis 85:1006–1011Google Scholar
  31. 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 13:860–868PubMedGoogle Scholar
  32. Mourgues F, Brisset M-N, Chevreau E (1998) Activity of different antibacterial peptides on Erwinia amylovora growth, and evaluation of the phytotoxicity and stability of cecropins. Plant Sci 139:83–91CrossRefGoogle Scholar
  33. 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
  34. Ohshima M, Mitsuhara I, Okamoto M, Sawano S, Nishiyama K, Kaku H, Natori S, Ohashi Y (1999) Enhanced resistance to bacterial disease of transgenic tobacco plants overexpressing sarcotoxin IA, a bactericidal peptide of insect. J Biochem 125:431–435PubMedGoogle Scholar
  35. 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
  36. Pichersky E, Bernazky R, Tanksley SD, Cashmore AR (1986) Evidence for selection as a mechanism in the concerted evolution of Lycopersicon esculentum (tomato) genes encoding the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 83:3880–33884PubMedGoogle Scholar
  37. Powell WA, Catranis CM, Maynard CA (1995) Synthetic antimicrobial peptide design. Mol Plant Microbe Interact 8:792–794PubMedGoogle Scholar
  38. Rao AG (1995) Antimicrobial peptides. Mol Plant Microbe Interact 8:6–13PubMedGoogle Scholar
  39. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring HarborGoogle Scholar
  40. Sheldrake R, Boodley JW (1973) Cornell peat-like mixes for commercial plant growing. Cornell Cooperative Extension Bulletin 1.B.43Google Scholar
  41. Smart CD, Myers KL, Restrepo S, Martin GB, Fry WE (2003) Partial resistance of tomato to Phytophthora infestans is not dependent upon ethylene, jasmonic acid, or salicylic acid signaling pathways. Mol Plant Microbe Interact 16:141–148PubMedGoogle Scholar
  42. Smith FD, Gadoury DM, Vaneck JM, Blowers A, Sanford JC, Van der Meij J, Eisenreich R (1998) Enhanced resistance to powdery mildew in transgenic poinsettia conferred by antimicrobial peptides. Phytopathology 88:S83Google Scholar
  43. Van Hoftsen P, Faye I, Kockum K, Lee JY, Xanthopoulos KG, Boman IA, Boman HG, Engstrom A, Andreu D, Merrifield RB (1985) Molecular cloning, cDNA sequencing, and chemical synthesis of cecropin B from Hyalophora cecropia. Proc Natl Acad Sci USA 82:2240–2243PubMedGoogle Scholar
  44. 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 84:5449–5453PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Department of Plant BreedingCornell UniversityIthacaUSA
  2. 2.Ball HelixWest ChicagoUSA

Personalised recommendations