3 Biotech

, 8:391 | Cite as

Fusion of a chitin-binding domain to an antibacterial peptide to enhance resistance to Fusarium solani in tobacco (Nicotiana tabacum)

  • Azam Badrhadad
  • Farhad Nazarian-FirouzabadiEmail author
  • Ahmad Ismaili
Original Article


An antibacterial peptide-encoding gene from alfalfa seeds, alfAFP, was fused to the C-terminal part of chitin-binding domain (CBD) of the rice chitinase-encoding gene (CBD-alfAFP) and introduced to tobacco by Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) technique was used to confirm the integration of the recombinant CBD-alfAFP encoding gene in transgenic tobacco plants. A number of transgenic lines and a non-transgenic control plant were selected for further molecular analyses. The result of analyzing the transgenic plants by semi-quantitative RT-PCR showed that the recombinant gene is expressed in transgenic plants and there is a difference between the transgenic plants in terms of the level of CBD-alfAFP expression. The total protein was extracted from a few selected transgenic plants and used to evaluate the antibacterial/antifungal of recombinant protein activity against some important plant and human pathogens. The results of this experiment showed that the total protein extract obtained from transgenic lines significantly (P < 0.05) inhibited the growth of various bacteria and fungi compared to the non-transgenic plants. Transgenic lines showed a significant (P < 0.01) difference considering their ability to inhibit bacterial and fungal pathogens growth. The recombinant CBD-alfAFP protein significantly (P < 0.01) increased the resistance of the transgenic plants against Fusarium solani. Transgenic lines showed no significant wilting symptoms and obvious wilting symptoms were not observed even 30 days post-inoculation (dpi) with Fusarium solani spores. These results suggest that transgenic tobacco plants are resistant to Fusarium solani wilt and fusion of CBD to the alfAFP antimicrobial peptide is an efficient approach to control fungal diseases.


Antibacterial Chitin-binding domain Fungi Genetic engineering Transformation 



We would like to show our gratitude to the member of Dr. Mostafa Darvishnia Plant disease LAB for in vitro fungal tests.

Compliance with ethical standards

Conflict of interest

Authors declare that there is no competing interest in the publication of this manuscript.


  1. Alastruey-Izquierdo A, Cuenca-Estrella M, Monzón A, Mellado E, Rodríguez-Tudela JL (2008) Antifungal susceptibility profile of clinical Fusarium spp. isolates identified by molecular methods. J Antimicrob Chemother 61(4):805–809CrossRefPubMedGoogle Scholar
  2. Aoki T, O’Donnell K, Homma Y, Lattanzi AR (2003) Sudden-death syndrome of soybean is caused by two morphologically and phylogenetically distinct species within the Fusarium solani species complex—F. virguliforme in North America and F. tucumaniae in South America. Mycologia 95(4):660–684PubMedGoogle Scholar
  3. Bauer A, Kirby W, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45(4):493CrossRefGoogle Scholar
  4. Becker-Ritt AB, Carlini CR (2012) Fungitoxic and insecticidal plant polypeptides. J Pept Sci 98(4):367–384CrossRefGoogle Scholar
  5. Bhattacharyya PN, Jha DK (2012) Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J Microbiol Biotechnol 28(4):1327–1350CrossRefPubMedGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254CrossRefGoogle Scholar
  7. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3(3):238–250CrossRefGoogle Scholar
  8. Chen S, Liu A, Wang F, Ahammed G (2009) Combined overexpression of chitinase and defensin genesin transgenic tomato enhances resistance to Botrytis cinerea. Afr J Biotechnol 8:(20)Google Scholar
  9. Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J (2012) The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13(4):414–430CrossRefPubMedGoogle Scholar
  10. Diaz AH, Kovacs I, Lindermayr C (2016) Inducible expression of the de-novo designed antimicrobial peptide SP1-1 in tomato confers resistance to Xanthomonas campestris pv. vesicatoria. PloS One 11(10):e0164097CrossRefGoogle Scholar
  11. Fantozzi P, Sensidoni A (1983) Protein extraction from tobacco leaves: technological, nutritional and agronomical aspects. Plant Food Hum Nutr J (Formerly Qualitas Plantarum) 32(3):351–368CrossRefGoogle Scholar
  12. FAO (2017) FAOSTAT data production, trade, food balance, food security. http://www.faoorg/faostat/en/#home. Accessed 2 Aug 2017
  13. Free SJ (2012) Fungal cell wall organization and biosynthesis. Adv Genet 81:33–82Google Scholar
  14. Furletti V, Teixeira I, Obando-Pereda G, Mardegan R, Sartoratto A, Figueira G, Duarte R, Rehder V, Duarte M, Höfling J (2011) Action of Coriandrum sativum L. essential oil upon oral Candida albicans biofilm formation. Evid Based Complement Alternat Med. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Gao A-G, 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 Biotech 18(12):1307CrossRefGoogle Scholar
  16. Gow NA, Hube B (2012) Importance of the Candida albicans cell wall during commensalism and infection. Curr Opin Microbiol 15(4):406–412CrossRefPubMedGoogle Scholar
  17. Guaní-Guerra E, Santos-Mendoza T, Lugo-Reyes SO, Terán LM (2010) Antimicrobial peptides: general overview and clinical implications in human health and disease. Clin Immunol 135(1):1–11CrossRefPubMedGoogle Scholar
  18. Jan P-S, Huang H-Y, Chen H-M (2010) Expression of a synthesized gene encoding cationic peptide cecropin B in transgenic tomato plants protects against bacterial diseases. J Appl Environ Microbiol 76(3):769–775CrossRefGoogle Scholar
  19. Jaynes JM, Nagpala P, Destéfano-Beltrán L, Hong Huang J, 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(1):43–53CrossRefGoogle Scholar
  20. Kaur J, Sagaram US, Shah D (2011) Can plant defensins be used to engineer durable commercially useful fungal resistance in crop plants? Fungal Biol Rev 25(3):128–135CrossRefGoogle Scholar
  21. Kazan K, Rusu A, Marcus JP, Goulter KC, JM M (2002) Enhanced quantitative resistance to Leptospharia maculans conferred by expression of a novel antimicrobial peptide in canola (Brassica napus L.). Mol Breed 10:63–70CrossRefGoogle Scholar
  22. Khan RS, Nakamura I, Mii M (2011) Development of disease-resistant marker-free tomato by R/RS site-specific recombination. Plant Cell Rep 30(6):1041–1053CrossRefPubMedGoogle Scholar
  23. King EO, Ward MK, Raney DE (1954) Two simple media for the demonstration of phycocyanin and fluorescin. J Lab Clin Med 44:301–307PubMedGoogle Scholar
  24. Komárek M, Čadková E, Chrastný V, Bordas F, Bollinger J-C (2010) Contamination of vineyard soils with fungicides: a review of environmental and toxicological aspects. Environ Int 36(1):138–151CrossRefPubMedGoogle Scholar
  25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685CrossRefPubMedGoogle Scholar
  26. Lee SC, Hwang IS, Choi HW, BK H (2008) Involvement of the pepper antimicrobial protein CaAMP1 gene in broad spectrum disease resistance. Plant Physiol 148:1004–1020CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lee H-H, Kim J-S, Hoang QT, Kim J-I, Kim YS (2018) Root-specific expression of defensin in transgenic tobacco results in enhanced resistance against Phytophthora parasitica var. nicotianae. Eur J Plant Pathol 151(3):811–823CrossRefGoogle Scholar
  28. Li Z, Zhou M, Zhang Z, Ren L, Du L, Zhang B, Xu H, Xin Z (2011) Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis. Funct Integr Genomics 11(1):63–70CrossRefPubMedGoogle Scholar
  29. Mangena T, Muyima N (1999) Comparative evaluation of the antimicrobial activities of essential oils of Artemisia afra, Pteronia incana and Rosmarinus officinalis on selected bacteria and yeast strains. Lett Appl Microbiol 28(4):291–296CrossRefPubMedGoogle Scholar
  30. Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13(6):614–629CrossRefPubMedGoogle Scholar
  31. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497CrossRefGoogle Scholar
  32. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8(19):4321–4326CrossRefPubMedPubMedCentralGoogle Scholar
  33. 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 Biotech 18(11):1162–1166CrossRefGoogle Scholar
  34. 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(2):181–190CrossRefPubMedGoogle Scholar
  35. Parashina E, Serdobinskii L, Kalle E, Lavrova N, Avetisov V, Lunin V, Naroditskii B (2000) Genetic engineering of oilseed rape and tomato plants expressing a radish defensin gene. Russ J Plant Physiol 47(3):417–423Google Scholar
  36. Sagaram US, Pandurangi R, Kaur J, Smith TJ, Shah DM (2011) Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum. PLoS One 6(4):e18550CrossRefPubMedPubMedCentralGoogle Scholar
  37. Salas CE, Badillo-Corona JA, Ramírez-Sotelo G, Oliver-Salvador C (2015) Biologically active and antimicrobial peptides from plants. BioMed Res Int 2015:1–11CrossRefGoogle Scholar
  38. Shoseyov O, Shani Z, Levy I (2006) Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol Rev 70(2):283–295CrossRefPubMedPubMedCentralGoogle Scholar
  39. Sindambiwe J, Calomme M, Cos P, Totte J, Pieters L, Vlietinck A, Berghe DV (1999) Screening of seven selected Rwandan medicinal plants for antimicrobial and antiviral activities. J Ethnopharmacol 65(1):71–77CrossRefPubMedGoogle Scholar
  40. Spelbrink RG, Dilmac N, Allen A, Smith TJ, Shah DM, Hockerman GH (2004) Differential antifungal and calcium channel-blocking activity among structurally related plant defensins. Plant Physiol 135(4):2055–2067CrossRefPubMedPubMedCentralGoogle Scholar
  41. Suarez V, Staehelin C, Arango R, Holtorf H, Hofsteenge J, Meins F (2001) Substrate specificity and antifungal activity of recombinant tobacco class I chitinases. Plant Mol Biol 45(5):609–618CrossRefPubMedGoogle Scholar
  42. Terras F, Schoofs H, De Bolle M, Van Leuven F, Rees SB, Vanderleyden J, Cammue B, Broekaert WF (1992) Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 267(22):15301–15309PubMedGoogle Scholar
  43. Vanden B, Vlietinck A (1991) Screening methods for antibacterial and antiviral agents from higher plants. In: Dey PM, Harborne JB (eds) Methods in plant biochemistry. Academic press, LondonGoogle Scholar
  44. Wang Y, Nowak G, Culley D, Hadwiger LA, B F (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
  45. Yokoyama S, Iida Y, Kawasaki Y, Minami Y, Watanabe K, Yagi F (2009) The chitin-binding capability of Cy-AMP1 from cycad is essential to antifungal activity. J Peptide Sci 15(7):492–497CrossRefGoogle Scholar
  46. Zaccardelli M, Vitale S, Luongo L, Merighi M, Corazza L (2008) Morphological and molecular characterization of Fusarium solani isolates. J phytopathol 156(9):534–541CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Azam Badrhadad
    • 1
  • Farhad Nazarian-Firouzabadi
    • 1
    Email author
  • Ahmad Ismaili
    • 1
  1. 1.Agronomy and Plant Breeding Department, Faculty of AgricultureLorestan UniversityKhorramabadIran

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