|
Expression and antimicrobial activity analysis of dermaseptin B1 recombinant peptides in tobacco transgenic plants
|
Mitra Khademi  , Farhad Nazarian-Firouzabadi * |
| Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran , nazarian.f@lu.ac.ir |
|
|
Abstract: (10861 Views) |
Recently, new molecular breeding and genetic engineering approaches have emerged to overcome the limitations of conventional breeding methods in generating disease-resistance transgenic plants. The use of antimicrobial peptides (AMPs) to produce transgenic plants resistant to a wide range of plant pathogens has achieved great success. Among huge number of AMPs, Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acids peptide, exhibits significant antimicrobial activities towards a wide range of pathogens. In order to increase the antimicrobial efficacy of DrsB1, the DrsB1 encoding DNA sequence was either fused to the N- or C-terminus of the sequence encoding chitin-binding domain (CBD) of Avr4 gene from Cladosporium fulvum and constructs (CBD-DrsB1 and DrsB1-CBD) were used for tobacco leaf disk Agrobacterium-mediated transformation. Polymerase chain reaction (PCR), semi-quantitative RT-PCR and SDS-PAGE analysis indicated the integration of transgenes in tobacco genome and expression of the recombinant genes in transgenic plants, respectively. The antimicrobial activity of extracted recombinant peptides were assessed against a number of plant and human pathogens. Both recombinant peptides had statistically significant (P<0.01) inhibitory effects on the growth and development of fungi pathogens. Also, CFU test result showed that extracted recombinant peptides from transgenic plants, had a relatively high inhibitory effect on plant pathogens. The CBD-DrsB1 recombinant peptide demonstrated a higher antibacterial activity, whereas the DrsB1-CBD recombinant peptide performed a greater antifungal activity. In addition, the expression of DrsB1-CBD recombinant peptide significantly inhibited R.solani fungal infection in comparison with Pythium sp. interestingly, fungi with a higher amount of cell wall chitin were more vulnerable to recombinant peptides, suggesting recombinant peptides present a higher affinity for cell wall chitin. Owing to the high antimicrobial activity and novelty of recombinant peptides, this strategy for the first time, could be used to generate transgenic crop plants resistant to devastating plant pathogens. |
|
| Keywords: Gene expression, Pathogens, Antimicrobial peptide, Chitin-binding domain, Genetic engineering |
|
|
Full-Text [PDF 1150 kb]
(1662 Downloads)
|
|
Type of Study: Research |
Subject:
Molecular genetics
|
|
|
|
|
|
|
| References |
|
1. Alibakhshi, A., Nazarian Firouzabadi, F. and Ismaili, A. (2018). Expression and antimicrobial activity analysis of a Dermaseptin B1 antibacterial peptide in tobacco hairy roots. Plant Protection (Scientific Journal of Agriculture), 41(3): 87-96 (In persion). 2. Barra, D. and Simmaco, M. (1995). Amphibian skin: a promising resource for antimicrobial peptides. Trends in Biotechnology, 13: 205-209. [ DOI:10.1016/S0167-7799(00)88947-7] 3. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254. [ DOI:10.1016/0003-2697(76)90527-3] 4. Cao, H., Li, X. and Dong, X. (1998). Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proceedings of the National Academy of Sciences, 95: 6531-6536. [ DOI:10.1073/pnas.95.11.6531] 5. Conlon, J.M., Kolodziejek, J. and Nowotny, N. (2004). Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1696: 1-14. [ DOI:10.1016/j.bbapap.2003.09.004] 6. Flavia Cancado Viana, J., Campos Dias, S., Luiz Franco, O. and Lacorte, C. (2013). Heterologous production of peptides in plants: fusion proteins and beyond. Current Protein and Peptide Science, 14: 568-579. [ DOI:10.2174/13892037113149990072] 7. Fujikawa, T., Sakaguchi, A., Nishizawa, Y., Kouzai, Y., Minami, E., Yano, S., Koga, H., Meshi, T. and Nishimura, M. (2012). Surface α-1, 3-glucan facilitates fungal stealth infection by interfering with innate immunity in plants. PLoS Pathogens, 8: e1002882. [ DOI:10.1371/journal.ppat.1002882] 8. Hajiahmadi, Z., Shirzadian-Khorramabad, R., Kazemzad, M. and Sohani, M.M. (2017). Expression of cryIAb Driven by a Wound Inducible Promoter (MPI) in Tomato to Enhance Resistance to Tuta absoluta. Plant Genetic Researches, 4(2): 1-16 (In Persian). [ DOI:10.29252/pgr.4.2.1] 9. Holaskova, E., Galuszka, P., Frebort, I. and Oz, M.T. (2015). Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology. Biotechnology Advances, 33: 1005-1023. [ DOI:10.1016/j.biotechadv.2015.03.007] 10. Hunter, P.A., Darby, G., Russell, N.J. and Russell, A.D. (1995). Fifty Years of Antimicrobials: Past Perspectives and Future trends. The Press Syndicate of the University Cambridge; New York, USA. 11. Khademi, M., Nazarian-Firouzabadi, F. and Ismaili, A. (2019a). Cloning and expression of two new recombinant antimicrobial dermaseptin b1 peptides in tobacco to control the growth of human bacterial pathogens. Journal Mazandaran University, 29(176): 47-60 (In persion). 12. Khademi, M., Nazarian-Firouzabadi, F., Ismaili, A. and shirzadian-khorramabad, R. (2019b). Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco Microbiologyopen online, Special issue: 1-11. [ DOI:10.1002/mbo3.837] 13. Latgé, J.P. and Beauvais, A. (2014). Functional duality of the cell wall. Current Opinion in Microbiology, 20: 111-117. [ DOI:10.1016/j.mib.2014.05.009] 14. Latgé, J.P. (2010). Tasting the fungal cell wall. Cellular Microbiology, 12: 863-872. [ DOI:10.1111/j.1462-5822.2010.01474.x] 15. Li, Z., Zhou, M., Zhang, Z., Ren, L., Du, L., Zhang, B., Xu, H. and Xin, Z. (2011). Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis. Functional & Integrative Genomics, 11: 63-70. [ DOI:10.1007/s10142-011-0211-x] 16. Osusky, M., Osuska, L., Hancock, R.E., Kay, W.W. and Misra, S. (2004). Transgenic potatoes expressing a novel cationic peptide are resistant to late blight and pink rot. Transgenic Research, 13: 181-190. [ DOI:10.1023/B:TRAG.0000026076.72779.60] 17. Oyama, L.B., Crochet, J.A., Edwards, J.E., Girdwood, S.E., Cookson, A.R., Fernandez-Fuentes, N., Hilpert, K., Golyshin, P.N., Golyshina, O.V. and Privé, F. (2017). Buwchitin: A Ruminal Peptide with antimicrobial potential against Enterococcus faecalis. Frontiers in Chemistry, 5: 51. [ DOI:10.3389/fchem.2017.00051] 18. Phoenix, D.A., Harris, F., Mura, M. and Dennison, S.R. (2015). The increasing role of phos-phatidylethanolamine as a lipid receptor in the action of host defence peptides. Progress in Lipid Research, 59: 26-37. [ DOI:10.1016/j.plipres.2015.02.003] 19. Nazarian-Firouzabadi, F. (2014). Manipulation of starch biosynthasis and in planta biopolymer production. Plant Genetic Researches, 4(2): 1-14 (In Persian). [ DOI:10.29252/pgr.1.2.1] 20. Sohlenkamp, C. and Geiger, O. (2016). Bacterial membrane lipids: diversity in structuresand pathways. FEMS Microbiology Letters, 40: 133-159. [ DOI:10.1093/femsre/fuv008] 21. Stone, S.L. and Gifford, D.J. (1997). Structural and biochemical changes in loblolly pine (Pinus taeda L.) seeds during germination and early-seedling growth. I. Storage protein reserves. International Journal of Plant Sciences, 158: 727-737. [ DOI:10.1086/297484] 22. Teixeira, V., Feio, M.J. and Bastos, M. (2012). Role of lipids in the interaction of antimicro-bial peptides with membranes. Progress in Lipid Research, 51: 149-177. [ DOI:10.1016/j.plipres.2011.12.005] 23. Thomma, B.P., Nürnberger, T. and Joosten, M.H. (2011). Of PAMPs and effectors: the blurred PTI-ETI dichotomy. The Plant Cell Online, 23: 4-15. [ DOI:10.1105/tpc.110.082602] 24. Van den Burg, H.A., Harrison, S.J., Joosten, M.H., Vervoort, J. and de Wit, P.J. (2006). Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection. Molecular Plant-Microbe Interactions, 19: 1420-1430. [ DOI:10.1094/MPMI-19-1420] 25. Van den Burg, H.A., Spronk, C.A., Boeren, S., Kennedy, M.A., Vissers, J.P., Vuister, G.W., de Wit, P.J. and Vervoort, J. (2004). Binding of the Avr4 elicitor of Cladosporium fulvum to chitotriose units is facilitated by positive allosteric protein-protein interactions. Journal of Biological Chemistry, 249: 16786-16796. [ DOI:10.1074/jbc.M312594200] 26. Vidaver, A.K. (2002). Uses of antimicrobials in plant agriculture. Clinical Infectious Diseases, 34: S107-S110. [ DOI:10.1086/340247] 27. Vlietinck, A. (1991). Screening methods for antibacterial and antiviral agents from higher plants. Methods in Plant Biochemistry, 6: 47-69. 28. Wohlkönig, A., Huet, J., Looze, Y. and Wintjens, R. (2010). Structural relationships in the lysozyme Superfamily: significant evidence for glycoside hydrolase signature motifs. PLoS One, 5: e15388. [ DOI:10.1371/journal.pone.0015388] 29. Yan, R., Hou, J., Ding, D., Guan, W., Wang, C., Wu, Z. and Li, M. (2008). In vitro antifungal activity and mechanism of action of chitinase against four plant pathogenic fungi. Journal of Basic Microbiology, 48: 293-301. [ DOI:10.1002/jobm.200700392] 30. Yokoyama, S., Iida, Y., Kawasaki, Y., Minami, Y., Watanabe, K. and Yagi, F. (2009). The chitin‐binding capability of Cy‐AMP1 from cycad is essential to antifungal activity. Journal of Peptide Science, 15: 492-497. [ DOI:10.1002/psc.1147] 31. Zasloff, M. (2002). Antimicrobial peptides of multicellular organisms. Nature, 415: 389-395. [ DOI:10.1038/415389a] 32. Zasloff, M. (2006). Defending the epithelium. Nature Medicine, 12: 607-608. [ DOI:10.1038/nm0606-607] |
|
| Send email to the article author |
|
|
|
Khademi M, Nazarian-Firouzabadi F. Expression and antimicrobial activity analysis of dermaseptin B1 recombinant peptides in tobacco transgenic plants. pgr 2019; 6 (1) :139-150 URL: http://pgr.lu.ac.ir/article-1-178-en.html
|
