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Expression and Antimicrobial Activity Assessment of CBD-alfAFP Recombinant Peptide Produced in Tobacco Hairy Roots Against Plant Pathogens
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Zahra Zarindast   , Farhad Nazarian-Firouzabadi * , Mitra Khademi |
| Department of Plant Production and Genetic Engineering, Faculty of Agriculture, Lorestan University, Khorramabad, Iran , nazarian.f@lu.ac.ir |
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Abstract: (993 Views) |
Expression of antimicrobial peptides (AMPs) in plants to resist plant pathogens as well as to produce novel AMPs for pharmaceutical applications has recently received much consideration. alfAFP, a defensin cationic peptide synthesizing in alfalfa seeds, exhibits a strong antimicrobial activity. In order to facilitate alfAFP access to the pathogen’s membrane and increase the activity of the alfAFP peptide, the alfAFP encoding sequence was fused to the C-terminal of a chitin-binding domain (CBD) from a rice chitinase encoding gene. First, the antimicrobial properties of the recombinant peptide were assessed using bioinformatics tools. Next, the pGSA1285 expression vector harboring the CBD-alfAFP heterologous DNA was transformed into Agrobacterium rhizogenes for hairy root (HR) production in tobacco. The presence of transgene, transcription, and the expression of recombinant peptide in the HRs were confirmed by PCR and semi-quantitative RT-PCR analysis, respectively. Bioinformatic analysis was used to predict the antimicrobial activity of the alfAFP recombinant peptide. The results of the 3D structure analysis revealed a β-sheet and an α-helix structure that corresponded well with the structure of plant defensins. A Knottin functional domain was also recognized, suggesting that the recombinant peptide retains its antimicrobial activity. The results of the in vitro antimicrobial activity of the alfAFP recombinant peptide using CFU test showed that the recombinant peptide had significant inhibitory effects on Pseudomonas syringae pathogen. Therefore, the chitin-binding domain provided a better access of the recombinant peptide to the pathogenic bacterial cell wall through binding to peptidoglycan, and probably the recombinant peptide was able to target the plasma membrane with better efficiency. The results of this study suggested that the expression of the CBD-alfAFP recombinant peptide in crop plants and HRs can be a promising approach to producing pathogen-resistant plants as well as to produce new recombinant pharmaceutical AMPs. |
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| Keywords: Bioinformatics, alfAFP peptide, Antimicrobial peptide, Hairy root |
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Full-Text [PDF 966 kb]
(279 Downloads)
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Type of Study: Research |
Subject:
Genetic engineering
Accepted: 2023/09/19
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1. Aleinein, R., Schäfer, H. and Wink, M. (2015). Rhizosecretion of the recombinant antimicrobial peptide ranalexin from transgenic tobacco hairy roots. RRJBS Phytopathol Gene Diseas, 1: 45-55. 2. Aslam, M.Z., Firdos, S., Li, Z., Wang, X., Liu, Y., Qin, X., Yang, S., Ma, Y., Xia, X., Zhang, B. and Dong, Q. (2020). Detecting the Mechanism of Action of Antimicrobial Peptides by Using Microscopic Detection Techniques. Foods, 11: 2809. [ DOI:10.3390/foods11182809] 3. Alibakhshi, A., Nazarian-Firouzabadi, F. and Esmaili, A. (2018). Expression and antimicrobial activity analysis of a Dermaseptin B1 antibacterial peptide in tobacco hairy roots. Plant Protection (Scientific Journal of Agriculture), 41: 87-97. 4. Badrhadad, A., Nazarian-Firouzabadi, F. and Ismaili, A. (2018). Fusion of a chitin-binding domain to an antibacterial peptide to enhance resistance to Fusarium solani in tobacco (Nicotiana tabacum). 3 Biotech, 8: 1-10. [ DOI:10.1007/s13205-018-1416-7] 5. Bogdanova, L., Valiullina, Y., Faizullin, D., Kurbanov, R.K. and Ermakova, E.A. (2020). Spectroscopic, zeta potential and molecular dynamics studies of the interaction of antimicrobial peptides with model bacterial membrane. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 242: 118785. [ DOI:10.1016/j.saa.2020.118785] 6. 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] 7. Cardon, F., Pallisse, R., Bardor, M., Caron, A., Vanier, J., Ele Ekouna, J.P., Lerouge, P., Boitel‐Conti, M. and Guillet, M. (2019). Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha‐L‐iduronidase. Plant Biotechnology Journal, 17: 505-516. [ DOI:10.1111/pbi.12994] 8. Chang, S., Puryear, J. and Cairney, J. (1993). A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter. 11: 113-116. [ DOI:10.1007/BF02670468] 9. Chiche, L., Heitz, A., Gelly, J.C., Gracy, J., Chau, P.T., Ha, P.T., Hernandez, J.F. and Le-Nguyen, D. (2004). Squash inhibitors: from structural motifs to macrocyclic knottins. Current Protein and Peptide Science, 5: 341-349. [ DOI:10.2174/1389203043379477] 10. Finn, R.D., Coggill, P., Eberhardt, R.Y., Eddy, S.R., Mistry, J., Mitchell, A.L., Potter, S.C., Punta, M., Qureshi, M. and Sangrador-Vegas, A. (2016). The Pfam protein families database: towards a more sustainable future. Nucleic Acids Research, 44: 279-285. [ DOI:10.1093/nar/gkv1344] 11. Gao, A.G., Hakimi, S.M., Mittanck, C.A., Wu, Y., Woerner, B.M., Stark, D.M., Shah, D.M., Liang, J. and Rommens, C.M. (2000). Fungal pathogen protection in potato by expression of a plant defensin peptide. Nature Biotechnology, 18: 1307-1310. [ DOI:10.1038/82436] 12. Giri, A. and Narasu, M.L. (2000). Transgenic hairy roots: recent trends and applications. Biotechnology Advances, 18: 1-22. [ DOI:10.1016/S0734-9750(99)00016-6] 13. Gururani, M.A., Venkatesh, J., Upadhyaya, C.P., Nookaraju, A., Pandey, S.K. and Park, S.W. (2012). Plant disease resistance genes: current status and future directions. Physiological and Molecular Plant Pathology, 78: 51-65. [ DOI:10.1016/j.pmpp.2012.01.002] 14. Gurusamy, P.D., Schäfer, H., Ramamoorthy, S. and Wink, M. (2017). Biologically active recombinant human erythropoietin expressed in hairy root cultures and regenerated plantlets of Nicotiana tabacum L. PloS One, 12: e0182367. [ DOI:10.1371/journal.pone.0182367] 15. 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] 16. Horsch, R.B., Fry, J.E., Hoffmann, N.L. and Eichholtz D. (1985). A simple and general method for transferring genes into plants. Science, 227: 1229-1231 [ DOI:10.1126/science.227.4691.1229] 17. Hu, Z.B. and Du, M. (2006). Hairy root and its application in plant genetic engineering. Journal of Integrative Plant Biology, 48: 121-127. [ DOI:10.1111/j.1744-7909.2006.00121.x] 18. Jafari, M. (2006). Tohidfar M. Bt transgenic plants: safety advantages and potential impacts in control insect pests. Paper presented at the First Agricultural Biotechnology Conference. Kermanshah, Iran. 19. Jones, P., Binns, D., Chang, H.Y., Fraser, M., Li, W., McAnulla, C., McWilliam, H., Maslen, J., Mitchell, A. and Nuka, G. (2014). InterProScan 5: genome-scale protein function classification. Bioinformatics, 30: 1236-1240. [ DOI:10.1093/bioinformatics/btu031] 20. Kapp, K., Schrempf, S., Lemberg, M.K. and Dobberstein, B. (2009). Post-targeting functions of signal peptides. In: Zimmermann R., Ed., Madame Curie Bioscience Database, pp.1-16. Austin, TX: Madame Curie Bioscience Database: Landes Bioscience, Austin, Texas, USA. 21. Karimi, S., Pazhouhandeh, M. and Azizpour, K. (2022). Evaluation of some characteristics of substantial equivalence of a salinity-resistant transgenic potato. Plant Genetic Researches, 9(1): 43-56 (In Persian). [ DOI:10.52547/pgr.9.1.4] 22. Khademi, M., Nazarian‐Firouzabadi, F., Ismaili, A. and Shirzadian Khorramabad, R. (2019). Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco. MicrobiologyOpen, 8(11):1-11. [ DOI:10.1002/mbo3.837] 23. Khademi, M., Varasteh-Shams, M. and Nazarian-Firouzabadi, F. (2022). Induction of DrsB1-CBDAvr4 Recombinant Protein in Hairy and Adventitious Roots of T1 Transgenic Plants. Plant Genetic Researches, 9: 27-42 (In Persian). [ DOI:10.52547/pgr.9.1.3] 24. Kim, Y., Wyslouzil, B.E. and Weathers, P.J. (2002a). Secondary metabolism of hairy root cultures in bioreactors. In Vitro Cellular & Developmental Biology-Plant, 38: 1-10. [ DOI:10.1079/IVP2001243] 25. Kim, Y.J., Weathers, P.J. and Wyslouzil, B.E. (2002b). Growth of Artemisia annua hairy roots in liquid‐and gas‐phase reactors. Biotechnology and Bioengineering, 80: 454-464. [ DOI:10.1002/bit.10389] 26. Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35: 1547-1549. [ DOI:10.1093/molbev/msy096] 27. Landon, C., Barbault, F., Legrain, M., Menin, L., Guenneugues, M., Schott, V., Vovelle, F. and Dimarcq, J.L. (2004). Lead optimization of antifungal peptides with 3D NMR structures analysis. Protein Science, 13: 703-713. [ DOI:10.1110/ps.03404404] 28. Lee, M., Yoon, E., Jeong, J. and Choi, Y. (2004). Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters. Plant Cell Reports, 22: 822-827. [ DOI:10.1007/s00299-004-0763-5] 29. Ma, Z. and Michailides, T.J. (2005). Advances in understanding molecular mechanisms of fungicide resistance and molecular detection of resistant genotypes in phytopathogenic fungi. Crop Protection, 24: 853-863. [ DOI:10.1016/j.cropro.2005.01.011] 30. Makarova, K., Slesarev, A., Wolf, Y., Sorokin, A., Mirkin, B., Koonin, E., Pavlov, A., Pavlova, N., Karamychev, V. and Polouchine, N. (2006). Comparative genomics of the lactic acid bacteria. Proceedings of the National Academy of Sciences, 103: 15611-15616. [ DOI:10.1073/pnas.0607117103] 31. Marchler-Bauer, A., Derbyshire, M.K., Gonzales, N.R., Lu, S., Chitsaz, F., Geer, L.Y., Geer, R.C., He, J., Gwadz, M. and Hurwitz, D.I. (2015). CDD: NCBI's conserved domain database. Nucleic Acids Research, 43: 222-226. [ DOI:10.1093/nar/gku1221] 32. Naik, S. and Prasad, R. (2006). Pesticide residue in organic and conventional food-risk analysis. Journal of Chemical Health and Safety, 13: 12-19. [ DOI:10.1016/j.chs.2005.01.012] 33. Nguyen, L.T., Haney, E.F. and Vogel, H.J. (2011). The expanding scope of antimicrobial peptide structures and their modes of action. Trends in Biotechnology, 29: 464-47. [ DOI:10.1016/j.tibtech.2011.05.001] 34. Nielsen, H. (2017) Predicting secretory proteins with SignalP. In: Kihara D., Ed., Protein Function Prediction, pp. 59-73. Springer, New York, USA. [ DOI:10.1007/978-1-4939-7015-5_6] 35. Richards, E., Reichardt, M. and Rogers, S. (1994). Preparation of genomic DNA from plant tissue. Current Protocols in Molecular Biology, 2(1): 2-3. [ DOI:10.1002/0471142727.mb0203s27] 36. Schleifer, K.H. and Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriological Reviews, 36: 407-477. [ DOI:10.1128/br.36.4.407-477.1972] 37. Strange, R.N. and Scott, P.R. (2005). Plant disease: a threat to global food security. Annual Review of Phytopathology, 43: 83-116. [ DOI:10.1146/annurev.phyto.43.113004.133839] 38. Syed, S. and Tollamadugu, N.P. (2019). Microbes in the generation of genetically engineered plants for disease resistance. In: Viswanath, B., Ed., Recent Developments in Recent Developments in Applied Microbiology and Biochemistry, pp. 235-248. Academic Press, Cambridge, UK. [ DOI:10.1016/B978-0-12-816328-3.00018-0] 39. Waghu, F.H., Gopi, L., Barai, R.S., Ramteke, P., Nizami, B. and Idicula-Thomas, S. (2014) . CAMP: Collection of sequences and structures of antimicrobial peptides. Nucleic Acids Research, 42: 1154-1158. [ DOI:10.1093/nar/gkt1157] 40. Webb, C.A. and Fellers, J.P. (2006). Cereal rust fungi genomics and the pursuit of virulence and avirulence factors. FEMS Microbiology Letters, 264: 1-7. [ DOI:10.1111/j.1574-6968.2006.00400.x] 41. 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: An Official Publication of the European Peptide Society, 15: 492-497. [ DOI:10.1002/psc.1147] 42. Yu, C.S., Cheng, C.W., Su, W.C., Chang, K.C., Huang, S.W., Hwang, J.K. and Lu, C.H. (2014). Cello2go: a web server for protein subCellular Localization prediction with functional gene ontology annotation. PloS One, 9: e99368. [ DOI:10.1371/journal.pone.0099368] 43. Zasloff, M. (2002). Antimicrobial peptides of multicellular organisms. Nature, 415: 389. [ DOI:10.1038/415389a] 44. Zasloff, M. (2006). Defending the epithelium. Nature Medicine, 12: 607-608. [ DOI:10.1038/nm0606-607] |
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Zarindast Z, Nazarian-Firouzabadi F, Khademi M. Expression and Antimicrobial Activity Assessment of CBD-alfAFP Recombinant Peptide Produced in Tobacco Hairy Roots Against Plant Pathogens. pgr 2023; 10 (1) :43-60 URL: http://pgr.lu.ac.ir/article-1-286-en.html
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