Transgenic tobacco expressing Medicago sativa Defensin (Msdef1) confers resistance to various phyto-pathogens

Abstract

This study was performed to evaluate the competency of transgenic plants expressing Medicago sativa defensin (MsDef1) towards developing self-resistance against the attack of pathogens. We raised fifteen lines of T2 transgenic tobacco plants expressing MsDef1 under the control of a strong constitutive promoter (M24) and transgene integration was confirmed. Plant-derived enriched MsDef1 at a concentration of 0.6 μg/μl showed 78, 53 and 71% anti-bacterial activities against Pseudomonas aeruginosa, Ralstonia solanacearum and Xanthomonas campestris respectively; alongside it demonstrated 53, 65 and 52% anti-fungal activity against Aspergillus niger, Pyricularia oryzae and Rhizoctonia solani respectively, at a concentration of 0.8 μg/μl in vitro. In addition, plant-derived MsDef1 showed significant anti-bacterial activity against Pseudomonas syringae pv tabaci. The LD50 values obtained from in vitro analyses clearly indicated the efficacy of MsDef1 against the above mentioned phyto-pathogens. The in vivo studies employing R. solanacearum and A. niger showed that transgenic plants could withstand the invasion of these virulent pathogens. Under this nascent study, we demonstrated the activity of plant-derived MsDef1 against plant pathogens; both in vitro and in vivo.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. 1.

    Abdallah NA, Shah D, Abbas D, Madkour M. Stable integration and expression of a plant defensin in tomato confers resistance to fusarium wilt. GM Crops. 2010;1(5):344–50. https://doi.org/10.4161/gmcr.1.5.15091.

    Article  PubMed  Google Scholar 

  2. 2.

    Aerts AM, Carmona-Gutierrez D, Lefevre S, Govaert G, Francois IE, Madeo F, et al. The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans. FEBS Lett. 2009;583(15):2513–6. https://doi.org/10.1016/j.febslet.2009.07.004.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Alabouvette C, Olivain C, Steinberg C. Biological control of plant diseases: the European situation. Eur J Plant Pathol. 2006;114(3):329–41.

    Article  Google Scholar 

  4. 4.

    Anderson NA. The genetics and pathology of Rhizoctonia solani. Annu Rev Phytopathol. 1982;20(1):329–47. https://doi.org/10.1146/annurev.py.20.090182.001553.

    Article  Google Scholar 

  5. 5.

    Boman HG. Peptide antibiotics and their role in innate immunity. Annu Rev Immunol. 1995;13:61–92. https://doi.org/10.1146/annurev.iy.13.040195.000425.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Cummings PL, Sorvillo F, Kuo T. Salmonellosis-related mortality in the United States, 1990–2006. Foodborne Pathog Dis. 2010;7(11):1393–9. https://doi.org/10.1089/fpd.2010.0588.

    Article  PubMed  Google Scholar 

  8. 8.

    Datla RSS, Bekkaoui F, Hammerlindl JK, Pilate G, Dunstan DI, Crosby WL. Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence. Plant Sci. 1993;94(1–2):139–49. https://doi.org/10.1016/0168-9452(93)90015-R.

    CAS  Article  Google Scholar 

  9. 9.

    Dey N, Maiti IB. Structure and promoter/leader deletion analysis of mirabilis mosaic virus (MMV) full-length transcript promoter in transgenic plants. Plant Mol Biol. 1999;40(5):771–82.

    CAS  Article  Google Scholar 

  10. 10.

    Dominiqueandijck DB, Depuydt Pieter, Offner Fritz, Blot Stijn, Van Tilborgh AK, Nollet Joke, Steel Eva, Noens Lucien, Decruyenaere Johan. Impact of recent intravenous chemotherapy on outcome in severe sepsis and septic shock patients with hematological malignancies. Intensive Care Med. 2008;34(5):847–55.

    Article  Google Scholar 

  11. 11.

    Duffy B, Schouten A, Raaijmakers JM. Pathogen self-defense: mechanisms to counteract microbial antagonism. Annu Rev Phytopathol. 2003;41(1):501–38.

    CAS  Article  Google Scholar 

  12. 12.

    Gao J, Wang Y, Wang CW, Lu BH. First report of bacterial root rot of ginseng caused by Pseudomonas aeruginosa in China. Plant Dis. 2014;98(11):1577. https://doi.org/10.1094/pdis-03-14-0276-pdn.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Hancock RE, Lehrer R. Cationic peptides: a new source of antibiotics. Trends Biotechnol. 1998;16(2):82–8.

    CAS  Article  Google Scholar 

  14. 14.

    Hikosaka S. Production of value-added plants. Smart plant factory. Berlin: Springer; 2018. p. 325–51.

    Google Scholar 

  15. 15.

    Hilpert K, Volkmer-Engert R, Walter T, Hancock RE. High-throughput generation of small antibacterial peptides with improved activity. Nat Biotechnol. 2005;23(8):1008–12. https://doi.org/10.1038/nbt1113.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Howell CR. Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 2003;87(1):4–10. https://doi.org/10.1094/PDIS.2003.87.1.4.

    CAS  Article  Google Scholar 

  17. 17.

    Jacob SS, Cherian S, Sumithra TG, Raina OK, Sankar M. Edible vaccines against veterinary parasitic diseases–current status and future prospects. Vaccine. 2013;31(15):1879–85. https://doi.org/10.1016/j.vaccine.2013.02.022.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Jha S, Tank HG, Prasad BD, Chattoo BB. Expression of Dm-AMP1 in rice confers resistance to Magnaporthe oryzae and Rhizoctonia solani. Transgenic Res. 2009;18(1):59–69. https://doi.org/10.1007/s11248-008-9196-1.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Jha S, Agarwal S, Sanyal I, Jain GK, Amla DV. Differential subcellular targeting of recombinant human alpha(1)-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants. Plant Sci. 2012;196:53–66. https://doi.org/10.1016/j.plantsci.2012.07.004.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Kennedy BW, Alcorn SM. Estimates of U.S. crop losses to procaryote plant pathogens. Plant Dis. 1980;64(7):674–6.

    Article  Google Scholar 

  21. 21.

    Ko K, Norelli J, Reynoird J-P, Boresjza-Wysocka E, Brown S, Aldwinckle H. Effect of untranslated leader sequence of AMV RNA 4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple. Biotech Lett. 2000;22(5):373–81. https://doi.org/10.1023/A:1005672601625.

    CAS  Article  Google Scholar 

  22. 22.

    Kovaleva V, Kiyamova R, Cramer R, Krynytskyy H, Gout I, Filonenko V, et al. Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings. Peptides. 2009;30(12):2136–43. https://doi.org/10.1016/j.peptides.2009.08.007.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Krishnakumari V, Singh S, Nagaraj R. Antibacterial activities of synthetic peptides corresponding to the carboxy-terminal region of human beta-defensins 1-3. Peptides. 2006;27(11):2607–13. https://doi.org/10.1016/j.peptides.2006.06.004.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Lacerda A, Vasconcelos ÉAR, Pelegrini PB, Grossi-de-Sa MF. Antifungal defensins and their role in plant defense. Front Microbiol. 2014;5:116.

    Article  Google Scholar 

  25. 25.

    Lehrer RI, Lichtenstein AK, Ganz T. Defensins: antimicrobial and cytotoxic peptides of mammalian cells. Annu Rev Immunol. 1993;11:105–28. https://doi.org/10.1146/annurev.iy.11.040193.000541.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Liu Z, Han J, Liu Z, Zhang X, Chen J, Dong A, et al. First report of Pseudomonas aeruginosa causing tumor disease of Populus koreana in China. J Plant Dis Prot. 2019;1–4.

  27. 27.

    Lobo DS, Pereira IB, Fragel-Madeira L, Medeiros LN, Cabral LM, Faria J, et al. Antifungal Pisum sativum defensin 1 interacts with Neurospora crassa cyclin F related to the cell cycle. Biochemistry. 2007;46(4):987–96. https://doi.org/10.1021/bi061441j.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Maiti IB, Murphy JF, Shaw JG, Hunt AG. Plants that express a potyvirus proteinase gene are resistant to virus infection. Proc Natl Acad Sci USA. 1993;90(13):6110–4.

    CAS  Article  Google Scholar 

  29. 29.

    Mason AB, He QY, Halbrooks PJ, Everse SJ, Gumerov DR, Kaltashov IA, et al. Differential effect of a his tag at the N- and C-termini: functional studies with recombinant human serum transferrin. Biochemistry. 2002;41(30):9448–54.

    CAS  Article  Google Scholar 

  30. 30.

    Mueller DS, Hartman GL, Pedersen WL. Development of Sclerotia and Apothecia of Sclerotinia sclerotiorum from infected soybean seed and its control by fungicide seed treatment. Plant Dis. 1999;83(12):1113–5. https://doi.org/10.1094/PDIS.1999.83.12.1113.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Neeleman L, Olsthoorn RC, Linthorst HJ, Bol JF. Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA. Proc Natl Acad Sci USA. 2001;98(25):14286–91. https://doi.org/10.1073/pnas.251542798.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Ogle H. Disease management: chemicals. Accessed Sept. 2016;11:326.

    Google Scholar 

  33. 33.

    Onsando M. Black rot of crucifers. In: Chaube HS, Kumar J, Mukhopadhyay AN, Singh VS, editors. Diseases of vegetables and oilseed crops—plant diseases of international importance. Englewood Cliffs: Prentice Hall; 1992. p. 243–52.

    Google Scholar 

  34. 34.

    Patro S, Maiti S, Panda SK, Dey N. Utilization of plant-derived recombinant human beta-defensins (hBD-1 and hBD-2) for averting salmonellosis. Transgenic Res. 2015;24(2):353–64. https://doi.org/10.1007/s11248-014-9847-3.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Rahme LG, Stevens EJ, Wolfort SF, Shao J, Tompkins RG, Ausubel FM. Common virulence factors for bacterial pathogenicity in plants and animals. Science. 1995;268(5219):1899–902.

    CAS  Article  Google Scholar 

  36. 36.

    Rahme LGTM, Le L, Wong SM, Tompkins RG, Calderwood SB, Ausubel FM. Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proc Natl Acad Sci USA. 1997;94:13245–50.

    CAS  Article  Google Scholar 

  37. 37.

    Rai V, Dey N. Identification of programmed cell death related genes in bamboo. Gene. 2012;497(2):243–8. https://doi.org/10.1016/j.gene.2012.01.018.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Rana D, Paul Y. Effect of biocontrol agents against Pyricularia oryzae causing paddy blast. Plant Dis Res. 2019;34(1):48–50.

    Article  Google Scholar 

  39. 39.

    Rosenfeld Y, Papo N, Shai Y. Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action. J Biol Chem. 2006;281(3):1636–43. https://doi.org/10.1074/jbc.m504327200.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Sahoo DK, Dey N, Maiti IB. pSiM24 is a novel versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants. PLoS ONE. 2014;9(6):e98988.

    Article  Google Scholar 

  41. 41.

    Scardaci SC WR, Greer CA, Hill JE, William JF, Mutters RG, Brandon DM, McKenzie KS, Oster JJ. Rice blast: a new disease in California. Davis: Department of Agronomy and Range Science, University of California. Agronomy Fact Sheet Series. 1997.

  42. 42.

    Schneider JJ, Unholzer A, Schaller M, Schafer-Korting M, Korting HC. Human defensins. J Mol Med (Berl). 2005;83(8):587–95. https://doi.org/10.1007/s00109-005-0657-1.

    CAS  Article  Google Scholar 

  43. 43.

    Schuster S, Marhl M, Hofer T. Modelling of simple and complex calcium oscillations. From single-cell responses to intercellular signalling. Eur J Biochem. 2002;269(5):1333–55.

    CAS  Article  Google Scholar 

  44. 44.

    Seon J-H, Szarka JS, Molongey MM. A unique strategy for recovering recombinant proteins from molecular farming: affinity capture on engineered oilbodies. J Plant Biotechnol. 2002;4:95–101.

    Google Scholar 

  45. 45.

    Shaaltiel Y, Bartfeld D, Hashmueli S, Baum G, Brill-Almon E, Galili G, et al. Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher’s disease using a plant cell system. Plant Biotechnol J. 2007;5(5):579–90. https://doi.org/10.1111/j.1467-7652.2007.00263.x.

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Silva PM, Gonçalves S, Santos NC. Defensins: antifungal lessons from eukaryotes. Front Microbiol. 2014;5:97.

    PubMed  PubMed Central  Google Scholar 

  47. 47.

    Spelbrink RG, Dilmac N, Allen A, Smith TJ, Shah DM, Hockerman GH. Differential antifungal and calcium channel-blocking activity among structurally related plant defensins. Plant Physiol. 2004;135(4):2055–67. https://doi.org/10.1104/pp.104.040873.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Spiegel H, et al. Ready‐to‐use stocks of Agrobacterium tumefaciens can simplify process development for the production of recombinant proteins by transient expression in plants. Biotechnol J. 2019;14(10):1900113.

    CAS  Article  Google Scholar 

  49. 49.

    Starkey M, Rahme LG. Modeling Pseudomonas aeruginosa pathogenesis in plant hosts. Nat Protoc. 2009;4(2):117–24. https://doi.org/10.1038/nprot.2008.224.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003;21(12):570–8. https://doi.org/10.1016/j.tibtech.2003.10.002.

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    van Dijck PW, Selten GC, Hempenius RA. On the safety of a new generation of DSM Aspergillus niger enzyme production strains. Regul Toxicol Pharmacol. 2003;38(1):27–35.

    Article  Google Scholar 

  52. 52.

    Vazquez F, Gonzalez EA, Garabal JI, Valderrama S, Blanco J, Baloda SB. Development and evaluation of an ELISA to detect Escherichia coli K88 (F4) fimbrial antibody levels. J Med Microbiol. 1996;44(6):453–63.

    CAS  Article  Google Scholar 

  53. 53.

    Vriens K, Cammue B, Thevissen K. Antifungal plant defensins: mechanisms of action and production. Molecules. 2014;19(8):12280–303.

    Article  Google Scholar 

  54. 54.

    Walker JT, Jhutty A, Parks S, Willis C, Copley V, Turton JF, et al. Investigation of healthcare-acquired infections associated with Pseudomonas aeruginosa biofilms in taps in neonatal units in Northern Ireland. J Hosp Infect. 2014;86(1):16–23. https://doi.org/10.1016/j.jhin.2013.10.003.

    CAS  Article  PubMed  Google Scholar 

  55. 55.

    Zainal Z, Marouf E, Ismail I, Fei CK. Expression of the Capsicuum annum (Chili) defensin gene in transgenic tomatoes confers enhanced resistance to fungal pathogens. Am J Plant Physiol. 2009;4:70–9.

    CAS  Article  Google Scholar 

  56. 56.

    Zasloff M. Antimicrobial peptides of multicellular organisms. Nature. 2002;415(6870):389–95. https://doi.org/10.1038/415389a.

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

We are very much grateful to the Directors of Institute of Life Sciences (ILS) and Kentucky Tobacco Research and Development Center (KTRDC) for facilities and support. This work was supported by the KY state KTRDC grant to Dr. Indu B. Maiti and ILS/Core fund to Dr. Nrisingha Dey. Ankita Shrestha and Debasish Deb are thankful to DST-INSPIRE, Govt. of India for their Ph.D. fellowship.

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Indu Bhushan Maiti or Nrisingha Dey.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Deb, D., Shrestha, A., Sethi, L. et al. Transgenic tobacco expressing Medicago sativa Defensin (Msdef1) confers resistance to various phyto-pathogens. Nucleus 63, 179–190 (2020). https://doi.org/10.1007/s13237-020-00307-2

Download citation

Keywords

  • Bio-farming
  • MsDef1
  • M24 promoter
  • Phyto-pathogen
  • Plant-derived defensin
  • Transgenic plant