Two Novel Defensin Genes from Brassica Juncea and Camelina Sativa Confers Antifungal Activity Against Pathogenic Fungi Alernaria brassicae

GDG Chaturani¹*, Zahoor Ahmad Mir² and Anita Grover³

¹PhD Scholar, National Institute of Plant Biotechnology, New Delhi, India
²Senior Research Fellow, National Institute of Plant Biotechnology, New Delhi, India
³Principle Scientist, National Institute of Plant Biotechnology, New Delhi, India

*Corresponding Author: GDG Chaturani, PhD Scholar, National Institute of Plant Biotechnology, New Delhi, India.

Received: November 02, 2021; Published: January 28, 2022

Abstract

Alternaria blight, incited by A. brassicae is one of the most common and destructive fungal pathogens in Indian mustard which accounts for 46% of yield loss. Efforts are being made in order to have sufficient knowledge about the genes induced during infection and their regulation measures. We hear report two novel defensins, BjDef and CsDef which confers antifungal activity against A. brassicae. During the search of defensins, cloning and sequencing resulted in two open reading frames of 234 bp with characteristic α-helix, triple strand antiparallel β-sheets, highly conserved eight cysteines and an identical backbone structure stabilized by four intra-molecular disulphide bridges in their peptide chains. Amplified fragments were cloned into pET29a vector system and were expressed in Escherichia coli. Recombinant defensin proteins were optimally expressed in isopropyl thio-galactoside concentration of 1.0 mM/ml and 6 h of incubation. Purified proteins with hexa-histidine tag by Ni-NTA column exhibited a 16 kDa single fragment. The purified proteins showed an inhibitory effect on hyphal growth indicating disrupted mycelial cells while percentage mycelial growth inhibition calculations indicate inhibition effect of proteins in a concentration dependent manner. Even though at lower concentration inhibition was high by BjDef, at higher concentrations CsDef conferred comparatively more effective inhibition. Relative expression of CsDef revealed a high level of expression at 48 h in response to Alternaria infection. Comparatively low level of expression was reported by BjDef. Our results confirmed the ability of above defensins to be used in construction of transgenic brassica to confirm resistance to A. brassicae.

Keywords: Alternaria Blight; Brassica Juncea; Defensin; Antifungal

References

1. Aerts AM., et al. “The mode of antifungal action of plant, insect and human defensins”. Cell Molecular and Life Sciences 65 (2008): 2069-2079.
2. Anuradha TS., et al. “Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens”. Plant Cell Reports 27 (2008): 1777-1786.
3. Aoun M. “Host defense mechanisms during fungal pathogenesis and how these are overcome in susceptible plants”. International Journal of Botany2 (2017): 82-102.
4. Brian L., et al. “A self-inducible heterologous protein expression system in Escherichia coli”. Scientific Reports 6 (2016): 33037.
5. Broguel KE., et al. “Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani”. Science 254 (1991): 1194-1197.
6. Buonaurio R. “Infection and plant defense responses during plant bacterial infection”. In: Barka, E. and Clement, C. (eds.) (2008)”. Plant Microbe Interactions (2008): 169-197.
7. Chhikara S., et al. “Combined expression of a barley class II chitinase and type I ribosome inactivating protein in transgenic Brassica juncea provides protection against Alternaria brassicae”. Plant Cell and Organ Culture1 (2011): 83-89.
8. Doughari JH. “An overview of plant immunity”. Journal of Plant Pathology and Microbiology 6 (2015): 322-333.
9. Gachomo EW., et al. “Structural characterization of plant defensing protein superfamily”. Molecular Biology Reports4 (2012): 4461-4469.
10. Gaiser RA., et al. “Production of eukaryotic antimicrobial peptides by bacterial”. (In) Vilas, M. (ed). Science against microbial pathogens. Formatex Research Center 3 (2011): 992-1002.
11. Ghag SB., et al. “Petunia floral defensins with unique prodomains as novel candidates for development of Fusarium wilt resistance in transgenic banana plants”. Plos One 7 (2012): e39557.
12. Glazebrook J. “Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens”. Annual Review in Phytopthology 43 (2005): 205-227.
13. Guest D and Brown J. “Plant defense against pathogens”. Australian vice chnacellors’ committee Canberra (1980): 263-285.
14. Heyn P., et al. “Introns and gene expression: Cellular constraints, transcriptional regulation, and evolutionary consequences”. Bioassays 37 (2015): 148-154.
15. Kumar D., et al. “Alternaria blight of oilseed Brassicas: A comprehensive review”. African Journal of Microbiology Reports30 (2014): 2816-2829.
16. Lacerda AF., et al. “Antifungal defensins and their role in plant defense”. Frontiers in Microbiology116 (2014): 1-10.
17. Lay FT., et al. “Isolation and properties of floral defensin from ornamental tobacco and petunia”. Plant Physiology 131 (2003): 1283-1293.
18. Liu YJ., et al. “Solution structure of the plant defensin VrD1 from mung bean and its possible role in insecticidal activity against bruchids”. Structure and Functional Bioinformatics 63 (2006): 777-786.
19. Loon LCV and Strien EAV. “The families of pathogenesis related proteins, their activities, and comparative analysis of PR-1 type proteins”. Physiology and Molecular Plant Pathology 55 (1999): 85-97.
20. Maarof E., et al. “Cloning and heterogeneous expression of CDef1, a ripening induced defensin in Capsicum annum”. Australian Journal of Crop Science 3 (2011): 123-134.
21. Marques L., et al. “Production of an Aabidopsishalleri foliar defensins in Escherichia coli”. Journal of Applied Microbiology 106 (2009): 1640-1648.
22. Meena PD., et al. “Alternaria blight: a chronic disease in rapeseed mustard”. Journal of Oilseed Brassica1 (2010): 1-11.
23. Mishra A., et al. “Molecular cloning and in silico analysis of functional homologous of hypersensitive response genes induced during pathogenesis of Alternaria Blight in two genotypes of Brassica”. Journal of Protein and Bio Informatics 3 (2010): 244-248.
24. Moosa A., et al. “Transgenic expression of antifungal pathogenesis-related proteins against phyto-pathogenic fungi- 15 years of success”. Israel Journal of Plant Sciences 6 (2017): 122-134.
25. Nanayakkara NMC., et al. “Differential expression of defense-related genes in Synapsis alba and Brassica juncea upon the infection of Alternaria brassicae”. Tropical Agriculture Research3 (2016): 123-136.
26. Nowicki M., et al. “Alternaria black spot of Crucifers: Symptoms, importance of disease, and perspectives of resistance breeding”. Transgenic Research 19 (2012): 373-384.
27. Padovan L., et al. “Techniques for plant defensin production”. Current Protein and Peptide Science3 (2010): 231-239.
28. Peng KS., et al. “Tissue distribution, expression, and antimicrobial activity of Anus platyrphynchos avian β-defensin 6”. Plant Science1 (2013): 97-104.
29. Rawat S., et al. “Isolation and expression analysis of defensin gene and its promoter from Brassica juncea”. Journal of Plant Diseases and Protection6 (2017): 591-600.
30. Sahdev S., et al. “Production of active eukaryotic proteins through bacterial expression system: a review of the existing biotechnology strategies”. Molecular Cell Biochemistry 307 (2008): 249-264.
31. Sels J., et al. “Plant Pathogenesis-related (PR) proteins: A focus on PR peptides”. Plant Physiology and Biochemistry 46 (2008): 941-950.
32. Stotz HU., et al. “Plant defensins: defense, development and application”. Plant Signal Behave 4 (2009): 1010-1012.
33. Terras FR., et al. “Small cysteine-rich antifungal proteins from radish: their role in host defense”. Plant Cell 7 (1995): 573-588.
34. Thevissen K., et al. “Interactionso antifungal plant defensins with fungal membrane components”. Peptides 24 (2003): 1705-1712.
35. Thomma BPHJ., et al. “The complexity of disease signaling in Arabidopsis”. Current Opinion in Immunology 13 (2001): 63-68.
36. Van der Weerden NL., et al. “The plant defensin NaD1 enters the cytoplasm of Fusarium oxysporum hyphae”. Journal of Biochemistry 283 (2008): 14445-14452.
37. Wang YP., et al. “Constitutive expression of pea defensin gene DRR206 confers resistance to blackleg (Leptosphaeriamaculans) disease in transgenic canola (Brassica napus)”. Molecular Plant Microbe Interaction 12 (1999): 410-418.
38. Wu J., et al. “A plant defensin gene from Orychophragmus violaceus can improve Brassica napus resistance to Sclerotinia sclerotiorum”. African Journal of Biotechnology22 (2009): 6101-6109.
39. Zhang Y., et al. “The genetic and molecular basis of plant resistance to pathogens”. Journal of Genetics and Genomics 40 (2013): 23-35.

Citation

Citation: GDG Chaturani., et al. “Two Novel Defensin Genes from Brassica Juncea and Camelina Sativa Confers Antifungal Activity Against Pathogenic Fungi Alernaria brassicae ". Acta Scientific Agriculture 6.2 (2022): 40-49.

Copyright

Copyright: © 2022 GDG Chaturani., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.