In Silico and In vitro Investigation of Plant Defensin as potent agents to combat antipathogen applications

Abstract

Plant health is significantly impacted by pathogen diseases, which has led to the development of complex defense mechanisms, such as the production of defensins, which are cationic antimicrobial peptides essential to a plant's innate immune system. Plant defensins are a primary source of innate immunity in plants. Structurally, they are highly stable, small, cysteine-rich antimicrobial peptides. Fungal cells have been reported as the most invading pathogen among biotic flora. The fungal cell wall is rich in receptors such as MAP Kinase receptor and Phosphatidic acid receptor. In this study, a bioinformatic approach has been made to study the interactions of different plant defensins with the fungal cell wall receptors. Around 20 plant defensins have been identified, and their protein sequences were retrieved from biological databases. Docking studies showed that plant defensins showed stronger interactions with cell wall receptors coding for MAPK and phosphatidic acid pathways in the cytosol with the lowest binding energies and zero rmsd values. The in vitro studies aimed to clone the MsDef1 plant defensin gene from Medicago sativa into E. coli to enable large-scale production and additional investigation. Using the phenol-chloroform technique, genomic DNA was extracted from Medicago seeds and analyzed using agarose gel electrophoresis. Gradient PCR was used to amplify the MsDef1 gene with an optimal annealing temperature of 59°C, producing a unique 252 bp DNA band. For quantitative RT-PCR analysis, total RNA was separated, subjected to DNase treatment, and converted into cDNA. Defensin gene expression was verified in comparison to the internal control, β-actin. The heat shock transformation method was used to insert the MsDef1 gene into E—coli after it was cloned into the pCambia1301 vector. The growth of white colonies served as an indicator of a successful transformation, and transformed colonies were chosen on an LB medium containing IPTG and Kanamycin. The recombinant bacterial cell was transfected into Arabidopsis plant and selected transgenic plants were selected on MS media supplemented with kanamycin. The ability to clone plant defensin genes into E. coli and its transfection into the host plant has been demonstrated by this work, offering a valuable method for mass-producing defensin proteins for use in agriculture and medicine.