Title:Comprehensive Structural and Functional Characterization of a Seed
γ-thionin as a Potent Bioactive Molecule Against Fungal Pathogens
and Insect Pests
Volume: 29
Issue: 42
Author(s): Binish Khaliq, Mohnad Abdalla, Sohaib Mehmood, Ahsan Saeed, Aisha Munawar, Muhammad Qamar Saeed, Qamar Saeed, Muhammad Ibrahim, Zahid Ali, Saber Hussain, Wafa Ali Eltayb, Christian Betzel and Ahmed Akrem*
Affiliation:
- Botany Division, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan
Keywords:
Brassica oleracea, γ-thionins, antimicrobial peptides, defensins, entomotoxin, pesticides.
Abstract:
Background: Fungi and insect pests ruin stored crop grain, which results in millions of
dollars of damage, presenting an ongoing challenge for farmers in addition to diminishing the safety
of stored food. A wide-range defensive system against pathogens is needed to reduce or even
eliminate the dependence of the crop yield upon the use of pesticides. Plant defensins (γ-thionins)
are antimicrobial peptides (AMPs) that are a component of the host defense system. They are
known to interact with cell membranes to exhibit antifungal and insecticidal activity. They exhibit
a broad range of activities against fungi and insects and are effective at low concentrations.
Thionins act on membranes, greatly reducing the development of pathogen resistance.
Objective: The aim of this study is to investigate a bioactive molecule that acts against fungal pathogens and
stored grain insect pests.
Methods: γ-thionin protein was extracted from Brassica oleracea L. var. capitata f. alba (white cabbage) seed
powder in phosphate buffer (100 mM, pH 7.0) and was identified by MALDI-TOF/TOF. The crude extract was
subjected to 70% ammonium sulfate saturation followed by gel filtration chromatography. The disc diffusion assay
along with a microtiter bioassay was used to determine the antifungal activity of the protein against phytopathogenic
fungi. The insecticidal efficacy was evaluated by feeding insect pests with food contaminated with
the purified protein. Additionally, an in silico molecular structure prediction study of the protein was performed
using Auto Dock Vina for molecular docking of the protein with either fungal membrane moieties or α-amylase
from Tenebrio molitor L. MD simulations of protein-ligand complexes were conducted using Schrodinger’s
Desmond module.
Results: γ-Thionin (BoT) was purified from white cabbage seeds and showed 100% homology with thionin
(Brassica oleracea L. var. viridis) and 80% homology with defensin-like protein 1 (Raphanus sativus L.), respectively.
BoT significantly inhibited the mycelial growth of Aspergillus niger van Tieghem and Aspergillus
flavus Link at a concentration of 2 μM. Similarly, 0.12 μM BoT treatment resulted in significant mortality of
Tribolium castaneum Herbst and Sitophilus oryzae L. Molecular docking and MD simulation of BoT confirmed
the strong binding affinity with fungal membrane moieties (phosphatidylinositol 4,5-bisphosphate and phosphatidic
acid), which causes disruption of the cell membrane and leakage of the cellular contents, leading to cell
death. BoT blocked the active site of α-amylase, and as a result of the inactivation of this gut enzyme, the digestive
systems of insects were disturbed, resulting in their deaths.
Conclusion: This study revealed that γ-thionin is a good antifungal and insecticidal agent that could be used as
an alternate to fungicides and insecticides.
