Molecular characterization of diverse defensins (γ-thionins) from Capsicum annuum flowers and their effects on the insect pest Helicoverpa armigera

Abstract

Plant defensins (γ-thionins) are antimicrobial peptides (AMPs) constituting the host defense system. They are known to interact with the cell membranes to exhibit antifungal and antibacterial activity. Though defensin genes are inducible under biotic and abiotic stress, their involvement in insect herbivore defense is not well studied. In this work we studied the phylogeny, structural diversity, biochemical and functional properties of defensins from Capsicum annuum L with particular emphasis on their interaction with insect and fungal pathogens. Phylogenetic analysis of defensin peptides from Solanaceae, Pinaceae, Fabaceae, Asteraceae, Brassicaceae and similar peptides from invertebrates divided them into ten groups and Capsicum peptides were represented in most of them. Three thionin (CanThio-1, −2, −3) and two defensin (CanDef-20, −21) genes isolated from the flowers of Capsicum annuum were cloned and recombinant proteins were expressed. The mature peptide amino acid sequences of CanThio and CanDef were 54–75 aa with 21.82 to 73.81% similarity. They contained the conserved features of defensin peptides like the gamma core region and the presence of eight cysteine residues. Predicted structural alignment indicated that CanThio and CanDef had structural diversity and structurally unaligned loop regions.The recombinant CanThio and CanDef inhibited 5% - 65% Aspergillus oryzae amylase activity at 20 μM and ~ 20% of Helicoverpa armigera gut amylase activity at 50 μM peptide concentrations. CanThio-2 displayed bovine trypsin inhibition (~14%) while all the CanThio and CanDef inhibited ~20% of H. armigera gut proteinase activity at 20 μM. H. armigera larvae fed on an artificial diet incorporated with recombinant CanThio-2 and CanDef-20 (125 μg/ml) exhibited a 15% reduction in larval mass and 13% reduction in pupal mass. The CanThio and CanDef peptides also exhibited antifungal properties against pathogenic fungus Fusarium oxysporum at 5 μg/ml, where CanThio-2 and CanDef-20 showed the strongest activity, which may be attributed to the positively charged amino acids in the γ-core region.We conclude that despite having relatively similar conserved three-dimensional structures CanThio and CanDef display functional variability, which can be utilized for enhancing insect and fungal resistance in plants.