An in vitro generated variant of Tephrosia villosa defensin (α-TvD1) enhances biotic stress tolerance in transgenic tobacco

Abstract

Plant defensins are small, cationic, cysteine-rich peptides with well conserved four disulphide bridges between an α-helix and three anti-parallel β-strands, produced by plants as a first line of innate immunity against the invading fungal pathogens. Some of the plant defensins exhibit strong antibacterial and anti-insect activities in vitro as well as in vivo. TvD1, a defensin from Tephrosia villosa was characterized in our previous studies with demonstrated inhibitory effect against many plant pathogenic fungi and some insects. To further enhance its activity, α-TvD1, a peptide variant of TvD1, was generated through site directed mutagenesis. It showed enhanced antifungal and Tenebrio molitor α-amylase inhibitory activity under in vitro condition. In the present study, we compared the antifungal and anti-insect activities of α-TvD1 with the native TvD1 in transgenic tobacco plants. α-TvD1 transgenic plants showed enhanced tolerance against the oomycetes pathogen Phytophthora parasitica var. nicotianae and the fungal pathogens Alternaria alternata and Rhizoctonia solani. Also, it showed enhanced anti-insect activity against the 1st and 2nd instar larvae of the generalist herbivore, Spodoptera litura. These results demonstrated that α-TvD1 appears to be a potent antimicrobial gene that can be used for generating superior transgenic crops with enhanced resistance against many phytopathogens and insect pests.