Antimicrobial Activity of a Cys-Rich Peptide Derived from a <i>Centrosema virginianum</i> Vicilin

Abstract

Many antimicrobial peptides (AMPs) have been identified in plants. These peptides are highly divergent at the primary sequence level and vary in their hierarchical structures. Some common biochemical features include the ability to form disulfide bonds, tandemly repeated amino acid sequences and a net charge at pH 7. Unusual Cysteine containing repeats has been identified in several plant seed storage proteins that may act as AMPs. We identified a Cys repeat within a vicilin (seed storage protein) of a wild legume, Centrosema virginianum . Cleavage of the vicilin protein during germination would generate a vicilin derived Cys peptide (VDCP). We investigated the antimicrobial properties of this VDCP and compared its efficacy as an antimicrobial agent to VDCPs from other species. We developed transgenic tobacco plants that expressed cloned sequences encoding the Cysteine repeat unit from C. virginianum, Theobroma cacao and Gossypium hirsutum . Extracts from fully expanded leaves were tested for antimicrobial activity against a fungal pathogen, Botrytis cinerea . The Cys motif from C. virginianum was also expressed in two E. coli cell lines (reducing or oxidizing cytoplasm) and peptide fusion protein fractions were tested for antimicrobial activity against a battery of fungal strains. The unique Cysteine repeat single unit from C. virginianum exhibited antimicrobial properties greater than or equal to the antimicrobial activity associated with expression of the multiple Cys-repeat VDCPs from G. hirsutum or T. cacao in transgenic tobacco. When expressed in bacteria, a C. virginianum VDCP fusion protein exhibited antifungal activity against 3 of the 4 fungi tested. Although the primary role of seed storage proteins is to provide a pool of amino acids and nitrogen for germinating seeds and developing plantlets, it is likely that seed storage protein proteolytic products also provide beneficial antimicrobial properties during germination and young plantlet development.