Limen, Non-Toxic Recombinant Plant Defensin and Its Effect against Pathogenic Yeast and Fungi

Abstract

Limen is a small cysteine-rich peptide that was originally isolated from lima beans, and its activity against Micromycetes was described. Recombinant expression of limen in both a prokaryotic and plant system was performed to demonstrate a wide selection of its activities with potential for further use. At first limen was expressed in E. coli cells and after purification steps, its antimicrobial activity was tested on pathogenic fungi. This showed that limen can significantly eliminate the growth of several pathogenic yeasts such as Candida albicans, while its hemolytic activity was not recognizable. To confirm limen’s activity in vivo, transformation of the barley Hordeum vulgare L. was chosen. Transgenic barley was obtained using Agrobacterium-mediated transformation and the production of limen peptide in plant tissues was verified by molecular techniques. Young barley plants were repeatedly exposed to Fusarium oxysporum spores. Increased resistance to the most important pathogen was followed using chlorophyll measurements as indicators of fusariosis. Focusing on the food safety aspect of the use of GM plants, cytotoxicity was followed in extracts from transgenic stressed plants. No or a lower cytotoxic effect was found on human keratinocytes and human embryonic kidney cells than extracts from the non-transformed plants.