Use of the Non-Essential Yeast Deletion Collection to Define the Antifungal Mechanisms of Plant Defensins

K. Parisi1, M. Bleackley1, N. van der Weerden2, M. Anderson1

Author address: 

Trobe Univ., Melbourne, Australia, 2Hexima Limited, Melbourne, Australia

Abstract: 

Background: Plant defensins are potent antifungal peptides that represent a novel class of antifungal drug with excellent potential for clinical application. Plant defensins share a similar three-dimensional structure but vary significantly in sequence. This variation in sequence is thought to account for the different mechanisms of action observed for plant defensins. We have identified several novel antifungal plant defensins with high sequence variability and potentially novel mechanisms of action.Methods: To define the mechanism of action of each defensin we employed a Saccharomyces cerevisiae gene deletion library that contains 5000 unique knockout strains. Each knockout strain is identifiable by an individual barcode incorporated into the gene during deletion. A pool of this library was treated in triplicate with eight different defensins alongside an untreated control. Following defensin treatment, genomic DNA was isolated from the treated yeast. The barcoded region was amplified and sequenced using next-generation sequencing to determine the abundance of each strain. From this analysis, we determined the fitness of 5000 yeast deletion strains in the presence of a specific defensin.Results: We identified several strains that were sensitive or resistant to all eight defensins indicating that elements of the activity of plant defensins are conserved. These strains may represent general stress response genes. However, the clustering revealed elements that are unique to each defensin and suggests at least three different mechanisms of action. Two of the defensins we tested appear to have a conserved mechanism of action which correlates with their degree of sequence identity.Conclusions: Understanding how plant defensins exert their antifungal activity will assist in selection of defensins with different antifungal mechanisms which can be used in therapeutics to create broad spectrum resistance to fungi.
2016

abstract No: 

SUNDAY-392

Full conference title: 

ASM Microbe 2016
    • ASM microbe 1st (2016)