Novel Antimicrobial Activities of Trichoderma hamatum GD12 Following Deletion of Heterochromatin Protein 1

Abstract

Heterochromatin Protein 1 (HEP1) is a highly conserved, chromatin re-modelling protein involved in activation and repression of secondary metabolite producing gene clusters. In-house genome sequencing of the plant growth promoting and biocontrol fungus Trichoderma hamatum GD12 has shown that ~40 % of the genome is unique to GD12 compared to its closest relatives, suggesting enormous genetic potential to encode novel bioactive compounds with antimicrobial and PGP activities. It is apparent that under axenic conditions, a substantial proportion of the bioactive potential of the fungus is not expressed. We therefore hypothesised that loss of HEP1 would lead to activation of cryptic gene clusters responsible for the production of novel bioactive secondary metabolites. Identification of compounds with antimicrobial activities might benefit a growing population faced with numerous multidrug resistant microorganisms. HEP1 was inactivated in T. hamatum GD12 using the split-marker method of homologous recombination and ΔThhepA::hph strains were confirmed via DIG-labelled Southern blot analysis. Phenotypic analysis revealed significantly reduced hyphal growth of hepA mutants compared to GD12. Confrontation assays of GD12 and three independent ΔThhepA::hph strains against fungal pathogens revealed a change in the biocontrol activities, with a zone of inhibition surrounding mutant strains suggesting the secretion of inhibitory bioactive compound(s). Liquid chromatography-mass spectrometry was used to determine the secretome profiles of hepA mutants. Analysis of the data revealed a number of key features which are differentially expressed in hepA mutants. One such feature of particular interest is Brefeldin A, which functions as an antimicrobial agent. This project would benefit from characterisation of key features to determine their antimicrobial potentials.