Dissecting the events of the Trichoderma-plant interaction through proteomics and metabolomics approaches: Trichoderma virens and maize as model systems : A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Lincoln University

In nature, almost every plant is colonised by fungi. Trichoderma virens is a biocontrol fungus which has the capacity to behave as an facutaltive plant endophyte. Even though many plants are colonised by this symbiont, the exact mechanisms by which it masks its entrance into its host plant remain unknown. They are likely to involve a) the secretion of different protein families into the apoplast and b) the modulation of the plant metabolome. Both mechanisms may play crucial roles in the suppression of plant immune responses to facilitate colonisation. The aim of this study was to investigate the molecular and cellular mechanisms of maize root colonisation by T. virens. To achieve this, the T. virens-maize interaction was investigated under hydroponic growth conditions using confocal microscopy, proteomics and metabolomics approaches, and functional characterisation of the 2-oxoglutarate/Fe (II)-dependent dioxygenase gene tvox1, from T. virens. This study demonstrated that upon host penetration, T. virens colonises inter- and intracellular spaces and modifies morphology and physiology of maize roots. Using a gel-free shotgun proteomics approach, secreted proteins from maize and T. virens were identified. A reduction in the maize secretome was induced by T. virens, including two major groups, glycosyl hydrolases and peroxidases. T. virens secreted proteins were mainly involved in cell wall hydrolysis, scavenging of reactive oxygen species and secondary metabolism, as well as putative effector proteins. Therefore, demonstrating that T. virens secretes an arsenal of proteins into the apoplast to facilitate colonisation of maize root tissues. In addition, the role of the T. virens tvox1 up-regulated gene in secondary metabolism and iron regulation during the T. virens-maize interaction was explored by gene deletion. The functional analysis of TVOX1 suggested that it participates in the regulation of siderophore production and iron homeostasis in T. virens. To further explore the relevance of tvox1 during the T. virens-maize interaction, an untargeted metabolic fingerprinting approach by HPLC-DAD-ESI-QTOF-MS/MS of maize roots following T. virens (WT and Δtvox1) colonisation revealed a modulation of the maize metabolome, showing general and specific metabolic responses of maize to both T. virens genotypes. Moreover, this work showed that the deletion of tvox1 which is involved in fungal secondary metabolism shifted the metabolic flux in T. virens. Overall, three main mechanisms are proposed as the molecular interplay between T. virens and maize: a) glycosyl hydrolases and lignification, b) reactive oxygen species homeostasis, and c) iron and secondary metabolism.