Regulation of reactive oxygen species metabolism in early infection stages of Fusarium graminearum on wheat

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Fusarium graminearum is a necrotrophic, filamentous ascomycete and one of the most important phytopathogens worldwide. It is pathogenic to all cereals, causing crop losses and food and feed poising by accumulation of mycotoxins. Fusarium graminearum forms infection cushions (IC), specialized complex infection structures to penetrate the surface of wheat floral leafs which represent the natural infection site. We separated epiphytically growing runner hyphae (RH) from infection cushions on colonized wheat floral leaves by laser dissecting microscopy. Transcriptional profiling of either structure revealed a large set of genes specifically up regulated in RH and IC, respectively. A subset of these genes encodes secreted enzymes, which play a role in the metabolism of reactive oxygen species (ROS), byproducts of many oxidative processes. ROS have different important functions, i.e. gene regulation, cell signaling and development, involvement in apoptosis, and pathogen defense. In necrotophic fungi, it may act as an aggressive agent. They can operate intra- and extracellular. Recent investigations in our group led us to the assumption that production and secretion of ROS represents an important factor for virulence of F. graminearum to wheat. Tight regulation of ROS-equilibrium during early infection stages seems to be crucial for the outcome of the interaction.In the proposed project we will concentrate on 35 secreted ROS-related enzymes (SREs), up regulated during infection cushion development. We will delete the genes starting with functionally related genes with low genetic redundancy, thereby focusing on ROS detoxification. Next, genes of functional groups will be disrupted; starting with the highest expressed ones. Mutants will be analyzed in a broad scale phenotypic screening including ROS metabolism, infection cushion development and penetration, virulence assays on wheat and maize, and DON-measurements. Mutants with interesting phenotypes will be complemented using an inducible expression system. This enables an in-situ complementation on the surface of wheat flower leafs. ROS-dynamics during the initial infection of F. graminearum in wheat will be visualized using the ratiometric hydrogen peroxide sensor protein HyPer, recently adapted for filamentous fungi in our lab.The proposed project is part of the F. graminearum virulence studies in our lab, and the proposed PhD student will be supported by technical assistances, bachelor and master students, and the applicant. The combination of transcriptomic studies, forward genetic tools and life cell imaging using ratiometric sensor proteins represents a powerful tool to identify proteins that are essential for ROS-homeostasis, development of complex infection structures, and for successful penetration and colonization of the plant.