Investigation of the reactive oxygen species metabolism during the life cycle of Fusarium graminearum

Abstract

F. graminearum is a necrotrophic filamentous ascomycete that is able to infect all major cereal crops causing plant diseases such as Fusarium Head Blight in wheat. The infection process is mediated mainly via differentiation of fungal cells into complex multicellular organs, so called infection cushions (ICs). In previous work an RNAseq-based transcriptomic and functional analysis indicated major transcriptional rearrangements between ICs and non-invasive cells. Data analysis revealed that expression of enzymes involved in the metabolism of reactive oxygen species (ROS) was elevated in ICs. ROS are integral components of every aerobic cell’s metabolism and are formed as by-products of oxygen-based cellular reactions. While being harmful to the cell structure when accumulating, ROS are also necessary for cellular functions serving as an important second messenger mediating cellular differentiation. Particularly, they are essential elements in plant-pathogen interactions, such as fungal infection processes. Only few ROS-related enzymes involved in these interactions are known to this day, however. The aim of this study was to gain further insights into the role of ROS and specific ROS-related enzymes, particularly secreted ones, in different aspects of the fungal life-cycle. Via gene deletions 9 monooxygenases (4 of them secreted), 5 secreted peroxidases, 2 secreted oxidases, 1 secreted dehydrogenase, 1 secreted reductase, 2 secreted cupredoxins, 3 metallothioneins, 1 NAD(P) transhydrogenase (NNT), and 1 secreted superoxide dismutase (SOD) were disrupted. Mutants were tested for virulence, vegetative growth, ROS-sensitivity, ROS-accumulation, and fertility. It could be shown that secreted peroxidases are involved in vegetative growth, ROS-accumulation, and fertility. Other ROS-related enzymes deleted in this study caused no effect on the tested parameters. This low number of phenotypes indicates a high resilience of F. graminearum against disruptions of its ROS-metabolism. It seems that the ROS-equilibrium which the fungus seeks to maintain during infection is a highly secured system, fortified by a large array of enzymes with redundant function. Further experiments for gaining insights into F. graminearum-related ROS fluctuations were based on the genetically encoded ratiometric H2O2 probe HyPer which reacts towards H2O2 with an increase of the ratio of its two fluorescence peaks. HyPer had been expressed in the cytosol of F. graminearum previously (cytHyPer). In this study a GPI-anchor was attached to the probe. Organelle-specific staining revealed that this modified HyPer (GPI-HyPer) is attached to the endoplasmic reticulum (ER) and mitochondria. GPI-HyPer was used in ER-stress experiments. Substitution of GPI-HyPer expressing strains with brefeldin A and tunicamycin showed no reaction from GPI-HyPer suggesting that ER-stress in F. graminearum is not linked to a significant change of the ER’s H2O2-level. Deletion of the NADP(H) oxidase regulator NoxR in cytHyPer and in GPI-HyPer expressing strains revealed a significant increase of the ratio of cytHyPer but not of GPI-HyPer. This showed, for the first time, that a NoxR deletion-mediated ROS-accumulation in F. graminearum is not caused by an increase of the ER’s H2O2-level.