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Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), are deployed as defense molecules across kingdoms, e.g., by plants in order to defeat invading pathogens like fungi. Necrotrophic plant pathogenic fungi themselves secrete H2O2 to induce host cell death and facilitate infection. Hydrogen peroxide is, to a certain extent, freely diffusible through membranes. To be able to monitor intracellular hydrogen peroxide dynamics in fungi, we recently established the versatile HyPer-imaging technique in the filamentous plant pathogen Fusarium graminearum (Mentges and Bormann, 2015). HyPer consists of a circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain (RD) of the prokaryotic H2O2-sensing protein, OxyR. The OxyR domain renders the sensor highly specific for H2O2. Oxidation of HyPer increases fluorescence of cpYFP excited at 488 nm and decreases fluorescence excited at 405 nm, thereby facilitating ratiometric readouts (Belousov et al., 2006). HyPer turned out to be pH-sensitive. A single amino acid mutation in the H2O2-sensing domain of HyPer renders the sensor insensitive to H2O2. This reporter is called SypHer and serves as a control for pH changes.By using the HyPer-imaging technique, we could demonstrate that: i) HyPer imaging enables the specific and accurate detection of rapid changes in the intracellular H2O2 balance, ii) F. graminearum reacts on external stimuli with the transient production of H2O2, and iii) faces increased H2O2 level during initial infection of wheat.The aim of this protocol is to guide the user through the basic setup of an in vitro HyPer imaging experiment in basically any fungus. It will provide the specific parameter for the fluorescence imaging as well as the construction of customized flow chambers for in vitro applications. |
