Real-time imaging of hydrogen peroxide dynamics in vegetative and pathogenic hyphae of Fusarium graminearum

Abstract

Balanced dynamics of reactive oxygen species in the phytopathogenic fungus Fusarium graminearum play key roles for development and infection. To monitor those dynamics, ratiometric analysis using the novel hydrogen peroxide (H2O2) sensitive fluorescent indicator protein HyPer-2 was established for the first time in phytopathogenic fungi. H2O2 changes the excitation spectrum of HyPer-2 with an excitation maximum at 405 nm for the reduced and 488 nm for the oxidized state, facilitating ratiometric readouts with maximum emission at 516 nm. HyPer-2 analyses were performed using a microtiter fluorometer and confocal laser scanning microscopy (CLSM). Addition of external H2O2 to mycelia caused a steep and transient increase in fluorescence excited at 488 nm. This can be reversed by the addition of the reducing agent dithiothreitol. HyPer-2 in F. graminearum is highly sensitive and specific to H2O2 even in tiny amounts. Hyperosmotic treatment elicited a transient internal H2O2 burst. Hence, HyPer-2 is suitable to monitor the intracellular redox balance. Using CLSM, developmental processes like nuclear division, tip growth, septation, and infection structure development were analyzed. The latter two processes imply marked accumulations of intracellular H2O2. Taken together, HyPer-2 is a valuable and reliable tool for the analysis of environmental conditions, cellular development, and pathogenicity.

Chasing stress signals – Exposure to extracellular stimuli differentially affects the redox state of cell compartments in the wild type and signaling mutants of Botrytis cinerea

“…Surprisingly, every stress signal seems to be transduced into a changed redox potential. Similar results were previously obtained for redox changes induced by osmotic stress agents in Fusarium graminearum (Mentges and Bormann, 2015).…”

Reactive oxygen species metabolism and plant-fungal interactions

“…Ronen et al (2013) used the pHyPer probe to monitor the time it took to reestablish redox homeostasis after challenge with oxidative stress in a Δchap1 mutant by measuring fluorescent excitation ratios. Subsequently, a more sensitive HyPer2 sensor was developed to measure redox balance in mutants impaired for oxidative stress regulation in F. graminearum (Mentges and Bormann, 2015). This sensor has been further codon optimized from Neurospora crassa for use in M. oryzae and other fungi (Huang et al, 2016).…”

Pathways of pathogenicity: The transcriptional stages of germination in the fatal fungal pathogen Rhizopus delemar

“…Furthermore, harsh environmental conditions can induce the production of ROS internally. For example, changes in osmolarity induce hydrogen peroxide bursts within the hyphae of F. graminearum (63). This remains to be studied in Mucor species.…”