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

摘要

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.