A new and reliable method for live imaging and quantification of reactive oxygen species in Botrytis cinerea: Technological advancement

• A method allowing simultaneous imaging of superoxide and peroxideis first applied in a filamentous fungus.
• The method allowsreliable fluorometrical ROS quantificatione.g. in different growth phases and different strains/mutants.
• Superoxide is produced in an oscillation like pattern within Botrytis cinerea.
• Superoxide accumulates in the hyphal tips during formation of appressoria and penetratinghyphae.
• Mutants in different subunits of NADPH oxidase complexes differ in production of hydrogen peroxide but not of superoxide.

Reactive oxygen species (ROS) are produced in conserved cellular processes either as by-products of the cellular respiration in mitochondria, or purposefully for defense mechanisms, signaling cascades or cell homeostasis. ROS have two diametrically opposed attributes due to their highly damaging potential for DNA, lipids and other molecules and due to their indispensability for signaling and developmental processes. In filamentous fungi, the role of ROS in growth and development has been studied in detail, but these analyses were often hampered by the lack of reliable and specific techniques to monitor different activities of ROS in living cells.Here, we present a new method for live cell imaging of ROS in filamentous fungi. We demonstrate that by use of a mixture of two fluorescent dyes it is possible to monitor H2O2 and superoxide specifically and simultaneously in distinct cellular structures during various hyphal differentiation processes. In addition, the method allows for reliable fluorometric quantification of ROS. We demonstrate that this can be used to characterize different mutants with respect to their ROS production/scavenging potential.