title: A new and reliable method for live imaging and quantification of reactive oxygen species in Botrytis cinerea: technological advancement.
abstract: 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.
authors.affiliation: Institut fuer Biologie und Biotechnologie der Pflanzen, Westf. Wilhelms-Universität Muenster, Hindenburgplatz 55, D-48143 Muenster, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Marschall
authors.affiliation: Institut fuer Biologie und Biotechnologie der Pflanzen, Westf. Wilhelms-Universität Muenster, Hindenburgplatz 55, D-48143 Muenster, Germany. Electronic address: tudzyns@uni-muenster.de.
authors.forename: Paul
authors.initials: P
authors.lastname: Tudzynski
mesh_terms: D020171:Botrytis / Q000378:metabolism*; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods; D006861:Hydrogen Peroxide / Q000378:metabolism; D025301:Hyphae / Q000378:metabolism; D019697:Onions / Q000378:metabolism / Q000382:microbiology; D061848:Optical Imaging / Q000379:methods*; D019441:Plant Epidermis / Q000378:metabolism / Q000382:microbiology; D017382:Reactive Oxygen Species / Q000032:analysis* / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D013481:Superoxides; D006861:Hydrogen Peroxide
keywords: Botrytis cinerea; Fluorometric quantification; ROS