Open AccessRedox‐sensitive GFP2: use of the genetically encoded biosensor of the redox status in the filamentous fungus Botrytis cinerea
Author(s) -
HELLER JENS,
MEYER ANDREAS J.,
TUDZYNSKI PAUL
Publication year - 2012
Publication title -
molecular plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.945
H-Index - 103
eISSN - 1364-3703
pISSN - 1464-6722
DOI - 10.1111/j.1364-3703.2012.00802.x
Subject(s) - botrytis cinerea , appressorium , glutathione , dithiothreitol , biology , hypha , reactive oxygen species , redox , microbiology and biotechnology , biochemistry , confocal microscopy , mutant , botrytis , biophysics , chemistry , botany , enzyme , gene , organic chemistry
SUMMARY The production of reactive oxygen species (ROS) is part of the defence reaction of plants against invading pathogens. The effect of ROS on filamentous fungi is still unclear. In this study, ratiometric redox‐sensitive green fluorescent protein (roGFP) was introduced as a tool for in vivo measurement of the cellular redox status in filamentous fungi. A fungal expression system for roGFP2 was constructed. Expressed in Botrytis cinerea , roGFP2 reversibly responded to redox changes induced by incubation with H 2 O 2 or dithiothreitol, which was determined by confocal laser scanning microscopy imaging and fluorometry. As the sensor detects the redox potential of the cellular glutathione pool, it was used to analyse the kinetics of GSH (glutathione, reduced form) recovery after H 2 O 2 treatment. The transcription factor Bap1 is the main transcriptional regulator of H 2 O 2 ‐scavenging proteins in B. cinerea. When compared with the wild‐type, GSH recovery in the Δbap1 deletion mutant was affected after repeated H 2 O 2 treatment. ROS and intracellular redox changes can be used by fungi for signalling purposes. In planta experiments, performed in this study, indicated that redox processes seem to be important for the differentiation of penetration structures. During the penetration of onion epidermal cells, the status of the cellular glutathione pool differed between appressoria‐like structures and infecting hyphae, being reduced in the presence of infecting hyphae and more oxidized around appressoria‐like structures.