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Münster, Germany

Heller J.,Westf Wilhelms University | Meyer A.J.,University of Bonn | Tudzynski P.,Westf Wilhelms University
Molecular Plant Pathology | Year: 2012

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 H2O2 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 H2O2 treatment. The transcription factor Bap1 is the main transcriptional regulator of H2O2-scavenging proteins in B. cinerea. When compared with the wild-type, GSH recovery in the Δbap1 deletion mutant was affected after repeated H2O2 treatment. ROS and intracellular redox changes can be used by fungi for signalling purposes. Inplanta 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. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD. Source

Tudzynski P.,Westf Wilhelms University | Heller J.,Westf Wilhelms University | Siegmund U.,Westf Wilhelms University
Current Opinion in Microbiology | Year: 2012

Reactive oxygen species (ROS) generated by NADPH-dependent oxidases (Nox) have been shown to function as signaling molecules and to be essential for many differentiation processes in mammals and plants. There is growing evidence that ROS are important for many aspects of fungal life including vegetative hyphal growth, differentiation of conidial anastomosis tubes, fruiting body and infection structure formation, and for induction of apoptosis. Recent results from studies in fungal saprophytic and pathogenic model systems have shed new light on the role of Nox in cytoskeleton organization, the structure of Nox complexes and links to components of the apical complex, and the localization of Nox to the endoplasmic reticulum. © 2012 Elsevier Ltd. Source

Heller J.,Westf Wilhelms University | Ruhnke N.,Westf Wilhelms University | Espino J.J.,Westf Wilhelms University | Massaroli M.,University of Cadiz | And 2 more authors.
Molecular Plant-Microbe Interactions | Year: 2012

The mitogen-activated protein kinase (MAPK) BcSak1 of Botrytis cinerea is activated upon exposure to H2O2 and, hence, might be involved in coping with oxidative stress during infection. However, beside osmotic and oxidative stress sensitivity, Δbcsak1 mutants have a pleiotropic phenotype, as they do not produce conidia and are unable to penetrate unwounded host tissue. In this study, the role of BcSak1 was investigated in the stress response and during infection of French beans by Botrytis cinerea. Using a macroarray approach, it was shown that BcSak1 is only marginally involved in the specific oxidative stress response. In fact, the induction of several genes after oxidative stress treatment is BcSak1-dependent, but most of these genes are also induced under conditions of osmotic stress. The majority of genes regulated by BcSak1 are not involved in the stress response at all. Using a translational fusion of BcSak1 to green fluorescent protein, it was shown clearly that the localization of this MAPK depends on the type of stress being applied; it associates rapidly to the nucleus only under osmotic stress. Therefore, a model is proposed in which BcSak1 acts in the cytosol by activation of one or more transcription factors under oxidative stress and, at the same time, it reacts to osmotic stress by migrating to the nucleus. Interestingly, the MAPK is also involved in the regulation of secondary metabolism, as the major phytotoxins secreted by this fungus are reduced in the Δbcsak1 deletion mutant. Experiments done in planta underlined the essential role of BcSak1 in the early stages of infection, when it translocates to the nucleus and then changes to cytosolic distribution during hyphal growth within the tissue. © 2012 The American Phytopathological Society. Source

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