Fig. 2

RoGFP2 fused to BcNoxA reports cytosolic redox state changes. The genetically encoded biosensor roGFP2 was fused to the N- and C-terminus of bcnoxA. a NoxA is predicted as protein containing six transmembrane domains with both ends located inside the cytosol. Prediction was accomplished by the program PRED-TMR [36]. b For roGFP2 measurements, conidia were incubated on microscopic CLSM slides overnight in B5 medium supplemented with glucose (2 %). Measurements were performed by using the excitation wavelengths 395 (first track) and 488 (second track) as well as a 505–530 bandpass filter for collecting images. The ratio of 395/488 images was build and compared to the reference strains (B05.10 with cytosolic or endoplasmic roGFP2). For the induction, the medium was removed and replaced by B5 medium supplemented with glucose (2 %) and 10 mM H₂O₂. c Microscopic analyses of the roGFP fusion constructs revealed that the protein (as described before) is localized to ER structures. For the microscopic analyses the strains were grown in B5 medium supplemented with 2 % glucose overnight on glass slides. White arrows are indicating perinuclear structures, which belong to the ER. Scale bars 10 µm