Redox-sensitive GFP2: use of the genetically encoded biosensor of the redox status in the filamentous fungus Botrytis cinerea

Abstract

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.

Figure 1

The redox‐dependent fluorescence of the ratiometric redox‐sensitive green fluorescent protein (roGFP2) expressed in the cytosol of Botrytis cinerea hyphae is fully reversible. Conidial suspensions were germinated overnight and analysed microscopically. Z‐Stacks of images were taken by confocal laser scanning microscopy (CLSM) with excitation at 405 nm and 488 nm, respectively. Average projections of these Z‐stacks (top and middle row) were used for the calculation of the ratio images (bottom row). The colour scale for the ratio values indicates reduced roGFP2 in blue and oxidized roGFP2 in yellow. Medium, hypha growing in minimal medium before the addition of H₂O₂; H₂O₂, hypha 10 min after the addition of 10 mm H₂O₂; DTT, hypha 10 min after the addition of 10 mm dithiothreitol. Scale bar, 10 µm.

Figure 2

Characterization of the ratiometric redox‐sensitive green fluorescent protein (roGFP2) expressed in Botrytis cinerea hyphae using a fluorometer. Germlings of a roGFP2‐expressing wild‐type (WT) strain were used for fluorescence measurements at a microplate reader. Relative fluorescence units were measured with 3 × 3 reads per well after excitation with a wavelength of 395 ± 5 nm for the oxidized state and 488 ± 5 nm for the reduced state of roGFP2. Colonies were initially grown in GB5 medium to which a stressor (stressor) and 100 mm dithiothreitol (DTT), respectively, were added. (A) Example of the measurement in one well. After the addition of the stressor (10 mm H₂O₂), the fluorescence intensity in the 488‐nm channel decreases and the fluorescence intensity in the 395‐nm channel increases. As a result, the ratio (395 nm/488 nm) increases. With the addition of DTT, the initial status is obtained. (B) Correlation of the amplitude of the ratio change with the H₂O₂ concentration. (C) Specificity of the roGFP2 reaction to oxidizing agents. (B, C) The indicated values are the means of five different experiments; standard deviations are indicated by error bars.

Figure 3

Washout experiment showing the independent reduction of the glutathione pool in Botrytis cinerea hyphae after treatment with H₂O₂. Z‐Stacks of images were taken by confocal laser scanning microscopy (CLSM) with excitation at 405 nm and 488 nm, respectively. Average projections of these Z‐stacks were used for the calculation of the ratio images. The colour scale for the ratio values indicates reduced roGFP2 (ratiometric redox‐sensitive green fluorescent protein) in blue and oxidized roGFP2 in yellow. Numerical ratio values were calculated using the software ImageJ and plotted against the time. Hyphae were initially grown in GB5 medium. Oxidative stress was induced by replacing the medium with GB5 containing 100 mm H₂O₂ (H₂O₂). To observe self‐sufficient reduction of the glutathione pool, this medium was replaced by fresh GB5 medium again (medium). A reducing environment was created using 2 mm dithiothreitol (DTT). The graph shows an example of five different experiments with similar results.

Figure 4

Comparison of the reaction of the ratiometric redox‐sensitive green fluorescent protein (roGFP2) and Grx1‐roGFP2 expressed in Botrytis cinerea hyphae using a fluorometer. The response of Grx1‐roGFP2 is faster than the response of roGFP2, as shown by the faster induction of the ratio to the maximum level after 3 min. The indicated values are the means of five different experiments; standard deviations are indicated by the error bars.

Figure 5

Characterization of the strain Δbap1:roGFP2.22. (A) H₂O₂ sensitivity of the strain Δbap1:roGFP2.22 (Δbap) in comparison with the strain B05.10:roGFP2.1 (wild‐type, WT). Strains were cultivated for 3 days on complete medium (CM) supplemented with 10 mm H₂O_2. (B) Redox‐dependent fluorescence ratio of the ratiometric redox‐sensitive green fluorescent protein (roGFP2) expressed in the cytosol of Botrytis cinerea strain Δbap1:roGFP2.22. Z‐Stacks of images were taken by confocal laser scanning microscopy (CLSM) with excitation at 405 nm and 488 nm, respectively. Average projections of these Z‐stacks were used for the calculation of the ratio images. Colour scale: reduced roGFP2, blue; oxidized roGFP2, yellow. Scale bar. 10 µm.

Figure 6

Comparison of the reaction of the ratiometric redox‐sensitive green fluorescent protein (roGFP2) expressed in Botrytis cinerea strain B05.10:roGFP2.1 (WT) and strain Δbap1:roGFP2.22 (Δbap) using a fluorometer. Reduction of roGFP2 is displayed after a single oxidation event using 10 mm H₂O₂. There is no difference in glutathione pool recovery between the two strains in this experiment. The indicated values are the means of six different experiments; standard deviations are indicated by the error bars.

Figure 7

Comparison of the reaction of the ratiometric redox‐sensitive green fluorescent protein (roGFP2) expressed in Botrytis cinerea strain B05.10:roGFP2.1 (wild‐type, WT) and strain Δbap1:roGFP2.22 (Δbap) using confocal laser scanning microscopy (CLSM). Z‐Stacks of images were taken by CLSM with excitation at 405 and 488 nm, respectively. Ratio images of average projections of these Z‐stacks were used for the calculation of numerical ratio values employing the software ImageJ. Oxidation of roGFP2 after H₂O₂ treatment (100 mm) progresses in a similar manner in both strains. However, after a second H₂O₂ treatment, a reduced state of roGFP2 in the strain Δbap1:roGFP2.22 is not achieved, even when dithiothreitol (DTT) is added to create reducing conditions. The indicated values are the means of four different experiments; standard deviations are indicated by the error bars.

Figure 8

Analysis of the redox status of the ratiometric redox‐sensitive green fluorescent protein (roGFP2) expressed in Botrytis cinerea during the penetration of onion epidermal cells. Z‐Stacks of images were taken by confocal laser scanning microscopy (CLSM) with excitation at 405 nm and 488 nm, respectively. Average projections of these Z‐stacks were used for the calculation of the ratio images. The colour scale for the ratio values indicates reduced roGFP2 in blue and oxidized roGFP2 in yellow. Numerical ratio values were calculated using the software ImageJ. Infecting hyphae were divided into three parts: 1, hyphae growing on the surface of the onion epidermis; 2, appressoria‐like structures; 3, penetrating hyphae. A selection from four experiments is shown (A–D). Further experiments were performed that gave similar results. Scale bars, 10 µm.