Fig. 4
Reactive oxygen species (ROS) accumulation in infected transgenic A. thaliana plants. a 3,3'-diaminobenzidine (DAB, indicating H2O2 accumulation) and nitroblue tetrazolium (NBT, indicating superoxide anion [·O2−] accumulation) staining at 24 hpi. One representative replicate from the three independent experiments is shown. Three leaves were stained in each experiment. b and c Quantification of H2O2 accumulation and ·O2− accumulation in the infected tissue (necrotic tissue including the tissue 0.5 cm around the lesion edge) of transgenic A. thaliana plants. Data are presented as means ± SEM (n = 6, independent experiments). d Relative expression level of AtPRXIIB in syn-ROS and control lines at 24 hpi by normalizing with the quantity of A. thaliana AtActin7 and AtActin2 cDNA. Data are presented as means ± SEM (n = 3, technical replicates). The relative expression of the target gene in CK was set as one. e Relative expression level of SsGpx3 in syn-ROS and control lines at 24 hpi by normalizing with the quantity of S. sclerotiorum Sstubulin and SsActin cDNA. Data are presented as means ± SEM (n = 3, technical replicates). The relative expression of the target gene in CK was set as one. f ROS accumulation in A. thaliana protoplasts of infected syn-ROS and control lines at 24 hpi as detected by the fluorescent probe 2',7'-dichlorofluorescein diacetate (H2DCFDA). Scale bars = 20 μm. g ROS accumulation in S. sclerotiorum protoplasts of infected syn-ROS at 24 hpi as detected by the fluorescent probe H2DCFDA. Scale bars = 5 μm. h Quantification of relative fluorescent intensity in f and g. Data are presented as means ± SEM (n = 3, independent experiments). The fluorescent images of the protoplasts were captured using a confocal microscope. Different letters and asterisks indicate significant difference at p < 0.05 (b and c, one-way ANOVA followed by Tukey’s post hoc test; d–h, two-sided Student’s t-test). CK: A. thaliana plants carrying the empty vector