Fig. 5
OsCBM1 interacts with OsRacGEF1 and their coexpression enhanced reactive oxygen species (ROS) production. A Split-ubiquitin yeast two-hybrid assays of the “bait” pGBKT7-OsRacGEF1 with the “prey” pGADT7-OsCBM1. B Firefly luciferase complementation imaging (LCI) assay. C MBP-pull down assay, showing the interaction of OsCBM1 with OsRacGEF1 in vitro. D Co-immunoprecipitation (Co-IP) assay, showing the physical interaction of OsCBM1-eGFP with OsRacGEF1-6 × cMyc in vivo. E Transient coexpression of OsCBM1 and OsRacGEF1 in the leaves of Nicotiana benthamiana. The 3,3′-diaminobenzidine (DAB)-stained N. benthamiana leaves were transiently transformed with cMyc (P35S-cMyc), OsCBM1 (P35S-OsCBM1), OsRacGEF1 (P35S-OsRacGEF1), and their combination, respectively. The DAB staining intensity in situ ROS levels of agroinfiltrated N. benthamiana leaves in each treatment was calculated based on the stain intensity of the control cMyc. Bars annotated with different letters represent values that are significantly different (p ≤ 0.05) according to a one-way ANOVA. F Detection of ROS production by H2DCFDA fluorescent probe in N. benthamiana protoplasts isolated from the leaves of N. benthamiana agroinfiltrated by different vectors. Bars = 10 μm. The intensity of fluorescent signals was calculated with ImageJ 1.8.0 software and presented with scatter diagrams (the bottom images in F)