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. 2019 Feb 19;180(1):543–558. doi: 10.1104/pp.18.01503

Figure 7.

Figure 7.

Overproduction of BAK1 D287A enhances SoBIR1-dependent cell death. A, Expression of BAK1 and BAK1D287A under different promoters in N. benthamiana. Leaves of 4-week-old N. benthamiana were hand-inoculated with Agrobacteria carrying 35S::GFP, 2x35S::BAK1, 2x35S::BAK1D287A, 35S::BAK1, 35S::BAK1D287A, pBAK1::BAK1, or pBAK1::BAK1D287A. Pictures were taken under UV light (left) or visible light (right) 3 d after inoculation. Two d after inoculation and before cell death progressed, protein levels of BAK1 were analyzed by western blot (WB) with an α-BAK1 antibody (bottom). BAK1 proteins from 2x35S::BAK1 (2) and 2x35S::BAK1D287A (3) were detected by WB using SuperSignal West Pico Chemiluminescent Substrate, and BAK1 proteins from 35S::BAK1 (4) and 35S::BAK1D287A (5) were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). B, The fluorescent signal from the circled areas in (A) was obtained under the UV light and was quantified as means ± sd from 10 biological repeats. The asterisks indicate statistical significance by using Student’s t test (P < 0.05). C, Measurement of electrolyte leakage of leaf discs from (A). Data are shown as means ± sd from three biological repeats. The asterisks indicate statistical significance by using Student’s t test (P < 0.05). D, Overexpression of BAK1 and BAK1D287A in Arabidopsis. Arabidopsis plants overexpressing 35S::BAK1 or 35S::BAK1D287A were photographed at the 4-week-old stage. Each phenotype of T1 generation plants was calculated as a percentage to the total transgenic plants of that genotype. E, Cell death and H2O2 accumulation in the leaves of 4-week-old plants were examined by trypan blue and DAB staining, respectively. B; normal-looking plants; S; small plants. F, Expression levels of PR genes in 1-week-old transgenic plants were determined by RT-qPCR. The data are shown as means ± sd from three biological repeats. The asterisks indicate statistical significance by using Student’s t test (P < 0.05). G, BAK1 and BAK1D287A cell death is SoBIR1-dependent and BIK1-independent. 35S::BAK1 and 35S::BAK1D287A were transformed into sobir1 and bik1 mutant backgrounds. Each phenotype of T1 generation at the 4-week-old stage was calculated as a percentage as in (D). The above experiments were repeated three times with similar results.