Extended Data Fig. 4: Rescuing TIRBB-loop-triggered cell death by FUS and condensation-active TIR proteins in N. benthamiana.
From: Substrate-induced condensation activates plant TIR domain proteins
a, Confocal images of N. benthamiana transiently expressing GFP-tagged RPP1-TIRBB-loop and FUS-RPP1TIRBB-loop. Scale bar = 50 μm. The nucleus was shown in the corner. Scale bar = 10 μm. Images show a projection of fluorescent images acquired along the z-axis. b, Cell death phenotype of N. benthamiana plants transiently expressing GFP-tagged RPP1TIRBB-loop and FUS-RPP1TIRBB-loop. Top: Ion leakage assay at 3 dai. Results from three independent experiments are shown (n = 18 biologically independent samples; analyzed by one-way ANOVA with Tukey’s HSD test, p = 0.001). Bottom: Photograph shows a representative leaf after agro-infiltration at 4 dai. c, The NADase activity of RPP1TIRBB-loop protein and FUS- RPP1-TIRBB-loop protein. The NADase activity of the wild type RPP1-TIR was normalized to 100%. Data are mean ± SD (n = 3 biologically independent samples). d, The schematic for the rationale of RBA1E86A-induced RBA1BB-loop condensation. The cartoon images were created with BioRender.com. e, Western blot analysis of total N. benthamiana leaf protein extracts at 2 dai probed with anti-GFP and anti-mCherry antibodies. Ponceau S indicates equal loading of total leaf proteins on the blot. f, Confocal images of N. benthamiana leaves infiltrated with GFP-tagged RBA1BB-loop and mCherry-tagged RBA1E86A at different ratios. The numbers indicate the concentration (OD600) of each Agrobacterium solution used for infiltration. Left: views under 10× objective. Scale bar = 50 μm. Right: views under 40× objective. Scale bar = 50 μm. Images show a projection of fluorescent images acquired along the z-axis. g, Cell death phenotype of N. benthamiana plants infiltrated with GFP-tagged RBA1BB-loop and mCherry-tagged RBA1E86A at different ratios. Top: Ion leakage assay at 3 dai. Results from three independent experiments are shown (n = 18 biologically independent samples; analyzed by one-way ANOVA with Tukey’s HSD test, p = 0.001). Bottom: Photograph shows a representative leaf after agro-infiltration at 4 dai. h, Western blot analysis of total N. benthamiana leaf protein extracts at 2 dai probed with anti-GFP and anti-mCherry antibodies, respectively. The long exposure panel of anti-mCherry displays the low expressions of mCherry-tagged RBA1E86A in 0.8:0.05 and 0.8:0.1 samples, which are invisible on the short exposure panel. Ponceau S indicates equal loading of total leaf proteins on the blot. i, Images of 3-week-old transgenic Arabidopsis plants RBA1p:RBA1/Col-0 and RBA1p:RBA1BB-loop/Col-0. Scale bar = 1 cm. For the box plots, the centre line indicates the median, the bounds of the box show the 25th and the 75th percentiles, and the whiskers indicate 1.5 × IQR. The experiments in a, e, f and h were repeated at least three times with similar results.
