Extended Data Fig. 3: The subcellular localization of TIR condensates and TIR condensation in epss N. benthamiana.
From: Substrate-induced condensation activates plant TIR ___domain proteins
a, Left, confocal images of DAPI-stained N. benthamiana transiently expressing RPP1-TIR-GFP, RBA1-GFP and TX14-GFP. Middle, Confocal images of N. benthamiana transiently co-expressing GFP-tagged TIRs and the plasma membrane marker SYP122-mScarlet. Right, Confocal images of N. benthamiana transiently co-expressing GFP-tagged TIRs and the endomembrane marker ARA6-RFP. Scale bar = 50 μm. Images on the left and right show a projection of fluorescent images acquired along the z-axis. b, Confocal images of N. benthamiana transiently co-expressing GFP-tagged TIRs and P-body marker DCP1-mCherry (left panel) or stress granule marker UBPA-mCherry after heat shock at 42 °C for 5 min (right panel). Scale bar = 20 μm. Images show a projection of fluorescent images acquired along the z-axis. c, Western blot analysis of total N. benthamiana leaf protein extracts at 2 dai probed with anti-GFP antibodies. Ponceau S indicates equal loading of total leaf proteins on the blot. d, Confocal images of N. benthamiana transiently expressing full-length RPP1 without or with the cognate effector ATR1. Left lane: views under 40× objective. Scale bar = 50 μm. Images show a projection of fluorescent images acquired along the z-axis. Right three lanes: enlarged views of nucleus shows colocalization of RPP1-FL-GFP and DAPI-stained nucleus. Scale bar = 10 μm. e, Confocal images of N. benthamiana transiently expressing RBA1-GFP and RBA1-NBD-LRR-GFP. Scale bar = 50 μm. Images show a projection of fluorescent images acquired along the z-axis. f, Western blot analysis of total N. benthamiana leaf protein extracts at 2 dai probed with anti-GFP antibodies. Ponceau S indicates equal loading of total leaf proteins on the blot. g, Cell death phenotype of N. benthamiana leaves transiently expressing TIR-NBD-LRR fusions. Photograph shows a representative leaf after agro-infiltration at 4 dai. h, Photographs of representative leaf zones of epss N. benthamiana plants transiently expressing GFP-tagged RPP1-TIR, RBA1 and TX14 at 4 dai. i, Confocal images of epss N. benthamiana transiently expressing GFP-tagged RPP1-TIR, RBA1 and TX14 at 2 dai. At least five leaves were detected for each infiltration, all showing the similar results. Scale bar = 50 μm. Images show a projection of fluorescent images acquired along the z-axis. j, Split-luciferase assay of TIR-induced EDS1-SAG101 interaction with NRG1. The Split-Luc N and Split-Luc C were fused to SAG101 and NRG1, respectively. RPP1 (wild type or different variants), ATR1, EDS1 (wild type or small molecule binding site mutant), SAG101 and NRG1 were co-expressed in epss N. benthamiana. Results from five independent experiments are displayed (n = 10 biologically independent samples; analyzed by one-way ANOVA with Tukey’s HSD test, p = 0.05). 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–i were repeated at least three times with similar results.
