Extended Data Fig. 10: The active SUMM2 induces the formation of ADR1-L1 resistosome clusters.
From: Assembly of helper NLR resistosome clusters upon activation of a coiled-coil NLR
a-e, SUMM2-GFP subcellular dynamics with and without activation. a, TIRF microscopy images (exposure time: 95 ms) of Arabidopsis epidermal cells expressing pSUMM2::SUMM2-GFP, two weeks after VIGS with a vector control (Ctrl) or MEKK1. Bottom panels show kymographs derived from time-lapse TIRF imaging over 10 sec (acquisition rate: 8 frames per sec), illustrating comparable lateral mobility of SUMM2-GFP in Ctrl and RNAi-MEKK1 conditions. Scale bar, 5 µm. b, Representative single-particle trajectories reconstructed from TIRF image sequences (8 frames per sec) show comparable diffusion behavior of SUMM2-GFP in Ctrl and RNAi-MEKK1 conditions. Scale bar, 5 μm. c, Distribution of diffusion coefficients (D) represented as log (D) reveals similar distributions of SUMM2-GFP mobility under both conditions. d, Quantification of peak D values (log-transformed) from normal distribution fits of individual cells. Data represent mean ± SEM with each dot representing a single cell. Total sample sizes were: RNAi-Ctrl (n = 21), RNAi-MEKK1 (n = 23). No significant difference was observed between Ctrl and RNAi-MEKK1 conditions. e, Mean square displacement (MSD) analysis over time for all tracked SUMM2-GFP particle trajectories, showing comparable diffusion behavior between Ctrl and RNAi-MEKK1 conditions. Data represent mean ± SEM from pooled trajectories. f-j, SUMM2-GFP and SUMM2ac-GFP exhibit similar mobility dynamics at the plasma membrane. f, TIRF microscopy images (exposure time: 95 ms) of Arabidopsis epidermal cells expressing DEX::SUMM2-GFP or DEX::SUMM2ac-GFP. Transgenic plants were treated with 50 μM DEX and imaged 3 days post-treatment. Bottom panels show kymographs derived from time-lapse TIRF imaging over 10 s (acquisition rate: 8 frames per sec), illustrating comparable lateral mobility of SUMM2-GFP and SUMM2ac-GFP particles at the plasma membrane. Scale bar, 5 µm. g, Representative single-particle trajectories of SUMM2-GFP and SUMM2ac-GFP molecules reconstructed from TIRF time-lapse recordings (acquisition rate: 8 frames per second). Scale bar, 5 µm. h, Probability distribution of diffusion coefficients (D), plotted as log (D), shows similar profiles for SUMM2-GFP and SUMM2ac-GFP particles. i, Quantification of peak D values (log-transformed) from normal distribution fits of individual cells. Data represent mean ± SEM with each dot representing a single cell. Total sample sizes were: SUMM2-GFP (n = 18); SUMM2ac-GFP (n = 18). No significant difference was observed between SUMM2-GFP and SUMM2ac-GFP. j, MSD analysis over time of all tracked particle trajectories, showing comparable diffusion behavior between SUMM2-GFP and SUMM2ac-GFP. Data represent mean ± SEM from pooled trajectories. k, Comparison of ADR1-L1 puncta under the TIRF microscope (top panel) and confocal microscope (bottom panel). Scale bar, 1 μm. l, SUMM2ac stimulates the progressive assembly of ADR1-L1-TagRFP resistosome clusters under the TIRF microscope. ADR1-L1-TagRFP was co-expressed with a vector (-) or DEX::SUMM2ac-HA in N. benthamiana. Two days post-infiltration, SUMM2ac was induced by 50 μM DEX treatment for 4 or 6 hr, and the images were taken using TIRF microscopy (see also Fig. 3c). Scale bar, 5 μm. m, 3D projections of the SUMM2ac-induced ADR1-L1-TagRFP puncta were visualized using a depth color code. Scale bar, 5μm. 3D projections were created from 12 images acquired with a z-step of 0.05 µm using the LAS X software and color-coded according to depth to highlight the vertical distribution of the structures. Insets denote the regions zoomed in on the bottom panel. n, Quadrat count analysis used to assess the spatial randomness of ADR1-L1-TagRFP particle distributions. Schematic illustrating the quadrat-based spatial analysis applied to TIRF-derived particle coordinates shown in Fig. 3j. The region of interest (ROI) was divided into a 9 × 8 grid of equal-area quadrats. The number of ADR1-L1-TagRFP particles (depicted as circles) within each quadrat was counted. The resulting observed frequency distribution was compared to a Poisson distribution expected under complete spatial randomness (CSR) using a chi-squared (χ²) goodness-of-fit test. Distributions were classified as significantly clustered when p < 0.05 and consistent with randomness when p > 0.05. o, Avrblb2/Rpi-blb2 induces SlNRC3-GFP puncta formation and resistosome clusters in the PM. SlNRC3-GFP was co-expressed with Rpi-blb2 or Avrblb2 and Rpi-blb2 in N. benthamiana nrc2/3/4 leaves. Confocal images were taken two days after Agrobacterium infiltration. Scale bar, 10 μm (top). TIRF images were taken three days post-infiltration using a Leica DMi8 Infinity microscope. Scale bar, 5 μm (bottom). Upon Avrblb2 recognition, SlNRC3-GFP transitions from a diffuse localization to form immobile punctate clusters. p, EDS1-PAD4 complex localizes in the nucleus and cytoplasm. EDS1-nYFP and PAD4-cYFP were co-expressed in N. benthamiana for two days by Agrobacterium infiltration, and the YFP signals were detected using a confocal laser scanning microscope. Scale bar, 25 μm. Experiments were repeated three times with similar results.
