Fig. 3: Active SUMM2 immobilizes ADR1-L1 puncta, forming a ring-like pattern.
From: Assembly of helper NLR resistosome clusters upon activation of a coiled-coil NLR
a, SUMM2ac stimulates ADR1-L1–TagRFP discontinuous distribution on the PM under confocal microscopy. ADR1-L1–TagRFP was co-expressed with DEX::SUMM2–HA or DEX::SUMM2ac–HA in N. benthamiana. Maximum-intensity projection represents 16 images with a z-step of 1 µm. b, ADR1-L1–GFP exhibits punctate signals on the PM in mekk1 under confocal microscopy. c, SUMM2ac stimulates ADR1-L1–TagRFP to form immobile spots with a ring-like pattern under TIRF microscope (×4 magnification; right). d, SUMM2ac restricts ADR1-L1–TagRFP mobility. Single-particle trajectories (top). Data of D values represent mean ± s.e.m. with each dot representing a single cell (ADR1-L1–TagRFP n = 13 and ADR1-L1–TagRFP+SUMM2ac n = 11; bottom left). Data from MSD analysis represent mean ± s.e.m. from pooled trajectories (bottom right). e, SUMM2ac promotes progressive assembly of ADR1-L1–TagRFP resistosome clusters. Images of clusters (dashed lines) containing different numbers of oligomers with mean cluster area quantified using ImageJ (n = 12; left). Distribution of resistosome clusters containing different numbers of oligomers (4 h, n = 157; 6 h, n = 193; right). f, Fusion of ADR1-L1–TagRFP molecules into resistosomes with timelapse TIRF images (white arrow). g, Violin plots of relative fluorescence intensity of ADR1-L1–TagRFP particles. The violin plot represents pooled data from 18–20 cells detected by TIRF microscopy without (−; n = 1,625) or with (4 h, n = 454; 6 h, n = 315) SUMM2ac. Plots display the median and interquartile range, with the violin shape representing the kernel density distribution of fluorescence intensities. h, ADR1-L1–GFP resistosomes assemble into clusters in mekk1 under a TIRF microscope. i, ADR1-L1–GFP clusters contain two to six oligomers in mekk1. n = 67 (right). j, SUMM2ac promotes spatial clustering of ADR1-L1–TagRFP. Heatmaps show the spatial distribution of ADR1-L1–TagRFP particles. Each white dot marks the centroid of a particle detected by TIRF microscopy. The warmer colours indicate regions of higher particle density (colour scale on the right). Experiments were repeated three times with similar results. Two-tailed Student’s t-test (d) or two-sided Kruskal–Wallis test, followed by pairwise Mann–Whitney U-tests with Bonferroni correction for multiple comparisons (g) were performed.
