Extended Data Fig. 5: GBPL droplets are unique LLPS structures conserved across plant species, cell types and during development.
From: A phase-separated nuclear GBPL circuit controls immunity in plants
a, Coomassie staining of purified rEGFP protein. b, Standard curve of rEGFP via confocal microscopy. c, Quantitative measurement (mean ± SD) of EGFP–GBPL3 concentration 24 h after salicylic acid (0.5 mM), Psm (OD600 = 0.001) and Pst (OD600 = 0.001) from multiple 4-week-old plant nuclei. Comparison of mean via one-way ANOVA test with Holm–Sidak post hoc test. Individual data points represent biologically independent samples. d, e, Colocalization analysis of EGFP–GBPL3 in N. benthamiana (d, live cell) and HeLa cells (e, fixed cell). The latter enabled parallel antibody detection of subnuclear structures. DSB, double strand break. DNA stained with Hoechst 33342 dye. Bar, 5 μm (d, N.B.) and 2 μm (e, HeLa). f, Immunofluorescence of native GBPL3 in Col-0 and Col-0/GBPL3OX plants. Nuclei were isolated from 3-week-old soil-grown plants and DNA stained with Hoechst 33342. chr, chromocentre (white arrows). Bar, 2 μm. Experiment repeated once with similar results. g, Left, GBPL3 condensates in Arabidopsis cotyledons stably expressing 35S::EGFP-GBPL3. Shown are maximum projection images. Dashed circle, nuclear boundary. Bar, 5 μm. Right, salicylic acid-induced GBPL3 condensates (mean ± SD, Student’s t-test, two-tailed) in guard cells from 10-day-old Arabidopsis cotyledons (n = 4 biologically independent plants with 100 guard cells each). Mock, water treatment. h, Localization of EGFP-tagged tomato and maize GBPs in HeLa cells. DNA stained with Hoechst 33342 dye. Bar, 5 μm (SlGBP2 and ZmGBP1) and 2 μm (SlGBP1 and ZmGBP2). i, Maximum intensity projection images of EGFP–GBPL3 transiently expressed in N. benthamiana (N.B.), human HeLa and HEK 293T cells. Bar, 2 μm. j, Immunogold transmission electron microscopy of spheroid GDACs (dashed circles) expressed in HEK 293T cells. NE, nuclear envelope. NPC, nuclear pore complex. Bar, 500 nm. k, GBPL3 condensates were sensitive to 1,6-hexanediol (5%) but not 1,2,6-hexanetriol (5%). Digitonin (10 μg/ml) was used to facilitate chemical delivery.
