Extended Data Fig. 1: Endodermis-specific FLS2 induces ectopic lignin but cannot replace endogenous SGN3 for Casparian strip (CS) domain fusion.
(a) CASP1-GFP overviews of 2-in-Endo after 1d treatments of H2O, 100 nM CIF2 or flg22 from initiation to maturation (7th, 11th, 16-17th endodermal cells after onset of elongation). (b) Schematics showing steps to quantify CS discontinuity in overviews to incorporate many more endodermal cells at once: 1. CASP1-GFP overviews: maximum projection of z-stacks capturing CS network in endodermal cell layer; 2. Particle Analysis (Fiji): count fragments and calculate corresponding number of holes; 3. Skeletonize (Fiji): convert area to lines and measure CS length in µm; 4. Calculate ‘Number of holes per 100 µm’ as in (C). (c) Quantification of CS discontinuity in 2-in-Endo CASP1-GFP overviews at mature CS stage after constitutive (5d) treatments with H2O, 100 nM CIF2 or 100 nM flg22 (n ≥ 8). (d) Suberization quantification of 2-in-Endo after constitutive (5d) treatments with H2O, 100 nM CIF2 or flg22. Suberin was stained with Fluorol Yellow and quantified along the root axis divided by three zones: unsuberized (near the root-tip); patchy; and continuous (near hypocotyl). Data presented as percentage of total root length (Mean ± SE; n = 10). Zones with the same letter are not significantly different among treatments (p < 0.05, one-way ANOVA and Tukey test). (e) Diffusion barrier of 2-in-Endo visualized by Propidium Iodide (PI, red) penetration at 25th endodermal cell after onset of elongation. Arrowheads mark PI signal exclusion from the inner side of an endodermal cell (PI block). (f) Transmission Electron Microscopy (TEM) showing in situ H2O2 (ROS) detection with CeCl3 (left) and lignin detection with KMnO4 (right) at the CS of 2-in-Endo after 1d H2O, 100 nM CIF2 or flg22 treatments. Micrographs obtained from sections ~1.5 mm away from the root tip. Brackets indicate CS: uniformly light grey cell wall areas with membrane attachment (left) and dense black staining (right). Arrowheads indicate ectopic accumulations of ROS (left) and lignin (right). (g) CS & CASP1 domain fusion phenotypes of pCASP1::FLS2-GFP fls2 sgn3 CASP1-GFP after 1d treatment with H2O, 100 nM flg22 or 1 µM flg22. Endodermal cell surface views at mature CS stage (16-17th cell) show CASP1-GFP (green), lignin stained with Basic Fuchsin (magenta) and overlaps of the two channels (Merged). Arrowheads highlight ectopic lignin. ‘outer’, cortex-facing, ‘inner’, stele-facing endodermal surface. (h) Quantifications for (G) showing the percentage of gaps in CASP1-GFP pCASP1::FLS2-GFP fls2 sgn3 after 1d treatments (n ≥ 9). (i) PI penetration assay quantifies the CS barrier function of pCASP1::FLS2-GFP fls2 sgn3 with 1d treatments of H2O and flg22 at 100 nM and 1 µM concentrations compared to controls. Roots with no barrier show PI penetration near root-hypocotyl junction, shown in the ‘near hypocotyl’ category and are excluded from numerical statistical test (n ≥ 10). For (C)(H)(I), groups with the same letter are not significantly different (P < 0.05, one-way ANOVA and Tukey test). Unbalanced Tukey test used for unequal replication.
