Volume 27, Issue 16, Pages 2522-2528.e4 (August 2017) A Novel Plasma Membrane-Anchored Protein Regulates Xylem Cell-Wall Deposition through Microtubule-Dependent Lateral Inhibition of Rho GTPase Domains  Yuki Sugiyama, Mayumi Wakazaki, Kiminori Toyooka, Hiroo Fukuda, Yoshihisa Oda  Current Biology  Volume 27, Issue 16, Pages 2522-2528.e4 (August 2017) DOI: 10.1016/j.cub.2017.06.059 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2017 27, 2522-2528.e4DOI: (10.1016/j.cub.2017.06.059) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 IQD13 Regulates Secondary Cell-Wall Pit Shape (A) Localization of IQD13-GFP (LexA:IQD13-GFP) in a cultured xylem cell. Secondary cell walls are labeled with fluorescent wheat germ agglutinin (WGA). (B) Localization of IQD13-GFP and cortical microtubules (35S:tagRFP-TUB6) in a cultured xylem cell. In (A) and (B), the lower panels show magnification of the boxed region in the upper panels. (C) Localization of IQD13-GFP (pIQD13:IQD13-GFP) in metaxylem vessel cells in roots. Cell walls are labeled with propidium iodide (PI). (D) IQD13 mRNA levels in LexA:IQD13-RNAi and LexA:GUS-RNAi plants. Values are mean ± SD (n = 3), ∗∗p < 0.01 (Student’s t test). (E) Differential interference contrast (DIC) images of xylem vessels in roots. Arrowheads indicate enlarged secondary cell-wall pits. (F and I) Aspect ratio and surface area of secondary cell-wall pits in the RNAi plants (F) and in the LexA:IOD13 plants (I). Values are mean ± SD (n > 300 pits), ∗∗∗p < 0.001 (Student’s t test). (G) IQD13 mRNA levels in LexA:IQD13 plants treated with (+Est) or without (−Est) 2 μM estradiol. Values are mean ± SD (n = 3), ∗∗p < 0.01 (Student’s t test). (H) Secondary cell walls of metaxylem vessels in roots. Secondary cell walls were stained with Safranin O. Arrowheads indicate narrow elongated secondary cell-wall pits. Scale bars, 10 μm (A and B) and 5 μm (C, E, and H). See also Figure S1 and Movie S1. Current Biology 2017 27, 2522-2528.e4DOI: (10.1016/j.cub.2017.06.059) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 IQD13 Increases the Density of Cortical Microtubules in Non-xylem Cells (A and C) Cortical microtubules (GFP-TUB6) and IQD13-tagRFP in cultured non-xylem cells. The maximum-intensity projection of the z stack image (A) and single plane of the cell cortex (C) are shown. Arrowheads indicate the ends of cortical microtubules that are not decorated with IQD13. In (A), the lower panels show magnification of the boxed region in the upper panels. (B) Density of cortical microtubules. Values are mean ± SD (n > 30 cells), ∗∗p < 0.01 (Student’s t test). (D) Time-lapse images of cortical microtubules and IQD13-tagRFP shown in the blue boxes in (C). White arrowheads indicate a growing microtubule not decorated with IQD13. (E) Kymograph of the microtubule indicated by yellow arrowheads in (D). (F) Kymograph of a microtubule in non-xylem cells expressing GFP-TUB6, but not IQD13-tagRFP. Scale bars, 10 μm (A and C) and 2 μm (D–F). See also Figure S2, Table S1, and Movie S2. Current Biology 2017 27, 2522-2528.e4DOI: (10.1016/j.cub.2017.06.059) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 IQD13 Interacts with the Plasma Membrane and Microtubules (A) Diagram of full-length IQD13 and truncated IQD13 fragments. PM, MT, and CY indicate plasma membrane, microtubule, and cytoplasm localization, respectively. Gray domains indicate conserved motifs among IQD family members [12]. Numbers indicate the positions of amino acid residues. (B) Truncated (84–166, 251–310, 381–456, and 301–412) IQD13-tagRFP and cortical microtubules (GFP-TUB6) in leaf epidermal cells of N. benthamiana. (C) Mid-plane of leaf epidermal cells expressing full-length (FL) or truncated (1–83 and 21–100) IQD13-GFP together with tagRFP-ROP11. N, nucleus. Intensity profiles along the yellow lines are shown (right). a.u., arbitrary units. (D) Cortical microtubules (GFP-TUB6) in leaf epidermal cells (Control) and in cells expressing full-length (FL), ΔMT, or ΔPM IQD13-tagRFP. (E) Cortical microtubule density. Values are mean ± SD (n > 50 cells), ∗∗∗p < 0.001 (ANOVA with Scheffe test). Scale bars, 10 μm. See also Figure S3. Current Biology 2017 27, 2522-2528.e4DOI: (10.1016/j.cub.2017.06.059) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 IQD13 Regulates the Shape of Reconstituted Active ROP Domains (A) Cortical microtubules (YFP-TUB6) and active ROP domains (tagRFP-MIDD1ΔN) reconstituted in leaf epidermal cells of N. benthamiana co-expressing ROP11, ROPGEF4PRONE, and ROPGAP3 (top), together with IQD13FL-ECFP (middle) or ΔPM-ECFP (bottom). Intensity profiles along the white lines are shown (right). Scale bars, 10 μm. (B) Aspect ratio of reconstituted active ROP domains. Values are mean ± SD (n > 200 domains from >50 cells), ∗∗∗p < 0.001 (ANOVA with Scheffe test). (C) Schematic model of the spatial control of active ROP domains by IQD13. Low IQD13 expression levels lead to the formation of sparse cortical microtubules, which weakly inhibit the diffusion of active ROP11, resulting in large, round active ROP domains (left). High IQD13 expression levels lead to the formation of dense cortical microtubules with tight associations to the plasma membrane, which strongly inhibit the diffusion of active ROP11, resulting in narrow active ROP domains (right). (D) Schematic illustration of IQD13 associating with cortical microtubules and the plasma membrane to spatially control the active ROP domains. See also Figure S4. Current Biology 2017 27, 2522-2528.e4DOI: (10.1016/j.cub.2017.06.059) Copyright © 2017 Elsevier Ltd Terms and Conditions