Figure 5
From: A Rho-based reaction-diffusion system governs cell wall patterning in metaxylem vessels
ROPGEF4PRONE interacts with GTP-ROP11 and inhibits the diffusion of ROP11 in tobacco leaf epidermal cells. (A) BiFC assay between nYFP-ROPGEF4PRONE and cYFP-ROP11, cYFP-ROP11G17V, cYFP-ROP11T22N, or cYFP-ROPGEF4PRONE. Scale bars = 20 µm. (B) FRET efficiency between CFP-ROPGEF4PRONE and YFP-ROP11 derivatives or YFP-ROPGEF4PRONE. Data are means ± SD (n = 12). *P < 0.05; ***P < 0.001; ANOVA with Scheffe’s test. (C and D) FRAP analysis of GFP-ROP11 and tagRFP-ROPGEF4PRONE. GFP-ROP11 was expressed alone (NONE) or together with tagRFP-ROPGEF4PRONE, tagRFP-ROPGEF4, or tagRFP-ROPGAP3 in tobacco leaf epidermal cells. Kymograph of photo-bleached area at the mid plane of leaf epidermal cells (C; top); intensity plots of photo-bleached area (C; bottom); and fluorescence recovery percentage 60 s after photo-bleaching (D) are shown. In (C), t indicates time after the onset of scan. x indicates position along the plasma membrane. Photo-bleach was executed at 6 s. Data are means ± SD (n = 12). Bar  = 5 µm. ***P < 0.001; **P < 0.01; ANOVA with Scheffe’s test. Note that the recovery of GFP signals at the edges of photo-breached area is faster than that at the centre of the area in C (None and +tagRFP-GAP3). (E) FRAP analysis of tagRFP-ROPGEF4PRONE and GFP-ROPGAP3 in ROP11-activated domains. ROP11 was co-expressed with tagRFP-ROPGEF4PRONE and GFP-ROPGAP3. Kymograph of photo-bleached area (left top) and intensity of photo-bleached area (left bottom), and recovery of fluorescence at 60 s after photo-bleaching (right). Data are means ± SD (n = 15). Scale bar = 1 µm. ***P < 0.001; Welch’s t test. (F to I) Schematic model of secondary cell wall pit patterning. (F) The ROP-activation cycle generates periodic ROP-activated domains by reaction-diffusion mechanism. Positive feedback is provided by ROPGEF4 dimers interacting with GTP-ROP11. (G) ROPGEFs and ROPGAPs determine the density of ROP-activated domains. (H) The size and shape of ROP-activated domains are determined through the action of microtubule-associated proteins (MAPs). ROP-activated domains promote depletion of cortical microtubules. (I) Secondary cell walls are deposited on the area outside of the ROP11-activated domains, resulting in the formation of secondary cell wall pits.
