Modulating Membrane Fluidity and Microtubules

The membrane “rigidifier” Dimethylsulphoxide (DMSO, 2% v/v; Roth, Karlsruhe, Germany), and the membrane “fluidiser” Benzylalcohol (BA, 10 mM, Roth, Karlsruhe, Germany) were used to modulate plasma-membrane fluidity^{66 (link)–68 (link)}. The microtubule compounds taxol and oryzalin were employed to stabilise or disrupt microtubules, respectively^{38 (link)}. Diphenyleneiodonium chloride (DPI, 100 µM; Sigma-Aldrich, Deisenhofen, Germany) was used to inhibit apoplastic oxidative burst by the plasma membrane located NADPH oxidases^{69 (link)}, and Gadolinium chloride (GdCl₃, 100 µM; Sigma-Aldrich, Deisenhofen, Germany) to block the calcium channels^{66 (link)}. All inhibitors were diluted from a stock solution in DMSO, the maximal concentration of the solvent was 0.1%, and the experimental design, therefore, included one set with 0.1% DMSO as solvent control. To probe for the effect of microtubule stability, cells were treated with 10 μM taxol (Sigma-Aldrich, Deisenhofen, Germany) prior to elicitation by harpin or flg22. To eliminate microtubules, we added 10 μM of oryzalin (Sigma-Aldrich, Deisenhofen; Germany) 1 h prior to elicitation. A solvent control with 0.1% DMSO was included throughout.