Oryzalin

The following compounds were used: oryzalin (36182; Sigma-Aldrich), 5-iodotubercidin (5-ITu) (I100; Sigma-Aldrich), PD-180970 (PZ0142; Sigma-Aldrich), PD-166326 (9000988; Cayman Chemical), PD-173955-Analog1 (SYN-1062; SYNkinase), ponatinib (CS-0204; CHEMSCENE), bosutinib (PZ0192; Sigma-Aldrich), bafetinib (A10119; AdooQ Bioscience), PP2 (P0042; Sigma-Aldrich), PP1 (BML-EI275; Enzo Life Sciences), 4-aminopyrazolo[3,4-d]pyrimidine (PP2-Analog1) (A1041; Tokyo Chemical Industry), 1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidine-4-amine (PP2-Analog2) (GF-0723; Key Organics), PP3 (A2737; Tokyo Chemical Industry), and Src inhibitor1 (sc-204303; Santa Cruz Biotechnology) (Karni et al, 2002 (link); Wisniewski et al, 2002 (link); Hum et al, 2014 (link); Rossari et al, 2018 (link)).
Each compound was dissolved in DMSO and used at a final concentration of 10 μM in 0.1% DMSO, except for the dosage assay, and in oryzalin, which was used at 1 μM. oryzalin (10 μM) was used in the P. patens assay.

The deaerated-leaf sections were immersed in a solution containing 10, 25, 50, or 100 μM oryzalin (Sigma-Aldrich, St Louis, MO, USA) or 0.1, 1.0, or 10 μM Latrunculin B (Wako Pure Chemical Co. Ltd., Osaka, Japan) for 3 h under white light conditions. Alternatively, leaf sections were kept at 4°C for 1 h. Images were captured as Z-sections by CLSM. The number of individual fibers visualized by sfGFP-TP in a cell was manually counted by using Cell Counter tool in Fiji (ImageJ, NIH public domain) (S5 Fig in S1 Data). At least 11 cells from three different sections were quantified.

As a control for potential false positive results from the fluorescent labelling of F-actin and microtubules, specimens were treated with probes that influence the polymerisation of actin and tubulin: jasplakinolide (JAS, Invitrogen; a toxin that stabilises actin filaments and induces actin polymerisation), cytochalasin D (Invitrogen, Czech Republic; a drug disrupting actin filaments and inhibiting actin polymerisation), and oryzalin (Sigma-Aldrich, Czech Republic; a dinitroaniline herbicide acting through the disruption/depolymerisation of microtubules). Drugs were reconstituted in dimethyl sulfoxide to prepare a 1 mM stock solution. The final concentration of these membrane-permeable probes lower than 5 μM had no obvious effect. To obtain reliable results on vital cells, final solutions of 10 and 30 μM JAS, cytochalasin D and oryzalin prepared in filtered (0.22 μm Millipore) sea water were applied. Controls were performed in pure filtered sea water as well as corresponding concentrations of dimethyl sulfoxide in filtered sea water.

Arabidopsis wild type (Col-0 accession), MKKK20 (SALK_021755 and SALK_124398), MPK18 (SALK_069399), and MKK3 (SALK_051970) T-DNA mutants were obtained from TAIR¹ (for T-DNA line details, see Supplementary Table S1). Seeds were grown in growth cabinets at 21°C with a light/dark cycle of 16 h/8 h photoperiod. Primers used to screen for homozygous lines are listed in Supplementary Table S2. For root growth assays (elongation rate and skewing angles), seeds were grown aseptically on Hoagland medium solidified with 1.2% agar. For oryzalin (Sigma–Aldrich) treatments, small volumes of a concentrated stock solution in DMSO were added to the molten agar; controls received the same amount of DMSO. Seeds sown on agar plates with or without oryzalin were kept for at least 7 days and placed vertically in a growth cabinet (21°C, light/dark cycle of 16 h/8 h and 120 μmol m^-2 s^-1).

Chemicals were exogenously applied by incubating 5-day-old seedlings in liquid 1/10th strength MS medium and supplementing them with 500 µM LaCl₃ (Sigma-Aldrich), 500 µM GdCl₃ (Sigma-Aldrich), 5 mM EGTA (Sigma-Aldrich), or 25 μM oryzalin (Sigma-Aldrich) for 2 h or 16 h, or with 250 µM bis-(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (BAPTA) (Sigma-Aldrich) or 1 µM latrunculin B (Abcam) for 2 h or 16 h. The duration of the treatments was based on the general toxicity caused by the different chemical compounds in plants. To visualize Hechtian strands, 5-day-old cotyledon epidermal cells expressing the different markers were plasmolyzed for 4 h using 0.4 M mannitol. The images are an overlay of propidium iodide-stained cell walls with the localization of the GFP fusion proteins in green.