Flagellin fragment

To ensure that 80% of the stomata were open at the onset of the experiments, A. thaliana plants were placed in the light (100 μmol m⁻² s⁻¹) for 3 h. The epidermis of 3 fully expanded young leaves from the VqTLP29 transgenic lines and Col-0 was peeled off manually and immediately immersed in 10 mM MgCl₂ (mock treatment), DC3000 suspension (OD₆₀₀ of 0.02 in MgCl₂), 5 μM flg22 (Flagellin Fragment, peak area by HPLC ≥95%, Anaspec, USA) in MES buffer (25 mM MES-KOH, pH 6.15 and 10 mM KCl) and 100 ng μl⁻¹ LPS (lipopolysaccharide, Sigma-Aldrich, dissolved in MgCl₂)^{40 (link), 75 (link)}. At 1 h and 3 h time points, treated epidermal peel samples were observed under a microscope (Olympus BX53). Stomatal transverse length and longitudinal width were measured using Image-J. Four treatments were conducted for the transgenic lines and Col-0 using 1 mL needle syringes. Treated leaves were sampled separately at 30 and 60 min and frozen in liquid nitrogen. Leaves of the transgenic lines and Col-0 18 h post treatments were stained with aniline blue to detect callose deposition.

To assure that most stomata were open before experiments were initiated, plants were kept under light (100 mEm⁻²s⁻¹) for at least 3 h [69 (link)]. The epidermis was peeled from fully expanded leaves and immediately immersed in MES buffer (25 mM MES-KOH (pH 6.15) and 10 mM KCl), 5 mM flg22 peptide (Flagellin Fragment, Anaspec, Fremont, CA, USA), or 100 ng/μL LPS (lipopolysaccharide, Sigma). Flg22 was dissolved in MES buffer. LPS was dissolved in MES buffer solution containing 0.25 mM MgCl₂ and 0.1 mM CaCl₂ [70 (link),71 (link)]. At 1 and 3 hpi, samples were placed on glass slides and observed under a microscope. The width and length of the stomatal aperture was measured using the Image-Pro (Olympus Corporation, Tokyo, Japan). For the callose experiment, solution (Mgcl₂ (10 mM), flg22 (5 mM) or LPS (100 ng/μL)) was injected into rosette leaves using 1 mL needleless syringes, respectively. Leaves were stained with aniline blue (dissolved in 150 mM k₂HPO₄ (pH 9.5)) at 18 hpi to detect callose deposition.