Oregon green 488 dextran

For in planta quantitation of leaf apoplastic pH, 25 μM of the ratiometric fluorescent pH indicator dye Oregon Green 488‐dextran (Invitrogen GmbH, Darmstadt, Germany) was loaded with or without 2 × 10⁸ CFU/mL of Pph into the leaf apoplast of intact plants using a needleless syringe. The 10 kDa dextran conjugation ensures that the dye does not access the cytoplasm from the apoplastic space (Geilfus & Mühling 2012). As established previously, all pH responses were monitored starting 2 hpi to ensure evaporation of excess infiltrated water and normal gas exchange within the apoplast (Geilfus & Mühling 2011). Fluorescence images at excitation wavelengths 440/20 and 490/10 nm were collected with a Leica inverted microscope via 50‐fold magnification (0.15 numerical aperture, dry objective; voxel size = 0.002 mm; HCX PL FLUOTAR L, Leica Microsystems) with the optical and instrumental settings described elsewhere (Geilfus & Mühling 2011). Exposure time was 75 ms for both channels. The fluorescence ratio F₄₉₀/F₄₄₀ was obtained as a measurement of pH on a pixel‐by‐pixel basis using LAS AF software (version 2.3.5; Leica Microsystems). Background values were subtracted at each channel. For conversion of the fluorescence ratio data into apoplastic pH, an in vivo calibration was conducted (Fig. S2).

A volume of 50 μL of a 25 μM Oregon Green 488 dextran (Invitrogen GmbH, Darmstadt, Germany) dissolved in deionized water was infiltrated into the leaf apoplast of intact Arabidopsis leaves with the aid of a syringe without needle, by pressing carefully onto the abaxial leaf side. The loaded area appeared darker than its surroundings. Images were taken 1h after the infiltrations as described in Geilfus and Mühling [54 (link)].

For means of in planta recording of leaf apoplastic pH values, 7.5 μg/ml of the fluorescent pH indicator Pt-GFP or 25 μM of the pH-sensitive dye Oregon Green 488-dextran (Invitrogen GmbH, Darmstadt, Germany) were loaded into the leaf apoplast of intact plants following the step-by-step instructions that were given elsewhere [34 (link)]. Measurements were started 2 hours after loading.

Treatments were prepared using the following chemicals: IAA (10 mM stock in ethanol; Sigma-Aldrich), FC (1 mM stock in ethanol; Sigma-Aldrich), estradiol (20 mM stock in DMSO; Sigma-Aldrich). Fluorescein Dextran 10,000 MW, Anionic (D1821, Thermo Fisher) - 10 mg/ml stock in miliQ H₂O, final concentration in media is 29 µg/ml. Oregon Green 488 Dextran, 10,000 MW (D7170, Invitrogen) - 1 mg/ml stock in miliQ H₂O, final concentration in media is 2.5 µg/ml. HPTS (H1529, Sigma-Aldrich), 100 mM stock in H₂O, final concentration in media is 1 mM. FS (F1130, Thermo Fisher), 100 or 50 mM stock in H₂O, final concentration in media is 50 µM.
Induction of PIN2>>AHA2delta95 and PIN2>>PP2C-D1 lines was done by incubating 5-day-old seedlings for 2.5 hr in 1/2 MS MES buffered media (g/l), 1% sucrose, pH 5.8 containing 2 µM estradiol before experiments.

Cells seeded on 35-mm glass-bottom dishes (MatTek, Ashland, MA, USA) were incubated with 5 mg/ml Oregon Green 488 dextran 10 000 MW (Life Technologies) overnight, followed by 2 h chase in dextran-free medium. At the end of the chase period, the cells were rinsed with and maintained in the imaging buffer (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 10 mM HEPES, 10 mM Glucose, pH 7.4). Fluorescence images were acquired using an inverted Nikon ECLIPSE TE200 microscope at excitation wavelengths of 440 nm and 490 nm and emission wavelength of 545±50 nm, with a CCD camera controlled by InCytIM-2 software (Intracellular Imaging Inc., Cincinnati, OH, USA). The 490/440 ratio was calculated for each region of interest representing individual lysosomes or lysosomal clusters and converted into a pH value using the pH calibration curve generated from the same coverslip after the initial ratio measurement. To generate the lysosomal pH calibration curve, potassium isotonic solution (10 mM HEPES, 10 mM MES, 140 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5 mM glucose) of pH 3.5, 4, 4.5, 5, 5.5, 6, 6.5, and 7.0 containing 10 μg/ml nigericin were sequentially added to cells and each was equilibrated for 10 min before ratio images were taken. Lysosomal pH values were obtained by fitting the obtained intensity ratios to the standard curves.

Cells were plated in 12‐well plate at a concentration of 50,000 cells/ml overnight. For IEC18 and RAS3 cells, 12.5 μg/ml of 70 kDa dextran‐Oregon Green 488 (Thermo Scientific) or 12.5 μg/ml of transferrin‐Alexa Fluor 488 (Thermo Scientific) were incubated for 18‐h. For DKO1 and DKS8 cells, 1 ml of culture media with or without 50 μM of EIPA was pre‐incubated for 1‐h and then 20 μg/ml of 70 kDa dextran‐Oregon Green 488 or 20 μg/ml of transferrin‐Alexa Fluor 488 were incubated for 18‐h with or without 50 μM of EIPA. The transferred fluorescent signal was quantified by flow cytometry BD FACSCalibur for IEC18 and RAS3 cells and BD LSRFortessa for DKO1 and DKS8 cells.