Silwet l 77

Bacterial strains used in this study were P. syringae pv. tomato DC3000 (Pst DC3000), P. syringae pv. tomato DC3000 hrcC (Pst hrcC⁻), and P. syringae pv. maculicola ES4326 (Psm ES4326). The strains were grown on NYGA media (5 g/L bactopeptone, 3 g/L yeast extract, 20 ml/L glycerol, and 15 g/L agar) containing rifampicin (50 mg/l) at 28°C for 48 h. For bacterial enumeration assays, plants were sprayed with the strains (inoculum: OD600 nm = 0.2 for Pst DC3000 and Pst hrcC⁻ and OD600 nm = 0.002 for Psm ES4326), suspended in 10 mM MgCl₂ containing 0.001% (v/v) Silwet L-77 (Bio world). Sprayed plants were covered with a transparent plastic lid for the remaining time of the experiment. For bacterial titers, leaf discs from three different leaves per plant were harvested and washed, and then bacteria were extracted using 10 mM MgCl2 containing 0.04% (v/v) Silwet L-77. Quantification was done by plating appropriate dilutions on LB agar media containing rifampicin (50 mg/l) and incubated at 28°C for 2 days, after which the bacterial colonies were counted.

GUS assay was performed as described previously^{70 (link)} with minor modifications. Briefly, seedlings were grown in 24-well plates containing liquid LS medium supplemented with 0.5% sucrose under 16 h/8 h day/night cycle in a Percival plant growth chamber at 22 °C under a light intensity of 50 μmol m⁻² s⁻¹. Plants were inoculated at day 12 with bacterial strains. Bacterial strains were grown on R2A plates at 22 °C for 3 d, resuspended in 10 mM MgCl₂ and added to seedlings in LS medium without sucrose at OD₆₀₀ of 0.002. After treatment with SynCom strains for 5 h, seedlings were rinsed with 0.5 ml 50 mM sodium phosphate buffer (pH 7) and submerged in 0.5 ml GUS staining solution (50 mM sodium phosphate (pH 7), 0.5 mM K₄[Fe(CN)₆], 0.5 mM K₃[Fe(CN)₆], 1 mM X-Gluc (GoldBio, G1281C) and 0.01% Silwet-L77 (Bioworld, 30630216)). After vacuum infiltration for 10 min, plates were incubated at 37 °C overnight. Plants were fixed with a 3:1 ethanol:acetic acid solution at 4 °C for 1 d followed by transfer to 95% ethanol.

Bacterial infection assay was done based on a modified protocol according to the previous report^{67 (link)}. Briefly, seven-day-old Arabidopsis seedlings were chosen to do the Xcc flood-inoculation assay. Bacteria were harvested and resuspended into 40 mL of 0.02% Silwet L-77 (bioWORLD, USA), containing 10 mM MgCl₂, to a final concentration at OD = 0.1. The inoculum was dispensed into the 1/2 MS plate containing Arabidopsis seedlings, and the plates were incubated for 1 min at room temperature. Then the bacteria were removed by decantation, and the plates were sealed with 3 M Micropore Surgical Tape (3 M United States). The plates were incubated at 22 °C in the long-day growth chamber (16 h light/8 h dark). Images of bacteria-infected plants were taken at the indicated time points.