GUS Assay for Bacterial-Plant Interaction

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.