Rifampicin

The four plasmids harboring different sgRNAs were transformed into Agrobacterium tumefaciens strain GV3101. The transformed GV3101 were incubated in yeast extract peptone (YEP) liquid media [10 g/l of yeast extract (Sigma-Aldrich, USA), 10 g/l of peptone (Sigma-Aldrich, USA), and 5 g/l of NaCl (Sigma-Aldrich, USA), pH 7.2] supplemented with 50 mg/l rifampicin (Duchefa, Netherlands) and 50 mg/l kanamycin (Biosesang, Korea) for overnight at 28°C. In total, 1 ml of overnight culture was transferred into 20 ml fresh YEP media supplemented with 50 mg/l rifampicin and 50 mg/l kanamycin and incubated for 16 h at 28°C. The bacterial solution was centrifuged at 3,500 rpm for 20 min, and the resultant bacterial pellet was resuspended in 10 mM MgCl₂ (Sigma-Aldrich, USA) solution supplemented with 200 μM acetosyringone (Sigma-Aldrich, USA) (final OD₆₀₀ = 0.8–0.9) and incubated at room temperature for 3 h. The bacterial solution was infiltrated into tomato cotyledons using a needless syringe. The ~50 infiltrated cotyledons were harvested 5 days after infiltration for genomic DNA extraction and further PCR analysis. The efficiency of four sgRNAs was evaluated by sequencing analysis of TA-cloned PCR products.

Pseudomonas syringae pv. tomato DC3000 was grown for 36 h in King Agar B (KB) plates under dark conditions. Cells were scraped from the plates and suspended in 10 mM MgCl₂ at a final concentration of 10⁸ colony forming units (CFU)·ml⁻¹, and 0.002% (vol/vol) Silwet L-77 was added prior inoculation. Three-week-old plants of wild-type genotype and of each transgenic (#3 and #7 of AtCA-YDA, #5 and #6 of AtYDA) or CRISPR/Cas9 mutant lines (#36 and #37 of Slyda1 and #23, #52, and #313 of Slyda2) were spray-inoculated with the bacterial suspension. Bacterial growth was determined at 3 h post inoculation (time 0) and 5 days post infection by averaging the CFU·cm⁻² isolated from four infected plants. Leaf disks were homogenized in 10 mM MgCl₂, and serial dilutions were plated on KB solid medium containing rifampicin (25 μg/ml; Duchefa Biochemie). These assays were repeated in triplicate.