Bulk RNA-seq of A. thaliana leaves infiltrated with P. syringae

Protoplasts of Col-0 ecototype and cue1-6 mutant infiltrated with either virulent (Pseudomonas syringae pv. tomato DC3000) or avirulent (DC3000 AvrRpt2) bacteria, sampled at 3 and 5 hours post infection were sequenced. Seeds were surface-sterilized by and stratified at 4 °C for one week before sowing on soil. Seven- to ten-day-old seedlings were transplanted into individual pots and grown under 23 °C, 8 h light /16 h dark condition. 5- to 6-week-old Col-0 plants and 9-week-old cue1-6 plants were used. Pseudomonas syringae pv. tomato (Pst) DC3000 (empty vector, EV) and Pst DC3000(AvrRpt2) were cultured overnight at 28 °C in LB medium with kanamycin (50 mg/L). Cells were pelleted by centrifugation, washed, and resuspended in 10 mM MgCl2. For transcriptomic assays, suspensions were adjusted to OD600 = 0.005; for bacterial growth curves, to OD600 = 0.0001 to avoid hypersensitive cell death. The abaxial surfaces of leaves were infiltrated with a needle-less syringe, blotted dry, and harvested at 4 hpi for bulk RNA assays. Each treatment comprised three biological replicates (2–3 leaves per Col-0 plant and 3-6 leaves per cue1-6 plant).