Arabidopsis Seedling Flood Inoculation

Ten-day-old seedlings were flood-inoculated essentially as in Ishiga et al. (59 (link)). Seedlings were grown in 12-well plates containing 1/2 LS salts and 0.5% (wt/vol) Phytagel solid media. On the day of infection, Pst DC3000 (pVSP61) inoculum was suspended in sterile water with 0.025% (vol/vol) Silwet L-77 (Lehle Seeds) to an OD_595nm = 0.01 (∼8.0 × 10⁶ CFU/mL of bacteria) and 3 mL of inoculum was added to each well for 3 min, decanted, and tissue harvested from 5 to 10 seedlings per biological replicate at 1 and 3 DPI. After harvesting, tissue fresh weight was measured, and the samples were surface-sterilized with 5% (vol/vol) H₂O₂ for 5 min and rinsed three times with sterile water. Tissue was placed in 1.5-mL microfuge tubes and macerated in sterile water for 5 min in a 1600 miniG (Spex SamplePrep) set at 1,000 strokes per minute. Macerated tissue was serially diluted in sterile water and spread onto LB plates containing 25 µM rifampicin. Colonies per plate were counted after incubating at 28 °C for 48 h.