G1Tn0-EP2
A 1.25 mL aliquot of the transposon mutant library glycerol stock was inoculated from -80 C into 25 mL of fresh KB medium supplemented with 50 g/mL kanamycin and cultured with shaking at 25 C for 7 hours until the culture reached the mid-log phase (OD600 between 0.5 and 0.7). Bacterial cells were harvested and washed twice with sterile deionized water. For leaf surface inoculation, the bacterial pellet was resuspended and diluted to an OD600 of 0.1 (approximately 2  10 CFU/mL) in sterile deionized water. The bacterial suspension was sprayed onto the leaf surfaces until run-off. The inoculated plants were then transferred to a growth chamber and maintained at 16 C for 48 hours. Following incubation, the leaves were collected and immersed in a glass dish containing double-distilled water (ddHO). The dish was subjected to ultrasonic treatment in a Branson 5510 ultrasonic water bath (output frequency: 40 kHz) for 10 min to dislodge surface-associated bacterial cells. The resulting leaf wash solution, which contained ultrasonically liberated cells, was centrifuged to collect the supernatant. The supernatant was sequentially filtered through a BS-40-CS coarse filter to remove plant debris and subsequently centrifuged at 17,000  g for 1 min to pellet the bacterial cells.