G1Tn0-AP2
The transposon mutant library was resuscitated using the same procedure as other samples. The bacterial cells were diluted to a concentration of 2  10 CFU/mL. Following surface sterilization of the leaves, they were immersed in the bacterial suspension and subjected to vacuum infiltration (0.1 MPa, 10 seconds, repeated 3 times). The leaves were then rinsed three times with sterile water, placed in trays containing sterile moist filter paper, and incubated in a growth chamber at 16 C for 48 hours. After incubation, the leaves were fragmented into pieces approximately 2 to 3 mm in diameter using a blender. The leaf fragments were transferred to a glass dish containing ddHO and subjected to ultrasonic treatment in an ultrasonic water bath for 15 minutes to lyse the apoplastic cells. The resulting homogenate was clarified either by centrifugation or filtration through a 40-m filter to reduce plant debris. Bacterial cells were concentrated by centrifugation at 5,000  g for 10 minutes. The pellet was resuspended in sterile deionized water to obtain a bacterial cell precipitate.