Transient expression, promoter activity analysis and cell death
assay in N. benthamiana
Expression vectors (pGRRFP ::PPik-H4 ::GFP and a series of
pGR-based promoter split or mutation vectors, pEAQ
P35S ::Pik1-H4 ::Flag ,
pEAQ
P35S ::Pik2-H4 ::HA ,
pEAQ P35S ::AvrPik-E ::Myc ) were
transformed into Agrobacterium tumefaciens GV3103. Each strain
was set to OD600=0.2 and incubated in an induction
buffer as described(Lapin et al. , 2019) in the dark at room
temperature for 1 h. Then, the suspension was injected into 3- to
4-week-old tobacco leaves with a syringe. Data were collected after 2-3
days of infiltration. For promoter activity analysis, fifteen tobacco
mesophyll cells from three seedlings were imaged with a confocal
microscope (CarlZeiss LSM 750), and the fluorescence intensity was
measured via the software ZEN. The excitation/emission wavelengths were
488 nm/490-560 nm for GFP, 543 nm/580-660 nm for RFP. Five 8-10 mm
leaves with three biological replications were cut and put in tubes with
10 mL milliQ water for 30 min, then transferred to a new tube with 1 mL
milliQ water at room temperature. Conductivity was measured at 0 h and 6
h with a conductivity meter (Lei-ci DDBJ-350).