Fig. 3

DORN1 directly interacts with RBOHD in vivo and in vitro. a DORN1 directly interacts with RBOHD N-terminal in vitro. Purified, recombinant proteins GST-DORN1-KD, GST-DORN1-KD-1 (kinase dead), GST-DORN1-KD-2 (kinase dead), or GST were incubated with His-RBOHD-N followed by GST-mediated pull-down. Purified His-Lyk4 kinase domain protein was used as a negative control. Lambda protein phosphatase (Lambda PP) was added to release phosphate groups from phosphorylated serine, threonine, and tyrosine residues. b DORN1 interacts with RBOHD in the protoplast plasma membrane. The indicated constructs were transiently expressed in wild-type protoplasts and the BiFC assay was performed. FM4-64 was added to stain the plant cell plasma membrane. Bar = 20 μm. c DORN1 interacts with RBOHD in Arabidopsis plants. Stable transgenic Arabidopsis plants (F1) generated from a cross between NP::DORN1-HA/dorn1-3 and 35s::RBOHD-Myc/Col-0 expressing lines were treated with or without 250 μM ATP for 30 min, and total protein extract was subjected to Co-IP. All above experiments were repeated three times with similar results