Fig. 3.

Identification of SUMO components controlling FLS2-mediated immune signalling. a SCE1 interacts with FLS2. Nicotiana benthamiana leaves transiently expressing FLS2-GFP/FLS2^K/R-GFP with SCE1-HA were treated with MgCl₂ or 1 µM flg22 (10 min). Immunoprecipitation was done with anti-GFP beads (IP: αGFP) and immunoblotted with anti-HA (IB: αHA) for SCE1-HA and anti-GFP (IB: αGFP) for GFP/GFP-fusion proteins. Total protein extracts were probed with anti-HA antibody (SCE1-HA input) for equal SCE1. b Desi3a co-localizes with FLS2-GFP to plasma membrane. N. benthamiana leaves co-infiltrated with mCherry-Desi3a and FLS2-GFP or GFP were detected for fluorescence after 3 days. Before imaging, cells were plasmolysed with 1 M NaCl (1 h). Images were obtained using confocal laser scanning microscope Carl Zeiss 880. Scale bar = 10 µm. Arrows indicate overlap of mCherry-Desi3a and FLS2-GFP signals. c FLS2 interacts with Desi3a. Coimmunoprecipitation from N. benthamiana leaves expressing FLS2-GFP/FLS2^K/R-GFP or GFP with Desi3a-HA were performed. Total proteins were probed with anti-HA antibody for equal Desi3a (Desi3a-HA input). d Desi3a is a bona fide SUMO protease. High molecular conjugates of His-SUMO1 chains were incubated with GST-Desi3a (Desi3a^WT) or Desi3a^C168S at 30 °C and subsequently immunoblotted with anti-SUMO1 (IB: αSUMO1) to detect SUMO chains (upper panel), and with anti-GST to detect GST-fusion proteins (lower panel). e FLS2 is hyper-SUMOylated in desi3a-1. Immunoprecipitates (IP: αGFP) from transgenic lines expressing proFLS2::FLS-GFP in fls2 or desi3a-1 were probed with anti-GFP (IB: αGFP) or anti-SUMO1/2 (IB: αSUMO1) antibodies. f HyperSUMOylation of FLS2 enhances ROS burst. Leaf discs from three-week-old plants of Col-0, FLS2-GFP (fls2), FLS2-GFP (desi3a-1) transgenic lines were treated with 1 µM flg22 (10 min) and ROS burst detected. Results shown are average ± SE (n = 3). g HyperSUMOylation of FLS2 increases MAPK activation. Col-0, FLS2-GFP (fls2) and FLS2-GFP (desi3a-1) transgenic lines were treated with water or 1 µM flg22 (10 min) and total proteins were probed with anti-p44/42 MAPK antibody to detect activated MPK3/6 (upper panel) and with anti-MPK3 antibody for equal protein loading. h flg22 degrades Desi3a. 10-day-old seedlings of 35S::Desi3a-HA (desi3a-1) transgenic lines were treated with 200 μM cycloheximide or a combination of 200 μM cycloheximide + 1 μM flg22 and total proteins extracted at indicated time-points were immunobloted with anti-HA (IB: αHA) antibody