Fig. 2

SUMOylation is required for BIK kinase release from activated FLS2-BAK1 immune complex to initiate immune signalling. a FLS2-GFP and FLS2^K/R–GFP interacts with BAK1-myc in a flagellin dependant manner. Nicotiana benthamiana leaves transiently expressing FLS2-GFP or FLS2^K/R-GFP with BAK1-myc were treated with MgCl₂ or 1 µM flg22 for 10 min. Subsequently, total protein was subjected to immunoprecipitation with anti-GFP immunoaffinity beads (IP: αGFP) followed by immunoblot analysis with anti-myc (IB: αmyc) antibodies to detect BAK1-myc and anti-GFP (IB: αGFP) antibodies to detect FLS2-GFP. BAK1 protein levels in all samples were determined by probing with anti-myc antibodies to detect BAK1-myc in total protein extracts (BAK1-myc input). GFP was used as a negative control. b Non-SUMOylatable FLS2^K/R-GFP remains bound to BIK1 after flagellin treatment. FLS2-GFP and FLS2^K/R-GFP were transiently expressed with BIK1-myc in N. benthamiana and coimmunoprecipitation and immunoblotting was done as described in (a) except that BIK1 was analysed by antibodies against c-myc. Total protein of all samples was probed with anti-myc antibody to determine BIK1 protein levels (BIK1-myc input). GFP was used as a negative control. c SUMOylation of FLS2 is required for the induction of immune related ROS burst. Leaf discs from three-week-old plants of Col-0, fls2, and two transgenic lines each of FLS2-GFP, FLS2^K/R-GFP were treated with 1 µM of flg22 for 10 min and ROS burst detected. Results shown are average ± SE (n = 3). d Non-SUMOylatable FLS2^K/R-GFP plants exhibit significantly reduced levels of MAPK activation. Different genotypes indicated were treated with water or 1 µM of flg22 for 10 min and total protein were extracted after treatment. Immunoblots were probed with anti-p44/42 MAPK antibodies to detect activated MPK3 and 6 (upper panel); the lower panel was immunoblotted with anti-MPK3 antibody to ensure equal protein loading