Fig 1: SUMOylation is required for BIK kinase release from activated FLS2-BAK1 immune complex to initiate immune signalling. a FLS2-GFP and FLS2K/R–GFP interacts with BAK1-myc in a flagellin dependant manner. Nicotiana benthamiana leaves transiently expressing FLS2-GFP or FLS2K/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 FLS2K/R-GFP remains bound to BIK1 after flagellin treatment. FLS2-GFP and FLS2K/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, FLS2K/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 FLS2K/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
Fig 2: Loss of both AP2C1 and MKP1 causes developmental defects and precocious cell death, mediated by MPK6 and MPK3. (A) Phenotypes of 5-week-old plants of the indicated genotypes grown under standard short-day conditions. (B) Phenotypes of plants of the indicated genotypes grown for 4 weeks under short-day conditions followed by 3 weeks under long-day conditions. (C) Phenotypes of plants of the indicated genotypes grown for 6 weeks under short-day conditions. Scale bars=1 cm.
Fig 3: AP2C1 and MKP1 control wound-induced MAPK activities. Analysis of wound-induced MPK6, MPK4, and MPK3 kinase activities and protein amounts of leaves from 6-week-old WT, ap2c1, mkp1, and ap2c1 mkp1 plants grown under short-day conditions. (A) MAPK activities were determined after immunoprecipitation by phosphorylation of MBP detected by autoradiography. The entire kinase assay was based on one common master mix containing MBP and γ-ATP. Loading is demonstrated by Coomassie blue staining (CBS); representative lanes are shown. The experiment was repeated twice with similar results. (B) MAPK protein amounts before and after wounding demonstrated by immunoblotting of MPK3, MPK4, and MPK6 from total protein extract using specific antibodies. Loading is demonstrated by Ponceau S staining (Rubisco protein). mpw, Minutes post wounding.
Fig 4: Trimers, long-OG mix (DP > 8) and flg22 induce MAPK3and MAPK6 phosphorylation. Seedlings were treated for 10 min with trimers (200 μM & 1 mM), long-OG mix (200 μM & 1 mM), flg22 (1 μM), or mock solution (1/2 MS). Each biological replicate is a pool of three wells of seedlings, each containing approximately ten plants. Thirty micrograms of each protein sample was loaded and phosphorylation levels were assessed by immunoblotting using a phospho-p44/42 specific antibody (top). MPK3, MPK4 and MPK6 total protein amounts were assessed using specific antibodies (bottom). The experiment was repeated with a second biological replicate, yielding identical results
Fig 5: CIPK6 negatively regulated MAPK signaling during PTI. (A) Time course of MPK3 and MPK6 phosphorylation in Col-0 and cipk6 plants upon flg22 treatment was assessed by western blot using antibody (pTEpY) specific for phosphorylated MAPKs. Total MPK3 and MPK6 proteins were detected by the respective protein-specific antibodies. Ponceau S staining of Rubisco was used as a loading control. (B–D) Expression analysis of NHL10, FRK1, and PHI-1 in Col-0 and cipk6 plants by qRT–PCR. The Col-0 and cipk6 plants were treated with flg22 (1 µM). The samples were collected at the indicated time points for qRT–PCR analysis. Actin 2 and Tubulin 4 were used as internal controls. SDs were determined using three biological replicates and two technical replicates for each.
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