Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1

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1 Supporting Online Material for Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1 Birgit Schulze, Tobias Mentzel, Anna Jehle, Katharina Mueller, Seraina Beeler, Thomas Boller, Georg Felix, Delphine Chinchilla* * Corresponding author: Delphine Chinchilla Botanical Institute University of Basel Hebelstasse 1 CH-4056 Basel, Switzerland delphine.chinchilla@unibas.ch Tel: Fax: This file includes: Legends for Figs S1-S7 References 1

2 Fig. S1. Characterization of the anti-bak1 antibodies in Arabidopsis. A, Polyclonal antibodies raised against the C-terminus of BAK1 were used in Western blot analysis with extracts from Arabidopsis bak1-4 mutant plants, wild-type Col-0 plants, and bak1-4 mutant plants expressing BAK1-Myc (1). B, Top: Immunoprecipitation (IP) with anti-bak1 on solubilized membrane proteins of Arabidopsis cell cultures in presence or not of an excess of antigenic peptide of different SERKs, as indicated. Bottom: Alignment of the five C-terminal peptides of the SERKs. The presence of BAK1 in the IPs was additionally confirmed by mass spectrometric analysis. C, Co-immunoprecipitation of a 175 kda protein (band B) with BAK1 using anti-bak1 antibodies after 2 min of elicitation with 1 μm flg22 (+flg22) or not (-flg22), from Arabidopsis cultured cells. After separation on SDS-PAGE, immunoprecipitates were stained with colloidal Coomassie. Liquid Chromatography tandem mass spectrometric analysis confirmed the identity of the 175 kda band as FLS2 (Mass Spectrometry Protein Sequence Database: Q9FL28_ARATH) in band B but not in band A. 2

3 Fig. S2. Chemical crosslinking of FLS2 and BAK1 in Arabidopsis cells treated with the active ligand flg22. Aliquots of cells were treated with flg22, flg22-δ2 or brassinolide (BL) for 3 or 5 min in the presence or absence of 2 mm bis(sulfosuccinimidyl)suberate (BS 3 ) as indicated. Solubilized membrane proteins were adsorbed to Concanavalin A-sepharose and analyzed on separate Western blots by immunodetection with anti-fls2 (left panel) and anti- BAK1 (right panel) antibodies. Data shown are representative for a total of 3 independent experiments with chemical crosslinkers. 3

4 Fig. S3. Phosphorylation of FLS2 and BAK1 in vitro. Immunoprecipitation (IP) with anti- FLS2 and anti-bak1 was performed from flg22-treated or untreated Arabidopsis cells. Samples were incubated at 25 C in presence of radioactive [ 32 P]-γ-ATP for 5 min. FLS2 and BAK1 were strongly phosphorylated when immunoprecipitated from flg22-treated cells, but not when FLS2 and BAK1 were simultaneously immunoprecipitated from untreated cells and flg22 was added or not to the IP in vitro. The protein Q9FIC2 which is additionally recognized by FLS2 antibodies (2) and unknown protein bands are marked by asterisks. 4

5 Fig. S4. Stability of the phosphorylation on the FLS2/BAK1 complex. A, Experimental scheme: In all samples Arabidopsis cells were stimulated with 1 μm flg22 at time point zero and incubated with [ 33 P]phosphate for 1 min at different time points (gray bars). This pulse of was followed by chases with 10 mm non-radioactive phosphate (white bars). B, Autoradiography and immunodetection of FLS2 (top) and BAK1 (bottom) after immunoprecipitation (IP) of solubilized membrane proteins with anti-bak1 antibodies. Phosphorylation signals were observed for BAK1 and FLS2 only in samples where [ 33 P]phosphate was given in the first minute of flg22 treatment. The chase of non-radioactive phosphate did not suppress phospho-labeling on FLS2 nor BAK1 while protein labeling in the total protein extract was reduced (lower panel). 5

6 Fig. S5. Effect of the phosphatase inhibitor calyculin A on phosphorylation of BAK1 and FLS2. Arabidopsis cells were pre-treated with [ 33 P]phosphate for 1 min and incubated with the phosphatase inhibitor CA (1 μm) and/or flg22 for 1 min. Solubilized membrane proteins were immunoprecipitated (IP) with anti-bak1 (top) or anti-fls2 (bottom) and analyzed by autoradiography and Western blot. Neither the FLS2 nor the BAK1 protein show labeling with radioactive phosphate after CA treatment in the absence of flg22 (lane 1, 2 and 7, 8). CA did not affect flg22 induced complex formation (3, 4 and 9, 10). There seemed to be an increase in the flg22 induced phosphorylation on BAK1 and FLS2 (3, 4 and 9, 10). However, CA had the same effect when 1 μm CA was added to homogenized extracts (lane 5, 6), suggesting that its main effect was to prevent dephosphorylation in vitro. 6

7 Fig. S6. Determination of the half maximal response (EC 50 ) of FLS2/BAK1 complex formation, phosphorylation, and of ph alkalinization reaction after flg22 treatment. A, Doseresponse curve for the amount of protein and phosphorylation of FLS2 immunoprecipitated with anti-bak1 antibodies. Samples were treated with increasing concentrations of flg22 ( nm) for 30 s together with a 1-min-pulse of [ 33 P]phosphate. FLS2 protein accumulation and phosphorylation were quantified from autoradiography (CNT, counts integrated from bands on the blots) and immunodetection with anti-fls2 antibodies (INT, intensity of bands on the Western blots). B, Dose response curve for the alkalinization reaction upon increasing concentrations of flg22 ( nm). Extracellular ph of the cell culture was recorded 20 min after the start of the experiment. 7

8 Fig. S7. Medium alkalinization response of Arabidopsis cell cultures in response to the peptide elicitors flg22, elf26, AtPep1, and the fungal elicitors Pen and chitin. Aliquots of cell culture were treated with the elicitor at time point zero and the ph-response was continuously recorded. References 1. Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nürnberger T, Jones J D G, Felix G, Boller T (2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, Chinchilla D, Bauer Z, Regenass M, Boller T, Felix G (2006) The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18,