Mutations in FLS2 Ser-938 Dissect Signaling Activation in FLS2-Mediated Arabidopsis Immunity

<div><p>FLAGELLIN-SENSING 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing <i>FLS2_S938A</i>, while the acidic phosphomimic mutants FLS2_S938D and FLS2_S938E conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2_S938A, demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2_S938A and FLS2_S938D mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic <i>Arabidopsis thaliana</i> plants expressing <i>FLS2_S938A</i> or <i>FLS2_D997A</i> (a kinase catalytic site mutant), but was normally induced in FLS2_S938D plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.</p> </div

Induced phosphorylation of BIK1 and its homologous proteins under flg22 treatment.

<p>cMyc tagged BIK1, PBS1, PBL1, and PBL2 were transiently expressed (under control of 35S promoters) in protoplasts made from Arabidopsis <i>fls2-101</i> lines stably transgenic for full-length <i>FLS2_WT</i>, <i>FLS2_S938A</i>, <i>FLS2_S938D</i>, and <i>FLS2</i>_D997A (under control of <i>FLS2</i> promoters; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003313#ppat.1003313.s002" target="_blank">Figure S2A</a>). Protoplasts were treated with (+) or without (−) 1 µM flg22 for 15 min prior to harvest. Total protein extracts were separated by SDS-PAGE and immunoblots were probed with anti-cMyc to detect protein size shift attributable to phosphorylation. Lower panel of each pair shows Ponceau S staining of same immunoblot to assess similarity of total protein levels.</p

Interaction of BAK1 or BIK1 with variants of FLS2.

<p>HA-tagged full-length FLS2 wild type (WT), S938A, S938D or D997A (as labeled), and cMyc-tagged full length BAK1 or BIK1, were coexpressed under control of <i>35S</i> promoters in Arabidopsis <i>fls2-101</i> protoplasts. Protoplasts were harvested prior to (−) or 15 min. after (+) treatment with 1 µM flg22. Coimmunoprecipitation was carried out using anti-cMyc antibody. Input blots are from SDS-PAGE of total protein extracts; each lane was loaded with equivalent volumes of total protoplasts. WB: antibody used to probe immunoblot. <b>A.</b> FLS2_WT, FLS2_S938A and FLS2_S938D interact with BAK1 upon flg22 treatment. <b>B.</b> BIK1 dissociates from FLS2_WT, FLS2_S938A and FLS2_S938D after flg22 treatment. <b>C.</b> FLS2-FLS2 association before and after flg22 exposure is not reduced when both FLS2 partners carry the <i>FLS2_S938A</i> mutation. FLS2-BAK1 interaction from the same experiment is shown as a control (all six lanes in C from same protoplast batch, gel, blot and immunodetection). <b>D.</b> FLS2_D997A interacts with BAK_D416A upon flg22 treatment. FLS2_D997A does not interact as well as FLS2_WT with BIK1_D202A before flg22 treatment, and flg22-elicited release of BIK1 is not detected. <b>E.</b> FLS2_S938D, and separately, FLS2_D997A, form FLS2-FLS2 associations before and after flg22 treatment.</p

Identification of Ser-938 as a candidate autophosphorylation site of FLS2 <i>in vitro</i> and <i>in vivo</i>.

<p><b>A</b> and <b>B.</b> Intact (A) and antarctic phosphatase-treated (B) intracellular domains of FLS2 (aa #840-1172) were analyzed by Mass Spectrometry (MS). M: predicted molecular weight; 1P: predicted peptide with one phosphate group; 2P: predicted peptide with two phosphate groups. <b>C.</b> Peptides containing phosphorylated amino acids, identified by mass spectrometry (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003313#ppat.1003313.s001" target="_blank">Figure S1</a>). <b>D–F.</b> Functional test of three serine sites identified by MS. Reactive oxygen species were measured in leaf discs from transgenic Arabidopsis <i>fls2-101</i> plants for 30 min. after treatment with 1 µM flg22. Stable transgenic plants carried <i>FLS2</i> serine mutant alleles as specified, with expression driven by native <i>FLS2</i> promoter. Data shown are mean ± SE for four to six independent T1 plants per construct. RLU: relative luminescence units; wt: wild-type Col-0 FLS2; S938A: FLS2_S938A; other <i>FLS2</i> alleles similarly labeled.</p

Statistics for the NMR Structure of C2 2A^pro.

a<p>Stretches of regular secondary structure: 7–9, 12–16, 28–30, 35–39, 55–60, 65–74, 78–79, 88–96, 108–110, 115–122, 127–131.</p

Properties of C2 2A^pro datasets.

<p>(A) Secondary structural features from the NMR solution structure: β-strands (<i>arrows</i>) and 3₁₀ helices (<i>boxes</i>). (B) The total number of constraints used for the structure calculation plotted as a function of residue number. (C) Rmsd values for backbone atoms (N, C^α, and C′) of the best 15 models relative to the average structure. Structurally compact regions have rmsd values below 2 Å.</p

DNA Primers used for Cloning and Mutating RV-C2 2A^pro.

<p>*Restriction sites are in bold; primer regions that anneal to 2A^pro gene are underlined; and lowercase letters show DNA bases at the sites of directed mutagenesis.</p

An RV RNA genome encodes a single polyprotein.

<p>The polyprotein is cleaved co- and post-translationally to release mature viral proteins. During infection, 2A^pro is excised at the N-terminus by self-catalysis and at the C-terminus by 3C^pro. The released protease cleaves cellular substrates including eIF4G and nucleoporins.</p

¹H-¹⁵N HSQC spectra of ¹⁵N-labeled wild-type 2A^pro (<i>purple</i>) and C₁₀₅A 2A^pro (<i>red</i>).

<p>The two spectra are very similar; however, that of the wild-type protease exhibits small signals attributed to self-cleavage products.</p

SDS-PAGE illustrating purification of RV-C2 2A^pro.

<p>The recombinant methods described above were used to prepare ¹³C/¹⁵N-labeled C2 2A^pro (C₁₀₅A) for NMR studies. Representative samples from the procedure were fractionated by SDS-PAGE then visualized with Bio-Rad Stain-Free. Lane 1, Bio-Rad Precision Plus protein standards; lane 2, protein pellet after (NH₄)₂SO₄ precipitation; lane 3, SUMO-2A^pro after IMAC elution; lane 4, 2Apro after SUMO cleavage and IMAC elution; lanes 5–6, final protein fractions after gel filtration.</p