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

Mass Spectrometry Compatible Surfactant for Optimized In-Gel Protein Digestion

Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)­undecyloxy)­carbonylamino)­propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5–2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20–30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment

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

Multi-sequence alignment and comparison of the predicted galectin-related protein (GREP) sequences with the BS-90 and NMRI <i>Biomphalaria glabrata</i> variant GREP proteins.

<p>Amino acid sequence predicted from an RNAseq assembly (RNAseq-GREP; KM975647) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006081#ppat.1006081.ref018" target="_blank">18</a>] is compared to 4 predicted proteins encoded within Scaffolds18083, 47310, 210741 and 5606. Near-complete ORFs for the GREP, encoding IgSF1/IgSF2 domains and galectin binding domain, were amplified from BS-90 (GREP1.1) and NMRI (GREP1.2) snail cDNA. Original peptide sequences (shaded grey) aligned with the translated scaffolds and the GREP1.1 and 1.2 proteins. Cysteines involved in the formation of Ig-loop domains are shown in boldface, while residues associated with the sugar-binding pocket of the galectin domain, as predicted by NCBI delta-blast, are shown as underlined bolded letters (Q/H, R, R, T/Q, N/G/S, W, A/S, V). Amino acid differences between GREP [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006081#ppat.1006081.ref018" target="_blank">18</a>], GREP1.1 and GREP 1.2 are highlighted in blue.</p

PCR amplification of NMRI and BS-90 <i>B</i>. <i>glabrata</i> galectin-related protein (GREP) transcripts.

<p>Complementary DNA synthesized from whole body RNA extracts of 10 individual NMRI and 10 BS-90 <i>B</i>. <i>glabrata</i> snails were used to generate amplification products of the near-complete coding region of the BS-90 GREP sequence. GREP amplicons for each snail sample (1–10) are shown. Primers to <i>B</i>. <i>glabrata</i> α-actinin served as a loading control. Note that GREP amplicons were generated using cDNA from all BS-90 samples tested, while only 4/10 NMRI snails produced amplicons, demonstrating differential GREP gene expression in the NMRI snail population.</p

Other immune-related proteins from NMRI and BS-90 <i>Biomphalaria glabrata</i> plasma eluted from <i>Schistosoma mansoni</i> sporocyst membrane-enriched (Mem) and larval transformation protein (LTP) affinity columns.

<p>Other immune-related proteins from NMRI and BS-90 <i>Biomphalaria glabrata</i> plasma eluted from <i>Schistosoma mansoni</i> sporocyst membrane-enriched (Mem) and larval transformation protein (LTP) affinity columns.</p

Alignment of a partial C-type lectin-related protein 2 (CREP2.1) sequence with the predicted CREP2 protein.

<p>Amino acid sequence predicted from an RNAseq assembly (CREP2; AKS26832.1) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006081#ppat.1006081.ref018" target="_blank">18</a>] is compared to a partial ORF encoding a CREP2, presenting an Ig domain, 2 internal repeat domains and a C-type lectin domain, from BS-90 and NMRI snail cDNA (CREP2.1). CREP2.1 presented an additional 50 aa sequence containing an internal repeat domain (internal repeat domain1) and a single aa difference (italics) when compared to CREP2.</p

PCR amplification of NMRI and BS-90 <i>B</i>. <i>glabrata</i> ADAM-TS, FREP12, and CREP2 transcripts.

<p>Whole body total RNA from 10 individual NMRI and 10 BS-90 <i>B</i>. <i>glabrata</i> snails were subjected to cDNA synthesis and used in PCR analysis of the ADAM-TS metalloproteinase, FREP12 and CREP2 transcript expression. Amplicons of the predicted size are shown for each snail sample (1–10). Primers to <i>B</i>. <i>glabrata</i> α-actinin served as a loading control.</p