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. 2016 Mar;17(3):441-54.
doi: 10.15252/embr.201540806. Epub 2016 Jan 14.

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Sharon C Mithoe  1 , Christina Ludwig  2 , Michiel J C Pel  1 , Mara Cucinotta  1 , Alberto Casartelli  1 , Malick Mbengue  3 , Jan Sklenar  3 , Paul Derbyshire  3 , Silke Robatzek  3 , Corné M J Pieterse  1 , Ruedi Aebersold  4 , Frank L H Menke  5
Affiliations

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Sharon C Mithoe et al. EMBO Rep. 2016 Mar.

Abstract

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN-SENSITIVE 2 (FLS2) induces the activation of mitogen-activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin-triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7-mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.

Keywords: Arabidopsis; innate immunity; phosphorylation; signaling; targeted proteomics.

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Figures

Figure EV1
Figure EV1. MS/MS spectra of peptides mapped to MKKK7
  1. A, B

    LTQ‐Orbitrap MS/MS spectra of MKKK7 peptides identified in FLS2‐GFP‐co‐immuno‐precipitated samples. Peptide sequence and fragmentation pattern are shown above the spectra together with the observed m/z and charge state of the precursor ion.

Figure 1
Figure 1. Flagellin receptor FLS2 co‐immunoprecipitates with MKKK7
Immunoprecipitation was performed with GFP‐binding protein immobilized on magnetic beads using the extracts of Arabidopsis seedlings, expressing either YFP‐MKKK7 or Lti6B. Seedlings were treated with 1 μM flg22 for the indicated times. YFP‐MKKK7 and Lti6B‐GFP were detected with an anti‐GFP antibody, while FLS2 was detected with an FLS‐specific antibody. Upper panel shows (co)‐immunoprecipitated proteins, and lower panel shows input levels of protein. Arrowheads indicate the position of proteins of interest.
Figure EV2
Figure EV2. MKKK7 domain structure and phosphorylated residues

  1. Protein structure of MKKK7 with the protein kinase domain shown in yellow and an ARM/HEAT repeat domain shown in blue. The position of the phosphorylated serine residues (S) is shown with triangles. The green triangles indicate non‐differentially phosphorylated sites. The red triangles indicate phosphorylated serine (pS) sites that were targeted for mutagenesis.

  2. Protein sequence of MKKK7, highlighted in yellow are all (phospho‐) peptides measured by mass spectrometry. Highlighted in green are modified residues, and the red box around the S residues indicates phosphorylated serine residues that were targeted for mutagenesis.

Figure EV3
Figure EV3. Multiple sequence alignment of MKKK7 and related MAP3K
Amino acid sequences for Arabidopsis thaliana MKKK6 (AtMKKK6) and MKKK7 (AtMKKK7), Arabidopsis lyrata MKKK7 (AlMKKK7), Brassica napus MAP3K epsilon protein kinase 1 (BnM3KE1), Camelina sativa MAP3K epsilon protein kinase (CsM3KE), Solanum lycopersicum MAP3K epsilon protein kinase (SlM3KE), Nicotiana benthamiana MAP3K epsilon protein kinase (NbM3KE), Malus domestica MAP3K epsilon protein kinase (MdM3KE), and Populus trichocarpa MAP3K epsilon protein kinase (PtM3KE) were aligned with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Only alignments of amino acid sequences surrounding the phosphorylated residues in MKKK7 are shown. Residues phosphorylated in AtMKKK7 are indicated with a red arrow and highlighted in red, and conserved residues in other MAP3Ks are highlighted in green. Protein names and GenBank accession numbers are indicated on the left side of the alignments, while to the right the number of the last amino acid in the alignment is indicated.
Figure 2
Figure 2. Transient phosphorylation of MKKK7 and other MAP kinases upon flg22 treatment
  1. A–I

    Application of selected reaction monitoring (SRM) mass spectrometry to quantify phosphorylated peptides in cell extracts treated with 1 μM flg22. Bars represent the mean ratio of endogenous phosphopeptide versus spiked‐in synthetic phosphopeptide normalized to t = 0 with error bars ± SEM (n = 3.). Asterisks indicate a significant difference level compared to t = 0 (Student's t‐test, *P > 0.05, **P > 0.01 and ***P > 0.001). The color of each bar corresponds to the different time points (0 min = dark blue, 5 min = red, 10 min = green, 20 min = purple, 30 min = light blue). Above each graph is the protein name and the phosphorylated residue (in brackets) is indicated and below the corresponding phosphopeptide is shown with the serine (S), threonine (T), or tyrosine (Y) phosphorylation site indicated by “[+80]”.

Figure EV4
Figure EV4. MAP kinase activation loop phosphorylation

  1. Selected reaction monitoring (SRM) of MPK6 activation loop phosphorylation in response to flg22 stimulation in cultured cells.

  2. SRM of MPK3 activation loop phosphorylation in response to flg22 stimulation in cultured cells. Monophosphorylated and doubly phosphorylated versions of the tryptic MPK activation loop peptides were monitored at 0, 5, 10, 20, and 30 min after stimulation with 1 μM flg22.

Data information: Sequences are shown on the left, with lower case p indicating phosphorylation of the residue to the right. Left panels show integrated peak area data for three biological replicates (A, B, and C). Middle panels show examples of transitions measured for endogenous 15N‐labeled peptides. Right panels show examples of total integrated peak area for endogenous 15N‐labeled peptides and 14N‐labeled synthetic peptides, which were spiked into the samples at a constant amount.
Figure 3
Figure 3. Flg22‐induced MAPK phosphorylation is enhanced in the mkkk7 mutant

  1. Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col‐0 and in mkkk7. Protein extracts were made from the seedlings treated with 1 μM flg22, and samples were taken at t = 0, 10, and 30 min post‐induction. The p44/42 antibody was used to detect phosphorylated MAPKs. Position of the individual phosphorylated MAPKs is indicated at the right. Equal loading of proteins is shown with an α‐actin antibody as a loading control (bottom panel). Three biological replicates were done with identical results.

  2. MPK6 phosphorylation is specifically enhanced in mkkk7. Comparison of phosphopeptide abundance from selected MAP kinases in Col‐0 (blue) and mkkk7 (red) seedlings at t = 0 min and t = 10 min after 1 μM flagellin treatment by selected reaction monitoring (SRM) mass spectrometry. Phosphopeptides corresponding to MKKK7 are only detectable in Col‐0 seedlings and are non‐detectable (ND) in mkkk7. Bars represent means of measured peptide areas (sum of all transition areas) for three biological replicates, with error bars ± SEM (n = 3). Asterisks indicate a significant difference between Col‐0 and mkkk7 at individual time points (Student's t‐test, *P > 0.05, **P > 0.01 and ***P > 0.001). ND indicates the integration of an area without transitions significantly above background. Above each graph the protein name and the phosphorylated residue (in brackets) is indicated as well as the corresponding phosphopeptide sequence. Serine (S), threonine (T), or tyrosine (Y) phosphorylation is indicated by “[+80]”.

Figure 4
Figure 4. Flg22‐induced defense gene expression is enhanced in mkkk7

  1. Transient expression analysis in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment. Protoplasts were isolated from 4‐week‐old plants and transfected with pWKRY29:fLUC (WRKY29) or pFRK1:fLUC (FRK1) constructs together with 35S:rLUC, as indicated in the graph. Protoplasts were treated for 4 h with 10 μM flg22 or mock‐treated as indicated. The horizontal axis indicates the treatment, while the vertical axis represents expression levels relative to the mock‐treated control sample, as fold induction. All measurements were normalized to the rLUC activity. Bars represent means ± SD (n = 2). Experiment was repeated 6 times with similar results.

  2. WRKY29 transcripts measured by qRT–PCR in flg22‐treated leaf material. Leaf strips of Col‐0 and mkkk7 were treated with 1 μM flg22 for t = 0, 1, 2, and 4 h. WRKY29 transcripts were normalized against Ubiquitin transcript as described before 62. Bars represent mean value, and error bars show SE (n = 3).

  3. FRK1 transcripts measured by qRT–PCR in flg22‐treated leaf material. Leaf strips of Col‐0 and mkkk7 were treated with 1 μM flg22 for t = 0, 1, 2, and 4 h. FRK1 transcripts were normalized against Ubiquitin transcript as described before 62. Bars represent mean value, and error bars show SE (n = 3).

Data information: For each qRT–PCR experiment shown in (B, C), at least 2 biological replicates were done showing the same trend. *P < 0.05, **P < 0.01, Student's t‐test.
Figure 5
Figure 5. Phosphorylation of MKKK7 on specific serine residues is required for negative regulation of flg22‐induced WRKY29 gene expression

  1. Transient co‐expression of MKKK7 in Arabidopsis mesophyll protoplasts shows the suppression of flg22‐induced WRKY29 gene expression. Protoplasts were transfected with pWRKY29:fLUC, 35S:rLUC and overexpression constructs of MKKK7 (OE‐MKKK7, OE‐MKKK7 AA or OE‐MKKK7 DD) as indicated on the horizontal axis. Protoplasts were treated with 10 μM flg22 or mock‐treated for 4 h. All measurements were normalized to the rLUC activity and expression is relative to the mock‐treated control sample, shown as fold induction on the vertical axis. Results shown are means ± SD (n = 2). At least two biological replicates were done with similar results.

  2. Protein structure of MKKK7 and mutated versions of MKKK7 with the protein kinase domain shown in yellow and an ARM/HEAT repeat domain shown in blue. The position of the phosphorylated serine residues is indicated with triangles and bold S below the protein structure. The red triangles indicate phosphorylated serines that were targeted for mutagenesis or the corresponding phosphomimic aspartic acid. Blue triangles indicate the substitution with the non‐phosphorylatable amino acid alanine. Amino acid substitute versions of MKKK7 are shown below the wild type. S, serine; A, alanine; D, aspartic acid.

Figure EV5
Figure EV5. Phosphorylation of MKKK7 on specific serine residues is required for negative regulation of flg22‐induced FRK1 gene expression

  1. Transient co‐expression of MKKK7 in Arabidopsis mesophyll protoplasts shows the suppression of FRK1 gene expression in protoplasts after flg22 treatment. Protoplasts were isolated from 4‐week‐old plants and transfected with pFRK1:fLUC, 35S:rLUC, and overexpression constructs of MKKK7 (OE‐MKKK7, OE‐MKKK7 AA, or OE‐MKKK7 DD) as indicated on the horizontal axis. Sixteen hours later, protoplasts were treated with 10 μM flg22 for 4 h. All measurements were normalized to the rLUC activity, and expression levels were calculated relative to the mock‐treated control sample as shown as fold induction represented on the vertical axis.

Figure 6
Figure 6. MKKK7 negatively regulates basal resistance to virulent bacterial infection

  1. Four‐week‐old seedlings were dipped into a suspension containing virulent Pst DC3000, and 72 h later, the disease symptoms were scored. Data represent mean values ± SEM (n = 20; ***P < 0.001; paired t‐test). Three biological experiments were done showing similar results.

  2. Quantification of bacterial growth in Arabidopsis lines Col‐0, mkkk7, and p35S:MKKK7‐GFP in the mkkk7 background. Four‐ to five‐week‐old plants were pressure‐infiltrated with virulent Pst DC3000, and at indicated time points, six samples were harvested and bacteria re‐isolated on selective media. The number of colony‐forming units (cfu/cm2) was determined at t = 0, 2, and 3 days post‐inoculation (dpi). Data represent mean values ± SEM (n = 6; **P < 0.01; paired t‐test). Experiments were done at least twice with similar results.

  3. Disease symptom development in Pst‐infected lines with estradiol‐inducible constructs of ind‐MKKK7 AA L8, ind‐MKKK7 AA L10, ind‐MKKK7 DD L1, and ind‐MKKK7 DD L3. Two independent transgenic lines for each construct were grown under short‐day conditions and disease symptoms were scored 3 dpi. Data represent mean values ± SEM (n = 20; *P < 0.05; **P < 0.01; paired t‐test). The vertical axis represents the percentage disease symptoms. Experiments were done at least twice with similar results.

Figure EV6
Figure EV6. MKKK7 negatively regulates basal resistance to virulent bacterial infection

  1. Example of symptom development at 2 dpi in the p35S:MKKK7‐GFP in mkkk7 background as compared to Col‐0 (left panel). Example of symptom development at 2 dpi in mkkk7 and p35S:MKKK7‐GFP in mkkk7 background (right panel).

  2. Overexpression of MKKK7 DD reduces resistance to Pst infection. Quantification of bacterial growth in Col‐0 and two independent iMKKK7 DD lines. Four‐ to five‐week‐old plants were pressure‐infiltrated with virulent Pst DC3000, and at 2 dpi, leaf disks were harvested and bacteria re‐isolated. The number of colony‐forming units (cfu/cm2) was determined at t = 2 days post‐inoculation (dpi). Data represent mean values ± SEM (n = 6; *P < 0.05; paired t‐test).

Figure 7
Figure 7. Overexpression of MKKK7DD reduces flg22‐induced ROS burst in leaves
Analysis of reactive oxygen species (ROS) production after treatment with flg22.

  1. Effect of 100‐nM flg22 treatment on the ROS burst measured in 5‐week‐old plants of Col‐0 and mkkk7.

  2. Effect of 100‐nM flg22 treatment on the ROS burst measured in 5‐week‐old plants of Col‐0 and two independent inducible MKKK7 AA transgenic lines.

  3. Effect of 100‐nM flg22 treatment on the ROS burst measured in 5‐week‐old plants of Col‐0 and two independent inducible MKKK7 DD transgenic lines.

Data information: Graphs represent means with error bars ± SEM (n = 24). The vertical axis represents the relative increase in ROS production (photon counts) after PAMP treatment. At least three biological replicate experiments were done with similar results.
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