1. Volume 54, Issue 1, Pages 43-55 (April 2014)
Direct Regulation of the NADPH Oxidase RBOHD by the PRR-Associated Kinase BIK1 during Plant Immunity 
Yasuhiro Kadota, Jan Sklenar, Paul Derbyshire, Lena Stransfeld, Shuta Asai, Vardis Ntoukakis, Jonathan DG Jones, Ken Shirasu, Frank Menke, Alexandra Jones, Cyril Zipfel 
Molecular Cell 
Volume 54, Issue 1, Pages (April 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 54, 43-55DOI: (10.1016/j.molcel.2014.02.021)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 RBOHD Is Part of the PRR Complex
(A) Identification of RBOHD tryptic peptides by immunoprecipitation of EFR-GFP and LC-MS/MS analysis. efr-1/pEFR:EFR-GFP seedlings were treated or not (−) with 200 nM elf18 for 5 min. Untransformed Col-0 seedlings were used as a negative control.
(B) Coimmunoprecipitation of EFR and RBOHD proteins in Arabidopsis. Stable transgenic Arabidopsis seedlings (F1) originated from crosses between Col-0/p35S:GFP-LTI6b or efr-1/pEFR:EFR-GFP and rbohD/p35S:FLAG-RBOHD were treated or not (−) with 200 nM elf18 for 5 or 30 min. Total proteins (input) were subjected to immunoprecipitation with anti-GFP magnetic beads followed by immunoblot analysis with anti-FLAG and anti-GFP antibodies. GFP-LTI6b was used as a negative control. These experiments were performed three times with similar results. See also Figure S1.
Molecular Cell  , 43-55DOI: ( /j.molcel )
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4. Figure 2 BIK1 Directly Interacts with and Phosphorylates RBOHD
(A) BIK1 directly interacts with the N-terminal domain of RBOHD in vitro. MBP, MBP-EFR-CD (cytoplasmic domain), MBP-BAK1-CD, and MBP-BIK1 were incubated with GST-RBOHD-N (N-terminal domain of RBOHD) and pulled down with MBP. Input and pull down proteins are separated by SDS-PAGE and stained with Coomassie brilliant blue (CBB).
(B) BIK1 trans-phosphorylates RBOHD-N in vitro. MBP-EFR-CD, MBP-EFR-CD∗ (kinase dead), GST-BAK1-CD, GST-BAK1-CD∗, GST-BIK1, and GST-BIK1∗ were incubated (+) or not (−) with MBP-RBOHD-N in kinase buffer, and the autophosphorylation and trans-phosphorylation were visualized with radioactive ATP and autoradiography (upper panel). The protein loading control was shown by CBB staining (lower panel).
(C) PAMP-activated BIK1 trans-phosphorylates RBOHD-N. Col-0/pBIK1:BIK1-HA seedlings were treated or not (−) with 200 nM flg22 or elf18 for 10 min. Total proteins were subjected to immunoprecipitation with anti-HA beads followed by immunoblot analysis with anti-HA antibody to check BIK1-HA band shift and in vitro kinase assay with (+) or without (−) MBP-RBOHD-N. The assays were performed in the presence of 10 mM of the Ca2+ chelator BAPTA. Untransformed Col-0 seedlings were used as a control.
(D) PAMP-activated BIK1 has a stronger binding affinity with RBOHD. Stable transgenic Arabidopsis seedlings (F1) originated from crosses between Col-0/p35S:GFP-LTI6b and rbohD/p35S:FLAG-RBOHD (rbohD/p35S:GFP-LTI6b/p35S:FLAG-RBOHD), and Col-0/pBIK1:BIK1-HA and rbohD/p35S:FLAG-RBOHD (rbohD/pBIK1:BIK1-HA/p35S:FLAG-RBOHD) were treated or not (−) with 200 nM flg22 or elf18 for 5 or 30 min. Total proteins (Input) were subjected to immunoprecipitation with anti-HA magnetic beads or anti-FLAG beads followed by immunoblot analysis with anti-FLAG and anti-HA antibodies. FLAG-RBOHD protein was eluted with FLAG peptides. Homozygous Col-0/pBIK1:BIK1-HA and rbohD/p35S:GFP-LTI6b/p35S:FLAG-RBOHD seedlings (F1) were used as controls. All experiments were performed three times with similar results. See also Figure S2.
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5. Figure 3 BIK1 Phosphorylates Specific Sites on RBOHD
(A) Conservation of BIK1-mediated RBOHD phosphorylation sites. ClustalW multiple alignments were visualized using JalView v2.8 and colored by percentage identity.
(B) SRM analysis in vitro identified BIK1-mediated RBOHD phosphorylation sites. Equal amounts of GST-BIK1, MBP-EFR-CD, GST-BAK1-CD, MBP-CPK4, MBP-CPK5, MBP-CPK6, and MBP-CPK11 were incubated with MBP-RBOHD-N in kinase buffer followed by separation by SDS-PAGE and stained with CBB (upper panel). After tryptic digestion, phosphorylated peptides of S39, S343, S347, and S163 (T161/S162) were quantified by SRM. Data are mean ±SE of three biological replicates, each of which contains three technical replicates. Asterisks indicate significant differences compared with values without kinase (t test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).
(C) RBOHD phosphorylation in vivo upon PAMP treatment. rbohD/p35S:FLAG-RBOHD seedlings were treated or not (−) with 200 nM flg22 or elf18 for 10 min or with 1 mg/mL chitin for 5 min. Total proteins were subjected to immunoprecipitation with anti-FLAG beads followed by separation by SDS-PAGE and stained with CBB (upper panel). After tryptic digestion, phosphorylated peptides are quantified by SRM. Data are mean ±SE of three biological replicates, each of which contains two to three technical replicates (one-way ANOVA, Dunnett post hoc test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).
(D) Dynamics of RBOHD phosphorylation in vivo upon elf18 treatment. rbohD/p35S:FLAG-RBOHD seedlings were treated or not with 200 nM elf18 for 1, 2, 3, or 4 min. Data are mean ±SE of three to four biological replicates, each of which contains two technical replicates (one-way ANOVA, Dunnett post hoc test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).
(E) PAMP-inducible RBOHD phosphorylation at S39 and S343 in vivo requires BIK1 and PBL1. rbohD/p35S:FLAG-RBOHD and bik1 pbl1 rbohD/p35S:FLAG-RBOHD seedlings were treated or not (−) with 200 nM flg22 for 10 min. Total proteins were subjected to immunoprecipitation with anti-FLAG beads followed by separation by SDS-PAGE. After tryptic digestion, phosphorylated peptides were quantified by SRM. Data are mean ±SE of four biological replicates (one-way ANOVA, Tukey post hoc test). Different letters indicate significantly different values at p < All experiments were performed three times with similar results. See also Figure S3.
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6. Figure 4 BIK1-Mediated Phosphorylation of RBOHD Is Ca2+ Independent
(A) Elf18-induced BIK1 phosphorylation is Ca2+ independent. pBIK1:BIK1-HA seedlings were washed extensively with ultrapure water and were vacuum infiltrated with ultrapure water or 10 mM EGTA. After incubation for 30 min, the seedlings were treated with 200 nM elf18 for 15 min, and BIK1-HA phosphorylation was analyzed by its mobility shift in SDS gel by immunoblotting analysis.
(B) Phosphorylation of S39 and S343 is Ca2+ independent. rbohD/p35S:FLAG-RBOHD seedlings were treated as in (A), and total proteins were subjected to immunoprecipitation with anti-FLAG beads followed by separation by SDS-PAGE and stained with CBB (upper panel). SRM analysis was performed as in Figures 3C–3E. Data are mean ±SE of five biological replicates, each of which contains two technical replicates (one-way ANOVA, Tukey post hoc test). Different letters indicate significantly different values at p < 0.05 for S39 phosphorylation and p < for S343 and S163 (T161/S162) phosphorylations. All experiments were performed three times with similar results. See also Figure S4.
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7. Figure 5 BIK1-Mediated Phosphosites Are Required for RBOHD Activity
(A) BIK1-mediated RBOHD phosphorylation sites are required for flg22-induced ROS production in N. benthamiana. 3×FLAG-RBOHD or its phosphorylation site mutants were transiently expressed under the control of the native promoter in N. benthamiana leaves silenced for endogenous NbRBOHB. GUS was also expressed in the same leaf as negative control. Flg22-induced ROS production in eight leaf discs from each region was measured over 40 min. The total photon counts are represented relative to the counts induced by wild-type (WT) 3×FLAG-RBOHD. Data are mean ±SE of seven biological replicates, each of which contains eight technical replicates. Different letters indicate significantly different values at p < 0.05 (one way ANOVA, Tukey post hoc test).
(B–E) Phosphorylation of S39, S339, and S343 is required for the ROS burst induced by flg22 (B), elf18 (C), chitin (D), and pep1 (E) in Arabidopsis. Leaf discs from rbohD, rbohD/pRBOHD:3×FLAG-RBOHD (WT), or S39A/S339A/S343A were treated with 200 nM flg22, 200 nM elf18, 1 mg/mL chitin, or 200 nM pep1, and the ROS production was measured over 40 min. Values are mean ±SE (n = 8). Different letters indicate significantly different values at p < (one way ANOVA, Tukey post hoc test). All experiments were performed four times with similar results. See also Figure S5.
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8. Figure 6
BIK1-Meditaed RBOHD Phosphorylation Is Required for Stomatal Immunity
(A) BIK1-mediated phosphosites are required for PAMP-induced stomatal closure. Stomatal aperture was measured 2 hr after treatment with 5 μM flg22 or elf18. Values are mean ±SE (n = 60; one-way ANOVA, Tukey post hoc test). Different letters indicate significantly different values at p < This experiment was repeated three times with similar results.
(B) Loss of RBOHD leads to increased expression of PATHOGENESIS RELATED PROTEIN-1 (PR-1) upon bacterial perception. Col-0, rbohD, rbohD/pRBOHD:3×FLAG RBOHD (WT), or rbohD/pRBOHD:3×FLAG RBOHD (S39A/S339A/S343A) plants were syringe infiltrated with Pto DC3000 hrcC− (OD600 = 0.1) or 10 mM MgCl2 solution (mock treatment). At 24 hr postinoculation, total RNA were extracted and reverse-transcribed, and transcript levels of the immune marker gene PR-1 gene were measured by quantitative PCR after normalization to the U-box housekeeping gene transcript (At5g15400). The values are relative to the expression level in Col-0 with mock treatment. Data are mean ±SE of three technical replicates. Different letters indicate significantly different values at p < 0.05 (one-way ANOVA, Tukey post hoc test). This experiment was repeated three times with similar results.
(C) BIK1-mediated phosphosites are required for immunity to bacteria. Pto DC3000 COR− (OD600 = 0.02) (D) or Pci (OD600 = 0.2) (C) were sprayed onto leaf surfaces, and plants were maintained uncovered. Bacterial numbers were determined 3 days postinoculation. Values are mean ±SE (n = 8). Asterisks indicate significant differences compared with WT values (one-way ANOVA, Dunnett post hoc test, ∗∗p < 0.01). cfu indicates colony-forming units. These experiments were performed five times with similar results. See also Figure S6.
Molecular Cell  , 43-55DOI: ( /j.molcel )
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