A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity

Abstract

Plants and animals rely on innate immunity to prevent infections by detection of microbe-associated molecular patterns (MAMPs) through pattern-recognition receptors (PRRs). The plant PRR FLS2, a leucine-rich repeat-receptor kinase, recognizes bacterial flagellin and initiates immune signaling by association with another leucine-rich repeat-receptor-like kinase, BAK1. It remains unknown how the FLS2/BAK1 receptor complex activates intracellular signaling cascades. Here we identified the receptor-like cytoplasmic kinase BIK1 that is rapidly phosphorylated upon flagellin perception, depending on both FLS2 and BAK1. BIK1 associates with FLS2 and BAK1 in vivo and in vitro. BIK1 is phosphorylated by BAK1, and BIK1 also directly phosphorylates BAK1 and FLS2 in vitro. The flagellin phosphorylation site Thr(237) of BIK1 is required for its phosphorylation on BAK1 and FLS2, suggesting that BIK1 is likely first phosphorylated upon flagellin perception and subsequently transphosphorylates FLS2/BAK1 to propagate flagellin signaling. Importantly, bik1 mutants are compromised in diverse flagellin-mediated responses and immunity to the nonpathogenic bacterial infection. Thus, BIK1 is an essential component in MAMP signal transduction, which links the MAMP receptor complex to downstream intracellular signaling.

Fig. 1.

flg22 induces BIK1 phosphorylation. (A) flg22 induces BIK1 transcripts. RT–PCR analysis of 8-day-old wild-type (Col-0) seedlings treated with 10 nM flg22. (B) flg22 treatment induces the mobility shift of BIK1, but not of OX1, BSK1, or Pto. Protoplasts were transfected with HA-epitope-tagged BIK1, OX1, BSK1, or Pto for 6 h and treated with 1 μM flg22 for 10 min. (C) flg22-induced BIK1 mobility shift is restored by the treatment of CIP or kinase inhibitor K252a. The control (Ctrl) was nontreatment. (D) BIK1 is phosphorylated by flg22 within the first minutes upon stimulation. The protoplasts were concentrated by a low-speed centrifuge 6 h after transfection and treated with 1 μM flg22 at the indicated time before adding protein sample loading buffer. (E) flg22 and elf18, but not chitin, induce BIK1 phosphorylation in BIK1-HA transgenic plant seedlings. Twelve-day-old BIK1-HA seedlings were treated with H₂O (Ctrl), 1 μM flg22, 1 μM elf18, or 50 μg/mL chitin for 10 min.

Fig. 2.

FLS2- and BAK1-dependent BIK1 phosphorylation. (A) flg22-induced BIK1 phosphorylation depends on FLS2 and BAK1. BIK1-HA transgenic plant seedlings in a Col-0 [wild type (WT)], bak1, or fls2 background were treated with 1 μM flg22 for 10 min. (B) The kinase activity of FLS2 and BAK1 is required for flg22-mediated BIK1 phosphorylation. Protoplasts were isolated from fls2, bak1, or serk4 mutant plants and cotransfected with HA-epitope-tagged BIK1 and FLAG-epitope-tagged FLS2, FLS2Km, BAK1, BAK1Km, or SERK4. The control (Ctrl) was a vector control. (C) AvrPto blocks flg22-induced BIK1 phosphorylation. Protoplasts were cotransfected with a control vector or AvrPto-FLAG and treated with 1 μM flg22 for 10 min.

Fig. 3.

BIK1 interacts with FLS2 and BAK1. (A) BIK1 associates with FLS2 in vivo. The protoplasts were coexpressed with FLS2-FLAG and BIK1-HA or a control vector. Co-IP was carried out with an anti-HA antibody (IP: α-HA), and the proteins were analyzed using Western blot with an anti-FLAG antibody (WB: α-FLAG). (Top) BIK1 coimmunoprecipitates with FLS2. (Middle and bottom) The expression of FLS2 and BIK1 proteins. Protoplasts were stimulated with 1 μM flg22 for 10 min. (B) BIK1 associates with BAK1 in vivo. Co-IP was performed with protoplasts coexpressing BAK1-FLAG and BIK1-HA or a control vector. (C) GST-BIK1 pulls down both FLS2 and BAK1. Protoplasts were transfected with FLS2-HA or BAK1-HA and stimulated with or without 1 μM flg22 for 10 min. The cell extracts were incubated with GST or GST-BIK1 beads (PD: GST). (Top) GST-BIK1 beads pull down FLS2 and BAK1. (Bottom) Protein expression of FLS2 and BAK1. (D) Kinase domains of BAK1 and FLS2 pull down BIK1. Protoplasts were transfected with BIK1-HA and stimulated with or without 1 μM flg22 for 10 min (bottom: protein expression). The cell extracts were incubated with GST, GST-BAK1K, or GST-FLS2K beads. The proteins were detected using Western blot with an anti-HA antibody (top). (E) The kinase domain of FLS2 associates with BIK1 with Co-IP assay. (F) The kinase domain of BAK1 associates with BIK1 with Co-IP assay. The control (Ctrl) was a vector control.

Fig. 4.

Transphosphorylation between BIK1 and the FLS2-BAK1 complex. (A) Alignment of the activation domain of BIK1 with several related kinases, including BSK1, TPK1b, PBS1, and Pto. The residues of BIK1 individually mutated to alanine are indicated in bold. (B) BIK1^T237A mutation completely eliminates the flg22-induced BIK1 phosphorylation detected by Western blot. (C) BAK1 phosphorylates BIK1. An in vitro kinase assay was performed by incubating MBP, MBP-BAK1CD, or MBP-BAK1CDKm with GST or GST-BIK1Km. Proteins were separated with SDS/PAGE and analyzed by autoradiography (Upper), and the protein loading control was shown by Coomassie blue staining (Lower). (D) BIK1 phosphorylates BAK1 and FLS2 with an immunocomplex kinase assay. HA-epitope-tagged BIK1 was immunoprecipitated with an anti-HA antibody and subjected to an in vitro kinase assay with GST, GST-BAK1K, or GST-FLS2K as substrate. (E) BIK1 phosphorylates BAK1 and FLS2 in vitro. An in vitro kinase assay was performed by incubating GST-FLS2K, GST-BAK1K, or their kinase mutants with GST-BIK1 or its mutants. Proteins were separated with SDS/PAGE and analyzed by autoradiography (Upper). (Upper) Autophosphorylated GST-BIK1, phosphorylated GST-BAK1K, and phosphorylated GST-FLS2K. The protein loading control was shown by Coomassie blue staining (Lower).

Fig. 5.

BIK1 is required in flg22-mediated immunity. (A) bik1 mutants show reduced sensitivity to flg22 in seedling growth assay. Wild-type (Col-0, WT), bak1, and bik1 seedlings were grown for 10 days in the presence of 200 nM flg22 (Upper) or in the absence of flg22 (Lower). (B) bik1 mutants are compromised in flg22-mediated immunity to Pst DC3000 infection. Twelve-day-old WT and bik1 seedlings were pretreated with or without flg22 and then infected with Pst DC3000. The bacterial growth assays were performed 3 and 5 days after infection. (C) bik1 mutants are compromised in plant immunity to Pst DC3000 hrcC. Results of a bacterial growth assay of WT, bik1 mutant, and BIK1 complementation (bik1+BIK1) seedlings 3 days after infection are shown. (D) A model of BIK1 in flagellin signaling. In the absence of flagellin (flg22), BIK1 associates with FLS2 and BAK1 in an inactive state. On flagellin binding to FLS2, flg22 induces FLS2 and BAK1 association and probably phosphorylation. The activated BAK1 phosphorylates BIK1, which in turn transphosphorylates the FLS2–BAK1 complex. The fully active FLS2–BAK1 may further phosphorylate BIK1 and other substrates (blue configuration), and then the active BIK1 is likely released from the FLS2–BAK1 complex to activate downstream intracellular signaling.