Inverse modulation of plant immune and brassinosteroid signaling pathways by the receptor-like cytoplasmic kinase BIK1

Abstract

Maintaining active growth and effective immune responses is often costly for a living organism to survive. Fine-tuning the shared cross-regulators is crucial for metazoans and plants to make a trade-off between growth and immunity. The Arabidopsis regulatory receptor-like kinase BAK1 complexes with the receptor kinases FLS2 in bacterial flagellin-triggered immunity and BRI1 in brassinosteroid (BR)-mediated growth. BR homeostasis and signaling unidirectionally modulate FLS2-mediated immune responses at multiple levels. We have shown previously that BIK1, a receptor-like cytoplasmic kinase, is directly phosphorylated by BAK1 and associates with FLS2/BAK1 complex in transducing flagellin signaling. In contrast to its positive role in plant immunity, we report here that BIK1 acts as a negative regulator in BR signaling. The bik1 mutant displays various BR hypersensitive phenotypes accompanied with increased accumulation of de-phosphorylated BES1 proteins and transcriptional regulation of BZR1 and BES1 target genes. BIK1 associates with BRI1, and is released from BRI1 receptor upon BR treatment, which is reminiscent of FLS2-BIK1 complex dynamics in flagellin signaling. The ligand-induced release of BIK1 from receptor complexes is associated with BIK1 phosphorylation. However, in contrast to BAK1-dependent FLS2-BIK1 dissociation, BAK1 is dispensable for BRI1-BIK1 dissociation. Unlike FLS2 signaling which depends on BAK1 to phosphorylate BIK1, BRI1 directly phosphorylates BIK1 to transduce BR signaling. Thus, BIK1 relays the signaling in plant immunity and BR-mediated growth via distinct phosphorylation by BAK1 and BRI1, respectively. Our studies indicate that BIK1 mediates inverse functions in plant immunity and development via dynamic association with specific receptor complexes and differential phosphorylation events.

Keywords: BRI1-associated receptor kinase; botrytis-induced kinase 1; brassinosteroid insensitive 1; bri1-Ems-Suppressor 1; flagellin sensing 2.

Fig. 1.

Elevated BR responses in bik1 mutant plants. (A) The bik1 mutant is partially insensitive to BRZ treatment. The seedlings of WT (Col-0), bak1-4, and bik1 mutants were grown in the dark for 8 d on 1/2 MS plates with or without 2 μM BRZ. (B) Quantification of hypocotyl length shown in A. (C) The bik1 mutant is hypersensitive to BL treatment. The seedlings were grown on 1/2 MS plates with or without 100 nM BL under the constant light for 14 d. (D) Quantification of root and hypocotyl length shown in C. The data are shown as mean ± SE from at least 25 seedlings. Asterisk indicates a significant difference with P < 0.05 compared with data from WT seedlings. (E) BIK1 complementation lines restore the BRZ insensitivity of the bik1 mutant. The above experiments were repeated three to four times with similar results.

Fig. 2.

BIK1 negatively regulates BR signaling. (A) BES1 phosphorylation in WT and bik1 mutant plants. The phosphorylated (pBES1) and dephosphorylated BES1 proteins were detected with an α-BES1 antibody (Upper). Equal loading was ensured by total protein quantification before loading and by Coomassie brilliant blue staining (CBS) of the membrane (Lower). The band intensity was quantified by ImageJ software and labeled under the gel. (B) Expression of BR responsive genes with qRT-PCR analysis. Ten-day-old seedlings were treated with 2 μM BL or H₂O for 2 h. The expression of BR6OX, CPD, or DWF4 was normalized to the expression of UBQ10. The data are shown as mean ± SE from three independent biology repeats. Asterisk indicates a significant difference with P < 0.05 compared with data from WT seedlings. The above experiments were repeated three times with similar results.

Fig. 3.

BIK1 associates with BRI1. (A) BIK1 associates with BRI1 in protoplasts. BIK1-FLAG was coexpressed with BRI1-HA in Arabidopsis protoplasts. Co-IP was carried out with an α-FLAG antibody (IP: α-FLAG), and the proteins were analyzed by using Western blot with α-HA antibody. Top shows that BIK1-FLAG coimmunoprecipitated with BRI1-HA (IP: α-FLAG; WB: α-HA). Middle and Bottom show the expression of BRI1-HA and BIK1-FLAG proteins (WB: α-HA or α-FLAG for input control). Protoplasts were treated with 2 μM BL for 2 h. (B) BIK1 associates with BRI1 in transgenic plants. The membrane proteins from 4-wk-old pBIK1::BIK1-HA/pBRI1::BRI1-GFP (#6 and #9) or pBRI1::BRI1-GFP plants were immunoprecipitated with α-HA antibody and analyzed with Western blot using α-GFP antibody (Top). The expression of BRI1-GFP and BIK1-HA in transgenic plants are shown (Middle and Bottom). (C) BIK1 interacts with BRI1 cytosolic domain in vitro. GST-BIK1 proteins were incubated with MBP or MBP-BRI1CD beads (PD:MBP), and the beads were collected and washed for Western blot of immunoprecipitated proteins with α-GST antibody. Asterisk indicates nonspecific bands. The above experiments were repeated three times with similar results.

Fig. 4.

BL-induced BIK1 phosphorylation by BRI1. (A) BRI1 phosphorylates BIK1 in vitro. An in vitro kinase assay was performed by incubating MBP-BRI1CD with GST, GST-BIK1Km, or GST-BAK1Km proteins. Proteins were separated by 10% SDS/PAGE and analyzed by autoradiography (Upper), and the protein loading control was shown by CBS (Lower). (B) BL treatment enhances BRI1 phosphorylation on BIK1. BRI1-HA was expressed in WT protoplasts for 10 h followed by 2 μM BL treatment for 2 h. BRI1-HA proteins were immunoprecipitated with α-HA antibody and subjected to an in vitro kinase assay with GST-BIK1Km or GST-BAK1Km proteins as substrates (Top). Middle shows the BRI1-HA expression, and Bottom shows GST-BIK1Km and GST-BAK1Km proteins. (C) BIK1Km-BRI1 association in protoplasts. Protoplasts were treated with 2 μM BL for 2 h. (D) BIK1-BRI1Km association in protoplasts. (E) BAK1 is required for flg22-induced BIK1-FLS2 dissociation. The BIK1–FLS2 interaction was performed with bak1-4 protoplasts. Protoplasts were treated with 1 μM flg22 for 15 min. (F) BAK1 is not required for BL-induced BIK1-BRI1 dissociation. The BIK1–BRI1 interaction was performed with bak1-4 protoplasts. Protoplasts were treated with 2 μM BL for 2 h. The above experiments were repeated three times with similar results.

Fig. 5.

BIK1 acts downstream of BRI1 in BR signaling. (A) The bik1bri1-5, bik1bri1-119, and bik1det2-1 double mutants partially rescued the growth deficiency of single mutants. The phenotypes of 4-wk-old (Upper) and 8-wk-old (Lower) soil-grown plants are shown. (B) The hypocotyls of dark-grown bik1bri1-5 and bik1bri1-119 seedlings were longer than those of single mutants. Representatives of 8-d-old seedlings grown in the dark are shown on Left, and the quantification of hypocotyl length is shown on Right. The data are shown as mean ± SE from at least 25 seedlings. Asterisk indicates a significant difference with P < 0.05 compared with data from the corresponding single mutants. (C) The bik1bri1-5 and bik1bri1-119 seedlings partially restored the leave growth and hypocotyl length of single mutants grown under the light. Representatives of 14-d-old seedlings under the constant light are shown on Left, and the quantification of hypocotyl length is shown on Right.