Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Mar 4;111(9):3632-7.
doi: 10.1073/pnas.1318817111. Epub 2014 Feb 14.

Tyrosine phosphorylation of protein kinase complex BAK1/BIK1 mediates Arabidopsis innate immunity

Wenwei Lin  1 , Bo Li, Dongping Lu, Sixue Chen, Ning Zhu, Ping He, Libo Shan
Affiliations

Tyrosine phosphorylation of protein kinase complex BAK1/BIK1 mediates Arabidopsis innate immunity

Wenwei Lin et al. Proc Natl Acad Sci U S A. .

Abstract

The sessile plants have evolved a large number of receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) to modulate diverse biological processes, including plant innate immunity. Phosphorylation of the RLK/RLCK complex constitutes an essential step to initiate immune signaling. Two Arabidopsis plasma membrane-resident RLKs, flagellin-sensing 2 and brassinosteroid insensitive 1-associated kinase 1 (BAK1), interact with RLCK Botrytis-induced kinase 1 (BIK1) to initiate plant immune responses to bacterial flagellin. BAK1 directly phosphorylates BIK1 and positively regulates plant immunity. Classically defined as a serine/threonine kinase, BIK1 is shown here to possess tyrosine kinase activity with mass spectrometry, immunoblot, and genetic analyses. BIK1 is autophosphorylated at multiple tyrosine (Y) residues in addition to serine/threonine residues. Importantly, BAK1 is able to phosphorylate BIK1 at both tyrosine and serine/threonine residues. BIK1Y150 is likely catalytically important as the mutation blocks both tyrosine and serine/threonine kinase activity, whereas Y243 and Y250 are more specifically involved in tyrosine phosphorylation. The BIK1 tyrosine phosphorylation plays a crucial role in BIK1-mediated plant innate immunity as the transgenic plants carrying BIK1Y150F, Y243F, or Y250F (the mutation of tyrosine to phenylalanine) failed to complement the bik1 mutant deficiency in immunity. Our data indicate that plant RLCK BIK1 is a nonreceptor dual-specificity kinase and both tyrosine and serine/threonine kinase activities are required for its functions in plant immune signaling. Together with the previous finding of BAK1 to be autophosphorylated at tyrosine residues, our results unveiled the tyrosine phosphorylation cascade as a common regulatory mechanism that controls membrane-resident receptor signaling in plants and metazoans.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
BIK1 interacts with BAK1 in vivo and in vitro. (A) BIK1 associates with BAK1 in transgenic plants. Total proteins from seedlings carrying pBIK1::BIK1-HA/pBAK1::BAK1-GFP or pBIK1::BIK1-HA were immunoprecipitated with an α-GFP antibody (IP: α-GFP) and analyzed with Western blot using an α-HA-HRP antibody (WB: α-HA) shown (Top). The expression of BIK1-HA and BAK1-GFP is shown in the Middle and Bottom, respectively. (B) BIK1 interacts with BAK1 in BiFC assay. The increased fluorescence achieved by coexpression of BAK1-nYFP and BIK1-cYFP is visible as the halo around the periphery of the plasma membrane that is only observed when both constructs are expressed. (C) BIK1 interacts with BAK1CD with in vitro pull-down assay. GST or GST-BIK1 immobilized on glutathione Sepharose beads was incubated with MBP or MBP-BAK1CD proteins. The beads were collected for Western blot with an α-HA-HRP antibody. The proteins were shown by Coomassie blue staining (CBS). The above experiments were repeated three times with similar results.
Fig. 2.
Fig. 2.
Transphosphorylation in FLS2/BAK1/BIK1 complex. (A) Schematic structure of BIK1. The position of phosphorylated amid acid detected from MS analysis of BIK1 phosphorylation is labeled. * indicates the residues identified from both BIK1 autophosphorylation and transphosphorylation by BAK1. # indicates the residues identified from transphosphorylation by BAK1. NT, N-terminal domain; I–XI, 11 kinase subdomains; CT, C-terminal domain. (B) BIK1 T237 is phosphorylated by BAK1 with MS analysis. Sequence of a doubly charged peptide ion at m/z 747.30 matches DGPMGDLSYVSpTR of BIK1. (C) BIK1 T237 is an essential phosphorylation site by BAK1 in vitro. The in vitro kinase assay was performed using MBP-BAK1CD as a kinase and BIK1Km variants as the substrates. Phosphorylation was analyzed by autoradiography (Upper), and the protein loading was shown by CBS (Lower). (D) BAK1 T450 and T455 are required for autophosphorylation and transphosphorylation of BIK1 in vitro. BIK1Km fusion proteins were used as the substrates for BAK1CD variants. (E) T455 is one of the phosphorylation sites of BAK1 by BIK1 in vitro. BAK1K variants were used as the substrates for BIK1 in an in vitro kinase assay. (F) BAK1 phosphorylates FLS2 in vitro. GST-FLS2K fusion proteins were used as the substrates for BAK1CD variants. The above kinase assays were repeated four times with similar results. The MS analysis was repeated twice.
Fig. 3.
Fig. 3.
Specific tyrosine phosphorylation of BIK1. (A) BIK1 Y250 is autophosphorylated with MS analysis. (B) BIK1 is autophosphorylated on tyrosine residues in vitro. GST-BIK1 and its variants were used in the in vitro phosphorylation assay and BIK1 phosphorylation was detected by immunoblotting with an α-pY Ab (Top) and an α-phosphotheronine (α-pT) antibody (Middle). The protein loading was shown by CBS (Bottom). (C) In vitro BIK1 autophosphorylation detected by [32P]-γ-ATP (Upper). The protein loading was shown by CBS (Lower).
Fig. 4.
Fig. 4.
BAK1-mediated tyrosine phosphorylation on BIK1. (A) BAK1 phosphorylates BIK1 on tyrosine residues in vitro. BIK1Km variants were used as the substrates for MBP-BAK1CD. The α-pY Ab was used to detect tyrosine phosphorylation (Upper) and the protein loading was shown by CBS (Lower). (B) BIK1 Y250 (Left) and Y243 (Right) are phosphorylated by BAK1 with MS analysis. BIK1T242A and BIK1Km were used for MS assays. (C) In vitro phosphorylation of BIK1Km variants by BAK1. BIK1Km variants were used as the substrates for MBP-BAK1CD. The phosphorylation was shown by autoradiography (Upper) and the protein loading was shown by CBS (Lower). The above experiments were repeated three times with similar results. MS analysis was repeated twice.
Fig. 5.
Fig. 5.
BIK1 tyrosine phosphorylation in flg22 signaling. (A) In vivo tyrosine phosphorylation of BIK1. BIK1-GFP or the empty vector control (Ctrl) was expressed in WT protoplasts for 8 h followed by 1μM flg22 treatment for 10 min. BIK1 tyrosine phosphorylation was detected by immunoblotting with α-pY Ab (Upper) and with an α-GFP antibody for protein expression (Lower). (B) Requirement of specific tyrosine residues in flg22-induced BIK1 phosphorylation. BIK1 variants were expressed in WT protoplasts for 8 h followed by 1 μM flg22 treatment for 10 min and subjected to immunoblotting with an α-HA antibody. The flg22-mediated BIK1 phosphorylation is indicated by the mobility shift (Top) and the protein loading is shown by Ponceau S staining of the membrane (Middle). The intensity of the shifted and unshifted bands was quantified by ImageJ software and % of their ratio is shown (Bottom). (C) Activation of pFRK1::LUC by BIK1 variants. The pFRK1::LUC was cotransfected with BIK1, BIK1 variants or a vector control in protoplasts. UBQ10-GUS was included as a transfection control and the luciferase activity was normalized with GUS activity. The above experiments were repeated three to four times with similar results.
Fig. 6.
Fig. 6.
Y150, Y243, and Y250 are required for BIK1 functions in plant immunity. (A) flg22-triggered oxidative burst. ROS production from leaf discs of 5-wk-old plants was presented as total photon counts during 30 min of 100 nM flg22 treatment. Values presented are mean ± SE (n = 36) and significant difference is shown as *P < 0.05, established by a one-way ANOVA compared with data from WT plants. (B) flg22-mediated restriction of bacterial growth. Four-week old plants were pretreated with H2O or 100 nM flg22 for 24 h and followed by hand inoculation of Pst at 5 × 105 cfu/mL. Bacterial growth was measured at 2 dpi. The data are shown as mean ± SE of three repeats and the different letters indicate a significant difference with P < 0.05 compared with data from WT plants. (C) The disease symptom of Pst infection. The similar experiments were performed as in B with the picture taken at 3 dpi. (D) Bacterial growth of Psm infection. Plants were hand inoculated with Psm at 5 × 105 cfu/mL and the bacterial growth was measured at 2 and 3 dpi. The data are shown as mean ± SE of three repeats and significant difference is shown as *P < 0.05, compared with data from WT plants. (E) Disease symptom of Psm infection. The similar experiments were performed as in D with the picture taken at 3 dpi. (F) Disease symptom of B. cinerea infection. Leaves from 5-wk-old plants were deposited with 10 μL of B. cinerea strain BO5 at a concentration of 2.5 × 105 spores per mL. Disease symptom was recorded at 2 dpi. (G) Lesion development of B. cinerea infection. Similar assays were performed as in F and the lesion diameter was measured at 2 dpi. The data are shown as mean ± SE of at least 30 leaves and significant difference is shown at *P < 0.05, compared with data from WT plants. The above experiments were repeated two times with similar results. The BIK1 complementation transgenic plants were pBIK1::BIK1Y150F-HA line 2-1, pBIK1::BIK1Y243F-HA line 1-3, and pBIK1::BIK1Y250F-HA line A-7.
See this image and copyright information in PMC

References

    1. Chisholm ST, Coaker G, Day B, Staskawicz BJ. Host-microbe interactions: Shaping the evolution of the plant immune response. Cell. 2006;124(4):803–814. - PubMed
    1. Jones JDG, Dangl JL. The plant immune system. Nature. 2006;444(7117):323–329. - PubMed
    1. Boller T, Felix G. A renaissance of elicitors: Perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol. 2009;60:379–406. - PubMed
    1. Schwessinger B, Ronald PC. Plant innate immunity: Perception of conserved microbial signatures. Annu Rev Plant Biol. 2012;63:451–482. - PubMed
    1. Monaghan J, Zipfel C. Plant pattern recognition receptor complexes at the plasma membrane. Curr Opin Plant Biol. 2012;15(4):349–357. - PubMed

Publication types

MeSH terms