Identification of BZR1-interacting proteins as potential components of the brassinosteroid signaling pathway in Arabidopsis through tandem affinity purification

Abstract

Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response.

Fig. 1.

The phenotype of epitope-tagged bzr1-1D and BZR1 transgenic plants. A, Diagrammatic representation of the constructs used to transform wild-type Col plants. bzr1-1D or BZR1 was fused to 7Myc-6His (MH) under the control of the 2×35S promoter. Blue boxes indicate Gateway recombination sites. B and C, Three-week-old 35S::bzr1-1D-MH (B) and 35S::BZR1-MH (C) plants displayed a similar phenotype to that of bzr1-1D or pBZR1::bzr1-1D-CFP, and Col-0 or pBZR1::BZR1-CFP control plants respectively. D, Dark growth of the indicated seedlings on 0.5× MS medium containing 2 μm propiconazole (PPZ) for 6 days revealed that the 35S::BZR1-MH seedlings were sensitive to PPZ whereas the 35S::bzr1-1D-MH seedlings were insensitive to PPZ, similar to pBZR1::BZR1-CFP or Col-0, and pBZR1::bzr1-1D-CFP or bzr1-1D plants, respectively.

Fig. 2.

Separation of purified proteins by SDS-PAGE. bzr1-1D-MH (A) and BZR1-MH (B) copurified proteins were separated on a 5–20% gradient gel then stained with Coomassie blue. The numbers to the left indicate the sizes of the protein markers (kDa), whereas those to the right indicate the gel bands used for LC-MS/MS. m, bzr1-1D-CFP; b, bzr1-1D-MH; W, BZR1-CFP; B, BZR1-MH; star, heavy and light chains of IgG.

Fig. 3.

BZR1 interacts with and is phosphorylated by components of the MAPK complex. A, MBP-BZR1, but not MBP-PRE1 (PRE1, a HLH protein used as a negative control) pulled down GST-MKK5 and GST-MAPK4. The blots were probed with anti-GST antibodies. B, GST-MAPK4, but not GST-CYP59, pulled down MBP-BZR1, MBP-BZR1N, and (weakly) MBP-BZR1C. The blots were probed with anti-MBP antibodies. Ponceau S staining shows the amount of bait protein on the beads. C and D, Co-immunoprecipitation (IP) of BZR1 with MKK5. Arabidopsis protoplasts transfected with 35S::MKK5-YFP and 35S::BZR1-MH were immunoprecipitated using anti-c-Myc antibody (C) or anti-YFP antibodies (D), and the immunoblot was probed with anti-YFP or anti-c-Myc antibodies. E, A yeast two-hybrid assay showed that MKK5 interacted with BZR1, amino acids 1–198 of BZR1, and bzr1-1D. Yeast grown on leucine-, tryptophan-, and histidine-deficient medium (-LTH) supplemented with 1 mm 3AT revealed the interaction. Empty ADT7 was used as a negative control. F, In vitro kinase assay of MBP-BZR1 phosphorylation by GST-MKK5 but not by kinase dead GST-MKK5^K99M. Upper panel, autoradiograph; bottom panel, Coomassie blue staining of the protein gel.

Fig. 4.

HDA19 interacts with BZR1 in vitro and in vivo. A, Pull-down assay showing that GST-HDA19 interacts with MBP-BZR1 and MBP-BZR1N, but not MBP-BZR1C. The blot was probed with anti-MBP antibodies. Ponceau S staining shows the amount of bait protein on the beads. B, HDA19 interacts with bzr1-1D in yeast. Yeast grown on -LTH medium supplemented with 50 mm 3AT revealed the interaction. C, Interaction of HDA19 with bzr1-1D in planta as shown by a co-immunoprecipitation (IP) assay. Extracts of transgenic Arabidopsis seedlings expressing 35S::HDA19-YFP in a bzr1-1D or 35S::bzr1-1D-MH background were immunoprecipitated with anti-c-Myc antibodies, and the immunoblot was probed using anti-YFP antibodies. D, Relative expression of BZR1 target genes in the HDA19 null allele mutant hda19 and wild type. Seven-day-old seedlings grown on 2 μm BRZ were treated with mock solution or 100 nm BL for 1 h. *: significant difference (<0.67 ratio, and p < 0.05) between wild-type and hda19 plants before BR treatment. (a) and (b) represent a significant difference (>1.5 ratio, and p < 0.05) in fold change between wild-type and hda19 plants.

Fig. 5.

CYSB interacts with BZR1 in vivo and in vitro. A, Pull-down assay results showing the interaction of GST-CYSB with MBP-BZR1, but not with MBP alone. The immunoblot was probed with anti-MBP antibodies. B, Co-immunoprecipitation (IP) assay results showing that CYSB interacts with BZR1 in vivo. Protein extracts from Arabidopsis seedlings co-expressing 35S:: :bzr1-1D-MH and 35S::CYSB-YFP treated with 100 nm BL or mock for 1 h, or expressing 35S:: :bzr1-1D-MH alone, were immunoprecipitated with anti-YFP antibodies, and the immunoblot was probed using anti-c-Myc antibodies. Arrows indicate phosphorylated and dephosphorylated BZR1. Numbers below indicate the relative intensity of BZR1-MH and CYSB-YFP bands. C, Detection of the BZR1 level in Col, CYSB overexpression and cysb-2 plants exposed to 1 μm eBL or mock for 1 h using anti-BZR1 antibodies. Ponceau S (PS) staining indicates loading control.

Fig. 6.

Diagrammatic representation of the BZR1-co-purified proteins identified in this study and their binding positions. 14–3-3, PP2A, and BIN2 (in green) are known BZR1-binding proteins. CYSB, HDA19, and MKK5 (in red) were shown by in vitro and in vivo experiments to be new BZR1-binding proteins. CAMTA5, UBP12, CYP59, MAPK4, RH8, RD21A, and RD21B (in yellow) were shown by in vitro experiments to be BZR1-interacting proteins. BSK1 and SYTA (in pink) interact indirectly with BZR1. The BZR1-binding regions for the interacting proteins are given above the bar.