Ligand-induced monoubiquitination of BIK1 regulates plant immunity

Abstract

Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens1,2,3. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants4. The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)–BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2–BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR–RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.

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Fig. 1: MAMP-induced BIK1 endocytosis and monoubiquitination.
Fig. 2: The E3 ligases RHA3A/B interact with and monoubiquitinate BIK1.
Fig. 3: Identification of sites of RHA3A-mediated BIK1 ubiquitination.
Fig. 4: RHA3A/B-mediated monoubiquitination of BIK1 contributes to its function in immunity and endocytosis.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information files. Source Data (gels and graphs) for Figs. 14 and Extended Data Figs. 110 are provided with the paper.

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Acknowledgements

We thank Q. Chen for the CRISPR–Cas9 system, J. Li and J. Sheen for the amiRNA system, P. de Figueiredo for mouse cDNA, and T. Devarenne, M. Dickman, C. Kaplan, T. Igumenova, J. Sheen,  and the members of the Shan and He laboratories for discussion and comments on this work. The work was supported by NIH (R01GM097247) and the Robert A. Welch foundation (A-1795) to L.S., National Science Foundation (NSF) (MCB-1906060) and NIH (R01GM092893) to P.H., NSF (IOS-1147032) to A.H., the Special Research Fund (BOF15/24J/048) to E.R., and NIH (R01GM114260) to J.P.

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Authors

Contributions

X.M., P.H. and L.S. conceived and designed the experiments; X.M. performed most of the molecular, biochemical and transgenic experiments; L.A.N.C. and D.V.S., under the supervision of E.R., conducted BIK1, BIK1(9KR) and FLS2 endocytosis experiments in the transgenic plants and N. benthamiana, colocalization with ARA6, and spatial and temporal dynamics of BIK1 and FLS2 endocytosis in N. benthamiana; M.E.L., under the supervision of A.H., performed BIK1 and FLS2 endocytosis in transgenic plants by spinning disc confocal microscopy; K.T., under the supervision of B.M.T., conducted BIK1(G2A) localization, BIK1 endocytosis and colocalization with ARA6 experiments in N. benthamiana; Z.W., under the supervision of J.P., identified BIK1 in vivo and in vitro ubiquitination sites with LC–MS/MS; J.L. performed co-IP and Botrytis infection assays; X.Y. performed co-IP and transgenic plant assays; B.L. generated 35S::BIK1-GFP transgenic plants; J.Z. performed in vitro kinase assays; E.R., A.H., B.T., and J.P. analysed data, provided critical feedback and helped to shape the research. All experiments were independently reproduced in different laboratories. X.M., P.H. and L.S. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Ping He or Libo Shan.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 BIK1–GFP is functional in plants and undergoes endocytosis.

a, BIK1–GFP is functional, as confirmed by BIK1 phosphorylation in 35S::BIK1-GFP-expressing Col-0 cotyledons after treatment with 1 μM flg22. MPK6 is a loading control and the black stippled line indicates discontinuous segments from the same gel. b, BIK1–GFP restored ROS production in bik1 leaves upon flg22 treatment. Leaf discs from wild-type, bik1 and BIK1–GFP complementation plants (lines 1 and 2) were treated with 100 nM flg22 for ROS measurement using a luminometer over 50 min. Data are shown as mean ± s.e.m. (wild-type, bik1: n = 42; BIK1–GFP/bik1: n = 45). c, Time-lapse SDCM shows that BIK1–GFP endosomal puncta are highly mobile with puncta that disappear (red circle), appear (yellow circle), and rapidly move in and out of the plane of view (white circle). Scale bar, 5 μm. dk, BIK1–GFP localizes to endosomal puncta and plasma membrane in cross-sectional images of epidermal cells. The abaxial epidermal cells of cotyledons expressing BIK1–GFP were imaged with SDCM with a Z-step of 0.3 μm. A subset of the cross-sectional images is shown at the indicated depths (3, 6, 9, 12, 15, 18 and 21 μm) along with the maximum-intensity projection (MIP) of all 67 images through the epidermis. BIK1–GFP localizes to both plasma membrane and endosomal puncta (white arrows) within all sections. kp, Method for quantification of BIK1–GFP puncta within MIPs of SDCM images. k, MIPs were generated using Fiji distribution of ImageJ 1.51 (https://fiji.sc/) for each Z-series captured by SDCM imaging of BIK1–GFP cotyledons. l, Regions of MIP with non-pavement cells (for example, stomata) were removed from the image using the line draw and crop functions. The total surface area (μm2) of the image was measured using the analyze measure function. m, Puncta within the cropped MIP were recognized using a customized model generated and applied with the Trainable Weka Segmentation plug-in for Fiji. The same model was applied to all images to generate binary images showing the physical locations of all BIK1–GFP puncta (black). no, Puncta within the size range 0.1–2.5 μm2 were highlighted in green (n) and counted (o) using the analyze particles function in Fiji. BIK1–GFP endocytosis was quantified as the number of puncta per 1,000 μm2. p, An overlay of the BIK1–GFP puncta (yellow highlight) over the cropped MIP confirmed correct identification of puncta. The experiments in ac were repeated three times with similar results. Source Data

Extended Data Fig. 2 MAMP-triggered monoubiquitination of BIK1-family RLCK proteins.

a, Flg22 induces monoubiquitination of BIK1. Protoplasts from wild-type plants were transfected with BIK1–HA and FLAG–UBQ or a vector (Ctrl), and then treated with 100 nM flg22 for 30 min. After immunoprecipitation with anti-FLAG agarose, ubiquitinated BIK1 was detected by immunoblotting using anti-HA antibody (top). Middle, BIK1–HA proteins; bottom, CBB staining for RuBisCO (RBC). b, Flg22 induces BIK1 monoubiquitination in pBIK1::BIK1-HA transgenic plants. Protoplasts from pBIK1::BIK1-HA/bik1 transgenic plants were transfected with FLAG–UBQ and then treated with 100 nM flg22 for 30 min. After immunoprecipitation with anti-FLAG agarose, ubiquitinated BIK1 was detected by immunoblotting with anti-HA antibody (top). Bottom, BIK1–HA proteins. c, BAK1 is constitutively polyubiquitinated in vivo. Protoplasts from wild-type plants were transfected with BAK1–HA and FLAG–UBQ or control, and then treated with 100 nM flg22 for 30 min. Immunoprecipitation was carried out with anti-FLAG agarose. Ubn-BAK1 proteins were detected as a smear with anti-HA immunoblotting (top). Middle, BAK1–HA proteins; bottom, CBB staining for RBC. d, Flg22 induces FLS2 polyubiquitination. Protoplasts from wild-type plants were transfected with FLS2–HA and FLAG–UBQ and then treated with 100 nM flg22 for 30 min. e, f, Monoubiquitination of BIK1 with UBQ(K0). Protoplasts from pBIK1::BIK1-HA (e) or 35S::BIK1-HA (f) transgenic plants were transfected with FLAG–UBQ(K0) (all lysine residues mutated to arginine) and then treated with 100 nM flg22 for 30 min. The mutations of lysine to arginine in UBQ(K0) are shown at the top of e with amino-acid positions labelled. g, PYR-41 blocks flg22-induced BIK1 monoubiquitination. PYR-41 (50 μM) was added 30 min before flg22 treatment. h, Flg22 induces BIK1 monoubiquitination in the presence of MG132. MG132 (2 μM) was added 1 h or 2.5 h before treatment with flg22. i, Flg22-induced BIK1 monoubiquitination depends on FLS2 and BAK1. Protoplasts isolated from wild-type, fls2 or bak1-4 plants were transfected with BIK1–HA and FLAG–UBQ and then treated with 100 nM flg22 for 30 min. j, elf18, pep1 and chitin induce BIK1 monoubiquitination. 1 μM elf18, 200 nM pep1 or 100 μg/ml chitin was added to protoplasts for 30 min. k, The BIK1 homologue PBL1 is monoubiquitinated upon treatment with flg22. PBL1–HA and FLAG–UBQ were expressed in protoplasts. l, Flg22 induces monoubiquitination of the BIK1-family RLCK PBL10 but not of BSK1. HA-tagged PBL10 or BSK1 was expressed with FLAG–UBQ in wild-type protoplasts. Experiments were repeated at least three times with similar results. Source Data

Extended Data Fig. 3 Plasma membrane localization and phosphorylation are required for BIK1 ubiquitination.

a, The kinase inhibitor K252a blocks flg22-induced ubiquitination of BIK1. Protoplasts transfected with FLAG–UBQ and BIK1–HA were treated with 1 μM K252a for 30 min and then with 100 nM flg22. b, BIK1(G2A) no longer localizes to the plasma membrane. BIK1–YFP or BIK1(G2A)–YFP was expressed in N. benthamiana for imaging analysis. c, BIK1(G2A) show compromised flg22-induced monoubiquitination. BIK1–HA or BIK1(G2A)–HA was co-expressed with FLAG–UBQ in protoplasts. d, Single K-to-R mutations of BIK1 fail to block flg22-induced ubiquitination without altering kinase activity. HA-tagged wild-type or mutant BIK1 was co-expressed with FLAG–UBQ in protoplasts. e, BIK1(K204R) exhibits reduced autophosphorylation and phosphorylation of BAK1. An in vitro kinase assay was performed using GST–BIK1 or GST–BIK1(K204R) as a kinase and GST or GST–BAK1K (BAK1 kinase domain without detectable autophosphorylation activity) as a substrate with [γ-32P] ATP. Top, proteins were separated with SDS–PAGE and analysed by autoradiography (Autorad.); bottom, protein loading shown CBB staining. Experiments were repeated at least twice with similar results. Source Data

Extended Data Fig. 4 RHA3A/B interacts with BIK1 in vivo.

a, BIK1 interacts with RHA3A in a co-IP assay. RHA3A–HA was co-expressed with BIK1–FLAG or control in protoplasts and then treated with 100 nM flg22 for 15 min. Left, the co-IP assay was carried out with anti-FLAG agarose and immunoprecipitated proteins were immunoblotted with anti-HA or anti-FLAG antibody. Right, BIK1–FLAG and RHA3A–HA proteins. b, RHA3A expression (mean ± s.e.m.) in pRHA3A::RHA3A-FLAG/pBIK1::BIK1-HA transgenic plants. qRT–PCR was carried out to detect RHA3A transcripts using ACTIN2 as a control. Relative gene expression in wild-type (set as 1), pBIK1::BIK1-HA (Ctrl) and two independent transgenic lines (lines 7 and 10) is shown. One-way ANOVA, n = 3. c, BIK1 associates with RHA3B independent of flg22 treatment. RHA3B–HA was co-expressed with BIK1–FLAG or control in protoplasts and then treated with 100 nM flg22 for 15 min. Left, co-IP assay was carried out with anti-FLAG agarose and immunoprecipitated proteins were immunoblotted with anti-HA or anti-FLAG antibody. Right, BIK1–FLAG and RHA3B–HA proteins before immunoprecipitation. d, FLS2 interacts with RHA3A and RHA3B in a co-IP assay. Experiments were repeated three times with similar results. Source Data

Extended Data Fig. 5 RHA3A/B ubiquitinate BIK1 in vivo.

a, GST–RHA3ACD possesses E3 ligase activity in vitro. An in vitro ubiquitination assay was performed with GST–RHA3ACD followed by deubiquitination reactions with GST–USP2-cc. N-ethylmaleimide (NEM) (10 mM), an inhibitor of deubiquitinases, and heat-inactivated (HI, 95 °C for 5 min) USP2-cc are controls. Samples were analysed by SDS–PAGE and silver staining. b, GST–RHA3ACD possesses multi-monoubiquitination activity in vitro. A ubiquitination assay was done as in a but using the ubiquitin mutant with all lysine residues mutated to arginine (UBQ(K0)). Ubiquitinated proteins were detected by immunoblotting with anti-UBQ (left) or anti-RHA3A (right) antibodies. c, RHA3 expression in T-DNA insertion mutants. RHA3A expression in the T-DNA knockout line SALK_052714 and RHA3B expression in SALK_064303 were analysed as in Extended Data Fig. 4b. Mean ± s.e.m. fold change (WT set as 1.0); two-tailed Student’s t-test, n = 3. d, Screen for the optimal amiR-RHA3A and amiR-RHA3B. Protoplasts were transfected with RHA3A-HA or RHA3B-HA with control, amiR-RHA3A or amiR-RHA3B. RHA3A or RHA3B proteins were examined by immunoblotting with anti-HA antibody. e, RHA3A and RHA3B are required for BIK1 ubiquitination in vivo. A BIK1 ubiquitination assay was carried out by co-expressing control, artificial microRNA targeting RHA3A (amiR-RHA3A) or amiR-RHA3A together with microRNA targeting RHA3B (amiR-RHA3A amiR-RHA3B). f, RHA3A and RHA3B expression in amiR-RHA3A/B transgenic plants. qRT–PCR was carried out to detect RHA3A and RHA3B transcripts with ACTIN2 as a control. Mean ± s.e.m. fold change in gene expression from two independent transgenic lines (lines 1 and 2); one-way ANOVA, n = 5. g, RHA3A and RHA3B are required for BIK1 ubiquitination in transgenic plants. Protoplasts from amiR-RHA3A/B transgenic plants were transfected with BIK1–HA and FLAG–UBQ for ubiquitination assay. Bottom, quantification of BIK1 ubiquitination in amiR-RHA3A/B transgenic plants. Intensity of Ub-BIK1 or BIK1 bands was quantified with Image Lab (Bio-Rad). The amount of BIK1 ubiquitination is the relative intensity of the Ub-BIK1 band to the BIK1 band (no treatment in wild-type set as 1.0). Mean ± s.e.m.; different letters indicate significant difference with others (P < 0.05, one-way ANOVA, n = 3). h, Sequencing analysis of RHA3A and RHA3B genes in the CRISPR–Cas9 rha3a/b mutant. PCR fragments corresponding to RHA3A and RHA3B in rha3a/b were amplified, sequenced, and aligned to wild-type coding sequences. The reverse complement of the PAM sequence is underlined in red, and red arrowheads indicate the theoretical Cas9 cleavage sites. The experiments were repeated three times with similar results. Source Data

Extended Data Fig. 6 BIK1 in vitro ubiquitination sites identified by mass spectrometry.

MS/MS spectra of peptides containing ubiquitinated lysine residues of BIK1. a, K31; b, K41; c, K95; d, K106; e, K170; f, K186; g, K286; h, K337; I, K366. MS spectra are outputs from the SEQUEST program. MS analysis was performed once.

Extended Data Fig. 7 BIK1 in vivo ubiquitination sites identified by mass spectrometry.

a, Ubiquitinated BIK1–GFP in planta was immunoprecipitated for LC–MS/MS analysis. BIK1–GFP and FLAG–UBQ were co-expressed in wild-type protoplasts (about 4 × 106 cells) and then treated with 200 nM flg22 for 30 min. Ubiquitinated BIK1 was immunoprecipitated with GFP-trap-agarose, separated by SDS–PAGE, digested with trypsin and subjected to LC–MS/MS analysis. Portions of cell lysates were examined for BIK1–GFP expression (left), and immunoprecipitates were analysed by SDS–PAGE following silver staining (middle; right for longer exposure of the same gel) and SDS–PAGE following CBB staining (right). The highlighted area was cut and analysed by MS. b, BIK1 is ubiquitinated in vivo. Ubiquitinated lysines containing a diglycine remnant identified by LC–MS/MS analysis are marked in red with amino acid positions. ch, MS/MS spectra of peptides containing ubiquitinated lysines of BIK1 are shown. c, K31; d, K41; e, K95; f, K337; g, K358; h, K366. MS spectra are outputs from the SEQUEST program. MS analysis was performed once.

Extended Data Fig. 8 BIK1 monoubiquitination is required for plant defence and flg22 signalling.

a, BIK1(9KR) undergoes phosphorylation similar to BIK1 upon flg22 treatment. BIK1–HA or BIK1(9KR)–HA was expressed in wild-type protoplasts which were then treated with 100 nM flg22 for the indicated times. Band-shift of BIK1 was examined by immunoblotting with anti-HA antibody. b, BIK1(9KR) interacts with RHA3A in a co-IP assay. RHA3A–HA was co-expressed with BIK1–FLAG or BIK1(9KR)–FLAG in protoplasts that were then treated with 100 nM flg22 for 15 min. Co-IP assay was carried out with anti-FLAG agarose and immunoprecipitated proteins were immunoblotted with anti-HA or anti-FLAG antibody (top two panels). Bottom two panels show BIK1–FLAG or BIK1(9KR)–FLAG and RHA3A–HA proteins. c, Transgenic plants with BIK19KR overexpression in wild-type background show similar MAPK activation to wild-type plants. Eleven-day-old seedlings of wild-type or 35S::BIK19KR-HA/WT transgenic plants (lines 55 and 56) were treated with 200 nM flg22 for 15 min. MAPK activation was analysed with anti-pERK antibody (top), and protein loading is shown by CBB staining for RBC (bottom). d, Transgenic plants with BIK19KR overexpression in wild-type background show similar flg22-induced ROS production to wild-type plants. Leaf discs from the indicated genotypes were treated with 100 nM flg22, and ROS production was measured as relative luminescence units by a luminometer over 50 min. Mean total photon count ± s.e.m. overlaid with dot plot (one-way ANOVA, n = 16). e, Growth phenotype of pBIK1::BIK1-HA/bik1 and pBIK1::BIK19KR-HA/bik1 transgenic plants. Five-week-old soil-grown plants are shown. Scale bar, 1 cm. f, Expression of BIK1–HA or BIK1(9KR)–HA in transgenic plants. Top, total proteins from leaves of four-week-old transgenic plants were subjected to anti-HA immunoblotting. Bottom, CBB staining for RBC. g, RHA3A and RHA3B are involved in resistance to Pst DC3000 hrcC infection. Plants were spray-inoculated with Pst DC3000 hrcC and bacterial growth was measured at 4 dpi. Mean ± s.e.m. overlaid with dot plots (one-way ANOVA, n = 6). h, RHA3A and RHA3B are involved in resistance to Botrytis. Four-week-old plant leaves were deposited with 10 μl B. cinerea BO5 at a concentration of 2.5 × 105 spores per ml. Disease symptoms were recorded, and the lesion diameter was measured at 2 dpi. Mean ± s.e.m. overlaid with dot plots (one-way ANOVA, n = 34). i, ROS production is reduced in rha3a/b plants. Leaf discs from wild-type or rha3a/b plants were treated with 100 nM flg22 and ROS production measured over 50 min. Mean ± s.e.m. total photon count overlaid with dot plots (two-tailed Student’s t-test, n = 36 for wild-type and n = 32 for rha3a/b). j, RHA3A and RHA3B are involved in resistance to Pst DC3000. Plants were spray-inoculated with Pst DC3000 and bacterial growth was measured at 3 dpi. Mean ± s.e.m. overlaid with dot plots (two-tailed Student’s t-test, n = 9). Experiments were repeated three times with similar results. Source Data

Extended Data Fig. 9 The BIK1(9KR) mutation impairs flg22-induced endocytosis of BIK1.

a, b, BIK1(9KR)–GFP puncta colocalize less than BIK1–GFP with FM4-64 upon treatment with flg22. a, Five-day-old 35S::BIK1-GFP or 35S::BIK19KR-GFP seedlings were pretreated with FM4-64 (2 μM) for 15 min and elicited with 100 nM flg22 for the indicated times; fluorescence was detected in epidermis using confocal microscopy. White arrows, colocalized endosomes. Scale bars, 20 μm. b, Percentage of endosomes positive for BIK1–GFP or BIK1(9KR)–GFP and FM4-64 over time per 100% of image area. Mean ± s.e.m. overlaid with dot plots (two-tailed Student’s t-test, n = 21 images for BIK1–GFP and n = 16, 15 images for 10, 15 min, respectively, for BIK1(9KR)–GFP). c, Flg22-induced endocytosis of BIK1, BIK1(9KR) and FLS2 in N. benthamiana. BIK1–TagRFP (BIK1–RFP) or BIK1(9KR)–TagRFP (BIK1(9KR)–RFP) was co-expressed with FLS2–YFP in N. benthamiana, infiltrated with 100 μM flg22 and imaged at the indicated time points by confocal microscopy. Images at 30–40, 40–50 and 50–60 min after flg22 treatment from Fig. 4e are shown here. Scale bars, 20 μm. For BIK1–RFP/FLS2–YFP, n = 14, 11, 7, 10, 10, 6, 7 images for 0, 10–20, 20–30, 30–40, 40–50, 50–60, 100–120 min; for BIK1(9KR)–RFP/FLS2–YFP, n = 19, 11, 11, 9, 16, 12, 7 images for 0, 10–20, 20–30, 30–40, 40–50, 50–60, 100–120 min, respectively. d, Percentage of BIK1–RFP puncta that colocalized with FLS2–YFP after treatment with flg22 for the indicated times in c and Fig. 4e. Mean ± s.e.m. overlaid with dot plots (n = 14, 11, 7, 10, 10, 6, 7 images for 0, 10–20, 20–30, 30–40, 40–50, 50–60, 100–120 min, respectively). e, f, BIK1(9KR)–RFP shows reduced colocalization with ARA6–YFP. e, BIK1–RFP or BIK1(9KR)–RFP was transiently expressed with ARA6–YFP in N. benthamiana, and the images were taken 48–72 h after infiltration. Scale bars, 10 μm. f, Percentage of BIK1–RFP puncta that colocalized with ARA6–YFP. Mean ± s.e.m. overlaid with dot plots (two-tailed Student’s t-test, n = 9 images for BIK1–RFP; n = 10 images for BIK1(9KR)–RFP). Experiments were repeated three times with similar results. Source Data

Extended Data Fig. 10 Monoubiquitination mediates release of BIK1 from the plasma membrane upon ligand detection.

a, PYR-41 impairs flg22-induced dissociation of BIK1 from FLS2. FLS2–HA was co-expressed with BIK1–FLAG or control in protoplasts. After pretreatment with 50 μM PYR-41 for 30 min, protoplasts were stimulated with 100 nM flg22 for 15 min. Co-IP and immunoblotting were performed as in Fig. 4g. b, A working model of RHA3A/B-mediated BIK1 monoubiquitination in plant immunity. Under non-activated, steady-state conditions (0 min), BIK1 remains hypo-phosphorylated and associates with FLS2 and BAK1. Upon flg22 detection, FLS2 dimerizes with BAK1, which stimulates BIK1 phosphorylation (<1 min). Phosphorylated BIK1 is monoubiquitinated by the E3 ligases RHA3A and RHA3B, leading to dissociation of BIK1 from the FLS2–BAK1 complex, accompanied by endocytosis (10–20 min). Ligand-induced monoubiquitination of BIK1 contributes to the activation of ROS and other defence responses. FLS2 is polyubiquitinated and endocytosed 40 min after detection of flg22 to attenuate signalling. c, BIK1(9KR) shows comparable protein expression to BIK1 in transgenic plants. 35S::BIK1-HA or 35S::BIK19KR-HA transgenic plants in wild-type background were used for immunoblotting to detect BIK1 proteins with anti-HA antibody. Control, empty vector. d, Stability of BIK1 and BIK1(9KR) proteins after treatment with cycloheximide (CHX). BIK1–HA or BIK1(9KR)–HA was expressed in wild-type protoplasts for 12 h followed by treatment with 500 μg/ml CHX for the indicated time. BIK1 or BIK1(9KR) proteins were analysed by immunoblotting with anti-HA antibody. Asterisk indicates that CHX was added immediately after transfection, thus blocking protein synthesis. Experiments were repeated three times with similar results.

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This file contains Supplementary Figure 1, the uncropped blots, and Supplementary Table 1, a list of primers used in the study.

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Ma, X., Claus, L.A.N., Leslie, M.E. et al. Ligand-induced monoubiquitination of BIK1 regulates plant immunity. Nature 581, 199–203 (2020). https://doi.org/10.1038/s41586-020-2210-3

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