Mechanistic study of SCOOPs recognition by MIK2–BAK1 complex reveals the role of N-glycans in plant ligand–receptor–coreceptor complex formation

Abstract

Ligand-induced receptor and co-receptor heterodimerization is a common mechanism in receptor kinase (RK) signalling activation. SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) mediate the complex formation of Arabidopsis RK MIK2 and co-receptor BAK1, triggering immune responses. Through structural, biochemical and genetic analyses, we demonstrate that SCOOPs use their SxS motif and adjacent residues to bind MIK2 and the carboxy-terminal GGR residues to link MIK2 to BAK1. While N-glycosylation of plant RKs is typically associated with protein maturation, plasma membrane targeting and conformation maintenance, a surprising revelation emerges from our crystal structural analysis of MIK2–SCOOP–BAK1 complexes. Specific N-glycans on MIK2 directly interact with BAK1 upon SCOOP sensing. The absence of N-glycosylation at the specific site in MIK2 neither affects its subcellular localization and protein accumulation in plant cells nor alters its structural conformation, but markedly reduces its affinity for BAK1, abolishing SCOOP-triggered immune responses. This N-glycan-mediated receptor and co-receptor heterodimerization occurs in both Arabidopsis and Brassica napus. Our findings elucidate the molecular basis of SCOOP perception by the MIK2–BAK1 immune complex and underscore the crucial role of N-glycans in plant receptor–coreceptor interactions and signalling activation, shaping immune responses.

Wu et al. elucidate the molecular basis for SCOOPs perception by the MIK2–BAK1 immune complex and demonstrate an unexpectedly pivotal role of N-glycans in plant receptor–coreceptor interactions and signalling activation, shaping immune responses.

Main

Sensing the extracellular environment is of fundamental importance to eukaryotic development and defence. Plants deploy a repertoire of cell surface-localized receptor kinases (RKs) that recognize endogenous and exogenous signals^1–4. RKs with an extracellular leucine-rich repeat (LRR) domain constitute the largest subgroup of LRR-RKs in plants with more than 220 members in Arabidopsis and over 300 members in rice^5,6. Some LRR-RKs function as pattern-recognition receptors (PRRs) that recognize pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) or host-derived damage-associated molecular patterns (DAMPs), leading to pattern-triggered immunity (PTI)^4,7–11. The Arabidopsis LRR-RK MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) is a bona fide receptor of plant endogenous SERINE-RICH ENDOGENOUS PEPTIDE (SCOOP) family peptides as well as SCOOP-LIKE (SCOOPL) sequences residing in proteins from a wide range of fungal Fusarium spp. and bacterial Comamonadaceae^12,13. Perception of SCOOPs induces the heteromerization of MIK2 with BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) or SOMATIC EMBRYOGENESIS RK 4 (SERK4) in triggering a series of PTI responses including mitogen-activated protein kinase (MAPK) activation, reactive oxygen species (ROS) burst and cytosolic Ca²⁺ concentration increase^12,13. Thus, MIK2 represents a unique PRR with a dual role in perceiving both MAMPs and DAMPs. However, the biochemical mechanism underlying this bifunctional PRR sensing of ligands from different kingdoms has yet to be elucidated.

Glycosylation of asparagine (N) residues (N-glycosylation) is widely observed in plant RKs and commonly essential for their activities^14–18. Previous studies have shown that deficiency in N-glycosylation could impair protein maturation, plasma membrane targeting and conformation maintenance^14–18. Exploring novel roles of N-glycosylation of plant RKs could greatly advance the mechanistic understanding of ligand-induced RK activation and signal transduction.

SCOOP perception by MIK2–BAK1 complex in Brassicaceae plants

MIK2 is present and conserved in a wide range of plant species, including Brassicaceae, Fabaceae, Poaceae and Solanaceae¹³ (Supplementary Fig. 1a,b). In particular, MIK2 orthologues in different Brassicaceae plants bear ≥80% amino acid identities to Arabidopsis MIK2 (AtMIK2) (Supplementary Fig. 1a). However, plant SCOOPs are only present in the Brassicaceae family^19,20. So far, about 50 SCOOPs have been identified in Arabidopsis, with AtSCOOP12 being the most studied one^{12,13,20–23}. Consistent with previous studies^12,13, glutathione S-transferase (GST) pull-down assay showed that purified GST-tagged AtSCOOP12 and FocSCOOPL from Fusarium oxysporum f. sp. conglutinans strain Fo5176 could induce the complex formation of ectodomains (ECDs) of AtMIK2 and AtBAK1 (Fig. 1a). In the oilseed crop Brassica napus, a 13-amino-acid peptide sequence (FAGPSSSGHGGGR) in two secreted proteins (CDY22858.1 and CDY33880.1) bearing 48% identities to AtSCOOP12 was inferred to be a SCOOP homologue²⁰ (hereafter designated as BnSCOOP) (Supplementary Fig. 1c). Similar to AtSCOOP12 and FocSCOOPL, BnSCOOP induces the heteromerization of B. napus MIK2^ECD (BnMIK2^ECD) and BnBAK1^ECD (Fig. 1b). In line with the observation that AtSCOOP12 triggers immune responses in Brassica napus²⁰, BnSCOOP also induced AtMIK2-dependent MAPK activation in Arabidopsis protoplasts (Fig. 1c). Moreover, similar to AtMIK2, BnMIK2 complemented AtSCOOP12- or BnSCOOP-triggered MAPK activation in Arabidopsis mik2-1 protoplasts (Fig. 1d). These data suggest that perception of BnSCOOP and microbial-derived SCOOPL peptides by BnMIK2 and BnBAK1 also exists in B. napus.

Fig. 1. SCOOP-induced Arabidopsis and Brassica napus MIK2–BAK1 interaction.

a,b, SCOOPs induce Arabidopsis (a) and Brassica napus (b) MIK2^ECD to form a complex with BAK1^ECD. GST-tagged SCOOPs (GST-AtSCOOP12, GST-FocSCOOPL and GST-BnSCOOP) bound to glutathione sepharose 4B resin was used to pull down MIK2^ECD-His and BAK1^ECD-His. Samples of the input and pull down were visualized using western blot. c, SCOOP-induced MAPK activation is compromised in Arabidopsis mik2-1 protoplasts. The protoplasts were treated with 1 µM AtSCOOP12 or BnSCOOP for 0, 15 or 30 min. Protoplasts generated from Arabidopsis Col-0 were used as positive controls. MAPK activation was analysed using α-pERK1/2 immunoblotting. Coomassie brilliant blue (CBB) staining of Rubisco (RBC) was used for protein loading. Molecular weights (kDa) are labelled on the left or right of all immunoblots. d, AtMIK2 and BnMIK2 restore SCOOP-induced MAPK activation in Arabidopsis mik2-1 protoplasts. mik2-1 protoplasts were transfected with plasmids as indicated. Protoplasts generated from Col-0 and mik2-1 transfected with empty vector (EV) were used as positive and negative controls, respectively. The assays were performed as described in Fig. 1c, except that the samples were treated with 1 µM AtSCOOP12 or BnSCOOP for 15 min. The assays (a–d) were repeated three times with similar results.

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MIK2 forms super helical conformation complexing with SCOOPs

To understand the mechanism of SCOOP recognition by MIK2 at the atomic level, we attempted to crystallize the MIK2^ECD–SCOOPs binary complexes. After a systematic effort, we determined the crystal structures of an engineered AtMIK2^ECD carrying L137E/D164K/S564F (hereafter AtMIK2^ECD-3M) in complex with AtSCOOP12 and BnMIK2^ECD in complex with FocSCOOPL at 2.20 Å and 2.80 Å (Extended Data Table 1), respectively. Sequence alignment, GST pull-down, analytical size-exclusion chromatography (SEC), structural analysis and MAPK activation assays collectively revealed that AtMIK2^ECD-3M is functional as the wild type, hence the crystal structure of AtMIK2^ECD-3M could represent the conformation of the wild type (Fig. 2a, Extended Data Figs. 1 and 2, and Supplementary Fig. 1; see Supplementary Text for more details). The structures of AtMIK2^ECD-3M–AtSCOOP12 and BnMIK2^ECD–FocSCOOPL complexes are almost identical with a root mean square deviation (RMSD) of 0.67 Å in the corresponding Cα positions (Fig. 2a). MIK2^ECD adopts a super helical conformation (Fig. 2a), which is similar to the ECDs of other LRR-RKs such as FLAGELLIN SENSING 2 (FLS2), BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and PLANT ELICITOR PEPTIDE RECEPTOR 1 (PEPR1)^24–26. Both SCOOP peptides in the binary complex structures form stretched loop conformation, with several residues at the carboxyl (C) terminus being invisible in the electron density map (Fig. 2a,b and Extended Data Fig. 2), suggesting that their C termini are of high conformational flexibility and probably non-essential for MIK2^ECD binding.

Extended Data Table 1.

X-ray diffraction data collection and refinement statistics

Fig. 2. Structures of MIK2^ECD–SCOOPs binary complexes and specific recognition of SCOOPs by MIK2^ECD.

a, Overall structures of AtMIK2^ECD-3M–AtSCOOP12 (left) and BnMIK2^ECD-FocSCOOPL (right) complexes in ribbon representation. The AtSCOOP12 and FocSCOOPL peptides are shown as sticks with a transparent surface. The AtMIK2^ECD-3M, BnMIK2^ECD, AtSCOOP12 and FocSCOOPL are coloured in green, gold, orange and blue violet, respectively. b, Close-up view of the AtSCOOP12 binding in the AtMIK2^ECD-3M–AtSCOOP12 complex. The side chains or backbones of residues involved in the AtMIK2^ECD-3M–AtSCOOP12 interactions are shown as sticks. Colour coding is the same as in Fig. 2a. Residues in AtMIK2^ECD-3M and AtSCOOP12 are labelled in black and orange, respectively. The Fo−Fc electron density (2.5 σ level) of residues of interest is shown in light blue. Black dashed lines indicate hydrogen bonds. c, Mutations of the AtSCOOP12-interacting residues in AtMIK2^ECD reduce interaction with GST-AtSCOOP12. The assays were performed as described in Fig. 1a. d, Mutations of the AtSCOOP12-interacting residues in AtMIK2 compromise AtSCOOP12-induced MAPK activation. The assays were performed as described in Fig. 1d, except that the samples were treated with 0.4 μM AtSCOOP12 for 0, 10 or 20 min. The assays (c, d) were repeated three times with similar results.

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Extended Data Fig. 1. Engineered AtMIK2^ECD-3M is functional.

a, BnMIK2^ECD-to-BnMIK2^ECD contacts in the crystal lattice. BnMIK2^ECD and four symmetry-related molecules are colored in cyan, pale green, orange, pink, and blue, respectively. The close-up views of two crystal packing interfaces are shown in black and blue squares, respectively. The side chains of Q120, K147, F547 and their contacting residues located on the two interfaces are shown as sticks and labeled. Black dotted lines indicate salt bridges and hydrogen bonds. b, c, Analytical size-exclusion chromatography (SEC) showed that both wild-type AtMIK2^ECD (b) and AtMIK2^ECD-3M mutant (c) can form complexes with AtBAK1^ECD in presence of AtSCOOP12. (Upper) SEC profiles of AtMIK2^ECD/AtMIK2^ECD-3M and AtBAK1^ECD in the presence or absence of AtSCOOP12 were color-coded. (Lower) Coomassie blue-stained SDS-PAGE gels of peak fractions. d, GST pull-down assay showed that AtMIK2^ECD and AtMIK2^ECD-3M mutant can heteromerized with AtBAK1^ECD in presence of AtSCOOP12. GST-tagged AtSCOOP12 (GST-AtSCOOP12) bound to Glutathione Sepharose 4B resin was used to pull down wild-type or mutant AtMIK2^ECD-His and AtBAK1^ECD-His. Samples of the input and pull down were visualized by western blot. e, HA-tagged AtMIK2 and AtMIK2^ECD-3M can restore SCOOP-induced MAPK activation in Arabidopsis mik2-1 protoplasts. mik2-1 protoplasts were transfected with plasmids as indicated. Protoplasts generated from Col-0 and mik2-1 transfected with empty vector (EV) were used as positive and negative controls, respectively. The assays were performed as described in Fig. 2d except that the samples were treated with 0.25 μM AtSCOOP12 for 0-, 10- or 20-min. The assays were repeated three times (b-e) with similar results.

Source data

Extended Data Fig. 2. Structural comparison of MIK2^ECD in apo form and in complex with SCOOPs.

a, Overall structure of AtMIK2^ECD-3M in ribbon representation. The L137E, D164K and S564F are shown as sticks and labeled. The AtSCOOP12 peptide is shown as sticks with a transparent surface. b, Superposition of AtMIK2^ECD-3M (salmon) and AtMIK2^ECD-3M-AtSCOOP12 complex (green and orange) structures. AtMIK2^ECD-3M is shown as cartoon. The AtSCOOP12 peptide is shown as cartoon with a transparent surface. c, Superposition of the apo BnMIK2^ECD (slate) and BnMIK2^ECD-FocSCOOPL complex (gold and blueviolet) structures. BnMIK2^ECD is shown as cartoon. The FocSCOOPL peptide is shown as cartoon with a transparent surface. d-e, The electron density map of AtSCOOP12 bound in the AtMIK2^ECD-3M-AtSCOOP12 complex (d) and FocSCOOPL bound in the BnMIK2^ECD-FocSCOOPL complex (e). Residues in SCOOPs are shown as sticks and labeled. The Fo-Fc electron density of AtSCOOP12 (2.0 σ level) and FocSCOOPL (2.5 σ level) is shown in light blue. f, A close-up view of the FocSCOOPL binding in BnMIK2^ECD-FocSCOOPL complex. The side chains or backbones of the residues involved in the BnMIK2^ECD-FocSCOOPL interactions are shown as sticks. The color coding is the same as in Fig. 2a. Residues in BnMIK2^ECD and FocSCOOPL are labeled in black and blueviolet, respectively. The Fo-Fc electron density (2.5 σ level) of residues of interest is shown in light blue. Black dotted lines indicate hydrogen bonds.

MIK2 recognizes diverse SCOOPs via extensive hydrogen bonds

SCOOPs bind to the amine (N)-terminal concave side of MIK2^ECD starting from LRR3 (Fig. 2a). In detail, the side chain hydroxyl groups of S5 in AtSCOOP12 and FocSCOOPL are stabilized by the side chains of D246 in AtMIK2^ECD and D229 in BnMIK2^ECD (Fig. 2b and Extended Data Fig. 2f). Meanwhile, in the AtMIK2^ECD-3M–AtSCOOP12 complex structure, the side chain hydroxyl group of AtSCOOP12 S7 forms hydrogen bonds with AtMIK2^ECD S292 and H316 (Fig. 2b). To confirm the requirement of contacting sites of AtMIK2 in binding with AtSCOOP12 and signalling, we generated AtMIK2 D246A, S292A, H316A and S292A/H316A mutants. As shown in Fig. 2c, AtMIK2^ECD D246A, H316A and S292A/H316A but not S292A attenuated the binding of AtSCOOP12 in GST pull-down assays. Unlike the wild type (WT), AtMIK2 D246A and S292A/H316A showed substantially reduced activities in mediating AtSCOOP12-induced MAPK activation in mik2-1 protoplasts (Fig. 2d). All these data support the critical role of D246 and H316 for AtMIK2 function, which is consistent with a recent report²⁷. In addition, these observations provide a structural basis to rationalize previous biochemical and genetic characterization that the two conserved serine residues in the SxS motif of AtSCOOP12 are critical for its binding with AtMIK2 and for its activity to inhibit Arabidopsis root growth through AtMIK2 (ref. ¹³).

Strikingly, different from other LRR-RKs such as AtFLS2 and AtPEPR1 which extensively make both hydrogen bonds and hydrophobic contacts with the peptide ligands’ side chains^24,26, AtMIK2^ECD/BnMIK2^ECD predominantly recognize the backbone of SCOOPs via hydrogen-bond interactions (Fig. 2b and Extended Data Fig. 2f). The main chain amide group of AtSCOOP12 R3/FocSCOOPL S3 and the main chain carbonyl group of AtSCOOP12 S5/FocSCOOPL S5 are stabilized by the side chains of AtMIK2^ECD Y198/BnMIK2^ECD Y181 and AtMIK2^ECD N268/BnMIK2^ECD N251, respectively. In addition, in the AtMIK2^ECD-3M–AtSCOOP12 complex structure, the main chain carbonyl group of AtSCOOP12 S8 and the main chain amide group of AtSCOOP12 A10 engage in hydrogen bonds with the side chains of AtMIK2^ECD Y318 and E340, respectively. Consistent with our structural observations, mutations of AtMIK2^ECD Y198 and E340 significantly impaired its ability to bind AtSCOOP12 (Fig. 2c) and its activity to mediate AtSCOOP12-induced MAPK activation in mik2-1 protoplasts (Fig. 2d). Previous studies have shown that AtSCOOP4, AtSCOOP6B, AtSCOOP8, AtSCOOP10B, AtSCOOP13–15, AtSCOOP20 and AtSCOOP23–28 can trigger immune responses in Arabidopsis^12,13,21. However, the equivalent residues of AtSCOOP12 R3, S8 and A10 in these SCOOPs are not conserved, which could be G/P/F/E/V/K/L/D/S/T, G/P/R/Q/K/D/H/T/V/E and G/S/D, respectively (Supplementary Fig. 1c). GST pull-down assays revealed that the AtSCOOP12 R3G/P/F, S8G/P/R and A10G/S/D mutants could still induce the formation of the AtMIK2^ECD–AtBAK1^ECD complex (Extended Data Fig. 3a), indicating that AtSCOOP12 R3, S8 and A10 contribute to but are not essential for its activity.

Extended Data Fig. 3. In vitro and in vivo functional analyses of the AtSCOOP12 mutants and variant AtSCOOPs.

a, AtSCOOP12 variants have different effects in promoting the MIK2^ECD-BAK1^ECD association. The assays were performed as described in Fig. 1a. b, AtSCOOP10A, AtSCOOP19 and AtSCOOP20 cannot induce MIK2^ECD-BAK1^ECD heterodimerization in vitro. The assays were performed as described in Fig. 1a. c, AtSCOOP10A, AtSCOOP19 and AtSCOOP20 have different effects in inducing MAPK activation. The assays were performed as described in Fig. 3c. The assays were repeated three times (a-c) with similar results.

Source data

Taken together, our data revealed that AtMIK2^ECD/BnMIK2^ECD mainly recognize the side chains of the serine residues in the SxS motif and the main chains of several other residues in AtSCOOP12 and FocSCOOPL. The SxS motif is strictly conserved in SCOOPs (Supplementary Fig. 1c), and all the residues in AtMIK2^ECD/BnMIK2^ECD responsible for SCOOP recognition are strictly conserved among MIK2 homologues in the Brassicaceae family (Supplementary Fig. 1a). Therefore, our results rationalize why and how Brassicaceae MIK2 can recognize the divergent families of SCOOPs.

Overall structure of MIK2^ECD–SCOOPs–BAK1^ECD ternary complex

Binding of SCOOPs promotes association of MIK2^ECD with BAK1^ECD13,14 (Fig. 1a,b and Extended Data Fig. 1b). To probe the structural basis for SCOOP-induced heteromerization of MIK2^ECD with BAK1^ECD, we solved the crystal structures of AtMIK2^ECD-3M–AtSCOOP12–AtBAK1^ECD and AtMIK2^ECD-3M–FocSCOOPL–AtBAK1^ECD ternary complexes at a resolution of 3.10 Å and 3.30 Å, respectively (Extended Data Table 1). AtMIK2^ECD, SCOOP and AtBAK1^ECD assembled in a stoichiometry of 1:1:1 (Fig. 3a and Extended Data Fig. 4a). AtBAK1^ECD is anchored to the C-terminal segment (LRRs 17–23) of AtMIK2^ECD, while SCOOP runs across the concave surface of LRRs 3–14 in AtMIK2^ECD with its C terminus bridging AtBAK1^ECD with AtMIK2^ECD (Fig. 3a and Extended Data Fig. 4a). Structural comparison showed that the conformations of AtMIK2^ECD in the AtBAK1^ECD-free and AtBAK1^ECD-bound forms are almost identical (Extended Data Fig. 4b), indicating that AtBAK1^ECD binding induces no striking conformational changes in AtMIK2^ECD. By contrast, SCOOPs showed dramatic differences, of which several residues in the C terminus that are invisible in the AtBAK1^ECD-free structures due to the flexibility are well defined by electron density in the AtBAK1^ECD-bound structures (Extended Data Fig. 4b–d), suggesting that the C-terminal segment of SCOOPs plays a key role in promoting AtMIK2^ECD–AtBAK1^ECD heteromerization.

Fig. 3. Structures of the MIK2^ECD–SCOOP–BAK1^ECD ternary complex and SCOOP-mediated interactions between MIK2^ECD and BAK1^ECD.

a, Overview of the AtMIK2^ECD-3M–AtSCOOP12–AtBAK1^ECD ternary complex. The AtMIK2^ECD-3M, AtSCOOP12 and AtBAK1^ECD are coloured in green, orange and cyan, respectively. A close-up view of the AtMIK2^ECD-3M–AtSCOOP12–AtBAK1^ECD interacting interface is shown in the black square. The side chains or backbones of residues in AtMIK2^ECD-3M and AtBAK1^ECD that are involved in AtSCOOP12 binding and the three C-terminal residues ‘GGR’ of AtSCOOP12 are shown as sticks. Residues in AtMIK2^ECD-3M, AtSCOOP12 and AtBAK1^ECD are labelled in black, orange and marine, respectively. The Fo−Fc electron density (2.5 σ level) of residues of interest is shown in light blue. Black dashed lines indicate hydrogen bonds. b, AtSCOOP12 variants have different effects in promoting the MIK2^ECD–BAK1^ECD association. ΔC1 indicates deletion of R13 in AtSCOOP12. +G, +A, +S and +F indicate addition of a glycine (G), an alanine (A), a serine (S) and a phenylalanine (F) at the C termini of AtSCOOP12, respectively. The assays were performed as described in Fig. 1a. c, AtSCOOP12 variants have different effects in inducing MAPK activation. The assays were performed as described in Fig. 1c, except that the samples were treated with 0.5 μM AtSCOOP12 variants for 0, 15 or 30 min. d,e, AtSCOOP12 variants have different effects on root growth inhibition. Images of 10-day-old seedlings with or without 1 μM AtSCOOP12 variants (d). Scale bar, 6 mm. Quantification of primary root length (e). Box plots show the first and third quartiles (bounds of box) and medians (lines), with whiskers extending to the maximum and minimum values. Different letters above boxes indicate statistically significant differences (P < 0.05, one-way ANOVA followed by Tukey’s test, n = 51). f, AtMIK2^ECD mutations on the AtMIK2^ECD–AtSCOOP12–AtBAK1^ECD interface compromise AtSCOOP12-induced AtMIK2^ECD–AtBAK1^ECD heteromerization. The assays were performed as described in Fig. 1a. g, AtMIK2 mutations on the AtMIK2^ECD–AtSCOOP12–AtBAK1^ECD interface compromise AtSCOOP12-induced MAPK activation. The assays were performed as described in Fig. 2d. The assays (b–g) were repeated three times with similar results.

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Extended Data Fig. 4. FocSCOOPL-mediated AtMIK2^ECD-AtBAK1^ECD interactions.

a, Overview of AtMIK2^ECD-3M-FocSCOOPL-AtBAK1^ECD ternary complex. The AtMIK2^ECD-3M, FocSCOOPL and AtBAK1^ECD are colored in green, blueviolet and cyan, respectively. A close-up view of the AtMIK2^ECD-3M-FocSCOOPL-AtBAK1^ECD interacting interface is shown in black square. The side chains or backbones of the residues involved in the FocSCOOPL binding and the C-terminal three residues of FocSCOOPL are shown as sticks. Residues in AtMIK2^ECD-3M, FocSCOOPL and AtBAK1^ECD are labeled in black, blueviolet and marine, respectively. The Fo-Fc electron density (2.5 σ level) of residues of interest is shown in light blue. Black dotted lines indicate hydrogen bonds. b, Superposition of the AtMIK2^ECD-3M-AtSCOOP12, AtMIK2^ECD-3M-AtSCOOP12-AtBAK1^ECD and AtMIK2^ECD-3M-FocSCOOPL-AtBAK1^ECD complex structures was performed using the AtMIK2^ECD-3M as references. A close-up view of the SCOOPs-binding interface is shown in black square. For simplicity, only AtMIK2^ECD-3M and AtBAK1^ECD in AtMIK2^ECD-3M-AtSCOOP12-AtBAK1^ECD complex structure are shown in the close-up view. c, d, The electron density map of AtSCOOP12 bound in the AtMIK2^ECD-3M-AtSCOOP12-AtBAK1^ECD complex (c) and FocSCOOPL bound in the AtMIK2^ECD-3M-FocSCOOPL-AtBAK1^ECD complex (d). The Fo-Fc electron density (2.5 σ level) of SCOOPs is shown in light blue. e, FocSCOOPL variants have different effects in promoting the MIK2^ECD-BAK1^ECD association. ΔC1 indicates deletion of R13 in FocSCOOPL. +G, +A, and +S indicate addition of a glycine (G), an alanine (A), and a serine (S) at the C-terminus of FocSCOOPL, respectively. The assays were performed as described in Fig. 1a. f, FocSCOOPL variants have different effects in inducing MAPK activation. The assays were performed as described in Fig. 3c. g, h, FocSCOOPL variants have different effects in root growth inhibition. Images of 10-day-old seedlings with or without 1 μM FocSCOOPL variants. Scale bar, 6 mm (g). Quantification of root length (h). Box plots show the first and third quartiles as bounds of box, split by the medians (lines), with whiskers extending to the maximum and minimum values. Letters indicate statistically significant differences with others (P < 0.05, one-way ANOVA followed by Tukey’s test, n = 51). i, GFP-tagged AtMIK2 mutant variants R434A and D414A/D415A remain plasma membrane-localized in mik2-1 protoplasts. The assays were repeated three times (e-f) and twice (g-i) with similar results.

Source data

Residues 11–13 of SCOOPs mediate MIK2–BAK1 interaction

Detailed examination of the AtMIK2^ECD-3M–AtSCOOP12/FocSCOOPL–AtBAK1^ECD ternary complex structures showed that a short-stretch fragment from residues G11 to R13 of AtSCOOP12/FocSCOOPL was tightly sandwiched between AtMIK2^ECD and AtBAK1^ECD (Fig. 3a and Extended Data Fig. 4a). G11 in AtSCOOP12/FocSCOOPL did not make any direct interaction with either AtMIK2^ECD or AtBAK1^ECD. GST pull-down assays showed that compared with WT SCOOPs, the AtSCOOP12/FocSCOOPL G11K mutants induced the formation of AtMIK2^ECD–AtBAK1^ECD complex at a similar level (Fig. 3b and Extended Data Fig. 4e), indicating that residues with large side chains at this site would not generate steric clashes with AtMIK2^ECD or AtBAK1^ECD despite the limited space. Mutation of G11 to proline (G11P) in AtSCOOP12/FocSCOOPL completely blocked its ability to induce AtMIK2^ECD–AtBAK1^ECD interaction (Fig. 3b and Extended Data Fig. 4e). Considering that proline confers unique conformational restraints, it is possible that proline substitution of AtSCOOP12/FocSCOOPL G11 dramatically altered the peptide backbone conformation. Several AtSCOOPs, including AtSCOOP2, AtSCOOP6A, AtSCOOP10A, AtSCOOP17–20, AtSCOOP22, AtSCOOP25–26 and AtSCOOP28, have P at position 11. Similar to the AtSCOOP12 G11P mutant, AtSCOOP10A, AtSCOOP19 and AtSCOOP20 failed to induce the heterodimerization of AtMIK2^ECD and AtBAK1^ECD in vitro (Extended Data Fig. 3b). In line with the above interaction analysis, AtSCOOP10A could not, and AtSCOOP20 weakly induced MAPK activation in Arabidopsis WT plants (Extended Data Fig. 3c). Intriguingly, AtSCOOP19 still activated MAPKs similar to AtSCOOP12 (Extended Data Fig. 3c). In addition, AtSCOOP19 has been shown to induce ROS burst, while AtSCOOP10A and AtSCOOP20 moderately induced ROS production²³. Notably, both AtBAK1 and AtSERK4 have been shown to bind to AtMIK2 as the co-receptors upon SCOOPs perception¹³. The apparent difference between in vitro pull-down assays and in vivo immune response-eliciting activities for AtSCOOP19 could be attributed to the involvement of AtSERK4 in planta. In support of this idea, AtSCOOP19-induced MAPK activation was dramatically reduced in bak1–5/serk4 compared with that in WT (Extended Data Fig. 3c). Taken together, it is possible that co-receptors BAK1 and SERK4 or other SERKs, as well as other mechanisms, may be involved in the SCOOPs-induced immune signalling transduction and contribute to those SCOOPs with P at position 11 losing the ability to induce MIK2–BAK1 complex formation in vitro but still triggering immune responses via SERKs in planta.

The main chain amine and carbonyl groups of AtSCOOP12/FocSCOOPL G12 are recognized by the backbones of AtBAK1^ECD T52 and V54, respectively, via hydrogen-bond interactions (Fig. 3a and Extended Data Fig. 4a). Mutation of AtSCOOP12/FocSCOOPL G12 to tyrosine but not alanine almost completely abrogated its ability to promote the association of AtMIK2^ECD with AtBAK1^ECD (Fig. 3b and Extended Data Fig. 4e), suggesting that the bulky side chain of tyrosine would generate steric clashes with AtMIK2^ECD and AtBAK1^ECD due to the limited space, and consequently attenuate their interactions. Consistently, an AtSCOOP12 G12Y mutant dramatically impaired its ability to induce MAPK activation in Arabidopsis protoplasts (Fig. 3c).

The main chain carbonyl group of R13 in AtSCOOP12/FocSCOOPL is stabilized by the side chains of R434 and R436 in the LRR14 of AtMIK2^ECD, while the positively charged side chain of R13 is tightly held by a negatively charged area consisting of D390, D414 and D415 in LRRs 12–13 of AtMIK2^ECD (Fig. 3a and Extended Data Fig. 4a). Consistent with our structural observations, alanine substitution and deletion of R13 (designated as R13A and ΔC1) in AtSCOOP12/FocSCOOPL showed compromised and almost no activity of inducing AtMIK2^ECD–AtBAK1^ECD interaction in vitro (Fig. 3b and Extended Data Fig. 4e), inducing MAPK activation in mik2-1 protoplasts (Fig. 3c and Extended Data Fig. 4f) and inhibiting Arabidopsis root growth (Fig. 3d,e and Extended Data Fig. 4g, h), respectively. Meanwhile, AtMIK2^ECD mutant variants R434A and D414A/D415A no longer formed the complexes with AtBAK1^ECD upon AtSCOOP12 treatment (Fig. 3f). In addition, AtMIK2 mutant variants R434A and D414A/D415A remain plasma membrane localized (Extended Data Fig. 4i) but failed to restore the AtSCOOP12-induced MAPK activation in mik2-1 mutant protoplasts (Fig. 3g).

The overall structures of AtMIK2^ECD-3M–AtSCOOP12/FocSCOOPL–AtBAK1^ECD resemble that of AtFLS2^ECD–flg22–AtBAK1^ECD with RMSDs of ~6.12 Å for over 700 aligned Cα atoms (Supplementary Fig. 2a). However, detailed investigation showed that the small channel composed of an inner-curved loop (residues 49–58) of AtBAK1^ECD, together with the concave surface of LRRs 12–14 in AtMIK2^ECD to accommodate the C termini of SCOOPs, forms a unique conformation (Supplementary Fig. 2b). First, due to the existence of AtMIK2^ECD R434 and R436 with bulky side chains and their equivalent residues in AtFLS2^ECD being T459 and A463, the SCOOP-binding channel is more crowded than the flg22-binding channel. Second, the side chains of AtMIK2^ECD R434 and R436 tightly grasp the backbone carbonyl group of R13 in AtSCOOP12/FocSCOOPL via electrostatic interactions, while AtFLS2^ECD T459 and A463 do not form any interaction with flg22. This probably results in the conformation of the R13 backbone in AtSCOOP12/FocSCOOPL being fixed and the orientation of the C termini of AtSCOOP12/FocSCOOPL being almost 180° different from that of flg22. Lastly, N-glycans attached to AtMIK2^ECD N410 coincidently acts as a lid to cover the exit of the SCOOP-binding channel, while the exit of the flg22-binding channel is fully open (Supplementary Fig. 2b). All these structural observations indicate that any extension at the C termini of AtSCOOP12/FocSCOOPL would weaken or even abolish their activities. Consistently, addition of a residue, such as G/A/S/F (designated as +G/+A/+S/+F), at the C termini of AtSCOOP12/FocSCOOPL abrogated the association of AtMIK2^ECD with AtBAK1^ECD in vitro (Fig. 3b and Extended Data Fig. 4e). Furthermore, the AtSCOOP12/FocSCOOPL +F/+A mutants significantly impaired their abilities to induce MAPK activation in Arabidopsis protoplasts (Fig. 3c and Extended Data Fig. 4f) and exhibited reduced inhibition of Arabidopsis root growth (Fig. 3d,e and Extended Data Fig. 4g,h).

Altogether, our data reveal the important role of AtSCOOP12/FocSCOOPL G11 to R13 in promoting AtMIK2–AtBAK1 heterodimerization, and demonstrated that any extension at the C termini of AtSCOOP12/FocSCOOPL would impair its activities.

Direct interactions between MIK2 and BAK1

In addition to SCOOP-mediated interactions, AtMIK2^ECD and AtBAK1^ECD also make extensive and direct contacts in the structures of AtMIK2^ECD-3M–AtSCOOP12/FocSCOOPL–AtBAK1^ECD complexes. Similar to AtFLS2^ECD25, AtMIK2^ECD interacts with several residues located at the concave side of AtBAK1^ECD, including H61, R72, N77, Y96, Y100 and R146 (Fig. 4a and Extended Data Fig. 5). In detail, Y577 and Q601 in AtMIK2^ECD stabilize R72 in AtBAK1^ECD by π-cation and hydrogen-bond interactions, respectively. AtMIK2^ECD E533, Q599, N623 and E625 recognize the side chains of AtBAK1^ECD H61, Y100, R146 and Y96, respectively, via hydrogen bonds. AtMIK2^ECD R575 forms a hydrogen bond with the main chain of AtBAK1^ECD N77. Intriguingly, F60 and F144 in AtBAK1^ECD have been shown to be crucial for the ligand-induced association with multiple cognate LRR-RKs, including AtFLS2 (ref. ²⁴), AtBRI1 (ref. ²⁸) and AtPEPR1 (ref. ²⁶); however, neither of them makes direct contact with AtMIK2^ECD.

Fig. 4. Direct MIK2^ECD–BAK1^ECD interactions.

a, The C-terminal segment of AtMIK2^ECD mediates its interaction with AtBAK1^ECD. A close-up view of the AtMIK2^ECD-3M-AtBAK1^ECD interacting interface is shown in the blue square. The side chains or backbones of residues involved in the AtMIK2^ECD-3M–AtBAK1^ECD interactions are shown as sticks. The colour coding is the same as in Fig. 3a. Residues in AtMIK2^ECD-3M and AtBAK1^ECD are labelled in black and marine, respectively. The Fo−Fc electron density (2.0 σ level) of residues of interest is shown in light blue. Black dashed lines and red dashed lines indicate hydrogen bonds and π-cation interaction, respectively. b, Mutations of the AtBAK1^ECD-interacting residues in AtMIK2^ECD compromise AtSCOOP12-induced MIK2^ECD–BAK1^ECD heteromerization. The assays were performed as described in Fig. 1a. c, Mutations of the AtMIK2^ECD-interacting residues in AtBAK1^ECD compromise AtSCOOP12-induced MIK2^ECD–BAK1^ECD heteromerization. The assays were performed as described in Fig. 1a. d, Mutations of the AtBAK1^ECD-interacting residues in AtMIK2^ECD compromise AtSCOOP12-induced MAPK activation. The assays were performed as described in Fig. 2d. e, Mutations of the AtMIK2-interacting residues in AtBAK1 compromise AtSCOOP12-induced MIK2–BAK1 interaction in Arabidopsis Col-0 protoplasts. Protoplasts co-expressing HA-tagged AtMIK2 and Flag-tagged AtBAK1 were treated with 1 µM AtSCOOP12 for 15 min. Protoplasts expressing AtMIK2-HA alone and protoplasts without AtSCOOP12 treatment were used as negative controls. HA-tagged AtMIK2 and Flag-tagged AtBAK1 in the input and immunoprecipitation samples were analysed by α-Flag and α-HA immunoblotting, respectively. f, Mutations of the AtMIK2-interacting residues in AtBAK1 compromise AtSCOOP12-induced MAPK activation. Arabidopsis bak1–5/serk4-1 protoplasts were transfected with plasmids as indicated. The samples were treated with or without 0.4 μM AtSCOOP12 for 10 min. MAPK activation was analysed by α-pERK1/2 immunoblotting. AtBAK1 and its mutant expression level was analysed by α-Flag immunoblotting. CBB staining of RBC was used for protein loading. The assays (b–f) were repeated three times with similar results.

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Extended Data Fig. 5. Direct MIK2^ECD-BAK1^ECD interactions in the AtMIK2^ECD-3M-FocSCOOPL-AtBAK1^ECD ternary complex.

A close-up view of the AtMIK2^ECD-3M-AtBAK1^ECD interacting interface is shown in blue square. The side chains or backbones of the residues involved in the AtMIK2^ECD-3M-AtBAK1^ECD interactions are shown as sticks. The color coding is the same as in Extended Data Fig. 4a. Residues in AtMIK2^ECD-3M and AtBAK1^ECD are labeled in black and marine, respectively. The Fo-Fc electron density (2.0 σ level) of residues of interest is shown in light blue. Black and red dotted lines indicate hydrogen bonds and π-cation interaction, respectively.

In line with our structural observations, the AtMIK2^ECD E553A, R575A, Y577A/Q601A and N623A/E625A mutants and the AtBAK1^ECD H61A, R72A, Y96A and R146A mutants, but not the negative control AtMIK2^ECD F60A/F144A, attenuated AtSCOOP12-induced AtMIK2^ECD–AtBAK1^ECD heterodimerization in GST pull-down assays (Fig. 4b,c). However, alanine substitution of AtMIK2^ECD Q599 and AtBAK1^ECD Y100 did not impair AtSCOOP12-induced AtMIK2^ECD–AtBAK1^ECD heterodimerization (Fig. 4b,c), indicating that the hydrogen bond between them is dispensable for the AtMIK2^ECD–AtBAK1^ECD interaction. Furthermore, the AtMIK2^ECD Y577A/Q601A and N623A/E625A mutants failed to complement the AtSCOOP12-induced MAPK activation in mik2-1 protoplasts (Fig. 4d). AtBAK1 R72A and Y96A mutants dramatically attenuated AtSCOOP12-induced AtMIK2–AtBAK1 heterodimerization in planta (Fig. 4e). Unlike WT AtBAK1, mutant variants AtBAK1 R72A and AtBAK1 Y96A could not trigger the AtSCOOP12-induced MAPK activation in bak1–5/serk4-1 mutant protoplasts (Fig. 4f), corroborating the functional requirement of these residues revealed by structural analysis.

N-glycans attached to MIK2 mediate its interaction with BAK1

N-glycosylation in plant RKs has been found to play crucial roles in their protein maturation, plasma membrane targeting and protein conformation maintenance^14–18. Given that several conserved N-glycosylation consensus N-X-S/T motifs are present in MIK2^ECD (Supplementary Fig. 1a), we speculate that MIK2 also undergoes N-glycosylation in eukaryotic cells. Indeed, in our crystal structures of both MIK2^ECD–SCOOPs binary complexes and MIK2^ECD–SCOOPs–BAK1^ECD ternary complexes, N-glycans are covalently attached to several conserved asparagine residues in AtMIK2^ECD and BnMIK2^ECD (Supplementary Fig. 1a and Extended Data Fig. 6a–c). Strikingly, mass spectrometry and structural analyses collectively revealed that the N-glycosylated AtMIK2^ECD N410/BnMIK2^ECD N393 locates at the inner surface of LRR14 and within the aforementioned SCOOP C-terminus-binding channel (Fig. 5a and Extended Data Fig. 6c,d–g), implying that N-glycosylation of this site might be related to SCOOP-induced MIK2^ECD–BAK1^ECD heterodimerization and is consequently important for inducing plant immune responses.

Extended Data Fig. 6. The N-glycosylation of MIK2^ECD and N-glycans mediated interactions in the AtMIK2^ECD-3M-FocSCOOPL-AtBAK1^ECD ternary complex.

a, The N-glycosylation of MIK2^ECD observed in the AtMIK2^ECD-3M, BnMIK2^ECD, AtMIK2^ECD-3M-AtSCOOP12, BnMIK2^ECD-FocSCOOPL, AtMIK2^ECD-3M-AtSCOOP12-AtBAK1^ECD and AtMIK2^ECD-3M-FocSCOOPL-AtBAK1^ECD complex structures. The N-glycans and related asparagine residues are shown as sticks. N-glycans attached to AtMIK2^ECD-3M N410/BnMIK2^ECD N393 and the other sites are colored in magenta and gray, respectively. b, The electron density map of AtMIK2^ECD-3M N410/BnMIK2^ECD N393 and the attached N-glycans. The Fo-Fc electron density (2.5 σ level) is shown in light blue. c, The N-glycans on AtMIK2^ECD N410 directly interact with AtBAK1^ECD in the AtMIK2^ECD-3M-FocSCOOPL-AtBAK1^ECD complex. A close-up view of the N-glycans-interacting interface in the AtMIK2^ECD-3M-FocSCOOPL-AtBAK1^ECD ternary complex is shown in magenta square. The N-glycans on AtMIK2^ECD-3M N410 and the side chains of AtMIK2^ECD-3M N410, AtBAK1^ECD D50 and T52 are shown as sticks. The Fo-Fc electron density (2.5 σ level) of residues of interest and the glycans is shown in light blue. Black dotted lines indicate hydrogen bonds. The color coding for the proteins is the same as in Extended Data Fig. 4a. d-g, MS/MS analysis of AtMIK2^ECD (d), AtMIK2^ECD N410D (e), BnMIK2^ECD (f), and BnMIK2^ECD N393D (g) expressed and purified from insect cells.

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Fig. 5. N-glycans mediate interactions between MIK2^ECD and BAK1^ECD.

a, The N-glycans on AtMIK2^ECD N410 directly interact with AtBAK1^ECD. A close-up view of the N-glycans-interacting interface in the AtMIK2^ECD-3M–AtSCOOP12–AtBAK1^ECD ternary complex is shown in the magenta square. The N-glycans on AtMIK2^ECD-3M N410 and the side chains of AtMIK2^ECD-3M N410, AtBAK1^ECD D50 and T52 are shown as sticks. The Fo−Fc electron density (2.0 σ level) of residues of interest and the glycans is shown in light blue. Black dashed lines indicate hydrogen bonds. b, Mutations of AtMIK2^ECD N410 compromise AtSCOOP12-induced MIK2^ECD–BAK1^ECD heteromerization. The assays were performed as described in Fig. 1a. c, ITC measurements of the binding affinity between AtBAK1^ECD and AtMIK2^ECD (wild type, left) or N410D mutant (right) in the presence of AtSCOOP12 (see Methods for details). Top: raw data curves. Bottom: fitted integrated ITC data curve. d, Molecular dynamics simulations of the AtMIK2^ECD-3M–AtSCOOP12-AtBAK1^ECD complex with or without glycans on AtMIK2^ECD N410. Top: time-dependent RMSDs for protein backbone atoms in different simulation runs. Bottom: snapshots from the molecular dynamic simulations at different time points. e, The AtMIK2 N410D mutant compromises AtSCOOP12-induced MAPK activation in Arabidopsis mik2-1 protoplasts. The assays were performed as described in Fig. 2d. f, The AtMIK2 N410D mutant compromises AtSCOOP12-induced MAPK activation in stable transgenic seedlings. The leaf discs from transgenic plants were treated with 0.2 μM AtSCOOP12 for 0 or 10 min. Specifically, four independent lines (L1–L4) of Arabidopsis MIK2 N410D/mik2-1 transgenic plants were used. The assays were performed as described in Fig. 1d. g, AtSCOOP12-induced ROS production in leaves from stable transgenic seedlings. The ROS production was measured as RLUs using a luminometre for 60 min. Box plots show the first and third quartiles (bounds of box) and the medians (lines), with whiskers extending to the maximum and minimum values. Different letters above boxes indicate statistically significant differences (P < 0.05, one-way ANOVA followed by Tukey’s test, n = 10). The assays were repeated three times (b, e) or twice (c, f, g) with similar results.

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To explore the exact function of the N-glycans on AtMIK2^ECD N410/BnMIK2^ECD N393, we generated a series of mutants that are incapable of being N-glycosylated at this site, including AtMIK2^ECD N410A/D and BnMIK2^ECD N393A/D. Gel filtration analysis showed that all these MIK2^ECD variants exhibited as monomers in solution similar to the wild type (Extended Data Fig. 7a,b). In addition, the crystal structures of BnMIK2^ECD N393A and N393D mutants were determined at 3.6 Å and 2.7 Å (Extended Data Table 1), respectively. BnMIK2^ECD N393A and N393D both adopted nearly identical conformation to the wild type (Extended Data Fig. 7c). These data collectively demonstrate that N-glycosylation at AtMIK2^ECD N410/BnMIK2^ECD N393 is not essential for native protein conformation.

Extended Data Fig. 7. N-glycosylation of MIK2 is crucial for MIK2^ECD-BAK1^ECD heteromerization.

a, b, Analytical size-exclusion chromatography showed that AtMIK2^ECD N410A/D (a) and BnMIK2^ECD N393A/D (b) exhibit as monomers in solution, which is the same as the wild type. Coomassie-blue stained SDS-PAGE gels of peak fractions are shown. c, Structural comparison of BnMIK2^ECD variants. The wild-type BnMIK2^ECD in the BnMIK2^ECD-FocSCOOPL complex structure (left), the BnMIK2 ^ECD N393D mutant (middle), and the BnMIK2 ^ECD N393A mutant (right) are shown in ribbon representation. The N-glycans and related asparagine residues are shown as sticks. The Fo-Fc electron density (2.5 σ level) of BnMIK2 ^ECD N393D and N393A is shown in light blue. N-glycans attached to BnMIK2^ECD N393 and the other sites are colored in magenta and gray, respectively. d, Mutations of BnMIK2^ECD N393 compromise AtSCOOP12-induced BnMIK2^ECD-BnBAK1^ECD heteromerization. The assays were performed as described in Fig. 1a. e, Analytical size-exclusion chromatography (SEC) showed that AtSCOOP12 failed to induce the heteromerization of AtMIK2^ECD N410D and AtBAK1^ECD. (Upper) SEC profiles of AtMIK2^ECD N410D and AtBAK1^ECD in the presence or absence of AtSCOOP12 were color-coded. (Lower) Coomassie-blue stained SDS-PAGE gels of peak fractions. f, ITC measurements of the binding affinity between AtSCOOP12 and AtMIK2^ECD (wild-type or N410D mutant). The top panels show raw data curves, and the bottom panels show the fitted integrated ITC data curve. g, AtBAK1 D50A/T52V mutant compromise AtSCOOP12-induced AtMIK2^ECD-AtBAK1^ECD heteromerization. The assays were performed as described in Fig. 1a. The assays were repeated three times (a, b, d-f) and twice (g) with similar results.

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In the AtMIK2^ECD-3M–AtSCOOP12/FocSCOOPL–AtBAK1^ECD complex structures, N-acetyl-β-d-glucosamine (NAG)-NAG-β-d-mannose (BMA) attached to AtMIK2^ECD N410 are defined unambiguously with evident electron density (Extended Data Fig. 6b). Strikingly, the NAG at position one forms two hydrogen bonds with the side chains of AtBAK1^ECD D50 and T52 (Fig. 5a and Extended Data Fig. 6c), implying a critical role in AtMIK2^ECD–AtBAK1^ECD heteromerization. Consistently, GST pull-down and gel filtration assays failed to detect the AtSCOOP12-induced BAK1^ECD interactions with AtMIK2^ECD N410A or N410D and BnMIK2^ECD N393A or N393D mutant variants (Fig. 5b and Extended Data Fig. 7d,e). Isothermal titration calorimetry (ITC) measurements revealed that the AtMIK2^ECD N410D variant exhibited a similar binding affinity to AtSCOOP12 as WT AtMIK2^ECD, but a markedly decreased binding affinity to AtBAK1^ECD in the presence of AtSCOOP12 at a saturating concentration by over 50-fold (Fig. 5c and Extended Data Fig. 7f).

In addition, molecular dynamics simulations of the AtMIK2^ECD–AtSCOOP12–AtBAK1^ECD complex with or without N-glycans on AtMIK2^ECD N410 showed notable differences in complex stability. In the presence of AtMIK2^ECD N410-attached N-glycans, the ternary complex remained stable and showed little conformational change after 100 nanoseconds (ns) (Fig. 5d, left panels). In contrast, the absence of N-glycosylation at AtMIK2^ECD N410 resulted in the ternary complex being much more dynamic, starting to dissociate after 56 ns and completely disintegrating after 100 ns (Fig. 5d, right panels). Moreover, the AtBAK1^ECD D50A/T52V mutant variant, which could not form hydrogen bonds with the AtMIK2^ECD N410-attached N-glycans, was unable to interact with AtMIK2^ECD upon SCOOP treatment in a GST pull-down assay (Extended Data Fig. 7g). Taken together, the data demonstrate that N-glycosylation of AtMIK2^ECD N410/BnMIK2^ECD N393 mediates the AtMIK2^ECD–AtBAK1^ECD interaction, which is essential for the formation and stabilization of the MIK2^ECD–SCOOPs–BAK1^ECD ternary complex.

Corroborating the structural and biochemical analysis and bioinformatics data, unlike WT AtMIK2, the AtMIK2 N410D mutant could not restore the AtSCOOP12-induced MAPK activation in mik2-1 protoplasts (Fig. 5e). Compared with WT AtMIK2, the N410D mutant dramatically compromised AtSCOOP12-induced ROS production in N. benthamiana leaves (Extended Data Fig. 8a). Moreover, AtSCOOP12/FocSCOOPL-induced MAPK activation and ROS production were substantially compromised in MIK2 N410D/mik2-1 transgenic seedlings compared with MIK2/mik2-1 seedlings (Fig. 5f,g and Extended Data Fig. 8b,c). The AtBAK1 D50A/T52V mutant dramatically attenuated AtSCOOP12-induced AtMIK2–AtBAK1 heterodimerization in planta (Extended Data Fig. 8d). Moreover, the AtBAK1 D50A/T52V variant could complement flg22-induced MAPK activation in bak1–4 protoplasts (Extended Data Fig. 8e) but failed to complement the AtSCOOP12-induced MAPK activation in bak1–5/serk4-1 protoplasts (Extended Data Fig. 8f).

Extended Data Fig. 8. N-glycosylation of MIK2 is crucial for SCOOPs-induced immune responses.

a, AtSCOOP12-induced ROS production in N. benthamiana leaves infiltrated the Agri-bacteria containing pMDC32-MIK2-GFP or pMDC32-MIK2 N410D-GFP vectors. These leaves were treated with 0.5 µM AtSCOOP12 or H₂O (labeled as Mock). RLU, relative light units. b, AtMIK2 N410D mutant compromises FocSCOOPL-induced MAPK activation in Arabidopsis MIK2 N410D-HA/mik2-1 transgenic plants. The assays were performed as described in Fig. 5f. c, FocSCOOPL-induced ROS production in leaves from stable transgenic seedlings. The assays were performed as described in Fig. 5g. d, AtBAK1 D50A/T52V mutant compromise AtSCOOP12-induced MIK2-BAK1 interaction in Arabidopsis Col-0 protoplasts. The assays were performed as described in Fig. 4e. e, AtBAK1 D50A/T52V mutant can restore flg22-induced MAPK activation in Arabidopsis bak1-4 protoplasts. bak1-4 protoplasts were transfected with plasmids as indicated. The samples were treated with or without 0.4 μM flg22 for 10 min. bak1-4 protoplasts transfected with empty vector (EV) and wild-type bak1 were used as negative and positive controls, respectively. f, AtBAK1 D50A/T52V mutant compromise AtSCOOP12-induced MAPK activation in Arabidopsis bak1-5/serk4-1 protoplasts. The assays were performed as described in Fig. 4f. Box plots in (a) and (c) show the first and third quartiles as bounds of box, split by the medians (lines), with whiskers extending to the maximum and minimum values. Letters indicate significant differences with others (P < 0.05, One-way ANOVA followed by Tukey’s test, n = 12 (a) and 13 in (c)). The assays were repeated twice (a-f) with similar results.

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STT3A (Staurosporin and temperature sensitive-3A) encodes the catalytic subunit of the ER oligosaccharyltransferase (OST) complex involved in N-glycosylation of nascent proteins²⁹. GFP-fused AtMIK2 (WT) expressed in N-glycosylation-deficient stt3a-2 protoplasts, as well as GFP-fused AtMIK2 (WT and N410D mutant) expressed in N. benthamiana leaves and mik2-1 protoplasts, all locate at the plasma membrane (Extended Data Fig. 9a–c), suggesting that N-glycosylation at N410 is dispensable for the subcellular localization of AtMIK2. The GFP-fused AtMIK2 protein level was not affected in stt3a-2 protoplasts (Extended Data Fig. 9d). Meanwhile, Arabidopsis transgenic plants expressing MIK2 N410D or WT MIK2 tagged with HA in the mik2-1 background also showed no apparent differences in terms of MIK2 protein abundance (Fig. 5f and Extended Data Fig. 8b). These results collectively indicate that N-glycosylation at AtMIK2 N410 exerts no effects on its protein quality control.

Extended Data Fig. 9. N-glycosylation of MIK2 is dispensable for its subcellular localization and protein abundance.

a. Subcellar localization of AtMIK2 in Col-0 and N-glycosylation -deficient stt3a-2 protoplasts. Col-0 and stt3a-2 protoplasts were transfected with indicated vectors. Images were taken under a confocal microscope at 3 dpi. Scale bar, 5 μm. b-c, GFP-fused AtMIK2 (WT and N410D mutant) expressed in N. benthamiana leaves (b) and mik2-1 protoplasts (c)all locate at the plasma membrane. N. benthamiana leaves were infiltrated with A. tumefaciens GV3101 carrying p35S::MIK2-GFP and p35S::MIK2 N410D-GFP. mik2-1 protoplasts were transfected with indicated vectors. Images were taken under a confocal microscope at 3 dpi. Scale bar, 20 μm (b) or 5 μm (c). d, The AtMIK2 protein abundance in Col-0 and N-glycosylation -deficient stt3a-2 protoplasts. Col-0 and stt3a-2 protoplasts were transfected with indicated vectors. GFP-tagged AtMIK2 was detected by α-GFP immunoblotting. Coomassie brilliant blue (CBB) staining of Rubisco (RBC) was used for protein loading. Molecular weight (kDa) was labeled on the left or right of all immunoblots. The assays were repeated twice (a-d) with similar results.

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Altogether, our data demonstrate that N-glycosylation at AtMIK2 N410/BnMIK2 N393 plays a pivotal role in the perception of SCOOPs via directly strengthening SCOOP-induced MIK2^ECD–BAK1^ECD heteromerization.

Mechanism of MIK2–BAK1 complex formation induced by SCOOPs

To study the mechanism of SCOOP-induced activation of the MIK2–BAK1 complex, we solved the crystal structures of BnMIK2^ECD and AtMIK2^ECD-3M at a resolution of 2.85 Å and 1.95 Å, respectively (Extended Data Table 1). Structural superposition of MIK2^ECD in apo form and in complex with SCOOPs revealed that MIK2^ECD adopts a nearly identical conformation in these structures, suggesting that, in the cellular milieu as well, SCOOP-binding induced no conformational change in MIK2^ECD (Extended Data Fig. 2b,c). In the MIK2^ECD–SCOOP binary complex structures, only the N-terminal segment of SCOOP is well defined and characterized to form extensive contacts with MIK2^ECD, while the C-terminal segment is invisible (Fig. 2a and Extended Data Fig. 2d,e), indicating that SCOOP may mainly utilize its N-terminal segment to interact with MIK2^ECD. In contrast, in the ternary structures of MIK2^ECD–SCOOP–BAK1^ECD complexes, the C-terminal segments of SCOOPs are also well defined (Extended Data Fig. 4c,d) and especially the last three residues act as a ‘glue’ to bridge MIK2^ECD and BAK1^ECD via extensive hydrogen-bond interactions (Fig. 3a and Extended Data Fig. 4a). Thus, the flexible C-terminal short segments of SCOOPs in the MIK2^ECD-bound binary complexes function as a fishing bait to catch BAK1^ECD, subsequently stabilizing the captured BAK1^ECD in complexing with MIK2^ECD. Remarkably, the glycans attached to a conserved asparagine residue of MIK2^ECD (AtMIK2^ECD N410/BnMIK2^ECD N393) located right at the MIK2^ECD–SCOOPs–BAK1^ECD interaction interface form hydrogen bonds with BAK1^ECD (Fig. 5a and Extended Data Fig. 6c), functioning as a lock to reinforce the ternary complexes. Taking together our eight MIK2^ECD structural snapshots (AtMIK2^ECD-3M, AtMIK2^ECD-3M–AtSCOOP12, AtMIK2^ECD-3M–AtSCOOP12–AtBAK1^ECD, AtMIK2^ECD-3M–FocSCOOPL–AtBAK1^ECD, BnMIK2^ECD, BnMIK2^ECD N393D, BnMIK2^ECD N393A, BnMIK2^ECD–FocSCOOPL), as well as biochemical, bioinformatics and genetic analyses, the data point to N-glycosylation-dependent MIK2–BAK1 complex activation by SCOOPs (Fig. 6).

Fig. 6. A working model of MIK2–BAK1 complex activation by SCOOPs.

In resting cells, MIK2 is N-glycosylated and the attached glycan chains are of high conformational flexibility. For simplicity, only the N-glycans attached to a conserved asparagine residue of MIK2 involved in ligand recognition (equivalent to AtMIK2 N410/BnMIK2 N393) are shown and coloured in pink. Once a SCOOP phytocytokine or a SCOOP-like peptide from the pathogen Fusarium or Comamonadaceae exists in the extracellular environment, the N-terminal segments of SCOOPs bind to the ectodomain of MIK2 while the C-terminal segments dangle over the side, enabling the ectodomain of BAK1 to be grasped by the C-terminal segments of SCOOPs and be further stabilized by the MIK2 ectodomain. The N-glycans at the aforementioned site of MIK2 interact with the ectodomain of BAK1 and tightly lock the MIK2–SCOOP–BAK1 ternary complex at the cell surface, presumably leading to intracellular signal transduction. To emphasize that the conformation of indicated N-glycans is fixed after BAK1 binding, they are coloured in magenta in the final step.

Discussion

N-glycosylation in proteins is a ubiquitous co- and post-translational modification process³⁰. Arabidopsis RKs FLS2 and EF-TU RECEPTOR (EFR) are well-studied PRRs sensing bacterial flagellin (flg22) and elongation factor-Tu (elf18), respectively^31,32. FLS2 and EFR have been observed to be N-glycosylated in plant cells and deficiency of N-glycosylation impairs their protein folding, quality control and subsequent transport to the cell surface, resulting in dysfunction of plant immunity^14–16. Arabidopsis RK BRI1 is essential and sufficient for the recognition of brassinosteroids³³. Mutational analysis of N-glycosylation sites in BRI 1 demonstrated their importance not only for protein maturation and plasma membrane targeting¹⁷ but also for the formation of native protein conformation¹⁸. In this study, we showed that the ectodomain of plant RK MIK2 is also subjected to N-glycosylation at several sites. Unexpectedly, our structural and biochemical data together with molecular dynamics simulations revealed that N-glycans attached to MIK2 can strengthen SCOOP-induced MIK2–BAK1 heteromerization via directly interacting with BAK1. Combined with the mutagenesis and genetic analysis, we propose a novel mechanism whereby SCOOP-induced activation of the MIK2–BAK1 complex could be mediated by protein N-glycosylation. Furthermore, we analysed 60 currently available structural models of plant LRR-RKs deposited in the Protein Data Bank (https://www.rcsb.org/) and found that 5 well-studied receptor–peptide–coreceptor complexes, including AtFLS2^ECD–flg22–AtBAK1^ECD, AtPEPR1^ECD–PEP1, AtHSL1^ECD–IDA–AtSERK1^ECD, AtGSO1^ECD–CIF2 and NbRXEG1^ECD–XEG1–NbBAK1^ECD, also bear N-glycans in the proximity of ligand-binding sites on receptors. N-glycans on receptors AtFLS2^ECD N388, AtPEPR1^ECD N252, AtHSL1^ECD N262 and AtGSO1^ECD N369 form hydrogen bonds with their corresponding ligands (Supplementary Fig. 3a–d), while N-glycans on NbRXEG1^ECD N562 interact with the co-receptor NbBAK1^ECD (Supplementary Fig. 3e). However, none of these were revealed and subjected to in-depth biochemical, genetic and functional analysis in the original research papers^{24,26,34–36}. This implies that in addition to previously reported protein–protein interaction, N-glycan–protein interaction may be a prevalent and evolutionarily conserved mechanism for activating receptor signalling in different plant species.

Arabidopsis RK MIK2 was identified as a receptor to perceive endogenous SCOOP and microbial-derived SCOOPL peptides, and could form a complex with co-receptor BAK1 upon treatment of SCOOPs^12,13. In this study, biochemical analysis revealed that SCOOP homologues in oilseed crop B. napus (BnSCOOP) and virulent F. oxysporum f. sp. conglutinans strain Fo5176 (FocSCOOPL) can also be recognized by the B. napus MIK2^ECD–BAK1^ECD complex. In fact, sequence alignment shows that MIK2 orthologues in different Brassicaceae plants share a high degree of amino acid identity (over 80%), and the key residues responsible for the SCOOP and BAK1 binding are strictly conserved among them (Extended Data Fig. 1a). Meanwhile, SCOOPs are widely identified in Brassicaceae genomes^19,20. Therefore, perception of SCOOPs in a MIK2-dependent manner to trigger PTI responses and regulate root growth might commonly exist in Brassicaceae plants.

In the structures of AtMIK2^ECD-3M–AtSCOOP12/FocSCOOPL–AtBAK1^ECD ternary complexes, the channels formed by the concave surface of MIK2 and an inner-curved loop of BAK1 to accommodate the C termini of SCOOPs are especially narrow. Our structural data combined with biochemical and genetic evidence demonstrated that any extension at the C termini of AtSCOOP12/FocSCOOPL would weaken or even abolish its activities, including promoting the association of AtMIK2^ECD with AtBAK1^ECD in vitro, inducing MAPK activation in Arabidopsis and inhibiting Arabidopsis root growth. These findings strongly suggest that in vivo proteolytic processing at the C-terminal sides happens during the maturation of SCOOPs. Coincidently, a similar mechanism was previously proposed for the maturation of Arabidopsis endogenous peptides AtPEPs on the basis of a modelled structure of the AtPEPR1^ECD–AtPEP1–AtBAK1^ECD complex and biochemical analysis²⁶. Altogether, proteolytic processing of phytocytokines at the C-terminal side for maturation might extensively exist in plants.

Recently, subtilases have been reported to mediate the release of Arabidopsis SCOOPs (AtSCOOPs) from PROSCOOPs²³. In this case, AtSCOOPs localize at the C terminus of the PROSCOOPs, while the subtilases can recognize and cleave the RxLx/RxxL motif preceeding the AtSCOOPs. However, this is unlikely to happen in the maturation of FocSCOOPL. FocSCOOPL resides in the very N terminus of an uncharacterized large protein without the RxLx/RxxL motif from Fusarium oxysporum. Intriguingly, the residue at the very C terminus of FocSCOOPL is R, and FocSCOOPL forms a flexible and stretched loop in the predicted three-dimensional structural model of its precursor¹³. It is well known that trypsin specifically cleaves the peptide bond on the carboxyl side of lysine or arginine residues. Especially, F. oxysporum trypsin has greater activity towards arginine peptides than towards lysine peptides^37,38. Future biochemical and genetic analyses will elucidate whether F. oxysporum trypsin is involved in the release of FocSCOOPL peptide.

Methods

Peptide synthesis

The wild type AtSCOOP12 and FocSCOOPL peptides used for crystallization and isothermal titration calorimetry assays were synthesized at GenScript. All AtSCOOP12 and FocSCOOPL variants used for analyses of MAPK activation, root growth inhibition and ROS production were synthesized at Biomatik. The sequences of synthesized peptides are listed in Supplementary Table 1.

Protein expression and purification

The coding frames of AtMIK2^ECD (residues 1–700, AT4G08850), BnMIK2^ECD (residues 1–690, XP_013711204.1), AtBAK1^ECD (residues 1–220, AT4G33430) and BnBAK1^ECD (residues 1–220, XP_013707345.1) were individually inserted into the pFastBac1 vector with a 10× His tag at the C terminus. The DNA sequences encoding AtSCOOP12 (PVRSSQSSQAGGR, AT5G44585), FocSCOOPL (ESSSSHSERAGGR) and BnSCOOP (FAGPSSSGHGGGR, CDY22858.1 and CDY33880.1) were synthesized (Tsingke Biotechnology) and individually inserted into the pGEX-6p-1 vector with a GST tag at the N terminus. All mutants were generated using the QuickChange Site-directed Mutagenesis system or Gibson Assembly. All the constructs were verified by DNA sequencing. The primer sequences are listed in Supplementary Table 2.

Baculoviruses (40 ml) for His-tagged AtMIK2^ECD, His-tagged BnMIK2^ECD, His-tagged AtBAK1^ECD and His-tagged BnBAK1^ECD were individually added to 1 l of High Five cells (≈1.8 × 10⁶ cells per ml) cultured in High Five medium (Sino Biological) at 22 °C. The medium was collected at 60 h after infection. His-tagged AtMIK2^ECD, His-tagged AtBAK1^ECD, His-tagged BnMIK2^ECD and His-tagged BnBAK1^ECD were purified by Ni-NTA affinity chromatography (Qiagen) with elution buffer E1 (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). The eluted proteins were further purified by ion-exchange chromatography (HiTrap Q FF, GE Healthcare) and size-exclusion chromatography (Superdex 200 Increase 10/300 GL column, GE Healthcare) in buffer G1 (20 mM Tris pH 8.0, 150 mM NaCl).

Plasmids of GST-tagged AtSCOOP12, GST-tagged FocSCOOPL and GST-tagged BnSCOOP were individually transformed into E. coli BL21 (DE3) cells. The transformed bacterial cells were grown at 37 °C to optical density at 600 nm (OD₆₀₀) of 0.6. Protein expression was induced with 0.2 mM isopropyl-β-d-thiogalactoside at 16 °C overnight. The cells were collected, resuspended in lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 10% glycerol and 1 mM Phenylmethanesulfonyl fluoride) and lysed with a high-pressure cell disrupter (JNBIO). The target protein was collected from the supernatant, purified by GST affinity chromatography (GE Healthcare) with elution buffer E2 (50 mM Tris pH 8.0, 500 mM NaCl and 25 mM reduced glutathione) and then desalted into buffer D1 (10 mM Bis-Tris pH 6.0, 100 mM NaCl) using a HiPrep 26/10 desalting column (GE Healthcare).

Crystallization

Crystallization experiments were performed using the sitting drop vapour diffusion method by mixing equal volumes (0.5 µl) of protein and reservoir solution at 16 °C. For obtaining crystals of MIK2^ECD–SCOOPs binary complexes, a mixture of AtMIK2^ECD-3M/BnMIK2^ECD (∼5 mg ml⁻¹) and chemically synthesized AtSCOOP12/FocSCOOPL (4 mg ml⁻¹) with a molar ratio of 1:8 was used for crystallization. For obtaining crystals of MIK2^ECD–SCOOPs–BAK1^ECD ternary complexes, the AtMIK2^ECD-3M (∼5 mg ml⁻¹), AtBAK1^ECD (∼1.5 mg ml⁻¹) and AtSCOOP12/FocSCOOPL (2 mg ml⁻¹) were pre-mixed at a molar ratio of 1:1:4 and incubated for 1 h at 4 °C before crystallization. Crystals of BnMIK2^ECD and BnMIK2^ECD–FocSCOOPL complex with good quality were obtained in buffer containing 0.2 M lithium sulfate, 0.1 M Tris pH 8.5 and 34% (w/v) PEG 400. Crystals of AtMIK2^ECD-3M and AtMIK2^ECD-3M–AtSCOOP12 complex with good quality were obtained in buffer containing 0.2 M sodium acetate, 0.1 M Tris pH 7.4 and 18% (w/v) PEG 3350. Crystals of AtMIK2^ECD-3M–AtSCOOP12–AtBAK1^ECD and AtMIK2^ECD-3M–FocSCOOPL–AtBAK1^ECD complex with good quality were obtained in buffer containing 0.1 M MES pH 5.9, 22% PEG 200 and 7% (w/v) PEG 3000. All crystals were transferred to cryoprotectants consisting of the crystallization buffer and 20% glycerol and flash cooled in liquid nitrogen.

Data collection and structure determination

X-ray diffraction data were collected at beamlines BL02U1 and BL19U1 of the Shanghai Synchrotron Radiation Facility³⁹, and processed using XIA2 (ref. ⁴⁰), DIALS⁴¹, HKL2000 (ref. ⁴²) and CCP4i^43,44. The structures were solved using the molecular replacement method in Phenix⁴⁵ and structures predicted using AlphaFold v.2.0 system⁴⁶ as search models. Model building was performed using Coot⁴⁷ and structure refinement was carried out using Phenix. Stereochemistry of the structure models was analysed using Molprobity⁴⁸. All graphics were generated using Pymol (http://www.pymol.org) and ChimeraX (https://www.cgl.ucsf.edu/chimerax). Statistics of the structure refinement and the structure models are summarized in Extended Data Table 1.

Analytical size-exclusion chromatography

Analytical SEC experiments were performed using a Superdex 200 Increase 10/300 GL column (GE Healthcare) and a running buffer containing 10 mM Bis-Tris pH 6.0 and 100 mM NaCl. The purified AtMIK2^ECD variants were incubated with AtBAK1^ECD and AtSCOOP12 at a molar ratio of 1:2.5:5 and incubated for 1 h at 4 °C before SEC analysis. The protein mixture was loaded onto the column and the peak fractions were analysed by SDS–PAGE, followed by Coomassie brilliant blue staining.

In vitro glutathione S-transferase pull-down assay

The wild type and mutants of His-tagged MIK2^ECD, His-tagged BAK1^ECD, GST-tagged AtSCOOP12, GST-tagged FocSCOOPL and GST-tagged BnSCOOP were purified as described above. The purified GST-tagged SCOOPs were used to capture the His-tagged MIK2^ECD and His-tagged BAK1^ECD. Approximately 20 μg of GST-tagged SCOOPs was loaded onto 30 μl glutathione sepharose 4B resin (GE Healthcare). After extensive washing with washing buffer (10 mM Bis-Tris pH 6.0, 100 mM NaCl), the beads were incubated with His-tagged MIK2^ECD and His-tagged BAK1^ECD proteins at 4 °C for 1 h, washed with 1 ml washing buffer 8 times and finally eluted with 50 µl elution buffer (50 mM reduced glutathione, 10 mM Bis-Tris pH 6.0, 100 mM NaCl). The eluted samples were analysed by western blotting. Anti-His antibody (1:5,000, AE003) and anti-mouse antibody (1:2,000, AS003) were purchased from ABclonal. Anti-GST antibody (1:5,000, 66001-2-Ig) was purchased from Proteintech.

Isothermal titration calorimetry assay

ITC experiments were performed at 25 °C using a MicroCal iTC200 system (Malvern Instruments). The wild type and mutants of His-tagged AtMIK2^ECD and His-tagged AtBAK1^ECD used in ITC assay were desalted into the ITC buffer (10 mM Bis-Tris pH 6.0, 100 mM NaCl). The concentrations of His-tagged AtMIK2^ECD and His-tagged AtBAK1^ECD were determined by absorbance at 280 nm. The concentration of synthesized AtSCOOP12 peptide was determined by the difference in spectrophotometric absorptions between 215 nm and 225 nm⁴⁹.

To measure the binding affinities of AtMIK2^ECD with AtSCOOP12, wild-type or mutant His-tagged AtMIK2^ECD (20 µM) was added to the sample cell while synthesized AtSCOOP12 peptide (200 µM) was filled in the syringe. To measure the binding affinities of AtBAK1^ECD with AtMIK2^ECD in the presence of saturated AtSCOOP12 peptide, His-tagged AtMIK2^ECD (20 µM) was pre-incubated with synthesized AtSCOOP12 peptide (200 µM) and then added to the sample cell while His-tagged AtBAK1^ECD (200 or 300 µM) was filled in the syringe. A background titration was performed using identical titrant with the buffer solution. The measured thermal data were analysed using the ORIGIN software (MicroCal). The first injection was discarded from each dataset to remove the effect of titrant diffusion across the syringe tip during the equilibration process. Thermodynamic parameters including stoichiometry (n) and dissociation constant (K_D) were obtained by fitting the data to an equilibrium binding isotherm using a ‘one set of sites’ binding model with a nonlinear least-squares method.

Mass spectrometry assay

His-tagged AtMIK2^ECD (WT and N410D) and His-tagged BnMIK2^ECD (WT and N393D) from insect cells were digested with trypsin (Thermo Fisher, MS grade) overnight at 37 °C. The final peptide samples were analysed using the Q Exactive Plus mass spectrometer equipped with nanoflow reversed-phase liquid chromatography (EASY nLC 1200, Thermo Scientific). The EASY nLC 1200 was fitted with a Thermo Scientific Acclaim Pepmap nano-trap column (C18, 5 μm, 100 Å, 100 μm × 2 cm) and a Thermo Scientific EASY-Spray column (Pepmap RSLC, C18, 2 μm, 100 Å, 50 μm × 15 cm) and run at 300 nl min⁻¹ with the following mobile phases (A: 0.1% formic acid; B: 80% CH3CN/0.1% formic acid). The liquid chromatography separation was carried out with the following gradient: 0~8% B for 3 min, 8~28% B for 42 min, 28~38% B for 5 min, 38~100% B for 10 min. Eluted peptides were electrosprayed directly into the mass spectrometer for MS and MS/MS analyses. One full MS scan (m/z 350–2,000) was acquired using the Orbitrap mass analyser; subsequently, the 10 highest-intensity ions were selected for fragmentation by higher-energy collisional dissociation. Carbamidomethylation of cysteine residues (+57.021 Da) was set as a static modification, while oxidation of methionine (+15.995 Da) and NAG-NAG-BMA on asparagine (+568.212 Da) was set as dynamic modifications.

Plant materials and growth conditions

The Arabidopsis thaliana accession Columbia-0 (Col-0) was used as the wild type. T-DNA insertion mutants of Arabidopsis mik2-1 (SALK_061769) was obtained from the Nottingham Arabidopsis Stock Centre (NASC). The Arabidopsis bak1–4 and bak1–5/serk4-1 have been reported previously⁵⁰. Plants were grown in soil (Metro Mix 366, Sunshine LP5 or Sunshine LC1, Jolly Gardener C/20 or C/GP) in a growth room at 20–23 °C, 50% humidity and 75–100 μE m⁻² s⁻¹ light with a 12 h light/12 h dark photoperiod for 4–5 weeks before protoplast isolation. Seedlings used for analyses of root growth inhibition were grown on half-strength Murashige and Skoog (½ MS) plates containing 0.5% (w/v) sucrose, 0.75% (w/v) agar and 2.5 mM MES pH 5.8, under the same conditions as plants grown in soil.

In vivo co-immunoprecipitation assay

Protoplasts from 4-week-old plant leaves grown in soil were transfected with indicated vectors and incubated for 12 h. After treatment with 1 µM peptides for 15 min, protoplasts were collected by centrifugation at 100 g for 2 min and lysed in IP buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100 and protease inhibitor cocktail) from Sigma. The supernatant was immunoprecipitated with anti-Flag agarose resin (Sigma-Aldrich) at 4 °C for 2 h. The beads were then sequentially washed with wash buffer A (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1% (v/v) Triton X-100) three times and with wash buffer B (50 mM Tris pH 7.5) once. Immunoprecipitated proteins and input proteins were analysed by immunoblotting with anti-HA antibody (1:2,000; 12013819001, Roche) or anti-Flag antibody (1:2,000; a8592, Sigma-Aldrich).

Generation of transgenic plants

AtMIK2 and AtBAK1 variants were generated by site-directed mutagenesis using the pHBT-MIK2-HA and pHBT-BAK1-FLAG constructs as templates, respectively. To generate the pCAMBIA1300-p35::MIK2-HA and pCAMBIA1300-p35::MIK2 N410D-HA constructs, the coding sequences of p35S::MIK2-HA and p35S::MIK2 N410D-HA were amplified from pHBT-MIK2-HA and pHBT-MIK2 N410D-HA, respectively, and then subcloned into the pCAMBIA1300 vector between the XbaI and EcoRI sites using the ClonExpress II One Step Cloning kit (Vazyme). The primer sequences are listed in Supplementary Table 2.

Agrobacterium tumefaciens strain GV3101 carrying binary vectors was used for Arabidopsis transformation by floral dipping. Transformants were selected with 25 µg ml⁻¹ hygromycin B. Multiple transgenic lines in the T₁ generation were analysed by immunoblotting for protein expression. Two lines with a 3:1 segregation ratio for hygromycin resistance in the T₂ generation were selected to obtain homozygous seeds.

MAPK activation assay

For the MAPK activation assay in Arabidopsis mik2-1, bak1–5/serk4-1 or bak1–4 protoplasts, protoplasts from 4-week-old plant leaves grown in soil were transfected with indicated vectors and incubated for 12 h. After treatment with peptides for indicated time, protoplasts were collected by centrifugation at 100 g for 2 min and lysed in 2× SDS loading buffer (125 mM Tris pH 6.8, 20% (v/v) glycerol, 2% (w/v) SDS, 0.05% (w/v) bromophenol and 2% (v/v) β-mercaptoethanol). For the MAPK activation assay in the transgenic plants, leaf discs from 4-week-old plants were inoculated in water overnight and then treated with indicated peptides. The leaf discs were ground in 2× SDS loading buffer.

After brief vortexing, the samples were boiled at 98 °C for 5 min. The supernatant, collected after centrifugation at 13,000 g for 2 min at 25 °C, was loaded on 10% (v/v) SDS–PAGE gel, transfered to a PVDF membrane, and then blotted with anti-pERK1/2 antibody (1:2,000; 9101, Cell Signaling) and anti-rabbit antibody (1:10,000; 7074, Cell Signaling) for the detection of phosphorylated MPK3, MPK4 and MPK6.

Reactive oxygen species assay

To measure the SCOOPs-induced ROS production in Arabidopsis, the third or fourth pair of true leaves from 4- to 5-week-old soil-grown Arabidopsis plants were excised into leaf discs (5 mm diameter). To measure the SCOOPs-induced ROS production in N. benthamiana, the Agri-bacteria containing pMDC32-MIK2-GFP or pMDC32-MIK2^N410D-GFP vectors were infiltrated into the leaves of 4- to 5-week-old soil-grown N. benthamiana. At 2 days post inoculation, leaf discs (5 mm diameter) were excised.

Leaf discs were incubated in 100 µl MilliQ H₂O in a 96-well plate with gentle shaking overnight to eliminate the wounding effect. Water was replaced by 100 µl of a reaction solution containing 50 µM luminol and 10 µg ml⁻¹ horseradish peroxidase (HRP, Sigma-Aldrich) supplemented with or without peptides. Luminescence was measured with a luminometre (Glomax Multi-Detection System, Promega) for 40 min. ROS production was indicated as means of relative light units (RLUs).

Root growth assay

Cold-stratified seeds were surface-sterilized with 70% (v/v) ethanol for 5 min and sown on ½ MS plates with or without peptides at indicated concentrations. Seedlings (10-day-old) grown on plates vertically in a growth chamber were photographed, and the root lengths of seedlings were measured using Image J (http://rsb.info.nih.gov/ij/).

Subcellular localization assay

For subcellular localization analysis in N. benthamiana, A. tumefaciens strain GV3101 containing pMDC32-35S::MIK2-GFP or pMDC32-35S::MIK2 N410D-GFP were grown overnight in LB medium at 28 °C. Cultures were collected by centrifugation, and cells were resuspended in 10 mM MES pH 5.7, 10 mM MgCl₂ and 200 μM acetosyringone, adjusted to OD₆₀₀ of 1.0 and infiltrated into leaves of 4-week-old soil-grown N. benthamiana using a needleless syringe. The plants were then cultivated for 72 h. For subcellular localization analysis in Arabidopsis, protoplasts isolated from 12-day-old seedlings of Col-0, mik2-1 and stt3a-2 were transfected with indicated constructs and then incubated for 12 h.

Fluorescence images were taken with the Leica SP8 confocal laser microscope. The excitation laser of 488 nm was used for imaging GFP.

Molecular dynamics simulations

Molecular dynamics simulations were performed using a well-established protocol⁵¹. The initial systems were prepared using the Glycan Reader and Modeler⁵² in CHARMM-GUI⁵³. the initial model of full-glycosylated AtMIK2^ECD–AtSCOOP12–AtBAK1^ECD ternary complex was prepared on the basis of our crystal structure, while the initial model of AtMIK2^ECD–AtSCOOP12–AtBAK1^ECD without N-glycosylation at AtMIK2^ECD N410 was generated by deleting the N-glycans attached to AtMIK2^ECD N410 from the full-glycosylated model. The CHARMM36m⁵⁴ force field was provided by CHARMM-GUI for proteins and carbohydrates. TIP3P water model⁵⁵ was applied along with 0.15 M NaCl. The input files for GROMACS were generated, and we used GROMACS 2022.2 for equilibration and production with LINCS. To maintain the temperature (310.15 K), a Nosé–Hoover temperature coupling method^56,57 with a tau-t of 1 ps was applied. For pressure coupling (1 bar), a semi-isotropic Parrinello–Rahman method with a tau-p of 2 ps and a compressibility of 4.5 × 10⁻⁵ bar⁻¹ were used. During the equilibration run, NVT dynamics was employed with 1 fs time step for 125 ps. Subsequently, the NPT ensemble was applied with 1 fs time step for 125 ps. Simulations of full-glycosylated and AtMIK2^ECD N410-deglycosylated models were repeated three times with 2 fs time step. ChimeraX was used to render the molecular graphics and display the 100 ns production movies.

Statistical analysis

All statistical tests were performed using GraphPad Prism (v.8). Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s test. Differences were considered statistically significant when P value was <0.05. The data shown in box plots display the first and third quartiles (boxes) and medians (centre lines), with whiskers extending to the maximum and minimum values.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

S.X conceived the project. S.X., L.S., P.H., L.X., H.W., L.W. and Z.L. designed the experiments. H.W. performed crystallization, GST pull-down and ITC experiments. S.X., L.W. and Y.H., determined the crystal structures. L.W. performed the molecular dynamics simulations. Z.L. and Y.J. prepared the transgenic plants and performed the MAPK, ROS, root growth and subcellular localization experiments. C.Z., X.M., Z.W. and Q.W. participated in protein purification. H.W., Z.X., J.X. and S.L. performed the mass spectrometry experiments. S.X., L.S. and H.W. wrote the paper with support from all authors.

Peer review

Peer review information

Nature Plants thanks Nitzan Shabek, Fengjiao Xin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Data availability

Atomic coordinates of AtMIK2^ECD-3M, BnMIK2^ECD, AtMIK2^ECD-3M-AtSCOOP12, BnMIK2^ECD-FocSCOOPL, AtMIK2^ECD-3M-AtSCOOP12-AtBAK1^ECD, AtMIK2^ECD-3M-FocSCOOPL-AtBAK1^ECD, BnMIK2^ECD N393A and BnMIK2^ECD N393D have been deposited in the Protein Data Bank (PDB) under accession numbers 8WEB, 8WEG, 8WEE, 8WEF, 8WEC, 8WED, 8WEH and 8WEI, respectively. Atomic coordinates of previously published AtFLS2^ECD-flg22-AtBAK1^ECD, AtPEPR1^ECD-PEP1, AtHSL1^ECD-IDA-AtSERK1^ECD, AtGSO1^ECD-CIF2 and NbRXEG1^ECD-XEG1-NbBAK1^ECD structures used in this study are also available in the PDB under accession numbers 4MN8, 5GR8, 7ODV, 6S6Q and 7DRC, respectively. The materials used in this study are available from the corresponding authors upon request. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Extended data

is available for this paper at 10.1038/s41477-024-01836-3.

Supplementary information

The online version contains supplementary material available at 10.1038/s41477-024-01836-3.