Perception of a divergent family of phytocytokines by the Arabidopsis receptor kinase MIK2

Plant genomes encode hundreds of receptor kinases and peptides, but the number of known plant receptor-ligand pairs is limited. We report that the Arabidopsis leucine-rich repeat receptor kinase LRR-RK MALE DISCOVERER 1-INTERACTING RECEPTOR LIKE KINASE 2 (MIK2) is the receptor for the SERINE RICH ENDOGENOUS PEPTIDE (SCOOP) phytocytokines. MIK2 is necessary and sufficient for immune responses triggered by multiple SCOOP peptides, suggesting that MIK2 is the receptor for this divergent family of peptides. Accordingly, the SCOOP12 peptide directly binds MIK2 and triggers complex formation between MIK2 and the BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) co-receptor. MIK2 is required for resistance to the important root pathogen Fusarium oxysporum. Notably, we reveal that Fusarium proteomes encode SCOOP-like sequences, and corresponding synthetic peptides induce MIK2-dependent immune responses. These results suggest that MIK2 may recognise Fusarium-derived SCOOP-like sequences to induce immunity against Fusarium. The definition of SCOOPs as MIK2 ligands will help to unravel the multiple roles played by MIK2 during plant growth, development and stress responses.


Results and discussion
In an effort to understand how MIK2 regulates stress responses, we tested whether Arabidopsis thaliana (hereafter, Arabidopsis) mik2 mutant plants are affected in the production of reactive oxygen species (ROS) upon treatment with different immune elicitors. A clear defect in apoplastic ROS production was observed in mik2 plants for some, but not all elicitors tested based on elicitor origin ( Supplementary Fig. 1). As a way to test the generality of these observations, other microbial and plant elicitors were tested, including the recently identified SERINE RICH ENDOGENOUS PEPTIDE 12 (SCOOP12) peptide 12 that is proposed to function as a phytocytokine (a secreted peptide that regulates plant immune responses by analogy to cytokines in metazoans 13 ). Surprisingly, unlike what was observed with flg22 or Pep1 ( Supplementary Fig. 1), mik2 plants were insensitive to SCOOP12 treatment for all cellular responses measured (Fig. 1a-f and Supplementary Fig. 2a-d), i.e. ROS production increase in cytosolic Ca 2+ concentration, mitogen-activated protein kinase phosphorylation, and seedling growth inhibition-all of which being hallmarks of immune signaling 14 . Importantly, the loss of MIK2 could be complemented with the expression of MIK2 under its native promoter ( Supplementary Fig. 2e, f). That mik2 plants are blind to SCOOP12 suggests that MIK2 is the receptor for SCOOP12.
To further test if MIK2 is the SCOOP12 receptor, we transiently expressed MIK2 or a chimera between the MIK2 ectodomain and the intracellular domain of EF-TU RECEPTOR (EFR) 26 (MIK2-EFR) in Nicotiana benthamiana. This plant species is insensitive to SCOOP12 12 and its genome lacks any obvious MIK2 ortholog ( Supplementary Fig. 4a-c). However, upon expression of MIK2 or MIK2-EFR, we could measure SCOOP12-induced ROS production in N. benthamiana, which was not observed when expressing a transformation control (β-glucuronidase, GUS) or when expressing the reverse chimera EFR-MIK2 that is functional in elf18 perception (Fig. 1g, h and Supplementary Fig. 4d-f). This data demonstrates that MIK2 is sufficient to confer SCOOP12 responsiveness, and indicates that the MIK2 ectodomain is involved in this recognition.
The current paradigm is that ligand-binding LRR-RKs undergo ligand-induced heterodimerization with a shape complementary co-receptor of the SERK family (or a related member of LRR-RK subfamily II) in order to activate signaling 17,27,28 . Having shown that SCOOP12-induced responses are BAK1-dependent (Supplementary Fig. 3a-c and ref. 12 ), we tested whether SCOOP12 could induce MIK2-BAK1 complex formation. Stably expressed MIK2 fused to fluorescent epitope tags (MIK2-FP) coimmunoprecipitated endogenous BAK1 specifically upon treatment with SCOOP12 ( Fig. 2a and Supplementary Fig. 5). In contrast, BAK1 co-immunoprecipitated with EFR-GFP upon elf18 but not SCOOP12 treatment in Arabidopsis (Fig. 2a). Consistent with this specific ligand-induced heteromerization, SCOOP12-induced BAK1-S612 phosphorylation was abolished in mik2 mutant plants (Fig. 2b). Finally, we tested if the MIK2 ectodomain (MIK2 ECD ) is sufficient for SCOOP12 binding in vitro. For this, we heterologously expressed MIK2 ECD (residues 1-709) in insect cells, and performed binding assays using isothermal titration calorimetry. MIK2 ECD directly senses SCOOP12 with a binding affinity of~4.6 µM. We next quantified the affinity of the co-receptor to the MIK2-SCOOP12 complex. In the presence of SCOOP12, BAK1 strongly binds MIK2 with a dissociation constant in the mid-nanomolar range (~275 nM), which is comparable to its bioactivity (Fig. 2c, d and Supplementary  Figs. 6, 7a). This suggests that SCOOP12 itself promotes receptorco-receptor association, as previously reported for other ligand-LRR-RK pairs 17,29 . This result is in good agreement with the formation of a stable, SCOOP12-dependent MIK2-BAK1 complex in size-exclusion chromatography experiments (Fig. 2e). Together with the SERK-dependency of SCOOP12-induced responses ( Supplementary Fig. 3a-c and ref. 12 ) and SCOOP12induced MIK2-BAK1/SERK association in planta ( Fig. 2a and Supplementary Fig. 5), these in vitro data demonstrate that MIK2 is sufficient for SCOOP12 perception, and thus that MIK2 is the SCOOP12 receptor.
PROSCOOP12 is part of a 14-member family in Arabidopsis defined by the presence of a signal peptide, a pro-peptide region, a putative protease cleavage site, and a predicted mature peptide encompassing a serine-and glycine-rich 13-amino-acid epitope   12 ). Importantly, deletion of the C-terminal arginine residue in SCOOP12 (SCOOP12Δ1) abolished its activity ( Supplementary Fig. 7b), suggesting that the predicted 13-mer corresponds to a minimal bioactive SCOOP epitope. Notably, while MIK2 is the SCOOP12 receptor, a proscoop12 loss-of-function mutant 12 did not phenocopy mik2, as tested for ROS production and root skewing ( Supplementary Fig. 8), suggesting that additional SCOOPs are perceived by MIK2. A multiple alignment of Arabidopsis SCOOP sequences corresponding to the defined bioactive epitope revealed poor conservation beyond two conserved serines at positions 5 and 7 Fig. 1 MIK2 is required and sufficient for SCOOP12-induced responses. a, b ROS production in leaf disks collected from 4-week-old A. thaliana plants induced by 1 μM SCOOP12 application (n = 16 leaf disks). a Points represent mean; error bars represent S.E.M. b Integrated ROS production over 40 min. Line represents mean; error bars represent S.D. c, d The mik2-1 mutation was crossed into the pUBQ10::AEQUORIN background, and calcium levels were measured in seedlings relative to maximum upon discharge following the addition of 100 nM SCOOP12 (n = 12 seedlings). c Points represent mean; error bars represent S.E.M. d Maximum relative SCOOP12-induced calcium levels ±S.D. e Western blot using α-p42/p44-ERK recognizing phosphorylated MPK6, MPK3, and MPK4/11 in seedlings treated with 100 nM SCOOP12 or mock for 15 min. Membranes were stained with CBB, as a loading control. f Fresh weight of 14-day-old seedlings grown in the presence of 1 μM SCOOP12 for 10 days relative to mock (n = 8 seedlings). A line represents mean; error bars represent S.D. g, h) ROS production in leaf disks of N. benthamiana transiently expressing the defined constructs induced by 1 μM SCOOP12 application (n = 24 leaf disks). g Points represent mean; error bars represent S.E.M. h Integrated ROS production over 60 min. A line represents mean; error bars represent S.D. P-values indicate significance relative to the WT control (or GUS transformed control) in a Tukey's multiple comparisons test following oneway ANOVA. All experiments were repeated and analyzed three times with similar results. ROS reactive oxygen species, CBB Coomassie brilliant blue.  (previously shown to be essential for SCOOP12 activity 12 ), and the presence of a glycine-rich C-terminal region for SCOOP family members (Fig. 3a). Interestingly, the C-terminal sequence of SCOOP peptides differs from that of other known serine/ glycine/proline-rich plant peptides (e.g. CLE, Pep, IDA, PIP, or CEP families 2 ), which often contain an arginine-histidineasparagine motif 17 suggesting a potential different mode of ligand-binding-something which will need to be tested in the future in structural studies. We synthesized peptides corresponding to all Arabidopsis SCOOPs with available sequence information on TAIR10 (Supplementary Table 1), and found that all but three-SCOOP6, 8, and 11-were active in at least one assay tested at either 1 µM or 10 µM (Fig. 3b, c and Supplementary Fig. 9a, b). Of note, in the    absence of current knowledge about the physiological concentration of SCOOP peptides released in the plant apoplast, these arbitrary concentrations were simply chosen to test for the ability of the peptides to induce the responses measured. Notably, upon careful examination of PROSCOOP6 and PROSCOOP11, we noticed additional SCOOP motifs in these proteins 12 -which we named SCOOP6♯2 and SCOOP11♯2 (in contrast to SCOOP6♯1 and SCOOP11♯1 previously tested). Synthetic peptides derived from these alternative SCOOP motifs turned out to be active in multiple assays ( Supplementary Fig. 9c-e). Having identified twelve bioactive SCOOP peptides, we then tested if their activities were also MIK2-dependent. Using ROS production and seedling growth inhibition as a readout, we found that, despite the low level of sequence similarity, all active SCOOP peptides required MIK2 (Fig. 3b, c). Furthermore, ligand-induced MIK2-BAK1 association was also observed in response to selected SCOOPs (Fig. 3d). Together, these experiments indicate that MIK2 is the receptor for the divergent family of SCOOP peptides.
The finding that a single receptor is sufficient and necessary for the perception of all members of a given (divergent) peptide family is so far unique to the SCOOP-MIK2 ligand-receptor system. Indeed, members of other plant peptides families (e.g. CLEs, RALFs) are often distinctively or conjunctively perceived by several related receptors 30,31 , or are perceived by partially redundant-related receptors with often distinct quantitative contributions (e.g. perception of CIFs, IDLs, and AtPeps) 2,32,33 .
MIK2 is required for resistance to F. oxysporum 9 and is required for immune responses to a Fusarium extract 11 . One hypothesis would thus be that perception of the Fusarium extract leads to SCOOP perception by MIK2, potentially via the control of PROSCOOP expression, PROSCOOP cleavage, and/or SCOOP secretion. While theoretically possible, this would however not be in line with the responses to the Fusarium extract that can be measured within minutes 11 . Homologous sequences to plant peptides have been reported in plant-associated organisms, whose recognition and bioactivity depend on the corresponding plant receptors [34][35][36][37][38][39][40][41][42] . Using BLAST and MAST algorithms, we identified several SCOOP-like motifs from Fusarium proteomes and ordered the corresponding synthetic peptides (Fig. 4a, Supplementary  Fig. 10, and Supplementary Table 2). Two of these peptides, derived from the proteins A0A0M9EVJ7 and A0A0D2XZ19, were able to induce immune responses in a MIK2-dependent manner (Fig. 4a-e and Supplementary Fig. 11a-e). Response to these peptides was also BAK1-dependent, and peptide treatmentinduced MIK2-BAK1 association (Fig. 4b-f and Supplementary  Fig. 11b, c), demonstrating that these synthetic peptides are sufficient as ligands for the MIK2-BAK1 complex. Notably, whilst the SCOOP-like motif present in A0A0M9EVJ7 is relatively poorly conserved, the A0A0D2XZ19 motif shows conservation within related fungal proteomes ( Fig. 4a and Supplementary Fig. 11f), consistent with the presence of a MIK2-dependent eliciting activity in extracts from different Fusarium species 11 . Together, these results indicate that the elicitor present in Fusarium extracts may derive from Fusarium proteins harboring a SCOOP-like motif recognized by MIK2. It however remains to be established in future studies whether these epitope(s) facilitate non-self recognition in the context of an infection, thus contributing to the enhanced susceptibility of mik2 mutants to Fusarium 9,11 .
In conclusion, the characterization of the SCOOP-MIK2 ligand-receptor module represents a significant advance in our understanding of the mechanism through which phytocytokines are perceived by plant cells. Given the multiple physiological functions of MIK2, the identification of SCOOPs as MIK2 ligands will enable the future dissection of the roles of these divergent peptides in diverse pathways and on the molecular basis of their perception.

Methods
No statistical methods were used to predetermine the sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment.
Plant material and growth conditions. Arabidopsis thaliana ecotype Columbia (Col-0) and Wassilewskija (Ws-2) were used as wild-type controls. Plants for ROS burst assays were grown in individual pots at 21°C with a 10-h photoperiod. Seeds grown on plates were surface sterilized using chlorine gas for 5-6 h, and sown on Murashige and Skoog (MS) media supplemented with vitamins, 1% sucrose, and 0.8% agar and stratified at 4°C for 2-3 days. Nicotiana benthamiana plants were grown on peat-based media at 24°C, with 16-h photoperiod.
Synthetic peptides. All synthetic peptides were ordered at >80% purity (physiological assays), >95% purity (biochemical assays) (EZBiolabs). Sequences of all peptides can be found in Supplementary Table 1. The gene models from which the peptide sequences were extracted are listed in Supplementary Table 4.
Molecular cloning. For overexpression of MIK2 in A. thaliana and N. benthamiana, the MIK2.1 coding sequence was PCR amplified from Arabidopsis Col-0 cDNA using gene-specific primers (Supplementary Table 3) and was cloned into pENTR using the D-TOPO kit (Invitrogen) subsequently recombined using LR Clonase II (Invitrogen) into the pEarleygate103 expression vector downstream of the 35S promoter and in frame with a C-terminal poly-His-GFP tag 48 . Generation of the chimeric receptors was performed using overlap extension PCR from EFR and MIK2 entry clones. The final PCR product was then recombined into the pEarleygate103 expression vector using LR Clonase II (Invitrogen). Native promoter sequences were amplified and cloned with C-terminal tags into pICSL86955 using BsaI restriction sites. All clones were verified by Sanger sequencing.
ROS measurement. Leaf disks were harvested from 4-week-old Arabidopsis plants or 3-week-old N. benthamiana using a 4-mm diameter biopsy punch (Integra™ Miltex™). Leaf disks were floated overnight on 100 μL of distilled water in white 96-well-plates (Greiner Bio-One). Prior to ROS measurement, the water was removed and replaced with ROS assay solution (100 μM Luminol (Merck), Fig. 3 Divergent SCOOP peptides induce MIK2-dependent responses. a Alignment of SCOOP motifs from A. thaliana. b ROS production in leaf disks collected from 4-week-old A. thaliana plants induced by application of 1 μM SCOOP peptides in Col-0 (black) or mik2-1 (green) (n = 8 leaf disks). A line represents mean; error bars represent S.D. P-values indicate significance relative to the WT control as indicated by two-tailed T test c Fresh weight of 14-day-old seedlings grown in the presence of 1 μM SCOOP peptides for 10 days relative to mock. A line represents mean; error bars represent S.D. (n = 12 seedlings). A two-way ANOVA indicated a significant effect of treatment and genotype (P < 0.0001); P-values indicate significance relative to Col-0 in a Sidak's multiple comparison test between genotypes within each treatment. d Co-immunoprecipitation of BAK1 with MIK2-GFP from mik2-1/MIK2-GFP seedlings treated with 1 μM SCOOP peptides, or water for 10 min. Western blots were probed with antibodies α-GFP and α-BAK1. These experiments were performed and analyzed three times with similar results. These experiments were performed and analyzed three times with similar results. ROS reactive oxygen species. c   Fig. 4 Fusarium-derived peptides are induced MIK2-dependent responses. a Alignments of the SCOOP-like motifs from A0A0M9EVJ7 and A0A0D2XZ191 in closely related fungal species with the most closely related Arabidopsis SCOOP based on amino acid identity. Residues that are not found in any Arabidopsis SCOOP are underlined b Relative fresh weight of 14-day-old seedlings grown in the presence of 1 μM A0A0M9EVJ7 or A0A0D2XZ19 for 10 days relative to mock (n = 12 seedlings). A line represents mean; error bars represent S.D. P-values are derived from a Dunnett's multiple comparison test comparing the treatment to mock for each genotype following a two-way ANOVA. c Western blot using α-p42/p44-ERK recognizing phosphorylated MPK6, MPK3, and MPK4/11 in seedlings treated with 1 μM A0A0M9EVJ7, A0A0D2XZ19 or mock for 15 min. Membranes were stained with CBB, as a loading control. d, e Integrated ROS production in leaf disks collected from 4-week-old A. thaliana plants induced by d 1 μM A0A0M9EVJ7 or e 1 μM A0A0D2XZ19 application over 40 min (n = 8 leaf disks). Line represents mean; error bars represent S.D. P-values are derived from a Dunnett's multiple comparison test comparing the treatment to mock for each genotype following a one-way ANOVA. f Co-immunoprecipitation of BAK1 with MIK2-GFP from mik2-1/MIK2-GFP and GFP-LTI6B from Col-0/GFP-LTI6B seedlings treated with 1 μM A0A0M9EVJ7, 1 μM A0A0D2XZ19, or water for 10 min. Western blots were probed with antibodies α-GFP and α-BAK1. All experiments were performed and analyzed three times with similar results. CBB Coomassie brilliant blue, ROS reactive oxygen species. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-20932-y ARTICLE NATURE COMMUNICATIONS | (2021) 12:705 | https://doi.org/10.1038/s41467-021-20932-y | www.nature.com/naturecommunications 20 μg mL −1 horseradish peroxidase (Merck)) with or without the addition of elicitors. Immediately following the addition of the assay solution light emission was measured from the plate using a HIGH-RESOLUTION PHOTON COUNTING SYSTEM (HRPCS218, Photek) equipped with a 20 mm F1.8 EX DG ASPHERICAL RF WIDE LENS (Sigma Corp).

F T G P S G S G H G G G R D T G P S T S G R G R G R D A D T S T S T R G R G R D A D T S T S T R G R G R E A N G A P A A R G G G R
Cytoplasmic calcium measurement. Arabidopsis seedlings were grown in 96-well plates (Greiner Bio-One) in 100 μL liquid MS for 5 days. The evening before calcium measurements the liquid MS was replaced with 100 μL 20 μM coelenterazine (Merck) and the seedlings incubated in the dark overnight. The following morning the coelenterazine solution was replaced with 100 μL water and rested for a minimum of 30 min in the dark. Readings were taken in a VARIOSKAN TM MUTIPLATE READER (ThermoFisher) using the injector to add 50 μL of 3× concentrated elicitor solution or mock. L max was determined as the maximum luminescence emission upon discharge with (1 M CaCl 2 , 10% ethanol).
Seedling growth inhibition. Four-day-old seedlings growing on MS plates were transferred individually into separate wells of transparent 48-well tissue culture plates (Greiner Bio-One) containing 500 μL of liquid MS media with/without elicitor addition. The plates were then transferred back to the growth conditions for an additional 10 days before seedlings were blot-dried and weighed.
Transient expression in Nicotiana benthamiana. Agrobacterium tumefaciens strain GV3101 transformed with the corresponding construct were grown overnight in L-media and spun-down. For ROS assays the bacteria were resuspended in 10 mM MgCl 2 and adjusted to O.D. 600 = 0.2 prior to infiltration into the youngest fully expanded leaves of 3-week-old plants. Leaf disks were collected 24 h later, and ROS assays were performed as described for Arabidopsis.
Root skewing. Seeds were sown directly on MS agar square plates and stratified for 2 days at 4°C. Plates were transferred to 22°C under a 16-h photoperiod, in an upright position for 9 days. The root angle was measured by the ImageJ software, as performed previously 9 .
Protein expression and purification. Codon-optimized synthetic genes for expression in Spodoptera frugiperda (Invitrogen GeneArt), coding for Arabidopsis thaliana MIK2 (residues 1-709) and BAK1 (1-220) ectodomains were cloned into a modified pFastBac (Geneva Biotech) vector, providing a TEV (tobacco etch virus protease) cleavable C-terminal StrepII-9xHis tag. For protein expression, Trichoplusia ni Tnao38 cells 49 were infected with MIK2 or BAK1 virus with a multiplicity of infection (MOI) of 3 and incubated for 1 d at 28°C and 2 d at 22°C at 110 rpm. The secreted proteins were purified from the supernatant by sequential Ni 2+ (HisTrap Excel column; GE Healthcare; equilibrated in 25 mM KP i pH 7.8, 500 mM NaCl) and StrepII (Strep-Tactin Superflow high capacity (IBA Lifesciences) equilibrated in 25 mM Tris pH 8.0, 250 mM NaCl, 1 mM EDTA) affinity chromatography. All proteins were incubated with TEV protease to remove the tags. Proteins were further purified by SEC on a Superdex 200 increase 10/300 GL column (GE Healthcare), equilibrated in 20 mM sodium citrate pH 5.0, 150 mM NaCl. For biochemical experiments, proteins were concentrated using Amicon Ultra concentrators (Millipore, molecular weight cutoff 10,000 and 30,000).
ITC experiments. Experiments were performed at 25°C using a MicroCal PEAQ-ITC (Malvern Instruments) with a 200-µL standard cell and a 40-μL titration syringe. MIK2 and BAK1 ectodomains were gel filtrated into pH 5 ITC buffer (20 mM sodium citrate pH 5.0, 150 mM NaCl). SCOOP12 peptide powder was dissolved in the same buffer to obtain the desired concentration. A typical experiment consisted of injecting 1 μL of a 200 μM solution of the ligand into 20 μM MIK2 solution in the cell at 150 s intervals. BAK1 vs MIK2-SCOOP12 experiments were performed by titrating 200 µM BAK1 onto the MIK2-SCOOP12 complex in the cell, using the same injection pattern. ITC data were corrected for the heat of dilution by subtracting the mixing enthalpies for titrant solution injections into protein-free ITC buffer. Experiments were done in triplicates and data were analyzed using the MicroCal PEAQ-ITC Analysis Software provided by the manufacturer. All ITC runs used for data analysis had an N ranging between 0.8 and 1.3. The N values were fitted to 1 in the analysis.

Data availability
For blot source images, see Supplementary Fig. 1. All other data or materials can be obtained from the corresponding author upon request. Source data are provided with this paper.
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