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

1Plants employ cell-surface localized receptors, such as receptor kinases (RKs), to sense their extracellular environment and coordinate their growth and development in response to endogenous and exogenous cues. Secreted plant peptides—encoded by hundreds of genes in plant genomes—have recently emerged as playing a major role in this autocrine and paracrine communication and are proposed as a new class of plant hormones. The number of known receptors for such peptides however remains scarce.

2The leucine-rich repeat receptor kinase (LRR-RK) MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) was initially identified for its proposed role in pollen tube guidance mediated by the cysteine-rich AtLURE1 peptides. In this context, it was shown to interact with the LRR-RK MALE DISCOVERER 1 (MDIS1), and was therefore proposed to be part of a receptor complex for AtLURE1 peptides,. At the same time, another set of LRR-RKs, POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 6 (PRK6) and related proteins, were shown to be involved in AtLURE1 perception. Structural and biochemical work then showed that AtLURE1 peptides bind to PRK6, but not to MIK2, and the genetic role of PRK6 in AtLURE1 perception was recently corroborated. These findings questioned whether AtLURE1s are ligands for a potential MDIS1–MIK2 complex. Accordingly, it was later revealed that MIK2 (also named LEUCINE-RICH REPEAT KINASE FAMILY PROTEIN INDUCED BY SALT STRESS, or LRR-KISS), but not MDIS1, is involved in other physiological processes, such as stress responses upon cell wall damage, salt tolerance, root growth, and resistance to the important vascular fungal pathogen Fusarium oxysporum–. None of these processes are known to involve AtLURE1 peptides, and thus the identity and origin of the MIK2 ligand(s) remain unknown.

3Here, we identify MIK2 as the receptor of the SCOOP family of phytocytokines. SCOOP12 binds directly to MIK2 to induce MIK2–BAK1 complex formation and activation of downstream signaling. All tested SCOOP peptides similarly induce MIK2-dependent responses and MIK2–BAK1 complex formation. Moreover, we identify SCOOP-like sequences within Fusarium proteomes, whose corresponding synthetic peptides induce MIK2-dependent responses and MIK2–BAK1 association, suggesting MIK2 may directly perceive Fusarium.

4In 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 that is proposed to function as a phytocytokine (a secreted peptide that regulates plant immune responses by analogy to cytokines in metazoans). 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 Ca2+ concentration, mitogen-activated protein kinase phosphorylation, and seedling growth inhibition—all of which being hallmarks of immune signaling. 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.

5Plant cell-surface immune receptors (called pattern recognition receptors) are either RKs or receptor proteins (RPs), the latter lacking any obvious intracellular signaling domain,. The majority of known receptors for plant-derived or exogenous peptides are LRR-RKs or LRR-RPs that form ligand-induced complexes with LRR-RK co-receptors belonging to the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family,. Notably, SCOOP12-induced seedling growth inhibition was impaired in a bak1 (BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1, BAK1 corresponds to SERK3) null mutant. Similarly, we found that SCOOP12-induced ROS production is impaired in bak1-5 (Supplementary Fig. 3a, b), a bak1 allele that has a dominant-negative effect on other SERKs–. Furthermore, SCOOP12 treatment-induced BAK1 phosphorylation on serine 612 (Ser612) (Supplementary Fig. 3c)—a post-translational modification required for immune signaling. By comparison, SCOOP12-induced ROS production was not affected in sobir1-13 (Supplementary Fig. 3a, b), a null allele of the adapter LRR-RK SUPPRESSOR OF BAK1-INTERACTING RECEPTOR-LIKE KINASE 1 (SOBIR1), required by LRR-RPs to form active signaling complexes with SERK co-receptors,. Together, these data suggest that the SCOOP12 receptor is an LRR-RK. Consistently, we also found that optimal SCOOP12-induced ROS production occurs via components involved downstream of other LRR-RK-type PRRs– (Supplementary Fig. 3d, e).

6To 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) (MIK2–EFR) in Nicotiana benthamiana. This plant species is insensitive to SCOOP12 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.

7The 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,,. Having shown that SCOOP12-induced responses are BAK1-dependent (Supplementary Fig. 3a–c and ref. ), we tested whether SCOOP12 could induce MIK2–BAK1 complex formation. Stably expressed MIK2 fused to fluorescent epitope tags (MIK2–FP) co-immunoprecipitated 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 (MIK2ECD) is sufficient for SCOOP12 binding in vitro. For this, we heterologously expressed MIK2ECD (residues 1–709) in insect cells, and performed binding assays using isothermal titration calorimetry. MIK2ECD 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 receptor-co-receptor association, as previously reported for other ligand-LRR-RK pairs,. 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. ) and SCOOP12-induced 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.

8PROSCOOP12 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 that was shown, at least for SCOOP12, to be biologically active in the nanomolar range when used as a synthetic peptide (Supplementary Fig. 7a, Supplementary Table 1, and ref. ). 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 did not phenocopy mik2, as tested for ROS production and root skewing (Supplementary Fig. 8), suggesting that additional SCOOPs are perceived by MIK2.

9A multiple alignment of Arabidopsis SCOOP sequences corresponding to the defined bioactive epitope revealed poor conservation beyond two conserved serines at positions 5 and 7 (previously shown to be essential for SCOOP12 activity), 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), which often contain an arginine–histidine–asparagine motif suggesting a potential different mode of ligand-binding—something which will need to be tested in the future in structural studies.

10We 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—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.

11The 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,, or are perceived by partially redundant-related receptors with often distinct quantitative contributions (e.g. perception of CIFs, IDLs, and AtPeps),,.

12MIK2 is required for resistance to F. oxysporum and is required for immune responses to a Fusarium extract. 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. Homologous sequences to plant peptides have been reported in plant-associated organisms, whose recognition and bioactivity depend on the corresponding plant receptors–. 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 treatment-induced 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. 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,.

13In 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.

14No 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.

15Arabidopsis 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.

16The following A. thaliana mutants in Col-0 background (except otherwise indicated) were used: mik2-1 (SALK_061769) (kindly provided by Wei-Cai Yang), mik2-2 (SALK_046987), mik2-4 (FLAG_518G04), bak1-5, sobir1−13 (SALK_009453) (kindly provided by Yuelin Zhang), cpk28-1 (GK‐523B08), rbohD, cpk5 cpk6 cpk11 (kindly provided by Jen Sheen), bik1 pbl1 (kindly provided by Jian-Min Zhou), pro35S::GFP-LTI6B, and proscoop12 (kindly provided by Jean-Pierre Renou). The transgenic lines efr/EFR-GFP and proUBQ10::AEQ (kindly provided by Justin Lee) lines were described,. The mik2-1/pro35S::MIK2–GFP line was generated in this study. mik2-1 plants were transformed with the MIK2 coding sequence in the pEARLEYGATE103 binary vector using floral dip transformation. Transformants were selected to homozygosity using phosphinotricin.

17All 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.

18For 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. 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.

19Leaf 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), 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).

20Arabidopsis 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 VARIOSKANTM MUTIPLATE READER (ThermoFisher) using the injector to add 50 μL of 3× concentrated elicitor solution or mock. Lmax was determined as the maximum luminescence emission upon discharge with (1 M CaCl2, 10% ethanol).

21Four-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.

22Two-week-old seedlings grown in liquid MS media (or N. benthamiana leaf tissue) were flash-frozen in liquid nitrogen. Plant tissue was ground in liquid nitrogen prior to boiling in 2× Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris-HCl; (10 μL.mg−1 tissue)) for 10 min at 95 °C. The samples were then spun at 13,000 × g for 5 min prior to loading and running on SDS-PAGE gels of an appropriate concentration. Proteins were transferred onto PVDF membrane (ThermoFisher) prior to incubation with appropriate antibodies (α-pBAK1 (Ser612) (1:2000); α-GFP-HRP (sc-9996, Santa Cruz; 1:5000); α-pMAPK (p44/42 MAPK (Erk1/2) antibody #9102; 1:4000); α-rabbit-HRP (A-0545, Merck; 1:10,000). Western blots were imaged with a LAS 4000 IMAGEQUANT SYSTEM (GE Healthcare). Staining of the blotted membrane with Coomassie brilliant blue was used to confirm loading.

23Fifteen to twenty seedlings were grown in wells of a 6-well plate for 2 weeks in liquid MS media with gentle agitation (6 wells were used per treatment). The MS media was replaced the night before treatment. Seedlings were treated with 1 μM elf18/SCOOP12 for 10 min before flash-freezing. Tissue was ground and proteins extracted in 1:1 (v/v) powdered tissue:extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM dithiothreitol, 1% protease inhibitor cocktail (P9599, Sigma Aldrich), 2 mM Na2MoO4, 2.5 mM NaF, 1.5 mM activated Na3VO4 and 1% IGEPAL) for 30 min at 4 °C. Cell debris was removed via centrifugation. For immunoprecipitation, GFP-TRAP/RFP-TRAP AGAROSE BEADS (ChromoTek) were incubated with extracts for 3 h at 4 °C and washed three times in wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM dithiothreitol, 1% protease inhibitor cocktail (P9599, Sigma Aldrich), 2 mM Na2MoO4, 2.5 mM NaF, 1.5 mM activated Na3VO4, and 0.1% IGEPAL) before adding 2× Laemmli sample buffer and incubating for 10 min at 95 °C. Detection was carried out by SDS-PAGE and western blots using α-BAK1 α-RFP (ab34771, Abcam) and α-GFP-HRP (sc-9996, Santa Cruz) antibodies. α-rabbit-HRP (A-0545, Merck) was used to detect α-BAK1 and α-RFP.

24Agrobacterium 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 MgCl2 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.

25Seeds 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.

26Codon-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 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 Ni2+ (HisTrap Excel column; GE Healthcare; equilibrated in 25 mM KPi 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).

27Experiments 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.

28SIAS was used to generate similarity and identity matrices (http://imed.med.ucm.es/Tools/sias.html).

29The timing of fungal species divergence was estimated using TimeTree–.

30Statistical analysis was performed in GraphPad Prism 7.0. (GraphPad Software, http://www.graphpad.com) unless stated otherwise. Dot plots were used to show individual data points wherever possible. P-values < 0.05 were considered nonsignificant. Sample sizes, statistical tests used, and P-values are stated in the figure legends.

31Further information on research design is available in the Nature Research Reporting Summary linked to this article.