Originally published 19 January 2017; revised 23, 24, and 26 January 2017

www.sciencemag.org/content/355/6322/287/suppl/DC1

Supplementary Materials for The receptor kinase FER is a RALF-regulated scaffold controlling plant

immune signaling

Martin Stegmann, Jacqueline Monaghan, Elwira Smakowska-Luzan, Hanna Rovenich, Anita Lehner, Nicholas Holton, Youssef Belkhadir, Cyril Zipfel*

*Corresponding author. Email: cyril.zipfel@tsl.ac.uk

Published 20 January 2017, Science 355, 287 (2017) DOI: 10.1126/science.aal2541

This PDF file includes

Materials and Methods Supplementary Text Figs. S1 to S18 Tables S1 and S2

Revision 1 (23 January 2017): In this revision, the acknowledgments have been expanded. The originally posted supplementary materials are available here.

Revision 2 (24 January 2017): In this revision, the RALF23 and RALF33 peptides in table S2 are correct. They were inadvertently swapped in the original.The second revision contained an error in the peptides.

Revision 3(26 January 2017): In this revision, the RALF23 and RALF33 peptides in table S2 are correct. They were inadvertently swapped in the original. The originally posted supplementary materials are available here.

Materials and Methods Plant material and growth conditions Arabidopsis thaliana ecotype Columbia (Col-0) was used as a wild type control for all plant

assays. Plants for ROS burst assays and pathogen infection experiments were grown in individual pots at 20-21 °C with an 8-hour photoperiod in environmentally controlled growth rooms. Seeds for PAMP-induced gene expression experiments were surface sterilized using chlorine gas for 6 hours and grown on Murashige and Skoog (MS) media supplemented with vitamins, 1% sucrose and 0.8% agar for five days at 22 °C and a 16 hour photoperiod. Subsequently, seedlings were transferred to multi-well plates containing liquid MS media and grown for another 7 days before performing the experiments. The bak1-5 and bak1-5 mob6 mutant were generated as previously described (2, 3). The s1p-3 mutant, which was previously described as a knockout mutant (21), was kindly provided by Sebastian Wolf. The ralf23-3 (SALK_135682), ralf33-1 (SALK_021110) and ralf33-2 (GK-862A06) mutants (fig. S6) were obtained from the European Arabidopsis Stock Centre in Nottingham, UK (NASC) and genotyped for homozygosity using allele-specific primers (Table S1). Additional T-DNA insertion lines for ralf23 were obtained (SALK_064994; SALK_000027), but none of these was a knockout allele. The fer-2 mutant (22) was kindly provided by Nana Keinath. The efr/EFR-GFP lines were described earlier (23). The fer-4, fer-4/FER-GFP, 35S::RALF23-GFP and Col-0/FLS2-GFP lines were recently published (7, 24, 25), and were kindly provided by Hen-Ming Wu, Pablo Vera and Silke Robatzek, respectively. The fer-2/35S::RALF23-GFP line was generated by crossing. Pictures of all the lines used in this study are shown in fig. S17.

Mapping of mob6 by whole-genome sequencing To map the mob6 locus, whole genome sequencing was performed as previously described

(2). In brief, bak1-5 mob6 was backcrossed to bak1-5 to fix the bak1-5 background mutation. The resulting F2 population was screened for individuals showing restoration of elf18-triggered seedling growth inhibition. For this purpose, around 1000 individual seedlings were screened and around 200 with the desired phenotype were pooled and analysed by Illumina sequencing. In parallel, the bak1-5 parent was re-sequenced and CandiSNP software (26) was used to identify candidate single nucleotide polymorphisms (SNPs) compared to the Arabidopsis TAIR10 genome, correcting for mutations identified in the bak1-5 genome. Sanger sequencing these SNPs in phenotypically homozygous F2 plants confirmed a cluster of homozygous SNPs on the top arm of chromosome 5. In addition, F2 plants resulting from a cross between bak1-5 mob6 and Col-0 were screened for individuals showing increased chitin-triggered ROS compared to Col-0. This resulted in the identification of 20 plants with a high ROS phenotype. Sequencing the cluster of SNPs described above indicated that only the SNP at position 6643698 located in At5g19660/S1P on chromosome 5 remained homozygous in each individual line with the high ROS phenotype (primer sequences in Table S1). Single mob6 mutants were isolated by genotyping out bak1-5 from the Col-0 cross. To confirm the causative locus, we tested for allelic non-complementation in the F1 generation of a cross between the mob6 single mutant and s1p-3, as well as trans-complementation by expressing 35S::S1P-cMyc in mob6.

Molecular cloning For transgenic complementation of mob6 mutants, S1P coding sequence was amplified from

Arabidopsis Col-0 cDNA using gene-specific primers (Table S1) and sub-cloned into pENTR

using the D-TOPO kit (Invitrogen). To generate Cauliflower Mosaic Virus (CaMV) 35S promoter-driven C-terminally-tagged S1P-cMyc fusions, the LR Clonase II kit (Invitrogen) was used to recombine S1P from pENTR into pGWB420 (27). Similarly, RALF23 was amplified by gene-specific primers, cloned into pENTR and recombined with pGWB420 to generate C-terminal cMyc fusions for transient protoplast experiments. All clones were verified by Sanger sequencing. The construct for generating N-terminally tagged FER ectodomain (MBP-ectoFER) was recently described (9), and was kindly provided by Michael Sussman.

For FER-V5-6xHis expression in Drosophila melanogaster Schneider 2 (S2) cells, the ectodomain of FER (37-440) was inserted into the pMT/BiP/V5/6xHis vector (Invitrogen). The extracellular domain of FER (26-446) was cloned by ligation independent cloning between the existing BiP signal sequence and the C-terminal V5 antibody epitope and hexahistidine tag. All clones were verified by Sanger sequencing. Primers (Table S1) were designed to have an amplicon homologous to the desired boundaries of FERONIA ectodomain and extensions for RecA-mediated SLIC strategy were attached. Amplification was done using Phusion Flash Mastermix (Thermo Scientific) according to the manufacturer’s instructions for 2-step PCR.

Synthetic peptides and elicitors The flg22, elf18, biotin-elf24, RALF17, RALF23, RALF32, RALF33, Biotin-RALF23 and

RALF34 peptides were synthesized by EZBiolab (United States) with a purity of >95%. All peptides were dissolved in sterile pure water. Sequences of all peptides can be found in Table S2. Chitin was obtained from Sigma Aldrich.

ROS burst measurement Eight leaf discs (4 mm in diameter) per individual genotype were collected in 96-well plates

containing sterile water. After collection, leaf discs were recovered overnight. The next day, the water was replaced by a solution containing 17 μg/mL luminol (Sigma Aldrich), 20 μg/mL horseradish peroxidase (HRP, Sigma Aldrich) and the PAMP in the appropriate concentration. Luminescence was measured for the indicated time period using a charge-coupled device camera (Photek Ltd., East Sussex UK).

To test the effect of RALF peptides on PAMP-triggered ROS, 8-16 leaf discs per treatment and/or genotype were collected in 96-well plates containing water and recovered overnight. The following day, the water was replaced by 75 µL 2 mM MES-KOH pH 5.8 to mimic the apoplastic pH. Leaf discs were incubated further for 4-5 hours before adding 75 μL 40 μg/mL HRP, 1 μM L-O12 (Wako Chemicals, Germany) and double concentrated elicitor RALF peptide solution (final concentration 20 μg/mL HRP, 0.5 µM L-O12, 1x elicitors).

ROS production is either displayed as the integration of total photon counts or as the progression of photon counts. Figure S18 displays all the curves over time used for integration of total photon counts as shown in the main figures and main figure legends.

Bacterial infections Pseudomonas syringae pv. tomato DC3000 COR- cells were streaked out from glycerol

stock on fresh King's B media plates containing 1% agar supplemented with 50 μg/mL rifampicin and 50 μg/mL kanamycin. Bacteria were collected from plates with a T-spreader and resuspended in water containing 0.02% Silwet L77 (Sigma Aldrich) to an OD600 = 0.2 (108 colony forming units per mL). This bacterial suspension was sprayed on 4- to 5-week-old plants, which were covered with vented lids for 3 days. Three leaf discs per sample from different plants

were collected in four microfuge tubes and ground with a drill-adapted pestle. Serial dilutions were plated on LB agar and colonies were counted 2 days later.

Induced resistance assay Four-to-five-week-old plants were infiltrated with an elicitor solution containing 1 μM elf18

and/or 1 µM RALF17 or RALF23 in 2 mM MES-KOH pH 5.8 and incubated for 24 hours. Subsequently, a suspension of Pseudomonas syringae pv. tomato DC3000 bacteria was prepared as above to an OD600 = 0.0002 (105 colony forming units per mL) and syringe-infiltrated in the pre-treated leaves. 2 days after inoculation, samples were collected as described above.

RALF23 cleavage assay For elf18-induced cleavage 2-week-old seedlings were transferred from MS medium into 2

mM MES-KOH, pH5.8 and incubated overnight. The next day, elf18 was added to a final concentration of 100 nM and the seedlings were harvested after time course incubation by flash freezing in liquid nitrogen. To analyse bacterial-induced RALF23 cleavage, 5- to 6-week-old soil grown plants were spray inoculated with a suspension of Pseudomonas syringae pv. tomato hrcC- at an OD600 = 0.2 and 0.02% Silwet L77 (Sigma Aldrich). Leaf discs of plants were harvested at the respective time points and flash frozen in liquid nitrogen. Protein extraction was performed by adding Laemmli sample buffer and incubating for 10 minutes at 95 °C. Analysis was carried out by SDS-PAGE and western blot using α-GFP antibodies (Santa Cruz biotechnology).

Protoplast transformation Protoplasts were transformed as previously described (28). Fifty micrograms DNA of

pGWB420-RALF23 were used to transform 1 mL of protoplasts (2 x 105 cells/mL). After transformation, protoplasts were aliquoted in microfuge tubes for performing elf18 treatment the following day. Protoplasts were frozen in liquid nitrogen and crude protein extract was boiled in Laemmli buffer before analysing samples with SDS-PAGE and western blot. Detection of RALF23-cMyc was performed using α-cMyc antibodies (Santa Cruz Biotechnology).

RNA isolation and quantitative RT-PCR RNA isolation, cDNA synthesis and quantitative real-time PCR (qRT-PCR) was performed

as previously described (29). In brief, four 2-week-old seedlings grown in liquid MS were treated with 100 nM elf18 for the indicated time and ground in liquid nitrogen. Total RNA was extracted using TRI reagent kit (Sigma-Aldrich) according to the manufacturer`s instructions. RNA samples were treated with Turbo DNA-free DNase (Ambion) and the quantity was measured using a Nanodrop spectrophotometer (Thermo Scientific). Semi-quantitative reverse transcription (RT) PCR to test transcript accumulation in ralf23-3, ralf33-1 and ralf33-2 was performed on leaf cDNA from 4- to 5-week-old plants with a 25 cycle PCR program and primers indicated in Table S1.

In vitro pull-down with biotin-RALF23 and MBP-ectoFER MBP-ectoFER was purified from E.coli as previously described (9). In brief, the p55K-TEV

MBP-ectoFER construct was transformed in BL21 DE3 and grown in liquid LB medium supplemented with 0.2% glucose until an OD600 = 0.8. Expression was induced by adding 0.1 mM IPTG for 2 hours. Bacteria were lysed in 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM

EDTA, 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and 1 mg/mL Lysoyzme 1. Purification was performed by pulling down MBP-ectoFER with Amylose Resin (New England Biolabs) for 1 hour at 4 °C. After washing 4 times, bound protein was eluted with extraction buffer containing 10 mM maltose. To produce MBP-ectoEFR, an extracellular fragment of EFR was generated by PCR with primers indicated in Table S1 and cloned into pMAL-c4e using EcoRI and XbaI restriction enzymes (Thermo Scientific). Purification of the protein was performed as described for MBP-ectoFER.

The in vitro pull-down of MBP-ectoFER/MBP-ectoEFR was performed with 1 ng/µL protein and 10 nM biotin-RALF23 in 500 µL 20 mM Tris-HCL pH 7.5, 1% IGEPAL using Neutravidin agarose resin (Thermo Scientific). The samples were incubated for 1 hour at 4 °C and washed 5 times with buffer. Pull-down was analysed with SDS-PAGE and western blot using α-MBP antibodies (New England Biolabs).

S2 cells expression and purification of ectoFER-V5-6xHis The recombinant ectodomain FER-V5-6xHis (ectoFER-V5-6xHis) was expressed in

Drosophila S2 cells and secreted in Schneider’s medium (Thermo Fisher) containing 10% fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, and 2 mM L-glutamine. S2 cells were transfected transiently with the pMT-BiP-FER-Fc-V5-6xHis vector using Effectene (Qiagen) following manufacturer’s instructions. Transfected cells were induced for protein expression with 1 mM CuSO4 24 hours after transfection, and supernatant was collected three days after induction. Protease inhibitors (Sigma) and 0.02% NaN3 was added to the collected media before storage at 4 °C. The cell supernatant was assessed for recombinant protein expression using α-V5 antibodies (Invitrogen). The culture medium was collected for protein purification with a metal affinity resin (Qiagen). The protein was eluted with 250 mM imidazole and dialyzed overnight at 4 °C using high-resolution seamless cellulose tubing system with a pore size of 12,400 MWCO (Sigma-Aldrich) against a binding buffer containing 20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 200 mM NaCl and 1% IGEPAL. Dialysed protein was subsequently concentrated via centrifugal filtration with a 20 kDa filter (Sartorius). Protein purity was confirmed by western blotting with α-V5 antibodies (Invitrogen).

ectoFER-V5-6xHis –RALF23 interaction assays To test an interaction of ectoFER-V5-6xHis with biotin-RALF23, 500 µL of the

concentrated protein was incubated at 4 °C for 2 hours on an orbital shaker with the labelled peptide. The mixed samples were then combined with 50 µL slurry of high-capacity Streptavidin resin (Pierce) and rotated for an additional 3 hours at 4 °C. For competition assays, non-labelled peptides were added simultaneously with the resin. Streptavidin resin was then pelleted at 1,000 g for 2 minutes at 4 °C and washed 4 times with binding buffer. Bound proteins were eluted with Laemmli buffer and analysed via SDS-PAGE and western blots using α-V5 antibodies.

ectoFER Expression and Purification from Baculovirus infected insect cells For FER expression in Hi5 cells, the ectodomain of FER was inserted into the baculovirus

transfer vectors pMelBac B1 (Invitrogen) by ligation independent cloning between the existing Honey bee melettin signal sequence and the C-terminal Strep II-9xhistidine tag. All clones were verified by Sanger Sequencing. Cloning Primers were designed to have an amplification part homologous to the desired boundaries of FER ectodomain and extensions for RecA-mediated SLIC strategy.

The C-ter StrepII-9xHis fused FER ectodomain was produced by secreted expression in baculovirus-infected insect cells, harvested 72 hours post-infection and purified by Ni-NTA affinity chromatography (Qiagen). The samples were then ran 2 consecutive times onto a Superdex 200 16/60 column (GE Healthcare) pre-equilibrated with 50 mM NaH2PO4/Na2HPO4 pH 7.5; 200 mM NaCl; 5% Glycerol. Protein purity was checked by SDS-PAGE. The protein was subsequently concentrated to 3-5 mg/ml via centrifugal filtration with a 20 kDa filter (Sartorius). Identity of the FER ectodomain was further confirmed by anti-His immunoblots.

Isothermal titration calorimetry (ITC) assays ITC was performed using an ITC200 calorimeter (Microcal). RALF23 peptides

concentration were determined to prior to all titrations by measuring absorbance of peptidic bonds at 205nm. FER ectodomain concentrations were determined by using tyrosine residue absorbance and the corresponding molecular extinction coefficient. All assays were performed in ITC buffer (50 mM NaH2PO4/Na2HPO4 pH 7.5; 200 mM NaCl; 5% Glycerol) The experiments were performed at 25°C. A typical titration consisted of injecting 8 μL aliquots of peptide (100 μM) into 3 μM protein solution at time intervals of 90 sec to ensure that the titration peak returned to the baseline. ITC data were corrected for the heat of dilution by subtracting the mixing enthalpies for titrant solution injections into protein-free buffer. ITC data were analyzed using the program Origin (version 7.0) as provided by the manufacturer. Single set of identical binding site models were used.

Microscale thermophoresis (MST) assays Prior to MST assays the FER ectodomain was labelled with a fluorescent dye by using a

labelling kit (MO-L001 Monolith™ Protein Labeling Kit RED-NHS (Amine Reactive). The fluorescently labelled FER ectodomain was kept at a constant concentration (0.235μM) in a buffer containing 50 mM NaH2PO4/Na2HPO4 pH 7.5; 200 mM NaCl; 5% Glycerol and 0.01% Tween, whereas varying peptide concentrations were added. Approximately 4-6 μL of each sample was loaded in a fused silica capillary (NanoTemper Technologies). Measurements were performed at room temperature in a Monolith NT.115 instrument at a constant LED power of 60% and varying MST power of 40%, 60% and 80%. Measurements were performed repeatedly on independent protein preparations to ensure reproducibility. The data were analyzed by plotting peptide concentrations against percent changes of normalized fluorescence (ΔFnorm [%] y axis). Curve fitting was performed by using the NT affinity analysis software from Nanotemper.

RALF-induced seedling growth inhibition Seeds were surface-sterilized and grown on MS Agar plates for 5 days before transferring

individual seedlings in each well of a 48-well plate containing MS medium with 1 µM RALF23, RALF33 or RALF32, respectively. Seedling fresh weight was measured 10 days after transfer.

Co-immunoprecipitation experiments Fifteen to twenty seedlings were grown in wells of a 6-well plate for 2 weeks, transferred to

2 mM MES-KOH, pH 5.8 and incubated overnight. The next day, flg22, elf18 (final concentration 100 nM) and/or RALF23 (final concentration 1 µM) were added and incubated for 10 minutes. Seedlings were then frozen in liquid N2 and subjected to protein isolation.

To analyse FLS2-BAK1 and EFR-GFP-BAK1 receptor complex formation, proteins were isolated in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM dithiothreitol, 1% protease inhibitor cocktail (Sigma Aldrich), 2 mM Na2MoO4, 2.5 mM NaF, 1.5 mM activated Na3VO4, 1 mM phenylmethanesulfonyl fluoride and 1 % IGEPAL. For immunoprecipitations α-rabbit Trueblot agarose beads (eBioscience) coupled with α-FLS2 antibodies (30) or GFP-Trap agarose beads (ChromoTek) were used and incubated with the crude extract for 2-3 hours at 4 °C. Subsequently, beads were washed 3 times with wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, 0.5 % IGEPAL) before adding Laemmli sample buffer and incubating for 10 minutes at 95 °C. Analysis was carried out by SDS-PAGE and western blots using α-FLS2 α-BAK1 (30) and α-GFP (Santa Cruz biotechnology) antibodies.

To test associations between FER-GFP and FLS2 or BAK1, total proteins were extracted in 50 mM Tris-HCl (pH 7.5, 50 mM NaCl, 10% glycerol, 5 mM dithiothreitol, 1% protease inhibitor cocktail (Sigma Aldrich), 2 mM Na2MoO4, 2.5 mM NaF, 1.5 mM activated Na3VO4, 1 mM phenylmethanesulfonyl fluoride and 0.5 % IGEPAL. For immunoprecipitation, GFP-Trap agarose beads (ChromoTek) were used and incubated with the crude extract for 2-3 hours at 4 °C. Subsequently, beads were washed 3 times with wash buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, 0.1 % IPEGAL) before adding Laemmli sample buffer and incubating them for 10 minutes at 95 °C. Analysis was carried out by SDS-PAGE and western blots using α-FLS2, α-BAK1 (30) and α-GFP (Santa Cruz biotechnology) antibodies.

Statistical analysis Statistical significance based on one-way ANOVA analysis was performed with Prism 5.01

(GraphPad Software).

Supplementary Text: revised Acknowledgments This research was funded by the Gatsby Charitable Foundation (C.Z.), the European Research Council (grant “PHOSPHinnATE” to C.Z.), the Austrian Academy of Science through the Gregor Mendel Institute (Y.B.), the Deutsche Forschungsgemeinschaft (fellowship STE 2448/1 to M.S.), the European Molecular Biology Organization and the U.K. Biotechnology and Biological Sciences Research Council (fellowships ALTF 459-2011 and BB/M013499 to J.M.), and the Erasmus Mundus program (H.R.). We thank R. Niebergall for the initial genotyping of the ralf23-3 allele, as well as L. Stransfeld, the John Innes Centre horticultural service and The Sainsbury Laboratory tissue culture service for technical assistance, and all members of the Zipfel laboratory for comments. We thank N. Keinath, M. Sussman, P. Vera, S. Wolf, S. Robatzek, and H.-M. Wu for providing biomaterials, the VBCF Protein Technologies Facility for excellent assistance, and T. Clausen for access to his Isothermal Titration Calorimetry platform. M.S., J.M., E.S.-L., Y.B., and C.Z. designed and conceived the experiments and analyzed the data. M.S., J.M., E.S.-L., H.R., A.L., and Y.B. performed the experiments. N.H. provided unpublished constructs. M.S., J.M., and C.Z. wrote the manuscript with input from all authors. Supplement contains additional data.

Fig. S1. MOB6 encodes the subtilisin-like serine protease S1P. (A) The mob6 mutation restores PAMP-triggered ROS in bak1-5. ROS burst was measured after elicitation of the indicated mutant lines with 100 nM elf18, 100 nM flg22, 1 μM AtPep1, or 100 μg/mL chitin. Values represent total photon counts over 60 min +/- SE, n=8. Mean values with different letters were significantly different from each other tested by one-way ANOVA (a-b, p<0.01; a-c, p<0.05; b-c, p<0.01). (B) Chromosome 5 single nucleotide polymorphism (SNP) density plot for bak1-5 mob6 using CandiSNP software (29). All SNPs with allele frequencies >70% are plotted in grey, while candidate causative SNPs (defined as those causing non-synonymous changes in gene-coding regions) with allele frequencies >70% are plotted in red. SNPs causing synonymous changes are plotted in orange. The position of S1P/At5g19660 is indicated. Bp, base pairs. (C) Gene structure of S1P. Exons are shown as black boxes and the T-DNA insertion site of s1p-3 and the location of the causative SNP in s1p-6 (mob6) are indicated. Bp, base pairs. (D) The s1p-6 (mob6) single mutant is allelic to s1p-3. The ROS burst was measured after elicitation of leaf discs from the indicated mutant lines with 100 nM elf18. Values are represented as total photon counts +/- SE, n=8. Mean values with different letters were significantly different from each other tested by one-way ANOVA (a-b, p<0.05; a-c, p<0.001; b-c, p<0.05). The cross was repeated once with similar results. (E) S1P-cMyc complements the s1p-6 mutant phenotype. The ROS burst was measured after elicitation with 100 nM elf18. Values are represented as total photon counts +/- SE, n=8 (one-way ANOVA; p<0.05). All experiments were performed 3 times with similar results.

Fig. S2. The subtilisin-like serine protease S1P negatively regulates immunity. (A) Reactive oxygen species (ROS) burst measured in leaf discs after 100 nM elf18 or water treatment. Shown are the kinetics of ROS production (left panel) and the integration (right panel) as mean values of total photon counts over 40 min. Mean values with different letters were significantly different from each other tested by one-way ANOVA (a-b, p<0.05; a-c, p<0.001; b-c, p<0.001; c-d, p<0.001; c-e p<0.05; d-e p<0.05). (B) Quantitative reverse-transcription polymerase chain reaction of immune marker genes after 100 nM elf18 treatment. Expression values relative to the U-BOX housekeeping gene are shown as fold change compared to mock-treated seedlings. Values are means +/- SD, n=3. (C) Colony forming units (cfu) of surface-inoculated Pseudomonas syringae pv. tomato (Pto) DC3000 COR- bacteria 3 days after inoculation. Values are means +/- SD, n=4 (one-way ANOVA; p<0.05). Similar results were obtained in 3 independent experiments.

Fig. S3. S1P negatively regulates responses triggered by multiple PAMPs. The s1p mutant lines show enhanced ROS in response to different PAMPs. The ROS burst was measured after elicitation of leaf discs from the indicated mutant lines with 100 nM flg22 (A) or 100 µg/mL chitin (B). Shown are the kinetics of ROS production (left panel) and the integration (right panel) as mean values of total photon counts over 40 min +/- SE, n=16. Mean values with different letters were significantly different from each other tested by one-way ANOVA (a-b, p<0.001; a-c, p<0.05; b-c, p<0.05). Similar results were obtained in 3 independent experiments.

Fig. S4. RALF23 is released in response to PAMP perception in a S1P-dependent manner. (A and B) Cleavage of PRORALF23-GFP peptide (~45 kDa) into RALF23-GFP (~30 kDa) in Arabidopsis seedlings treated with 100 nM elf18 (A) or plants sprayed with Pto DC3000 hrcC-(which corresponds to a mixture of PAMPs). Western blots with α-GFP. CBB, Coomassie brilliant blue. (C) Protoplasts derived from Arabidopsis Col-0 or s1p-6 were transfected with RALF23-cMyc and treated with 100 nM elf18 for the indicated time. In response to the treatment free RALF23-cMyc peptide (~20 kDa) is released from its precursor protein (~35kDa) in Col-0 but to a lesser degree in s1p-6. Similar results were obtained in 3 independent experiments.

Fig. S5. IC50 value for RALF23-dependent inhibition of elf18-triggered ROS. The ROS burst was measured after elicitation of Col-0 leaf discs with 100 nM elf18 and an increasing dosage of RALF23. Values are represented as total photon counts relative to the mock treated control as an average of five independent experiments (n=8) +/- SE. The dose response curve and IC50 value was calculated via non-linear regression using Prism 5.01 software (GraphPad software).

Fig. S6. Genetic characterization of ralf23 and ralf33 lines.

Gene structures of RALF23 (A) and RALF33 (B). Exons are shown as black boxes. T-DNA insertion sites are shown. Transcript accumulation in ralf23-3 (A), ralf33-1, and ralf33-2 (B) is not detectable. Total RNA was isolated from two-week-old seedlings and reverse transcription was performed followed by PCR using primers at positions indicated by arrows. Bp, base pairs.

Fig. S7. RALF23 negatively regulates flg22-triggered ROS. (A) Co-treatment of 100 nM flg22 with 1 µM synthetic RALF23 peptide impairs PAMP-triggered ROS. The ROS burst was measured after elicitation of Col-0 leaf discs with buffer (2 mM MES-KOH pH 5.8), 100 nM flg22, 1 µM RALF23, or co-treatment. Shown is the kinetic of ROS production (left panel) and the integration of total photon counts (right panel) as mean values over 30 min +/-SE, n=16 (one-way ANOVA; p<0.05). (B) Over-expression of RALF23-GFP impairs flg22-triggered ROS production. The ROS burst was measured after elicitation with 100 nM flg22. Shown is the kinetic of ROS production (left panel) and the integration of total photon counts (right panel) as mean values over 40 min +/- SE, n=8 (one-way ANOVA; p<0.05). (C) The T-DNA insertion line ralf23-3 shows enhanced flg22-triggered ROS production. The ROS burst was measured after elicitation with 100nM flg22. Shown is the kinetic of ROS production (left panel) and the integration of total photon counts (right panel) as mean values over 30 min +/- SE, n=8 (one-way ANOVA; p<0.05). Experiments were performed 3 times with similar results.

Fig. S8. RALF23 can complement the high ROS phenotype of s1p mutants. ROS measured after elicitation of Col-0 and s1p-6 leaf discs with buffer (2 mM MES-KOH pH 5.8), 100 nM elf18, 1 μM RALF23, or co-treatment. Shown is the kinetic of ROS production (left panel) and the integration (right panel) as mean values of total photon counts over 30 min +/- SE, n=8. Mean values with different letters were significantly different from each other as tested by one-way ANOVA (a-b, p<0.05; a-c, p<0.001; b-c, p<0.05). Similar results were obtained in 3 independent experiments.

Fig. S9. RALF33 also inhibits immunity. (A) Phylogenetic tree of the Arabidopsis RALF gene family based on the predicted mature peptide sequence. The tree was generated using MUSCLE software (http://www.ebi.ac.uk/Tools/msa/muscle/) followed by Clustal2_Phylogeny Neighbour-joining method. RALF1, RALF23 and RALF33 are highlighted in red. AtRALF1, At1g02900. AtRALF2, At1g23145. AtRALF3, AT1G23147. AtRALF4, AT1G28270. AtRALF5, AT1G35467. AtRALF6, AT1G60625. AtRALF7, AT1G60815. AtRALF8, AT1G61563. AtRALF9, AT1G61566. AtRALF10, AT2G19020. AtRALF11, AT2G19030. AtRALF12, AT2G19040. AtRALF13, AT2G19045. AtRALF14, AT2G20660. AtRALF15, AT2G22055. AtRALF16, AT2G32835. AtRALF17, AT2G32885. AtRALF18, AT2G33130. AtRALF19, AT2G33775. AtRALF20, AT2G34825. AtRALF21, AT3G04735. AtRALF22, AT3G05490. AtRALF23, AT3G16570. AtRALF24, AT3G23805. AtRALF25, AT3G25165. AtRALF26, AT3G25170. AtRALF27, AT3G29780. AtRALF28, AT4G11510. AtRALF29, AT4G11653. AtRALF30, AT4G13075. AtRALF31, AT4G13950. AtRALF32, AT4G14010. AtRALF33, AT4G15800. AtRALF34, AT5G67070. AtRALF35, AT1G60913. AtRALF36, AT2G32785. AtRALF37, At2g32788. (B). Co-treatment with synthetic RALF33 peptide impairs elf18-triggered ROS. The ROS burst was measured after elicitation of Col-0 leaf discs with buffer (2 mM MES-KOH pH 5.8), 100 nM elf18, 1 μM RALF23, 1 μM RALF33, or co-treatment. Shown is the kinetic of ROS production (top panel) and the integration of total photon counts (bottom panel) as mean values over 30 min +/- SE, n=16 (one-way ANOVA; p<0.001). (C) The ralf33 T-DNA insertion lines show enhanced elf18-triggered ROS. The ROS burst was measured after elicitation with 100 nM elf18. Shown is the kinetic of ROS production (left panel) and the integration of total photon counts (right panel) as mean values over 30 min +/- SE, n=16 (one-way ANOVA; p<0.001). Experiments were performed 3 times with similar results.

Fig. S10. S1P-cleaved RALF peptides inhibit PAMP-triggered ROS.

(A) Alignment of Arabidopsis RALF peptide sequences surrounding the propeptide-peptide transition site. Colouring indicates degree of conservation (black: highly conserved, white: not conserved). The important residues arginine (R) and leucine (L) for S1P recognition are highlighted in blue (Conserved motif: RXLX/RXXL) (15). The red box indicates all RALF peptides that have the recognition motif and are likely to be cleaved by S1P. (B) Co-treatment with S1P-processed RALF peptides inhibits elf18-triggered ROS. The predicted S1P non-processed RALF peptide RALF32 served as a control. The ROS burst was measured after elicitation of Col-0 leaf discs with 100 nM elf18 or co-treatment with 1 μM RALF23, 1 μM RALF33, 1 μM RALF34, or 1 μM RALF32. Shown are the kinetic of ROS production (left panel) and the integration of total photon counts as mean values over 30 min (right panel) +/- SE, n=16 (one-way ANOVA; p<0.05). (C) Seedling growth inhibition experiment comparing growth of Col-0 and fer-2 in the presence of 1 µM RALF23, 1 µM RALF33, or 1 µM RALF32. Shown is the relative fresh weight of seedlings compared to the MS medium control +/-SE, n=12 (one way ANOVA, p<0.01). Similar results were obtained in 3 independent experiments.

Fig. S11. RALF23, RALF33, and FER have an overlapping expression pattern. Tissue-specific expression patterns of RALF1, RALF23, RALF33, RALF17, and FER were obtained using Genevestigator (www.genevestigator.com). FER is ubiquitously expressed throughout most tissues, including leaves and roots, unlike its first described peptide ligand RALF1 (24) which has a root-specific expression pattern. Both RALF23 and RALF33 are strongly expressed in leaves, suggesting that these peptides may be perceived by the FER receptor kinase in leaves. RALF17 expression is overlapping with RALF23 and RALF33, and its expression levels are lower than those of RALF23 and RALF33.

Fig. S12. RALF23 and RALF33-induced inhibition of elf18-triggered ROS production is FER-dependent. (A) fer mutants are insensitive to the inhibitory effect of RALF33 on elf18-triggered ROS. The ROS burst was measured after elicitation of the indicated mutant lines with 100 nM elf18 or co-treatment with 1 μM RALF33. Shown are the kinetic of ROS production (left panel) and the integration of total photon counts (right panel) as mean values over 30 min +/- SE, n=16. Mean values with different letters were significantly different from each other tested by one-way ANOVA (a-b, p<0.001; a-c, p<0.01; b-c, p<0.05). Similar results were obtained in 3 independent experiments. (B) Overexpression of RALF23-GFP does not suppress ROS in the fer-2 mutant. ROS burst was measured after elicitation of the indicated genotypes with 100 nM elf18. Shown are the kinetic of ROS production (left panel) and the integration of total photon counts (right panel) as mean values over 30 min +/- SE, n=8. Letters indicate statistical significance in one-way ANOVA (a-b, p<0.05; a-c, p<0.001; b-c, p<0.05).

Fig. S13. RALF23 binds to insect cell purified FER ectodomain.

Isothermal titration calorimetry of the FER extracellular domain against the RALF23 peptide. Similar results were obtained in at least 3 independent experiments. Table summary for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for the RALF23 peptides binding to the FER extracellular domain are included in the bottom panel (± fitting errors).

Fig. S14. FER is required for the ROS burst triggered by flg22 and chitin. The fer mutant lines show reduced PAMP-triggered ROS in response to flg22 and chitin treatment. (A) ROS after elicitation of leaf discs from the indicated mutant lines with 100 nM flg22. Shown are the kinetics of ROS production (left panel) and the integration (right panel) as mean values of total photon counts over 30 min +/- SE, n=8 (one-way ANOVA; p<0.05). (B) ROS after elicitation of leaf discs from the indicated mutant lines with 100 µg/mL chitin. Shown are the kinetic of ROS production (left panel) and the integration (right panel) as mean values of total photon counts over 40 min +/- SE, n=16 (one-way ANOVA; p<0.05).

Fig. S15. Overexpression of RALF23 inhibits FLS2-BAK1 complex formation. Co-immunoprecipitation of FLS2 in Col-0 or 35S::RALF23-GFP. Seedlings were treated with water (mock) or 100 nM flg22 for 10 min. Western blots were probed with α-BAK1 and α-FLS2. CBB, Coomassie brilliant blue. Similar results were obtained in 3 independent experiments.

Fig. S16. The non-S1P cleaved RALF17 peptide actives immunity in a FER-dependent manner. (A) ROS burst was measured after elicitation of Col-0 leaf discs with buffer (2 mM MES-KOH pH 5.8), 100 nM elf18, 1 μM RALF23, 1 μM RALF17, or co-treatment. Shown is the ROS burst kinetic (left panel) and the integration of total photon counts as mean values over 30 min +/-SE, n=16. Mean values with different letters were significantly different from each other tested by one-way ANOVA (a-b, p<0.001; a-c, p<0.001; b-c, p<0.001). (B) ROS measured after elicitation of Col-0 and fer-4 with buffer (2 mM MES-KOH pH 5.8), 100 nM elf18, 1 μM RALF17, or co-treatment. Shown are the ROS burst kinetic (left panel) and the integration of total photon counts as mean values over 30 min +/- SE, n=16. Mean values with different letters were significantly different from each other tested by one-way ANOVA (a-b, p<0.01; a-c, p<0.001; b-c, p<0.05). (C) Growth of Pto DC3000 in leaves pre-treated with buffer (2 mM MES-KOH pH 5.8), 1 μM elf18, 1 μM RALF17, or co-treatment for 24 hours. Bacterial growth was determined 2 days after syringe-infiltration. Values are means +/- SD, n=4 (one-way ANOVA; p<0.05). Similar results were obtained in 3 independent experiments.

Fig. S17. Growth phenotype of s1p, ralf23, ralf33, 35S::RALF23-GFP and fer mutant alleles. Representative pictures of the growth phenotype of six-week old plants of the respective mutant alleles compared to the Col-0 control.

Fig. S18 ROS burst kinetics related to main figures. Shown are the respective ROS burst kinetics related to total photon count data shown in the main figures. (A) is related to fig. 1A, (B) to fig. 1C, (C) to fig. 1E, (D) to fig. 2A and (E) to fig. 3A.

Table S1. Oligonucleotides used in this study. For RT-PCR Primer name Sequence (5`-3`) RALF33 semi qRT F CTTTTGAAACCTCCAAAAAAAAAAACCTC RALF33 semi qRT R GCAATGCAAAGTATAAACATAGTAAACACC RALF23 semi qRT F CATAGTTCGTGCACAGAGAGAGCTAAAGC RALF23 semi qRT R CTATTAATTATAATGTATTTATTCCATAG

For RT-qPCR Primer name Sequence (5`-3`) FRK1 F ATCTTCGCTTGGAGCTTCTC FRK1 R TGCAGCGCAAGGACTAGAG PHI1 F TTGGTTTAGACGGGATGGTG PHI1 R ACTCCAGTACAAGCCGATCC U-box F TGCGCTGCCAGATAATACACTATT U-box R TGCTGCCCAACATCAGGTT

Cloning primers Primer name Sequence (5`-3`) S1P F CACCATGAAGGTGCTCGGAGAAG S1P R GGCTAATCGATTCGAC RALF23 F CACCATGAGAGGACTCTCCAGAAACTCCGGC RALF23 R TGAGCGCCGGCAGCGAGTG ectoEFR F ATGAATTCCTGTCGACCGTTGATTTATCGTC ectoEFR R TATCTAGACTAAAGATTCAGATTTCGCAG

S2ectoFER F GGTCGTATACATTTCTTACATCTATGCGTTGAATTGCGGTGGTGGTGCTTC

S2ectoFER R GCACCCTGGAAGTACAGGTTCTCCCTAGTAGTCGGGCGTAGGACTTTAG

Genotyping primers Primer name Sequence (5`-3`) SNP sequencing S1P F GATGTTATATGGCCGTGG SNP sequencing S1P R CCCCAAACGCAATCCCAA SNP sequencing BAK1-5 AAACTTGCTTCGGCTTCGTGG SNP sequencing BAK1-5 GACATCATCATCATTCGCGAGGC ralf23-3 LP GTTGGCATTTTAATAGGTAG ralf23-3 RP GGCCTGAGCGCCACGTCGAC ralf33-2 LP CCTCCAAGTGGCTCCATGAC ralf33-2 RP AGCTCAGGCTAATCCTTACT CT ralf33-1 LP TTGAATGAATCAAGGGCTGAG

ralf33-1 RP AGCTTGGTTGTTGGTTCATTG SALK Left border Primer LBb1 GCGTGGACCGCTTGCTGCAACT

GK Left border Primer ATATTGACCATCATACTCATTGC

Table S2. Peptide sequences used in this study.

Peptide name Sequence

elf18 SKEKFERTKPHVNVGTIG

biotin-elf24 Acetyl-SKEKFERTKPHVNVGTIGHVDHGK-biotin

flg22 QRLSTGSRINSAKDDAAGLQIA

RALF17 NSIGAPAMREDLPKGCAPGSSAGCKMQPANPYKPGCEASQRCRGG

RALF23 ATRRYISYGALRRNTIPCSRRGASYYNCRRGAQANPYSRGCSAITRCRRS

Biotin-RALF23 Biotin-ATRRYISYGALRRNTIPCSRRGASYYNCRRGAQANPYSRGCSAITRCRRS

RALF33 ATTKYISYGALRRNTVPCSRRGASYYNCRRGAQANPYSRGCSAITRCRR

RALF32 QAHKLSYGALRRNQPACDGGKRGESYSTQCLPPPSNPYSRGCSKHYRCGRDS

RALF34 YWRRTKYYISYGALSANRVPCPPRSGRSYYTHNCFRARGPVHPYSRGCSSITRCRR

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