An extracellular network of Arabidopsis leucine-rich repeat receptor kinases

Abstract

The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, we investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay, and produced an LRR-based cell surface interaction network (CSI^LRR) that consists of 567 interactions. To demonstrate the power of CSI^LRR for detecting biologically relevant interactions, we predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, we show that CSI^LRR operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses.

Extended Data Figure 1. Expression profiles of LRR-RK ECDs produced as recombinant baits with the Drosophila S2 cells protein expression system.

a-o, Western blot analyses of raw supernatants from S2 cells transfected with ECD expression vectors (WB: anti-V5). Blots were cropped and arranged to match the phylogenetic tree of the LRR-RK gene family. The family subclasses and AGIs are indicated on the top of the blots. For lanes showing no obvious anti-V5 signals, a mild concentration of the S2 cell media and/or purification on protein-A coated 96-well plates allowed for confirmation of expression and secretion of the ECDs. This experiment was conducted one time with the full set of 200 ECDs. The expression of 130 independently expressed ECDs was tested one additional time with similar results.

Extended Data Figure 2. Calibration of the CSI^LRR screen conditions on ligand-dependent (FLS2-BAK1) and ligand-independent (BAK1-BIR4) interaction pairs.

a-b, Western blot analyses of raw supernatants from S2 cells transfected with prey and bait expression vectors for the ECD of FLS2 (Bait, WB: anti-V5; Prey, WB: anti-FLAG). Drosophila S2 cells left untreated (-) or treated with CuSO4 (+). Days post induction are indicated on top (dpt). The experiment was repeated independently two times with similar results. c, Binding of the FLS2 ECD to the protein-A coated 96-well plates. A 4-fold dilution (4X) of the insect cell media containing the ECD of FLS2 saturates the binding sites of protein-A coated wells as indicated by immunoblots of the flow-through (FT). The experiment was repeated independently two times with similar results. d-f, same as a-c- but for BAK1. The experiment was repeated independently two times with similar results. g, Plate interaction assays between the ECDs of BAK1 (prey) and FLS2 (bait) represented as cumulative absorbance (Abs 650) over 18 hours. Dots represent individual observations at each hour from five technical replicates. Box plots display the 1st and 3rd quartiles, split by the median (red line); whiskers extend to include the max/min values. The presence of flg22 (+) in 4-fold diluted CSI^LRR screening conditions promotes weakly the interaction between the two ECDs. h, Technical replicates and box plots as in g, but with BAK1 (bait) and FLS2 (prey). i, Technical replicates and box plots as in g but with BAK1 (prey 8-fold diluted) and FLS2 (bait 4-fold diluted). In these conditions, the binding between the ECDs of BAK1 and FLS2 is largely enhanced by the presence of flg22 (+), indicating that the proteins produced in our expression system can interact in a ligand-dependent manner and are thus functional. j, Technical replicates and box plots as in g, but using a prey variant of BAK1 that can no longer pentamerize due to the deletion of the COMP domain (BAK1 mono-prey). Binding between the two ECDs is still observed, but at a reduced level, thus indicating the importance of the pentamerization motif for detecting transient and low affinity interactions in the absence of ligand. k-l, Binding of FLS2 and BAK1 ECDs to protein-A coated 96-well plates (as indicated by immunoblots of the flow-through (FT)) when proteins are produced from S2 cells growing either at 21°C or 27°C. Immunoblots show a slight increase in protein production at 27°C with similar binding capacities to the protein-A coated plate. The protein expression levels at the two temperatures were assessed more than three times with similar results. The plate saturation experiment for proteins produced at 27°C was conducted once. m, Plate interaction assays between BAK1 (prey) and FLS2 (bait) (in 4-fold diluted conditions) represented as cumulative absorbance (Abs 650) over a 150-mins time course. Dots represent individual observations made every 10 mins from four technical replicates. Box plots display the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. While slightly more abundant, proteins produced at 27°C do not interact as well as when produced at 21°C. Protein expression for the CSI^LRR screen was performed at 21°C. n, The FLS2-BAK1 interaction is insensitive to changes in pH conditions. Left panel, the interaction between FLS2 (bait) and BAK1 (prey) was observed in the pH range from 5.5 to 7.5. This experiment was conducted once. Right, plate interaction assays between BAK1 (prey) and FLS2 (bait) (in 4-fold diluted conditions) represented as cumulative absorbance (Abs 650) over a 3-hour time course. Dots represent individual observations at each hour from one technical replicate. The CSI^LRR screen was performed at the pH of the conditioned S2 cells supernatant (~pH 7.5). o, Plate interaction assays between BAK1 (as mono-prey (blue dots) or penta-prey (black dots)) and BIR4 represented as cumulative absorbance (Abs 650) over a 3-hour time course. Dots represent individual observations at each hour from one technical replicate. This experiment was conducted once. The data indicates that the pentamerization of the prey is a key requirement for enhancing the interaction detection sensitivity, without disrupting the functionality of the ECDs. BAK1 and BIR4 are ligand-independent interaction partners and the screening conditions used are also appropriate to detect this interaction.

Extended Data Figure 3. Comparison of the primary and retest screens parameters.

a, Geometric mean of the normalized absorbance values for the HCi (red dots) and LCi (yellow dots) obtained from the primary screen (CSI), the validation screen (Retest) and the negative prey controls (NC) associated with the two screens. n = numbers of bidirectional interactions: HCi CSI (n=567), HCi Retest (n=567), LCi CSI (n=248), LCi Retest (n=248), and NC (n=618). The box plots contain the 1st and 3rd quartiles, split by the median (yellow or red lines indicated by the arrow on the left of the boxes); whiskers extend to include the max/min values. Statistical significance was determined using unbalanced one-way ANOVA by Tukey’s HSD for all pairwise comparisons. Data sets with the same letter are indistinguishable at >95% confidence. b, Plots of a linear regression for the entire set of normalized absorbance values obtained from the retest screens (Absorbance Retest; y-axis) and the corresponding values from the from the primary screen (Absorbance CSI; x-axis). The thick, straight red line is the linear regression that best describes the entire set of data points (Spearman r: 0.7696; indicated on top). The fine red dashed lines represent the 95% confidence intervals of the regression. n = 815 bidirectional interactions. c, Comparison of the geometric mean of normalized absorbance values for selected interactions. Values from the primary screen (Absorbance CSI; y-axis) and the validation screen (Absorbance Retest; x-axis) are shown for the LCi set (yellow dots) and for 20 interactions selected at random from the HCi set (red dots). The number of interactions shown for each set was selected to approach the numbers present in the entire interaction search space. The red lines show the absorbance values corresponding to the FLS2-BAK1 interaction in both screens. d, Retest assay performance parameters interpreted within the performance window measured by PRS and LCi calibration. To estimate the reliability of the estimates provided by the retest, the observed rate of interactions found in the HCi and LCi sets were used for a Monte Carlo simulation. n =100,000 independent sets of observations selected at random from these populations, with the number of observations equal to the number present in the retest sets. These values were used to calculate the mean and standard deviations of the samplings, which are presented as error bars.

Extended Data Figure 4. Characterization of BRI1 interaction partners.

a, Quantitative real-time PCR analyses showing altered gene expression in T-DNA lines targeting the interaction partners of BRI1 (Fig. 1b). Genotypes are indicated at the bottom of the chart. Relative expression levels were calculated and ACTIN was used as reference gene to control for cDNA amount in each reaction. The box plots contain the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. n = 4 biologically independent mRNA samples for all genotypes, except for bak1-4, skm1, and sobir1 where n=3. Statistical significance was estimated by an unpaired two-sided t-test and is indicated on top of the boxes: ns (not significant), erl2 *P =0.0012, fir *P =5.3508 x 10^-6, bak1-4 *P =3.08212 x 10^-7, bam3 *P =1.9378 x 10^-5, serk4 *P =0.0108, hsl2 *P =2.06945 x 10^-5, sark *P =0.0259, rlk *P =2.12971 x 10^-10, rul1 *P =7.49918 x 10^-5, srf4 *P =3.08212 x 10^-7, skm1 *P =5.5911 x 10^-6, sobir1 *P =0.0001. b, T-DNA insertions targeting the HCi (Top interactions) and LCi (bottom interaction) partners of BRI1. Morphology of representative seedlings grown for 7-days in the absence (NT) or presence of 500 nM brassinolide (BL), the most potent brassinosteroid. Genotypes are indicated on the top of the picture. The experiment was conducted six times with similar results.

Extended Data Figure 5. Characterization of FLS2 interaction partners.

a, Quantitative real-time PCR analyses showing altered gene expression in T-DNA lines targeting the interaction partners of FLS2 (Fig. 1c). Genotypes are indicated at the bottom of the chart. Relative expression levels were calculated and ACTIN was used as reference gene to control for cDNA amount in each reaction. The box plots contain the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. n = 9 biologically independent mRNA samples for all tested genotypes. Statistical significance was estimated by an unpaired two-sided t-test and is indicated on top of the boxes: ns (not significant), mik1 *P =8.17192 x 10^-6, pskr1 *P =0.007, pepr2 *P =0.007, at3g14840 *P =0.005, at2g01210 *P =0.0032, pepr1 *P =1.16519 x 10^-5, fei1 *P =0.005, nik3 *P =0.0015. b, Oxidative burst represented as total photon counts, triggered by 1μM flg22 in wildtype (WT; black) and in mutant lines targeting the HCi (top; red) and LCi (bottom, yellow) partners for FLS2. Genotypes are indicated on the bottom of each graph. Dots represent individual observations from four independent experiments; Box plots display the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. n = numbers of biologically independent leaf discs: WT (n=36), mik1 (n=36), fls2 (n=28), pskr1 (n=27), pepr2 (n=38), at3g46350 (n=39). Statistical significance was determined using linear mixed effect modelling. The symbols on top of the boxes indicate the results of a post hoc unpaired two-sided t-test corrected with the Holm method for multiple testing: ns (not significant), mik1 *P =4.32 x 10^-2, fls2 *P =1 x 10^-15. c, as in (b,) except: WT (n=32), fls2 (n=27), bak1 (n=39), at3g14840 (n=33), at2g01210 (n=38), pepr1 (n=40). ns (not significant), bak1 *P =1 x 10^-15, fls2 *P =1 x 10^-15. d, as in b, and c, except: WT (n=43), fls2 (n=29), bam3 (n=33), fir (n=39), srf9 (n=32), fei2 (n=45), nik3 (n=32). ns (not significant), fir *P =1.38 x 10^-3, fls2 *P =1.2 x 10^-15, nik3 *P =1.38 x 10^-3. b-d, The ROS burst assays were performed on independent plates (set number) and every plate contained WT and fls2 controls, as well as randomly assigned mutant lines. e, flg22–induced peroxidase assay (POX) in wildtype (WT; black bar) and in mutant lines targeting the HCi (top interactions; red) and LCi (bottom interactions, yellow) partners for FLS2. Genotypes are indicated on the bottom of the graph. Leaf disks from 4-week-old plants were treated with water (NT) or 1 µM flg22 (T). The level of flg22-induced POX was normalized to the corresponding NT control. The level of POX present in WT was set to 100 for easier interpretation. Box plots contain the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. n = numbers of biologically independent leaf discs from two independent experiments: WT (n=44), mik1 (n=10), fls2 (n=17), bak1 (n=31), bam3 (n=42), srf9 (n=18), fir (n=55), pskr1 (n=24), pepr2 (n=12), at3g46350 (n=36), at3g14840 (n=12), at2g01210 (n=18), pepr1 (n=12), fei2 (n=11), nik3 (n=15). Statistical significance was estimated using a paired two-sided t-test for each genotype, corrected for multiple tests using the Holm-Bonferroni correction and is indicated in top of the boxes: ns (not significant), mik1 *P =5.71 x 10^-4, fls2 *P =0.046, bak1 *P =0.0039, fir *P =0.0048, pskr1 *P =9.49 x 10^-5.

Extended Data Figure 6. FIR regulates flg22-induced responses.

a, Seedlings of the genotypes indicated on the bottom were treated with either water (NT) or flg22 (T) and changes in FRK1 transcripts quantified by quantitative real-time PCR analyses. Dots represent individual observations from three independent experiments. n = numbers of biologically independent mRNA samples: WT (n=9 (NT), n=9 (T)), fir (n=9, n=9) and fls2 (n=6, n=6). Box plots display the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. Statistical significance was determined using linear mixed effect modelling followed by comparison of each genotype to the WT control using unpaired two-sided t-test followed by multiple testing correction using the Holm method and is indicated on top of the boxes: ns (not significant), fir *P =1.42 x 10^-7, fls2 *P =4 x 10^-16. b, Growth of Pto DC3000 on the genetic backgrounds indicated at the bottom of the chart. Four-week-old plants were infiltrated with 10⁵ cfu/ml in the absence (black bars) or presence (grey bars) of 1 μM flg22. The number of bacteria per area of leaf (cfu/ml) was plotted on a log10 scale for day 0 (open bars) and day 3 (closed bars). Dots represent individual observations from two independent experiments. n = numbers of samples each including 4 biologically independent leaf discs : For Day 0- WT(n=6), fir (n=6), fls2 (n=6); Day 3- WT (n=6), fir (n=6), fls2 (n=6); for Day 3 + flg22- WT (n= 6), fir (n= 6), fls2 (n=6). Box plots display the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. Statistical significance for bacterial growth was estimated by two-way ANOVA and is indicated on top of the boxes: ns (not significant), relevant P values are indicated in the chart. A third experiment performed at an inoculum of 10⁶ cfu/ml corroborated these results. c, Morphology of 7-day-old seedlings grown in the absence (-) or presence (+) of 1 μM flg22. Genotypes are indicated on top of the panel. The experiment was conducted two times with similar results. d, Primary root length (cm) from seedlings grown in the presence (T) or absence (NT) of 1μM flg22. Fold changes are T/NT ratios. Dots represent individual observations from two independent experiments. n = the following numbers of biologically independent roots: WT (n=32 (NT), n=36 (T)), fir (n=34, n=32), fls2 (n=27, n=26). Box plots display the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. Statistical significance for two biological replicates was determined using linear mixed effect modelling followed by comparison of each genotype to the WT control using unpaired two-sided t-test followed by multiple testing correction using the Holm method and is indicated on top of the bars: fir *P =2.02 x 10^-6, fls2 *P =2.02 x 10^-6.

Extended Data Figure 7. CSI^LRR network representation and table of nodes with their corresponding identification numbers or acronyms.

The network construction and other features are the same as shown in Fig. 2b. The nodes surrounded by white halos are articulation points. The numbers in each node corresponding to the ECD of specific LRR-RKs IDs are detailed in the bottom table.

Extended Data Figure 8. Characterization of independent apex mutant and 35S::APEX transgenic lines.

a, Top: Rosette morphology of 4-week-old wildtype (WT), apex-1 and apex-2 knockdown, and apex-3 knockout, lines grown under long-day photoperiod, at 22°C. Genetic backgrounds are indicated on the top. No obvious changes in rosette morphology are observed. The experiment was conducted three times with similar results. Bottom: Quantitative real-time PCR analyses showing fold reduction of APEX transcripts in the independent mutant lines. Relative expression levels were calculated and ACTIN was used as reference gene to control for cDNA amount in each reaction. Dots represent individual observations from three independent experiments. n =9 biologically independent mRNA samples for each genotype. Box plots display the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. Statistical significance was determined using linear mixed effect modelling followed by comparison of each genotype to the WT control using unpaired two-sided t-test followed by multiple testing correction using the Holm method and is indicated on top of the boxes: apex-1 *P =6 x 10^-16, apex-2 *P =5.33 x 10^-15, apex-3 *P =6 x 10^-16. b, Top: Rosette morphology of 3-week-old WT, 35S::APEX line1 and line2 lines grown under long-day photoperiod, at 22°C. Genetic backgrounds are indicated on the top. Rosettes of 35S::APEX lines are slightly larger than WT under long-day photoperiod, at 22°C. The experiment was conducted three times with similar results. Middle: Quantitative real-time PCR analyses showing fold induction of the APEX transgene in the overexpression lines used in this study. Relative expression levels were calculated and ACTIN was used as reference gene to control for cDNA amount in each reaction. Dots represent individual observations from two independent experiments. n=6 biologically independent mRNA samples for each genotype. Box plots display the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. Statistical significance was determined using linear mixed effect modelling followed by comparison of each genotype to the WT control using an unpaired two-sided t-test followed by multiple testing correction using the Holm method and is indicated on top of the boxes: 35S::APEX line 1 *P =3.38 x 10^-14, 35S::APEX line2 *P =7.77 x 10^-14. Bottom: Detection of APEX-YFP in stable transgenic T3 lines by Western Blot using anti-GFP antibody. c, Modulation of BRI1 signalling by APEX gene dosage. Morphology of representative seedlings corresponding to Fig. 4a. Genotypes are indicated on the top of the panel. The experiment was conducted over three times with similar results. d, Hypocotyl length ratios of seedlings grown in the presence (T) or absence (NT) of 500 nM brassinolide (BL). Genotypes are indicated on the top. Dots represent individual observations from three independent experiments. n = numbers of biologically independent hypocotyls: WT (n=43 (NT), n=33 (T)), apex-1 (n=31, n=35), apex-2 (n=32, n=33), apex-3 (n=39, n=38), bri1 (n=28, n=32). Box plots display the 1st and 3rd quartiles, split by the median (red line); whiskers extend to include the max/min values. Statistical significance was determined using linear mixed effect modelling followed by comparison of each genotype to the WT control using unpaired two-sided t-test followed by multiple testing correction using the Holm method and is indicated on topof the boxes: apex-1 *P =2.53 x 10^-14, apex-2 *P =1.10 x 10^-5, apex-3 *P =1.55 x 10^-12, bri1 *P =8 x 10^-16. e, flg22-induced oxidative bursts represented as total photon counts over 40 mins. Genetic backgrounds are indicated on the top. Dots represent individual observations from three independent experiments. n= numbers of biologically independent leaf discs: WT (n=31), apex-1 (n=19), apex-2 (n=23), apex-3 (n=25), fls2 (n=15). Box plots display the 1st and 3rd quartiles, split by the median (red line); whiskers extend to include the max/min values. Statistical significance was determined using linear mixed effect modelling followed by comparison of each genotype to the WT control using an unpaired two-sided t-test followed by multiple testing correction using the Holm method and is indicated on top of the boxes apex-1 *P =2.99 x 10^-3, apex-2 *P =2.84 x 10^-2, apex-3 *P =2.84 x 10^-2, fls2 *P =8 x 10^-16.

Extended Data Figure 9. Modulation of BR signalling by AT5G51560.

a, Morphology of representative seedlings grown for 7 days in the absence (NT) or presence of 500 nM brassinolide (BL), the most potent brassinosteroid. Genotypes are indicated on the top of the picture. The experiment was conducted two times with similar results. b, Hypocotyl length fold changes corresponding to panel (a,). Genotypes are indicated on the top of the chart. Dots represent individual observations from two independent experiments. n= numbers of biologically independent hypocotyl: WT (n=39 (NT), n=29 (T)), at5g51560 line 1 (n=36, n=26), at5g51560 line 2 (n=39, n=34), bri1 (n=25, n=27). Box plots display the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. Statistical significance was determined using linear mixed effect modelling followed by comparison of each genotype to the WT control using an unpaired two-sided t-test followed by multiple testing correction using the Holm method and is indicated on top of the boxes: at5g51560 line 1 *P =3.75 x 10^-6, at5g51560 line 2 *P =2.26 x 10^-12, bri1 *P =6 x 10^-16.

Figure 1. CSI^LRR interaction map and functional validation.

a, Interaction heat map organized by phylogenetic subgroups of LRR-RKs (roman numeral, XIV and XV are merged). The colour scale bar on top shows interaction score values. b, Hypocotyl length ratios of seedlings grown in the presence (T) or absence (NT) of 500 nM brassinolide (BL). n= the numbers of biologically independent hypocotyls for all genotypes are in the Supplementary Methods. rlk *P =3.17 x 10^-3, all others *P =3.2 x 10^-15 and not significant (ns). c, flg22–induced SGI. n= numbers of biologically independent seedlings are indicated in the chart. mik1 *P =3.14 x 10^-12, fls2 *P =2.8 x 10^-15, bak1 *P =2.8 x 10^-15, fir *P =2.88 x 10^-10, pskr1 *P =2.88 x 10^-10 and not significant (ns). b-c, Wild-type (WT; black), mutant lines targeting the HCi (top interactions; red) and LCi (bottom interactions; yellow) partners for BRI1 and FLS2 are indicated on the bottom and ordered by decreasing interaction score from left to right; Dots represent individual observations from six independent experiments; Box plots display the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. Statistical significance was determined using linear mixed effect modelling. The symbols on top of the boxes indicate the results of a post hoc unpaired two-sided t-test corrected with the Holm method for multiple testing. See Method section for information on genotypes. d, Western blot analyses of FLS2–BAK1 co-immunoprecipitations (Co-IP/IP) in seedlings treated with either water (-) or flg22 (+) for 10 min. anti-BAK1 or anti-FLS2 antibodies were used to analyse lysates from the genotypes indicated on the top. This experiment was repeated three times with similar results. e, flg22-induced oxidative bursts represented as total photon counts over 40 mins. Genetic backgrounds are indicated on the bottom. Dots represent individual observations from three independent experiments. Box plots and statistical significance as in b-c. n= numbers of biologically independent leaf discs and P values are indicated in the chart.

Figure 2. CSI^LRR is defined by four distinct subnetworks and two critical nodes.

a, Expected and observed percentages of interactions organized by interaction types and ECD sizes. The expected % were calculated assuming random interaction between observed proteins. b, WalkTrap subnetworks are shown in orange. The diameter of the nodes (red circles) is proportional to their PageRank score. Numbers in each node are detailed in Extended Data Fig. 7. Edges (black lines) show interactions between nodes. BAK1 and APEX are marked in black and cyan, respectively. APs are surrounded by a white halo. c, Small ECDs have higher PageRank scores than large ECDs n= numbers of independent nodes (dots) and statistical significance determined by an unpaired two-sided t-test are indicated in the chart. The box plots contain the 1st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. d, Representative rosettes of n=20 biologically independent 3-week-old Arabidopsis plants. Genetic backgrounds are indicated on the top.

Figure 3. APEX interacts with PEPR1 and PEPR2 to regulate danger peptide signalling.

a-b, Nicotiana benthamiana leaves expressing FLAG-tagged variants of PEPR1/2 either alone or together with a YFP-tagged APEX were treated with water (-) or Pep2 (+). Western blot analyses of PEPR1/2-APEX (co-)immunoprecipitations (Co-IP/IP). anti-FLAG and anti-YFP antibodies were used to analyse lysates. These experiments were repeated three times with similar results. Full scans of the blots in Supplementary Fig.1. c, Pep2-induced oxidative bursts represented as total photon counts over 40 mins. Genetic backgrounds are indicated on the bottom. Dots represent individual observations from three independent experiments. n= numbers of biologically independent leaf discs are indicated in the chart. Box plots display the 1st and 3rd quartiles, split by the median (red line); whiskers extend to include the max/min values. Statistical significance was determined by linear mixed effect modelling. The letters on top of the boxes indicate the results of a post hoc Tukey test. Genotypes with the same letter are indistinguishable at >95% confidence.

Figure 4. CSI^LRR functions as a unified regulatory network.

a, Hypocotyl length ratios of seedlings grown in the presence (T) or absence (NT) of 500 nM brassinolide (BL). Genotypes are indicated on the top (Wild-type (WT; black)). Dots represent individual observations from three independent experiments. n = numbers of biologically independent hypocotyls are indicated in the chart n=T/n=NT. b, flg22-induced oxidative burst in leaf discs of the genetic backgrounds indicated on the top. Dots represent individual total photon counts over a 40-min time course; observations are from five independent experiments. n= numbers of biologically independent leaf discs are indicated in the chart. a-b, Box plots display the 1st and 3rd quartiles, split by the median (red line); whiskers extend to include the max/min values. Statistical significance was determined by linear mixed effect modelling. The letters on top of the boxes indicate the results of a post hoc Tukey test. Genotypes with the same letter are indistinguishable at >95% confidence. c, Western blot analyses of FLS2–BAK1 co-immunoprecipitations (Co-IP/IP) in seedlings treated with either water (-) or flg22 (+). anti-BAK1 or anti-FLS2 antibodies were used to analyse lysates from the genotypes indicated on the top. This experiment was repeated three times with similar results. d, flg22-induced activation of MAPKs in the genotypes indicated on top. The phosphorylated MPK3/6 proteins were detected with an anti-pERK antibody. This experiment was repeated four times with similar results. Colloidal brilliant blue (CBB) shows equal loading of the samples. c-d, Full scans of the blots are presented in Supplementary Fig. 1 e- Seedlings of the genotypes indicated on the top were treated with either water (NT) or flg22 (T) and changes in FRK1 transcripts quantified by qPCR. Dots represent individual observations from three independent experiments. n = numbers of biologically independent mRNA samples are n=9 (NT) and n=9 (T) for all genotypes. Box plots as in a-b. Statistical significance determined by an unpaired two-sided t-test, followed by multiple testing correction using the Holm method is indicated in the chart.