Evolutionary trajectory of pattern

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The origin and expansion of cell-surface receptors in the plant lineage

Plant cell-surface receptors that are known to participate in immunity, development, and reproductive processes include the LRR-, G-lectin-, Wall-associated kinase (WAK)-, Domain of Unknown Function 26 (Duf26)-, L-lectin-, Lysin motif (LysM)-, and Malectin-containing RLKs and RLPs (Fig. 1a–h). There are additional RLK families with different ectodomains, such as the proline-rich extensin-like receptor kinases (PERKs) and thaumatin-like protein kinases (TLPKs)9,13. However, their function in immunity is not well-characterized. Cell-surface receptors with LRR-, G-lectin-, WAK-, and LysM-ectodomains have been reported to recognise PAMPs, while others perceive self-molecules or unidentified ligands (Fig. 1h; Supplementary Fig. 1). Recognition of the diverse array of ligands is likely to be accomplished by variable structures and combinations of different ectodomains (Fig. 1a–g). To trace the origins of different receptor classes within the plant lineage, we first identified RLKs and RLPs in 350 genomes from Glaucophyta, red algae, green algae, Bryophytes, and Tracheophytes. We define here RLKs as any proteins with both 1–2 TMs and KDs, and RLPs as any protein with 1–2 TMs, but lack KDs. In total, we identified 177,645 RLKs, almost up to 70% of which possess either LRR-, G-lectin-, WAK-, Duf26-, L-lectin-, LysM- and Malectin-ectodomains (Fig. 1i). Next, we searched for proteins with these ectodomains and TMs that lack KDs and found 41,144 RLPs (Fig. 1j). We further examined which of the identified RLKs and RLPs families are likely to be involved in immunity. A previous report suggested a positive correlation between the gene family sizes of cell-surface immune receptors and intracellular immune receptors (the NB-ARC family) across the angiosperms4. We examined the correlation between the relative size (%; number of identified genes in the family/numbers of searched genes × 100; see methods) of the RLK families, the RLPs families, and the NB-ARC family in each genome. Notably, most RLK families (except for the LysM-RLKs) exhibit positive correlations with the NB-ARC family, while most RLP families (except for the LRR-RLPs) do not exhibit positive correlation with the NB-ARC family (Main Fig. 1k). Furthermore, we checked the expression level of these receptor families in Arabidopsis thaliana during immunity. Notably, the RLKs, except for LRR- and Malectin-RLKs, generally exhibit higher expression levels compared to the RLPs during immunity (Main Fig. 1k; Supplementary Fig. 2). These data collectively suggest that the RLKs are more likely to be involved in immunity than the RLPs.

Fig. 1: The distribution of cell-surface receptors in plants.figure 1

a–g Ectodomain structure of an LRR receptor. a a G-lectin receptor, b an L-lectin receptor, c a LysM receptor, d a Malectin receptor, e a WAK receptor, f and a Duf26 receptor g. Structures of FLS2, CERK1, and FERONIA were published55,95,96. Structures of LORE, DORN1, WAK1 and CRK28 were predicted by Alphafold2*97. Ectodomains are visualized in iCn3D98. h Schematic displays the domain architecture of different classes of receptor-like kinase (RLKs) and receptor-like proteins (RLP) in plants. Arrows represent the ligands that these receptor classes have been reported to perceive or recognize. The upper box defines the ligands recognized by different receptors. The lower box defines the domains within the receptor classes. Note that these receptors may be able to recognise other unidentified ligands. For more information, see Supplementary Fig. 1. i Ectodomain distribution of RLKs in plants. Each fraction represents the percentage (%) of ectodomain out of all the RLKs from 350 species (177,645). j Ectodomain distribution of RLP in plants. Each fraction represents the percentage (%) of ectodomains out of all the RLPs with those seven ectodomains (41,144). k Table of RLKs and RLPs with LRR (red), G-lectin (orange), WAK (turquoise), Duf26 (blue), L-lectin (purple), LysM (green), and Malectin (magenta) ectodomains. Characterised receptors involved in microbial interaction (bacteria icon), reproduction (flower icon), and development (leaf icon) are indicated with light green boxes. Grey boxes indicate that the receptor class has not been reported to be involved in that biological process. For details, refer to Supplementary Fig. 1. Correlations between different classes of cell-surface receptors and NB-ARC in 300 angiosperms are indicated with bars. Strong positive correlations are indicated by extension to the light green area (Pearson’s r > 0.6) and medium positive correlations are within the yellow area (Pearson’s r between 0.3 and 0.6). Expression level^ refers to the expression of each class of cell-surface receptors during NLR-triggered immunity (NTI) in Arabidopsis thaliana. Light blue area represents increased expression and light pink area represents decreased expression during NTI. X-axis values represent log2 (fold change during ETI relative to untreated samples). Boxplot elements: center line, median; bounds of box, 25th and 75th percentiles; whiskers, 1.5 × IQR from 25th and 75th percentiles. Number of cell-surface receptors (n) analysed in the RNA-seq data: LRR-RLK, n  =  159; LRR-RLP, n  =  42; G-lectin-RLK, n  =  29; G-lectin-RLP, n  =  1; WAK-RLK, n  =  18; WAK-RLP, n  =  10; Duf26-RLK, n  =  33; Duf26-RLP, n  =  7; L-lectin-RLK, n  =  21; L-lectin-RLP, n  =  4; LysM-RLK, n  =  5; LysM-RLP, n  =  2; Malectin-RLK, n  =  13; Malectin-RLP, n  =  4. RNA-seq data analysed here were reported previously, where NTI was activated by estradiol-induced expression of AvrRps4 in A. thaliana for 4 h94. For the expression of each class of cell-surface receptors during PTI in A. thaliana, refer to Supplementary Fig. 2.

Next, we examined the presence or absence of ectodomains (LRR-, G-lectin-, WAK-, Duf26-, L-lectin-, LysM- and Malectin-ectodomains lacking TM or KD; ectodomain-only proteins), RLPs (TM-bound ectodomains) and RLKs (ectodomains encompassing both TM and KD) in the plant lineage (Fig. 2; Supplementary Fig. 3; Supplementary Data 1a–c). Ectodomains exhibit an ancient heritage, with LRR-, WAK-, LysM-, Malectin-, and L-lectin-domains dating back to the era of Glaucophyta. Similarly, relatively ancient counterparts such as LRR-RLPs, WAK-RLPs, LysM-RLPs, Malectin-RLPs, and L-lectin-RLPs are found in both Glaucophyta and Rhodophyta. In contrast, RLKs emerged more recently. Green algae harbour WAK-RLKs, Malectin-RLKs, and G-lectin-RLKs, and LysM-RLKs, L-lectin-RLKs, and Duf-26-RLKs are exclusive to Embryophytes (Fig. 2). Except for LRR-RLPs, all six families of RLP are basal to the RLK families. This suggests the intriguing possibility that some RLKs may have evolved directly from RLPs through the integration of kinase domains. To test this hypothesis, we aligned the G-lectin-, WAK-, Duf26-, L-lectin-, LysM- and Malectin-ectodomains from the ectodomain-only proteins, RLPs, and RLKs within either a subset of 25 species or all 350 species (Supplementary Fig. 4). In some cases, we observed proportions of RLKs (such as WAK-RLKs) that likely have directly evolved from the RLPs (Supplementary Fig. 4a). In other cases (such as the LysM-RLKs; Supplementary Fig. 4c), it is not clear whether the RLKs evolved from an ectodomain-only protein or an RLP. Thus, we concluded that RLKs have emerged from either the RLPs or directly from ectodomain-only proteins. Within the global ectodomain sequence similarity trees, we also observed RLKs (the LysM-RLKs and Malectin-RLKs; Supplementary Fig. 4d, f) that have emerged from two independent events. While other RLKs (WAK-, G-lectin-, L-lectin- and Duf26-RLKs; Supplementary Fig. 4b, h, j, l) likely to have emerged from RLPs or ectodomain-only proteins through a series of domain-swapping events. Such events likely occurred at various stages of plant evolution, given the presence of RLKs in diverse plant lineages14.

Fig. 2: The origin and expansion of cell-surface receptors in plants.figure 2

The top panel represents a sequence similarity tree of multiple algal and plant lineages. Circles (○) and stars (☆) indicate the origin and expansion of receptor families. The timescale (in millions of years; MYA) of the sequence similarity tree was estimated by TIMETREE599. The bottom panel represents the presence or absence of different receptor classes in algal and plant lineages. ‘-’ represents ectodomains with no transmembrane or kinase domain, ‘RLP’ represents ectodomains with a transmembrane domain but no kinase domain, ‘RLK’ represents ectodomains with both transmembrane and kinase domains. *M/C/K represents Mesostigmatophyceae, Chlorokybophyceae, and Klebsormidiophyceae. The number of species available from each algal and plant lineage is indicated by the numbers within respective boxes. A grey box indicates the absence of receptors and a green box indicates their presence in each lineage. The origin of a receptor is indicated with a circle (○). The origins of ‘-’, ‘RLP’, and ‘RLK’ are connected by black lines. Expansion rates of receptor classes are indicated by boxplots. The percentages (%) of cell-surface receptors from each genome were calculated as (number of identified genes/number of searched genes × 100). Next, the percentages from each species within a lineage (e.g., Rhodophtya or green algae) were grouped and the median percentage was calculated. Median value was used instead of mean to avoid outliers within the lineages. The expansion rate within a species is calculated by ((% cell surface receptors in that species)-(median))/(median). The cyan boxplot represents the expansion rate from Glaucophyta and Rhodophyta to green algae (LRR, n = 20; LRR-RLP, n = 13; LRR-RLK, n = 9; WAK, n = 16; WAK-RLP, n = 19; WAK-RLK, n =  0; LysM, n = 20; LysM-RLP, n = 16; LysM-RLK, n = 0; Malectin, n = 9; Malectin-RLP, n =  6; Malectin-RLK, n =  0; G-lectin, n = 0; G-lectin-RLP, n = 0; G-lectin-RLK, n = 0; L-lectin, n = 8; L-lectin-RLP, n = 2; L-lectin-RLK, n = 0; Duf26, n = 0; Duf26-RLP, n = 0; Duf26-RLK, n = 0). The yellow boxplot represents the expansion rate from green algae to Embryophytes (LRR, n = 324; LRR-RLP, n = 324; LRR-RLK, n = 324; WAK, n = 316; WAK-RLP, n = 324; WAK-RLK, n = 323; LysM, n = 323; LysM-RLP, n = 314; LysM-RLK, n = 0; Malectin, n = 321; Malectin-RLP, n =  294; Malectin-RLK, n = 319; G-lectin, n = 319; G-lectin-RLP, n = 315; G-lectin-RLK, n = 322; L-lectin, n = 277; L-lectin-RLP, n = 314; L-lectin-RLK, n = 0; Duf26, n = 0; Duf26-RLP, n = 0; Duf26-RLK, n = 0) and the orange boxplot represents the differences between early land plants to Tracheophytes (LRR, n = 314; LRR-RLP, n = 314; LRR-RLK, n = 314; WAK, n = 311; WAK-RLP, n = 314; WAK-RLK, n = 314; LysM, n = 313; LysM-RLP, n = 304; LysM-RLK, n = 312; Malectin, n = 311; Malectin-RLP, n =  292; Malectin-RLK, n = 311; G-lectin, n = 309; G-lectin-RLP, n = 307; G-lectin-RLK, n = 313; L-lectin, n = 267; L-lectin-RLP, n = 306; L-lectin-RLK, n = 312; Duf26, n = 312; Duf26-RLP, n = 313; Duf26-RLK, n = 0). Light blue area represents expansion and light pink area represents contraction of the gene family. X-axis values represent expansion rate (×). Values larger than 0 indicate expansion; values equal to 0 indicate no expansion, and values below 0 indicate contraction. Boxplot elements: centre line, median; bounds of box, 25th and 75th percentiles; whiskers, 1.5 × IQR from 25th and 75th percentiles. For details, refer to the methods.

We also examined the expansion patterns of different receptor classes across various plant lineages. Our analysis involved calculating the median percentage (%) of cell-surface receptors families in (i) Glaucophyta and Rhodophyta, (ii) green algae, and (iii) Bryophytes to determine the percentage increase (% increase; see methods and Supplementary Note 2) from Glaucophyta and Rhodophyta to green algae; from green algae to Embryophytes; and between Bryophytes and Tracheophytes. We observed substantial expansions in specific receptor families across these lineages. Green algae exhibited a significant expansion of LRR-RLKs, while Embryophytes displayed expansions in LRR-RLPs, LRR-RLKs, WAK-RLKs, and G-lectin-RLKs. Tracheophytes had further expansions in LRR-RLPs, WAK-RLKs, Malectin-RLKs, G-lectin-RLKs, and Duf26-RLPs (Fig. 2; Supplementary Fig. 3). Overall, RLKs demonstrate greater expansion compared to RLPs, with notable expansions observed in LRR-RLK, WAK-RLK, and G-lectin-RLK. In addition, the LRR-RLP family has also significantly expanded throughout the plant lineage. These findings align with the substantial size of these receptor families and their involvement in recognising pathogens (Fig. 1i; Supplementary Fig. 1b). LRR-RLKs are classified into 20 subgroups based on their kinase domains, with subgroup XII specifically implicated in PAMP recognition15. In particular, the LRR-RLK-XII subgroup exhibits a considerably higher expansion rate compared to other subgroups (Supplementary Fig. 5), reinforcing the idea that cell surface immune receptors underwent extensive expansions as the plant lineage diversified and evolved to adapt to a wide range of environments.

The origin and expansion of PTI-signalling components in the plant lineage

Upon ligand or elicitor perception, PRRs undergo dimerisation or form heteromeric complexes with other LRR-RLK co-receptors, such as BAK1 (a member of the Somatic embryogenesis receptor-like kinases (SERK) family) and SOBIR1. This spatial arrangement brings the cytoplasmic kinase domains (from co-receptors and/or receptors) in close proximity, initiating a cascade of auto- and trans-phosphorylation events16. The activated receptor complex subsequently phosphorylates members of the cytoplasmic receptor-like kinases subgroup VII (RLCK-VII)17, which, in turn, phosphorylate various cytoplasmic kinases, such as the mitogen-activated protein kinase kinase kinases (MAPKKKs), calcium‐dependent protein kinases (CDPKs) and plasma membrane-associated proteins, such as cyclic nucleotide-gated channels (CNGCs), hyperosmolality-gated calcium-permeable channels (OSCAs), and NADPH oxidases (RBOHs)16. The phosphorylation of these proteins collectively triggers transcriptional reprogramming and physiological changes, such as cytoplasmic calcium influx and the accumulation of reactive oxygen species (ROS)18. These physiological responses effectively hinder pathogen proliferation during infection (Fig. 2a; Supplementary Figs. 6 and 7).

We identified cell-surface co-receptors and signalling components from the 350 genomes and determined their absence or presence across the plant lineage (Supplementary Fig. 7). SERKs, acting as cell-surface co-receptors for multiple LRR-RLKs and LRR-RLPs are present in Zygnematophyceae and Embryophytes19 (Fig. 3b; Supplementary Fig. 8a, b), suggesting their emergence during or prior to the appearance of land plants. Immune-related LRR-RLPs lack intracellular kinase domains, thus require another LRR-RLK co-receptor, SOBIR1, to activate downstream signalling20. Similar to BAK1, SOBIR1 is also present in Embryophytes (Fig. 3b). Thus, co-receptors for cell-surface receptors likely evolved during or before the emergence of land plants. On the other hand, cytoplasmic kinases (RLCKs, CDPKs, MAPKKKs, MAPKKs, and MAPKs) are ancient, as are the PM-localised downstream signalling components (CNGCs, OCSAs, and RBOHs), found across all plant lineages (Fig. 3b; Supplementary Fig. 8). Although the exact function of these proteins in algal species remains unclear, their immune-related orthologs are present in green algae (Fig. 3b; Supplementary Fig. 8. This suggests that they underwent specialisation within the immune activation pathway prior to the emergence of land plants. The EP proteins (EDS1, PAD4, and SAG101) and RPW8-NLRs (NRG1 and ADR1) that are essential for both TIR-NLR and LRR-RLP mediated-immunity21,22, are only present in gymnosperms and angiosperms (seed plants)23. Considering the ancient nature of the LRR-RLPs, it is plausible that EP-protein and helper-NLRs were integrated into the LRR-RLP-signalling pathway, forming a robust immune network in seed plants.

Fig. 3: The origin and evolution of cell-surface receptor signalling component in plants.figure 3

a Schematic figure represents the simplified PTI signalling pathway in plants. Coloured hexagons on RLKs indicate activated kinases. For details, refer to Supplementary Fig. 6. b The top panel is a sequence similarity tree of multiple algal and plant lineages. Circles (○) and stars (☆) indicate the origins and expansion of receptor families, respectively. The timescale (in million years; MYA) of the sequence similarity tree was estimated by TIMETREE599. The bottom panel displays the presence or absence of receptor classes in different algal and plant lineages. *M/C/K represents Mesostigmatophyceae, Chlorokybophyceae, and Klebsormidiophyceae. The number of available species from each algal and plant lineage is indicated within the respective boxes. A grey box indicates the absence, while a green box indicates the presence of a given protein family in each lineage. Dark green indicates the presence of orthologs of immunity-related (PTI) signalling components within that protein family (see also Supplementary Fig. 8). The origin of a protein family is indicated with a circle (○), followed by another circle indicating the origin of the orthologs of PTI-signalling component. Expansion rates of PTI-signalling component families are indicated by boxplots. The percentages (%) of signalling components from each genome were calculated as (number of identified genes/number of searched genes × 100). Next, the percentages from each species within a lineage (e.g, Rhodophtya or green algae) were grouped and the median percentage was calculated. Median value was used instead of mean to avoid outliers within the lineages. The expansion rate within a species is calculated by ((%signalling components in that species)-(median))/(median). The cyan boxplot represents the expansion rate from Glaucophyta and Rhodophyta to green algae (SERKs, n = 0; SOBIR1, n = 0; RLKCs (VII), n = 0; CDPKs, n = 20; MAPKKKs, n = 20; MAPKKs, n = 20; MAPKs, n = 20; CNGCs, n = 19; OSCAs, n = 19; RBOHs, n = 10; EP proteins, n = 0; RPW8-NLRs, n = 0). The yellow boxplot represents the expansion rate from green algae to Embryophytes (SERKs, n = 316; SOBIR1, n = 0; RLKCs (VII), n = 324; CDPKs, n = 324; MAPKKKs, n = 324; MAPKKs, n = 324; MAPKs, n = 324; CNGCs, n = 324; OSCAs, n = 322; RBOHs, n = 324; EP proteins, n = 0; RPW8-NLRs, n = 0) and the orange boxplot represents the differences between early land plants to Tracheophytes (SERKs, n = 307; SOBIR1, n = 309; RLKCs (VII), n = 314; CDPKs, n = 314; MAPKKKs, n = 314; MAPKKs, n = 314; MAPKs, n = 314; CNGCs, n = 314; OSCAs, n = 312; RBOHs, n = 314; EP proteins, n = 0; RPW8-NLRs, n = 0). Light blue area represents expansion and light pink area represents contraction of the gene family. X-axis values represent expansion rate (×). Values larger than 0 indicate expansion; values equal to 0 indicate no expansion, and values below 0 indicate contraction. Boxplot elements: centre line, median; bounds of box, 25th and 75th percentiles; whiskers, 1.5 × IQR from 25th and 75th percentiles. For details, refer to the methods.

Our investigation of the expansion rate of signalling components within the plant lineage indicated an expansion of CDPKs in green algae and expansion of RLCK-VIIs in Tracheophytes (Fig. 3b; Supplementary Fig. 8; Supplementary Note 2). However, other families of signalling components exhibit more limited expansions, compared to cell-surface receptors. This is also consistent with the considerably larger family sizes of cell-surface receptors and NLRs in comparison to the signalling components (Supplementary Fig. 9). Furthermore, we examined the correlation between the percentages of signalling components and PRRs (LRR-RLK-XIIs + LRR-RLPs) across genomes. Except for CNGCs, EP proteins, and RPW8-NLRs (0.6 > Pearson’s r > 0.3), most signalling component families do not exhibit co-expansion or co-contraction with PRRs (Supplementary Fig. 9). Thus, we concluded that plants are more likely to evolve new receptors rather than downstream signalling components for adaptation. The RLCK-VIIs are further classified into ten subgroups which are differentially required for RLKs and RLPs to activate downstream responses17,24,25,26,27 (Supplementary Figs. 6 and 7). Similarly, CDPKs fall into 4 subgroups (Supplementary Fig. 8). RLCK-VII and CDPK subgroup members are differentially required by different PRRs to activate downstream responses28,29,30,31. Pathogens often target RLCK-VIIs through secreted effectors to suppress immunity32,33,34. Thus, redundancy among RLCK-VII subgroups serves as a protective mechanism for the downstream signalling pathway against effector targeting. In addition, plants have evolved RLCK-VII pseudokinases, or ‘decoys’, to guard functional RLCKs through NLRs34,35,36,37,38. Together, it has become apparent that the expansion of RLCK-VII families may have been driven by pathogenic pressure, thereby contributing to the enhanced robustness of the immune signalling network.

Immunity- and development-related cell surface receptors share a common origin

We sought to understand how cell-surface receptors evolved to be specialised in immunity. To achieve this, we decided to trace the evolutionary origin of LRR-RLPs in plants. Among RLPs, LRR-RLPs constitute the largest family, comprising more than twenty characterised members that perceive PAMPs or apoplastic effectors to activate immunity (Supplementary Fig. 10). The ectodomain of LRR-RLPs encompasses additional domains known as N-loop outs (NLs) and island domains (IDs) interspersed between the LRR motifs39,40. Typically, NLs are located closer to the N-termini of the ectodomain, whereas IDs are positioned closer to C-termini39,40. NLs are present in most immunity-related LRR-RLPs while IDs are present in all immunity-related LRR-RLPs (Supplementary Figs. 10 and 11). NL positioning is relatively more flexible, occurring either before the first LRR motif or between the first few LRR motifs. Conversely, ID positioning is less flexible, located mostly before the last 4 LRR motifs within the ectodomain (Supplementary Figs. 10 and 11). This observation implies a functional necessity for the specific placement of IDs in LRR-RLPs.

To investigate the functional necessity of IDs, we analysed ectodomains of LRR-RLPs and LRR-RLKs from 350 species (113,794) (Fig. 4a and Supplementary Fig. 12a, b). Employing multiple prediction programs, we identified gaps between LRR motifs ranging from 10–29 or 30–90 amino acids (AA) (Supplementary Fig. 12a). Since NLs typically span 6–30AA and IDs range from around 40–75AA, we focused on small gaps (10–29AA) corresponding to NLs, and large gaps (30–90AA) indicative of IDs (Supplementary Fig. 10). Small or large gaps are relatively infrequent in LRR-RLKs (10.6% and 5.43%, respectively) (Fig. 4a). In contrast, both small and large gaps are more prevalent in LRR-RLPs (28.3% and 61.6%, respectively). Furthermore, both LRR-RLKs and LRR-RLPs typically have only one gap, which can be either small or large (Supplementary Fig. 12c, d). Our analysis also showed that small gap positions within the ectodomains of both LRR-RLKs and LRR-RLPs are not fixed, but may be distributed randomly. Conversely, larger gaps are predominantly positioned before the last four LRR motifs in the ectodomain (51.2% for LRR-RLKs and 86.9% for LRR-RLPs) (Fig. 4b, c and Supplementary Fig. 13). Thus, our findings suggest functional requirement for IDs to be positioned before the last four LRRs.

Fig. 4: The origin and evolution of LRRID + 4LRR in plants.figure 4

a Distribution of LRR-RLKs and LRR-RLPs in LRR-containing cell surface receptors (LRR-TM) from 350 plant species, and the distribution of LRR-RLPs and LRR-RLKs with or without gaps of 10-29 amino acids (10-29) or 30-90 (30-90) amino acids. b, c Position of large gaps (IDs; 30–90 AA) in b LRR-RLKs and c LRR-RLPs with a single large ID. N1 represents the number of LRR motifs before the IDs and N2 represents the number of LRR motifs after the ID. For positions of gaps in LRR-RLKs and LRR-RLPs with multiple gaps, refer to Supplementary Fig. 13. d The concentric ring pie chart presents the percentage of LRR-containing cell-surface receptors (PRRs) from 350 species. The inner ring represents all LRR-containing cell-surface receptors (113,794); the middle ring represents LRR-containing PRRs with ID (20,556); the outer ring represents LRR-containing PRRs with an ID preceding the last 4 LRR (ID + 4LRR) at the C terminus (16,885). e The presence or absence of receptor classes in various algal and plant lineages. *M/C/K represents Mesostigmatophyceae, Chlorokybophyceae, and Klebsormidiophyceae. A grey box indicates the absence, and a green box indicates the presence of a given receptor class in each lineage. The origin of LRR-RLP and LRR-RLK-Xb with ID + 4LRR is indicated with a circle (○). f Sequence similarity tree of the C3 region (last 4 LRRs) from all LRR-containing cell-surface receptors of 350 species. Branches are colour-labelled as indicated. The inner ring and middle ring indicate the lineage and subclass/order of the corresponding protein (species) from the branch. Outer ring represents the LRR-RLP or LRR-RLK classification, which is indicated in d. The light grey area indicates clustering of LRR-RLK-Xb and LRR-RLP with ID + 4LRR. The pruned sequence similarity tree on the right g corresponds to the light grey area in the left tree, with clades labelled in dark grey areas accordingly. Characterized LRR-RLK-Xb and LRR-RLP members are labelled. The BRI/BRL-clade and the PSKR/PSY1R-clades are also labelled.

Analysis of the distribution of LRR-RLK subgroups with IDs indicated that over 55% belong to the Xb subgroup (Fig. 4d). Furthermore, 94.7% of LRR-RLKs with IDs positioned before the last four LRRs (ID + 4LRR) belong to the Xb subgroup (Fig. 4d), suggesting that both LRR-RLK-Xb and LRR-RLPs share the ID + 4LRR motif. Among the characterised members of LRR-RLK-Xb are important components of growth and development regulation, including the BRASSINOSTEROID INSENSITIVE 1 (BRI1) family members, PHYTOSULFOKIN RECEPTOR 1 (PSKR1) and PSY1 RECEPTOR (PSY1R) family members, EXCESS MICROSPOROCYTES1 (EMS1) and NEMATODE-INDUCED LRR-RLK 1 (NILR1/GRACE)41,42,43,44,45. Interestingly, we observed that both LRR-RLPs and LRR-RLK-Xbs with ID + 4LRR motifs are present in land plants (Embryophyte) but not in other lineages (Fig. 4e). Considering the similarity in structural motifs between LRR-RLPs and LRR-RLK-Xbs, it is likely that these two receptor classes share a common origin.

To test this hypothesis, we conducted phylogenetic and structural analyses on the ectodomains of LRR-RLPs and LRR-RLKs. First, we aligned the last four LRR motifs (referred to as the C3 region46) from LRR-RLKs and LRR-RLPs (60,240). LRR-RLPs and LRR-RLK-Xbs with an ID + 4LRR cluster together in the C3 sequence similarity tree, indicating a common origin (Fig. 4f). Within this cluster (indicated as the ID + 4LRR clade), we observed distinct subclades, including the BRI1/BRI1-LIKE (BRL) clade, the PSKR/PSY1R clade, and mostly LRR-RLPs (Fig. 4g). The closely similar C3 regions indicate a conserved function. BRI1 recognises brassinosteroids (BRs) and interacts with the co-receptor BAK1 to induce BR responses47,48. Similarly, PSKR1 perceives phytosulfokine (PSK) and interacts with the co-receptor SERK1/2. LRR-RLPs, on the other hand, perceive PAMPs or apoplastic effectors and engage both co-receptor BAK1 and SOBIR1 to initiate immune responses49. Structural studies have elucidated the interaction mechanism of BRI1, PSKR1, and the LRR-RLP RXEG1 with SERKs48,50,51,52,53,54 (Fig. 5a–c, Supplementary Fig. 14). Interestingly, the C3 regions of BRI1, PSKR1, and RXEG1 play a crucial role in SERK interactions48,50,52,53, differentiating them from other LRR-RLKs. BRI1, PSKR1 and RXEG1 contain specific amino acid residues within the terminal two LRR motifs (within the C3 region) to interact with BAK1/SERK1 (Fig. 5a–c), while the flagellin peptide (flg22) receptor FLS2 (an LRR-RLK-XII), relies on the 3rd–12th last LRR for BAK1 (SERK3) interactions55 (Supplementary Fig. 14a). The N-terminal region (or N-terminal cap) of BAK1 or SERK1 is important for FLS2, BRI1, PSKR1, and RXEG1 interactions, while the 1st LRR inner surface of BAK1 or SERK1 is primarily involved in BRI1, PSKR1, and RXEG1 interactions, and the 2nd and the 4th LRR inner surface of BAK1 is involved in FLS2 interactions (Supplementary Fig. 14b). There are also striking similarities in interaction network maps between PSKR1-SERK1 and RXEG1-BAK1 interactions (Fig. 5a, b). Additionally, the C3 region of BRI1 participates in both BR binding50,52 and co-receptor binding, whereas the C3 region of PSKR1 and RXEG1 exclusively engages in co-receptor interactions (SERK1/2 and BAK1, respectively)48,53. By aligning the C3 regions of various LRR-RLKs, including BRI1/BRL-orthologs, PSKR orthologs, PSY1R, and multiple LRR-RLPs, overlapping residues that are required for SERK interactions within PSKR/PSY1R, and LRR-RLP clades can be discerned (See Results section ‘Specialisation of cell-surface receptors in different biological processes’ for alignment). For example, the glutamic acid e residue at the second position of the penultimate LRR motif is involved in both PSKR1-SERK1 and RXEG1-BAK1 interactions53. This E residue is highly conserved in both clades (PSKR/PSY1R clade: 69.9%, LRR-RLP: 86%). Similarly, the phenylalanine (F) residue at the last position of the penultimate LRR motif contributes to both PSKR1-SERK1 and RXEG1-BAK1 interactions48. This F residue is conserved in the BRI1/BRL (62.2%), PSKR/PSY1R (99.7%), and LRR-RLP (64.1%) clades. There is also a conserved motif crucial for SERKs interactions within the last LRR motifs of BRI/BRL, PSKR/PSY1R, and LRR-RLP clades. This motif, Glutamine-x-x-Threonine/Serine (QxxT/S) loop, is conserved in BRI1/BRL (Q:86.6%; T/S:98.9%), PSKR/PSY1R (Q:99.9%; T/S:92.5%) and LRR-RLP (Q:91.8%; T/S:88.4%) clades, but it is not conserved in other LRR-RLPs outside of the ID + 4LRR clade or other LRR-RLK subgroups. Structural analysis of BRI1-BAK1, PSKR1-SERK1/2, and RXEG1-BAK1 further supports the importance of residues within and around the QxxT/S loop for SERK interactions48,50,52,53 (Fig. 5a–c). In conclusion, our findings indicate that the C3 regions of LRR-RLK-Xb and LRR-RLPs share a conserved function for interacting with SERKs.

Fig. 5: Functional characterization of the C3 region in LRR-RLPs and LRR-RLK-Xb.figure 5

a–c Structures and interaction interfaces of LRR-RLKs and LRR-RLPs with SERKs. Published structures of a NbRXEG1-NbBAK153, b AtPSKR1-AtSERK148, and c AtBRI1-AtBAK150 and are shown. The left panels show the full structure, and the middle panels show the interaction sites between LRR-RLKs or LRR-RLP and SERKs. Hydrogen bonds are indicated by green dotted lines, and salt bridges are shown as cyan dotted lines. The positions of LRR residues (counting from N to C for SERKs and counting from C to N for LRR-RLKs and LRR-RLP) are shown. Amino acid residues that are important for the interactions are labelled and the QxxT motifs are highlighted in yellow (red text). The right panel represents the 2D interaction network between SERKs and the receptors. Contacts/interactions are shown in grey lines, hydrogen bonds are shown in green lines, and salt bridges are shown in cyan lines. Amino acids are labelled in colours according to their positions in the LRR motifs (counting from N to C for SERKs and counting from C to N for LRR-RLKs and LRR-RLP l). Residues around and within the QxxT motifs in BRI1, PSKR1, and RXEG1 are highlighted in yellow. Residues in SERKs that are involved in the interactions with QxxT motifs are also highlighted in yellow. Structures were visualized in iCn3D98. For a–c, the interaction sites are calculated by iCn3D with the following thresholds: hydrogen bonds: 4.2 Å; salt bridges/ionic bonds: 6 Å; contacts/interactions: 4 Å. d Structure of the terminal LRR motif of N. benthamiana (Nb)RXEG1, A. thaliana (At)RLP23, AtPSY1R, AtPSKR1, AtPSKR2, AtBRI1 and AtEFR. Structures of NbRXEG1, AtPSKR1, and AtBRI1 were published48,50,51,52,53,54. Structures of AtRLP23, AtPSY1R, AtPSKR2 and AtEFR were predicted by Alphafold297. Ectodomains are visualised in iCn3D98. e Alignment of amino acids in the last LRR motifs from NbRXEG1, AtRLP23, AtPSY1R, AtPSKR1, AtPSKR1T (G > T), AtPSKR2, AtPSKR2T (G > T), AtBRI1, AtBRI1T (G > T), and AtEFR. Amino acid residues involved in the interaction between NbRXEG1 and BAK1 are highlighted in green. The QxxT motif positions are highlighted in yellow. Amino acids with similar properties to AtRLP23 are highlighted in grey. f Design of AtRLP23 chimeras. The last LRR motif of AtRLP23 is exchanged with either AtPSY1R, AtPSKR1, AtPSKR2, AtBRI1, or AtEFR. The glycine g residues in AtPSKR1, AtPSKR2, AtBRI1 have also been mutated to threonine (T). g, l Immuno-precipitation to test interactions between AtRLP23 chimeras, AtBAK1 and AtSOBIR1. Nb leaves expressing the indicated constructs were treated with either mock or 1 μM nlp20 for 5 min. h–k Functionality testing of AtRLP23 chimeras. Nb leaves expressing the indicated constructs were treated with 1 μM nlp20 and samples were collected at indicated time points. Phosphorylation of NbSIPK and NbWIPK was detected with p-P42/44 antibody. i, k Nb leaf discs expressing the indicated constructs were collected and treated with either mock or 1 μM nlp20, and ROS production was measured for indicated time points. For i and k, solid line, mean; shaded band, s.e.m. RLU, relative light units. For details of experiential design in g–l, refer to the methods section. For g, h, j and l, the experiments were repeated at least twice with similar results.

To further validate the functional conservation of C3 regions in LRR-RLK-Xb and LRR-RLPs, we performed functional analysis of the QxxT/S motif in the LRR-RLP RLP23 from A. thaliana. RLP23 forms heteromeric complexes with the LRR-RLK co-receptor SOBIR1, and upon the perception of the nlp20 peptide, BAK1 is recruited into the complex, leading to activation of the SOBIR1 KD to induce immunity20,49,56. Similar to RXEG1 (with TQLQT), RLP23 possesses a TQITG motif (TQxxx), while LRR-RLK-Xb members, such as PSY1R, PSKR1, PSKR2, and BRI1, feature TQFDT, GQFQT, GQFYS, and GQFET motifs, respectively (all QxxT/S). Notably, the LRR-RLK-XII member EFR lacks the QxxT/S motif in that position, having GVFRN instead (Fig. 5d, e). We generated chimeric constructs of RLP23 with the terminal LRR motifs swapped between PSY1R, PSKR2, BRI1, and EFR (Fig. 5e, f). By immuno-precipitation assays, we tested the ability of these chimeras to interact with BAK1 upon ligand perception. Both wildtype RLP23 (WT) and RLP23-PSY1R (PY) chimeras can interact with BAK1 upon nlp20 treatment, whereas RLP23-BRI1 (BR) and RLP23-EFR (EFR) cannot (Fig. 5g). This suggests that the terminal LRR motif of RLP23 and PSY1R bind BAK1 in a similar manner. All chimeras can interact with SOBIR1 regardless of the presence of nlp20, indicating that the last LRR motif may not be involved in SOBIR1 interactions (Fig. 5g). Furthermore, upon nlp20 treatment, both WT and PY can trigger immune responses, while K2 and BR cannot (Fig. 5h, i). We speculate that this may be due to the absence of a specific T residue before the Qxxx motif in PSKR2 and BRI1 (G instead of T), which is relatively less prevalent within the LRR-RLP clade (31.1%). Multiple A. thaliana LRR-RLPs contain this residue, but it is not in other studied LRR-RLPs, such as the tomato Cf proteins, suggesting that this residue evolved in some species after the divergence of LRR-RLK-Xb and LRR-RLPs. To test this hypothesis, we generated chimeric constructs of RLP23 with the terminal LRR motifs swapped to PSKR1 (K1), PSKR2 (K2), BRI1 (BR), and mutated the G residue into T (thereby K1T, K2T, BRT; Fig. 5e, f). We tested the immune responses triggered by these chimeras following nlp20 treatment. Both K1, K1T, and K2T induce relatively immune responses compared to WT and PY, while K2, BR, and BRT are unable to induce any immune responses (Fig. 5j, k). Both K1 and K1T weakly interact with BAK1 upon nlp20 treatment, while K2 does not interact with BAK1 (Fig. 5k). Thus, the residues around and within the TQxxx motif are both crucial for SERK interactions. We concluded that the C3 region in LRR-RLPs and some LRR-RLK-Xbs (such as PSY1R and PSKR1) interact with SERKs in a similar manner, while some LRR-RLK-Xbs (PSKR2 and BRI1) have evolved to interacts with SERKs in a slightly different manner. Nevertheless, our results strongly support the functional conservation of C3 regions in LRR-RLK-Xbs and LRR-RLPs, specifically their ability to interact with SERKs.

On the origin of LRR-RLPs and LRR-RLK-Xbs

The functional conservation of C3 regions in LRR-RLK-Xbs and LRR-RLPs implies that the ectodomains of these two receptor families might share a common origin. To dissect the ectodomain origin of LRR-RLPs and LRR-RLK-Xbs, we investigated the relatedness of the IDs between these two groups of receptors. First, we aligned the IDs extracted from both LRR-RLKs and LRR-RLPs (20,246). Remarkably, the ID clusters of LRR-RLPs and LRR-RLK-Xb are found in close proximity, mirroring the C3 sequence similarity tree (Fig. 6a). Again, the BRI1/BRI1-LIKE (BRL) and PSKR/PSY1R clusters are in proximity to the LRR-RLPs (Fig. 6a, b). These results are also consistent with a previous report that PSKRs are closely related to some LRR-RLPs in Arabidopsis and rice46. To dissect the ectodomain origin of LRR-RLPs and LRR-RLK-Xbs. A recent review identified two conserved lysine k-containing motifs, Yx8KG and Kx5Y, in the ID of LRR-RLPs40. The lysine residue in the Kx5Y motif from RXEG1 is required for its interaction with BAK153. We identified both Yx8KG and Kx5Y motifs in the extracted IDs (before the last 4LRR motifs) of LRR-RLKs and LRR-RLPs. More than 75% of IDs from LRR-RLPs contain at least one of these lysine motifs, while less than 5% of IDs from LRR-RLK-Xb have either motif (Fig. 6c). This is consistent with structural data indicating that IDs from BRI1 and PSKR1 employ distinct residues for their interactions with BAK148,50,52. However, IDs from LRR-RLPs that are closely related to those from LRR-RLK-Xbs retain Kx5Y motifs (Fig. 5b). It is therefore possible that the common ID ancestor may have originally harboured lysine motifs that were subsequently lost from the BRI1 and PSKR/PSY1R clades.

Fig. 6: On the origin of LRR-RLPs and LRR-RLK-Xbs.figure 6

a Sequence similarity tree of IDs extracted from all LRR-containing PRRs. Branches are labelled in colours as indicated. Grey clade represents clade that contains both PSKR/PSY1R & BRI/BRL family, this clade is shown in b. In b, characterized LRR-RLK-Xb and LRR-RLP members are labelled. The BRI/BRL-clade, the PSKR clade, the PSY1R clade, and a clade with monocot-only IDs are labelled in different colours. In a, b, inner ring (Inner), middle ring (Middle) and outer ring (Outer) are labelled as indicated. Outer ring represents LRR-RLP and LRR-RLKs classification shown in Fig. 4d. In the outmost three rings, the presence of Kx5Y (Kx5Y), Yx8K (Yx8K), or either Kx5Y or Yx8K (Either) in the IDs are indicated in green, and absence of these motifs is indicated in grey. c Percentage (%) of IDs (before the last four LRR motifs) from LRR-RLP and LRR-RLK-Xb with the Kx5Y, Yx8KG, or either Kx5Y or Yx8KG (*) motifs. The Fisher test (2-sided) was performed to compare the number/fraction of IDs with either Kx5Y or Yx8KG in LRR-RLPs against LRR-RLK-Xb. The calculated p-value stated here (<1e-16) is too low to be given exact number. Thus, the upper bound limit is stated instead. d, g Structures and interaction interfaces of AtBRI1-Brassinosteroid (BR) and AtPSKR1-Phytosulfokine (PSK). Published structures of d AtBRL1-BR100, g AtPSKR1-PSK48 are shown. The left panels show the interaction sites between the LRR-RLK-Xb receptors and their ligands. Contacts are indicated by grey lines, hydrogen bonds are indicated by green dotted lines, and salt bridges are shown as cyan dotted lines. The positions of LRR residues (counting from C to N) are shown. Amino acid residues that are important for the interactions are highlighted in yellow and green, respectively. The right panel represents the 2D interaction network between the LRR-RLK-Xb receptors and their ligands. Contacts/interactions are shown in grey lines, hydrogen bonds are shown in green lines, and salt bridges are shown in cyan lines. Amino acids are labelled in colours according to their positions in the LRR motifs (counting from C to N). Structures were visualized in iCn3D98. e, h Sequence similarity trees of the full-ectodomains of e BRI1/BRL clade and h PSKR clade from 350 species. Branches are labelled in colours as indicated in a. These trees are extracted from the BRI1/BRL and PSKR branches from Supplementary Fig. 16. In e, h, characterized LRR-RLK-Xb members are labelled. The LRR-RLK-Xb and LRR-RLP members taken for the alignment in f and i are also labelled in blue numbers (LRR-RLK-Xb) and pink numbers (LRR-RLP), respectively. f, i Ectodomain and alignment of multiple LRR-RLK-Xb and LRR-RLP members within the BRI1/BRL-clade extracted from e and the PSKR-clade extracted from h. f The alignment of ectodomain from LRR-RLK-Xb (blue) and LRR-RLP (pink) members taken from the sequence similarity tree in e. The orange highlights indicate the amino acids residues required from BR binding in AtBRI1, and the yellow highlights indicate the amino acid residues required for BR binding in AtBRL1100. The yellow highlights are corresponding to the amino acids highlighted in the structure in d. The LRR motifs and ID in the alignment are indicated in colours shown in d (the interaction network; right panel). i The alignment of ectodomain from LRR-RLK-Xb (blue) and LRR-RLP (pink) members taken from the sequence similarity tree in h. The green highlights indicate the amino acids residues required from PSK binding in AtPSKR148. The green highlights are corresponding to the amino acids highlighted in the structure in g. The LRR motifs and ID in the alignment are indicated in colours shown in g (the interaction network; right panel). Due to space limitation, the last 9 amino acids in each LRR motif are presented as * in the alignment. For the full alignment, refer to Supplementary Fig. 17a.

To further explore the relatedness of the IDs between LRR-RLK-Xb group and LRR-RLPs, we formed clusters of highly similar IDs and examined their subgroup/family affiliations. Overall, we identified more than 2,822 clusters, with the majority (2,734) consisting of LRR-receptors of a single subgroup/family (Supplementary Fig. 15). Among the 61 clusters containing LRR-receptors from two different subgroups/families, 80% (49 clusters) consist of LRR-receptors from LRR-RLK-Xb and LRR-RLPs (Supplementary Fig. 15). The enrichment of LRR-RLK-Xb and LRR-RLP pairings provides further evidence for the relatedness of their ectodomains. The substantial number of LRR-RLP-only ID clusters (2,383) also suggests that the IDs of LRR-RLPs have undergone extensive evolution and diversification, in the process providing a broad scope for the recognition of PAMPs and apoplastic effectors. Most clusters contain relatively a small number of IDs, predominantly from species within the same order or family (Supplementary Data 2). Consequently, tracing back the origin of IDs is challenging due to their considerable diversity. We therefore propose that the IDs of LRR-RLK-Xbs and LRR-RLPs likely originated from a common ancestor, with the IDs of LRR-RLPs expanding and diversifying after the divergence of LRR-RLK-Xb and LRR-RLPs.

Within the BRI1/BRI1-LIKE (BRL) and PSKR/PSY1R clusters, we found LRR-RLP counterparts of BRI1, PSKR, and PSY1R. To further test their relatedness, we aligned the full ectodomains from LRR-RLKs and LRR-RLPs and extracted the clades containing BRI1, PSKR, and PSY1R from the sequence similarity tree (Supplementary Fig. 16a, b). Within the BRI1/BRL- and PSKR-ectodomain clades (Fig. 6e, h), we found ectodomains from LRR-RLPs that are highly similar to BRL1/BRL3 and PSKR2, with residues essential for BR and PSK binding, respectively48,54 (Fig. 6d–l). Within the PSY1R-ectodomain subclade, we found multiple IDs from LRR-RLPs that share remarkable similarity with AtPSY1R (Supplementary Fig. 17b, c). AtRLP3 and AtPSY1R have over 70% sequence identity and 85% similarity in their ectodomains. Although PSY1R is not the receptor for PSY peptide57, it is possible that RLP3, and PSY1R recognise similar or identical ligands58. Currently, the functions of these BRL-, PSKR- and PSY1R-like LRR-RLPs remain unclear. AtRLP3 confers resistance against the vascular wilt fungus Fusarium oxysporum f. sp. matthioli58, while AtPSY1R is involved in growth and development43. We propose that these RLPs may either recognise endogenous molecules to activate growth and development, or participate in the recognition of pathogen-mimicking molecules to trigger immune signalling.

Specialisation of cell-surface receptors in different biological processes

Given the common origin of the ectodomains of LRR-RLPs and LRR-RLK-Xbs, these receptors must have undergone specialisation in immune- and developmental processes following their divergence. While LRR-RLK-Xbs activate downstream responses through the Xb kinase domain, LRR-RLPs recruit SOBIR1 to trigger immunity49,56. Because LRR-RLPs lack a kinase domain, the juxtamembrane (JM) and TM regions may activate immune responses. We therefore aligned the C3, eJM (JM region before TM), TM, and cJM (JM region after TM, but with the absence of kinase domain from RLKs) regions from LRR-RLKs and LRR-RLPs (with a subset of 40, or the full set of 62,896 cell-surface receptors; Fig. 7a–c). Interestingly, the eJM-TM-cJM region of LRR-RLPs is not closely related to LRR-RLK-Xbs (Fig. 7c). Previous studies have reported the requirement of Glycine-X-X-X-Glycine (GxxxG) motifs in SOBIR1 for its association with LRR-RLPs, and the potential contribution of negatively charged amino acids in the eJM region of LRR-RLPs to their interaction with SOBIR159. Consistent with previous reports59, LRR-RLPs are strongly negatively charged at the end of the eJM region, whereas LRR-RLKs, including SOBIR1, are positively charged at the end of the eJM region (Fig. 7a). Most LRR-RLPs have a single GxxxG motif, with some having two (GxxxGxxxG) or three (GxxxGxxxGxxxG) consecutive motifs (Fig. 7a). Conversely, GxxxG motifs are relatively less common in LRR-RLKs, but can be found in SOBIR1, BAK1, and the PSKR/PSY1R clade (Fig. 7a). We further examined overall charges in the eJM region of the LRR-RLP and LRR-RLK families. Most LRR-RLK subgroups feature positively charged eJM regions, whereas LRR-RLPs have negatively charged eJM regions. Importantly, negatively charged eJM regions are only present in LRR-RLPs within the ID + 4LRR clade (Fig. 7d). More than 80% of LRR-RLPs within the ID + 4LRR clade contain GxxxG motifs in their TMs. Moreover, GxxxGxxxG motifs are primarily found in LRR-RLPs and are relatively rare in LRR-RLKs (Fig. 4d). Interestingly, both GxxxG and GxxxGxxxG motifs are relatively enriched in the PSKR/PSY1R clade, indicating that the TM regions of LRR-RLPs and PSYR/PSY1R clade might also share a common origin. (Fig. 7d).

Fig. 7: Alignment and features of the terminal four C3, eJM, and TM region in LRR-RLKs and LRR-RLPs.figure 7

a Alignment of the C3, eJM, and TM region in LRR-RLKs (from subgroups II, XII, XI, Xa, Xb) and LRR-RLPs. The BRI1/BRL clade is highlighted in purple; PSKR/PSY1R clade is highlighted in cyan and LRR-RLP with the ID + 4LRR clade (see Fig. 4c) is highlighted in light green. Amino acid residues involved in brassinosteroid (BR) binding for BRI/BRL and residues required for SERK interaction for BRI1/BRL, PSKR1, and RXEG1 (LRR-RLP) are highlighted. The colour code for each highlight is indicated in the box on top. For the eJM region, amino acid residues with negative charges are highlighted in red, and amino acids with positive charges are in highlighted blue. The GxxxG motif in the TM region is highlighted in yellow. The interaction sites were calculated using iCn3D98 with the following thresholds: hydrogen bonds: 4.2 Å; salt bridges/ionic bonds: 6 Å; contacts/interactions: 4 Å. For details, please refer Fig. 5. b Percentages of LRR-RLKs or LRR-RLPs with the stated amino acid residues in the corresponding position in a. Percentages (%) were calculated by the number of LRR-RLKs or LRR-RLPs in the subgroup with the stated residue divided by the number of LRR-RLKs or LRR-RLPs in the subgroup without the stated residue × 100. c Sequence similarity tree of the eJM-TM-cJM region from all LRR-containing cell-surface receptors of 350 species. Branches are colour-labelled as indicated. The inner ring and middle ring indicate the lineage and subclass/order of the corresponding protein (species) from the branch. Outer ring represents the LRR-RLP or LRR-RLK classification, which is indicated in Fig. 4d. Characterized LRR-RLK-Xb and LRR-RLP members are labelled. The BRI/BRL-clade and the PSKR/PSY1R-clades are also labelled. d Overall charge distribution in eJM (left), percentage of receptors with GxxxG (middle), and GxxxGxxxG (right) in the TM region. For b, d RLP/RLK-Xb (outside clade) refers to receptors outside the light grey clade in Fig. 4f. RLP/RLK-Xb (within clade) refers to receptors inside the light grey clade in Fig. 4f. Number of cell-surface receptors (n) in each LRR-RLK subgroup: I, n  =  752; II, n = 682; III, n = 6572; IV, n = 1033; V, n = 8; VI-1, n = 84; V1-2, n = 146; VII, n  =  1720; VIII-1, n  =  195; VIII-2, n  =  411; IX, n  =  70; Xa, n  =  96; Xb, n  =  3182; XI, n  =  8807; XII, n  =  12863; XIIIa, n  =  739; XIIIb, n  =  465; XIV, n  =  241; XV, n  =  548; Xb (outside clade), n  =  580; Xb (within clade), n = 2527; BRI/BRL clade, n = 1170; PSKR/PSY1R clade, n = 1347. Number of cell-surface receptors (n) in each LRR-RLP subgroup: LRR-RLP (RLP), n = 24970; RLP (outside clade), n = 5000; RLP (within clade), n = 19970. Boxplot elements: center line, median; bounds of box, 25th and 75th percentiles; whiskers, 1.5 × IQR from 25th and 75th percentiles.

To assess the functionality of eJM-TM-cytosolic regions in LRR-RLK-Xbs and LRR-RLPs, we generated multiple chimeras of eJM, TM, and cytosolic regions of BRI1 and RLP23 (Fig. 8a). In RLP23c-BRI1, the cytosolic region (following TM) of BRI1 was swapped into RLP23. In RLP23TM-BRI1, the TM+ cytosolic region of BRI1 was swapped into RLP23. In RLP23eJM-BRI1, the eJM + TM + cytosolic region of BRI1 was swapped into RLP23 (Fig. 8a). Immuno-precipitation assays showed that both RLP23WT and RLP23c-BRI1 exhibit constitutive interactions with SOBIR1, and specifically interact with BAK1 upon nlp20 treatment. RLP23TM-BRI1 does not interact with SOBIR1 but still interacts with BAK1 upon nlp20 treatment, suggesting that the RLP23 TM region with GxxxG motifs is necessary for SOBIR1-, but not BAK1-interactions (Fig. 8b). Consistent with these immunoprecipitation assays, RLP23WT and RLP23c-BRI1 can activate immune responses, while RLP23c-BRI1 and RLP23TM-BRI1 can activate developmental responses as indicated by the dephosphorylation of BRI1-EMS-SUPPRESSOR 1 (BES1)60 (Fig. 8c; Supplementary Fig. 18). This confirms that the BRI1 kinase domain is specifically required for the activation of BR responses. Interestingly, RLP23TM-BRI1 can also activate weak immune responses, likely independent of SOBIR1 (Fig. 8c, d). RLP23eJM-BRI1 does not interact with SOBIR1 or BAK1, and is unable to activate either immune or developmental responses (Fig. 8c, d). These data suggest that the eJM region of RLP23 is necessary for RLP23-BAK1 interactions. This conclusion was reinforced by generating RLP23eo-BRI1 chimera, in which the eJM region of RLP23 is replaced with the BRI1 eJM (Fig. 8a). RLP23eo-BRI1 consistently accumulates less protein than RLP23WT. Moreover, RLP23eo-BRI1 constitutively interacts with SOBIR1, but fails to interact with BAK1 with nlp20 treatment (Fig. 8e). RLP23eo-BRI1 only weakly activates MAPKs and fails to trigger ROS production with nlp20 treatment (Fig. 8f, g). Thus, we conclude that the eJM regions of LRR-RLPs are important for protein accumulation and interaction with BAK1 upon ligand perception. The eJM, TM, and cytosolic region of LRR-RLPs and LRR-RLK-Xbs have indeed specialized to recruit different proteins into the receptor complex, which allows them to activate differential downstream responses (Fig. 8h).

Fig. 8: Adaptation of LRRID + 4LRR to differential downstream signalling pathways.figure 8

a Design of AtRLP23-BRI1 chimeras. Different regions (cytosolic, TM+cytosolic, and eJM+TM+cytosolic) of BRI1 were swapped into AtRLP23 as indicated. The alignment of amino acids in the eJM and TM regions from AtRLP23 and AtBRI1 is shown. Amino acid residues with negative charges are in red and amino acids with positive charges are in blue. The GxxxG motif in the TM region is highlighted in yellow. b, e Immuno-precipitation to test interactions between AtRLP23 chimeras, AtBAK1 and AtSOBIR1. Nb leaves expressing the indicated constructs were treated with either mock or 1 μM nlp20 for 5 min. c, d, f, g Functionality testing of AtRLP23 chimeras. Nb leaves expressing the indicated constructs were treated with 1 μM nlp20 and samples were collected at indicated time points. Dephosphorylation of BESI1-HA was detected with HA antibody. Phosphorylation of NbSIPK and NbWIPK was detected with p-P42/44 antibody. For f, twice the sample of RLP23oe-BRI1 was loaded as a reference, because RLP23oe-BRI1 protein accumulation is weaker than that of RLP23WT. d, g Nb leaf discs expressing the indicated constructs (without BES1-HA) were collected and treated with either mock or 1 μM nlp20 and ROS production was measured for 90 min. For d and g, solid line, mean; shaded band, s.e.m. RLU, relative light units. For details of experiential design, refer to the methods section. h Schematic model of the interaction between LRR-RLK-XbID+4LRR and LRR-RLPID + 4LRR with co-receptors to induce differential downstream signalling. Both receptor classes utilise the last 4 LRRs (highlighted in yellow) to interact with SERKs (BAK1). LRR-RLP evolved to interact with SOBIR1 with the GxxxG motifs in TM (highlighted in yellow outline). Coloured hexagons on RLKs indicate activated kinases and black hexagon indicates an inactivated kinase. i Modular evolution of different domains in cell-surface receptors to allow diverse ligand recognition and specificity of downstream signalling. Domains or regions that evolved different functions are highlighted in yellow. Bold arrows represent large expansions and diversifications. K* represents the lysine in Kx5Y or Yx8KG motifs in ID from LRR-RLPs. Domain or region structures (from left to right) are obtained from: BRI1 ectodomain (3RGX); RXEG1 ectodomain (7W3X); PSKR1 ID (4Z63); RXEG1 ID (7W3X); PSKR1 C3 (4Z63); RXEG1 C3 (7W3X); PSKR1 ejM-TM (predicted from Alphafold220); RLP23 eJM-TM (predicted from Alphafold220); BRI1 kinase (4OH4). Structures were visualized in iCn3D98. For b, c, e and f, the experiments were repeated at least twice with similar results.

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