Extracellular alkalinization has long been recognized as a hallmark of plant cell-surface receptor activation, including during pattern-triggered immunity (PTI), yet the mechanisms driving elicitor-induced alkalinization and its role in plant signaling remain unclear. Here, we demonstrate that inhibition of autoinhibited H+-ATPases (AHAs) is required for elicitor-induced extracellular alkalinization. This alkalinization is essential for immune and cell-wall damage signaling mediated by diverse plasma membrane-localized receptor kinases (RKs), likely through modulation of ligand-receptor interactions. Mechanistically, RKs transduce elicitor-triggered signaling via the receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1), which inhibits AHA activity by disrupting AHA-GENERAL REGULATORY FACTOR (GRF) interactions through a conserved phosphorylation event. This phosphorylation-driven extracellular alkalinization module is required for disease resistance and cell-wall damage responses initiated by ligand-RK pairs. Our findings uncover a conserved phosphorelay circuit that broadly regulates extracellular alkalinization to coordinate RK signaling, illuminating a general mechanism for RK activation and stress resilience.

The wheat resistance gene Pm4 encodes a kinase fusion protein and has gained particular attention as it confers race-specific resistance against two major wheat pathogens: powdery mildew and blast. Here we describe the identification of AvrPm4, the mildew avirulence effector recognized by Pm4, using UV mutagenesis, and its functional validation in wheat protoplasts. We show that AvrPm4 directly interacts with and is phosphorylated by Pm4. Using genetic association and quantitative trait locus mapping, we further demonstrate that the evasion of Pm4 resistance by virulent mildew isolates relies on a second fungal component, SvrPm4, which suppresses AvrPm4-induced cell death. Surprisingly, SvrPm4 was previously described as AvrPm1a. We show that SvrPm4, but not its inactive variant svrPm4, is recognized by the nucleotide-binding leucine-rich repeat immune receptor Pm1a. These multiple roles of a single effector provide a new perspective on fungal (a)virulence proteins and their combinatorial interactions with different types of immune receptors.

Plant cell surface pattern recognition receptors (PRRs) perceive non- or altered-self elicitors to induce immune responses. PRRs relay information across the plasma membrane and trigger downstream signalling via receptor-like cytoplasmic kinases such as BOTRYTIS-INDUCED KINASE 1 (BIK1). BIK1 associates with several PRRs and acts as a key executor of immune responses through the phosphorylation of substrate proteins. However, a comprehensive understanding of how BIK1 targets specific substrates and a full repertoire of these substrates are lacking. Here we defined the substrate specificity of BIK1 and used these data to predict candidate substrates in Arabidopsis. Using high-throughput biochemical and genetic screening of these candidates, we confirmed many as direct BIK1 substrates in vitro and novel regulators of plant immunity. Among the BIK1 substrates identified are MULTIPLE C2 DOMAIN AND TRANSMEMBRANE REGION PROTEIN 3, which we reveal regulates flagellin 22 (flg22)-induced plasmodesmata closure and immunity, and members of the largely uncharacterized CYCLIN-DEPENDENT KINASE-LIKE family, which we uncover as novel negative regulators of immunity. In parallel, we interrogated intracellular NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT (NLR) immune receptors for potential BIK1 phosphorylation motifs and identified multiple NLRs as direct BIK1 substrates. We reveal that BIK1 phosphorylation regulates NLR oligomerization, thus controlling a key activation step for these immune receptors. Together, our unbiased biochemical screens shed light on the central role of BIK1 as a key kinase shaping multiple layers of plant immune signalling.

Pattern-triggered immunity (PTI) is initiated when plants detect pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRRs). How moderate increases in temperature affect this plant immune signalling remains unclear. We explored this by using flg22 and the leucine-rich repeat receptor kinase (LRR-RK) FLS2 as a model receptor-ligand system and Ca2+ signaling as a representative PTI output. A pre-treatment at 28 °C significantly impaired the flg22-induced [Ca2+]cyt influx, leading to a reduced expression of calcium-dependent defence genes, ICS1 and EDS1. This effect correlated with a temperature-dependent reduction in FLS2 abundance. A qualitatively similar inhibition of these responses was observed when membrane fluidity was artificially increased using benzyl alcohol. This suggests that the effect of elevated temperature might act through changes in membrane properties. Artificially restoring FLS2 protein levels rescued flg22-dependent Ca2+ signalling and ICS1 and EDS1 expression in seedlings pre-treated at 28°C or with benzyl alcohol. Together, these findings indicate that increased membrane fluidity reduces FLS2 protein levels, thereby compromising Ca2+ signalling, and probably other, flg22-indcued responses. This highlights a potential mechanistic link between temperature perception, membrane fluidity, and FLS2-dependent calcium signalling, providing insight into how an increase in global temperatures may compromise plant immune responses in the future. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, 2182091 Gatsby Charitable Foundation, https://ror.org/0290hax27 University of Zurich, https://ror.org/02crff812

Precise ligand recognition by closely related leucine-rich repeat receptor kinases (LRR-RKs) is essential for plants to coordinate immunity, development, and environmental adaptation. Here, we show how HAESA-LIKE 3 (HSL3) specifically recognizes the folded, disulfide-stabilized CTNIP/SCREW phytocytokines in Arabidopsis. Quantitative binding assays define a minimal CTNIP4 region required for high-affinity HSL3 interaction and signaling activation. A 2.12 Å crystal structure of the HSL3-CTNIP4 complex reveals a unique C-terminal receptor pocket that accommodates the peptide’s cyclic architecture through a combination of hydrophobic and polar contacts, a feature absent in the closely related HAE/HSL LRR-RKs. The cyclic CTNIP4 fold further establishes a largely hydrophobic interface that bridges HSL3 to the SERK co-receptor, forming a distinct activation surface. Together, these structural, biochemical and physiological insights uncover a previously unrecognised mechanism of peptide perception and receptor activation, highlighting how subtle architectural variations enable precise ligand selectivity among highly conserved plant receptor kinases. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, https://ror.org/00yjd3n13, 310030_204526, 10001549 EMBO European Research Council, https://ror.org/0472cxd90, 773153 EMBO, ALTF 1061-2023

Plants have evolved an elaborate cell wall integrity (CWI) sensing system to monitor and modify cell wall formation. LRR-extensins (LRXs) are cell wall-anchored proteins that bind RAPID ALKALINIZATION FACTOR (RALF) peptide hormones and induce compaction of cell wall structures. LRXs also form a signaling platform with RALFs and the transmembrane receptor kinase FERONIA (FER) to maintain cell wall integrity. LRX1 of Arabidopsis thaliana is predominantly expressed in root hairs, and lrx1 mutants develop defective root hairs. Here, we identify a regulator of LRX1-RALF-FER signaling as a suppressor of the lrx1 root hair phenotype. The repressor of lrx1_23 (rol23) gene encodes PP2C12, a clade H phosphatase that interacts with FER and dephosphorylates Thr696 in the FER activation loop in vitro. Expression of FER phospho-mimetic and phospho-null mutants in an lrx1 fer-4 background demonstrates that phosphorylation of FER at Thr696 is essential for suppression of lrx1 phenotypes by rol23. The LRX1-related function of PP2Cs appears clade H-specific. Collectively, our data suggest that LRX1 acts upstream of the RALF-FER signaling module and that PP2C12 inhibits FER via activation-loop dephosphorylation.

Extracellular alkalinization has long been recognized as a hallmark of plant cell-surface receptor activation, including during pattern-triggered immunity (PTI); yet the mechanisms driving elicitor-induced alkalinization and its role in immune signaling remain unclear. Here, we demonstrate that inhibition of autoinhibited H+-ATPases (AHAs) is required for elicitor-induced extracellular alkalinization. This alkalinization is essential for immune signaling mediated by diverse plasma membrane-localized receptor kinases (RKs) through modulation of ligand-receptor interactions. Notably, RKs transduce elicitor-triggered signaling via BOTRYTIS-INDUCED KINASE 1 (BIK1), which inhibits AHA activity by disrupting AHA-GENERAL REGULATORY FACTOR (GRF) interactions through a conserved phosphorylation event. Interestingly, this pathway is crucial for cell wall damage (CWD) responses involving the RK MALE DISCOVERER 1-INTERACTING RECEPTOR LIKE KINASE 2 (MIK2) and its ligand, SERINE RICH ENDOGENOUS PEPTIDE 18 (SCOOP18). Our findings reveal a conserved phospho-regulatory pathway that governs extracellular alkalinization to coordinate plant immune signaling, offering new insights into plant stress resilience. ### Competing Interest Statement The authors have declared no competing interest. University of Zurich, https://ror.org/02crff812, - Gatsby Charitable Foundation, https://ror.org/0290hax27, - European Research Council, 773153 Swiss National Science Foundation, 31003A_182625, 310030_212382, 320030_228294

In the ongoing plant–pathogen arms race, plants use pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs), while in successful pathogens, PAMPs can evolve to evade detection. Engineering PRRs to recognize evading PAMPs could potentially generate broad-spectrum and durable disease resistance. Here we reverse-engineered two natural variants of the PRR FLAGELLIN SENSING 2 (FLS2), VrFLS2XL and GmFLS2b, with extended recognition specificities towards evading flg22 variants. We identified minimal gain-of-function residues enabling blind FLS2s to recognize otherwise evading flg22 variants. We uncovered two strategies: (1) optimizing FLS2–flg22 interaction around flg22’s key evasion sites and (2) strengthening direct FLS2–BAK1 interaction to overcome weak agonistic and antagonistic flg22s, respectively. In addition, we leveraged polymorphisms that enhance recognition through unknown mechanisms to engineer a superior recognition capability. These findings offer basic design principles to engineer PRRs with broader recognition spectra, paving the way for PRR engineering to generate precisely gene-edited disease-resistant crops. By dissecting natural variants, Zhang et al. uncover design principles for engineering the FLS2 immune receptor with broad recognition spectra to detect evasive bacterial pathogens, offering a promising strategy to improve crop disease resistance.

Summary Phytocytokines regulate plant immunity via cell-surface receptors. Populus trichocarpa RUST INDUCED SECRETED PEPTIDE 1 (PtRISP1) exhibits an elicitor activity in poplar, as well as a direct antimicrobial activity against rust fungi. PtRISP1 gene directly clusters with a gene encoding a leucine-rich repeat receptor protein (LRR-RP), that we termed RISP- ASSOCIATED LRR-RP (PtRALR). In this study, we used phylogenomics to characterize the RISP and RALR gene families, and functional assays to characterize RISP/RALR pairs. Both RISP and RALR gene families specifically evolved in Salicaceae species (poplar and willow), and systematically cluster in the genomes. Two divergent RISPs, PtRISP1 and Salix purpurea RISP1 (SpRISP1), induced a reactive oxygen species (ROS) burst and mitogen- activated protein kinases (MAPKs) phosphorylation in Nicotiana benthamiana leaves expressing the respective clustered RALR. PtRISP1 triggers a rapid stomatal closure in poplar, and both PtRISP1 and SpRISP1 directly inhibit rust pathogen growth. Altogether, these results suggest that plants evolved phytocytokines with direct antimicrobial activities, and that the genes coding these phytocytokines co-evolved and physically cluster with their cognate receptors.

Secreted signaling peptides are central regulators of growth, development, and stress responses, but specific steps in the evolution of these peptides and their receptors are not well understood. Also, the molecular mechanisms of peptide-receptor binding are only known for a few examples, primarily owing to the limited availability of protein structural determination capabilities to few laboratories worldwide. Plants have evolved a multitude of secreted signaling peptides and corresponding transmembrane receptors. Stress-responsive SERINE RICH ENDOGENOUS PEPTIDES (SCOOPs) were recently identified. Bioactive SCOOPs are proteolytically processed by subtilases and are perceived by the leucine-rich repeat receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) in the model plant Arabidopsis thaliana. How SCOOPs and MIK2 have (co)evolved, and how SCOOPs bind to MIK2 are unknown. Using in silico analysis of 350 plant genomes and subsequent functional testing, we revealed the conservation of MIK2 as SCOOP receptor within the plant order Brassicales. We then leveraged AI-based structural modeling and comparative genomics to identify two conserved putative SCOOP-MIK2 binding pockets across Brassicales MIK2 homologues predicted to interact with the "SxS" motif of otherwise sequence-divergent SCOOPs. Mutagenesis of both predicted binding pockets compromised SCOOP binding to MIK2, SCOOP-induced complex formation between MIK2 and its coreceptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1, and SCOOP-induced reactive oxygen species production, thus, confirming our in silico predictions. Collectively, in addition to revealing the elusive SCOOP-MIK2 binding mechanism, our analytic pipeline combining phylogenomics, AI-based structural predictions, and experimental biochemical and physiological validation provides a blueprint for the elucidation of peptide ligand-receptor perception mechanisms.

Plants have evolved an elaborate cell wall integrity (CWI) sensing system to monitor and modify cell wall formation. LRR-extensins (LRXs) are cell wall-anchored proteins that bind RAPID ALKALINIZATION FACTOR (RALF) peptide hormones and induce compaction of cell wall structures. At the same time, LRXs form a signaling platform with RALFs and the transmembrane receptor kinase FERONIA (FER) as a means to relay changes in CWI to the protoplast. LRX1 of Arabidopsis thaliana is predominantly expressed in root hairs and lrx1 mutants develop defective root hairs. Here, we identify a regulator of LRX1-RALF-FER signaling as a suppressor of the lrx1 root hair phenotype. The repressor of lrx1_23 ( rol23 ) gene encodes PP2C12, a type 2C phosphatase of clade H that interacts with FER and dephosphorylates Thr696 in the FER activation loop in vitro. The LRX1-related function of PP2Cs appears clade H-specific and was not observed for other PP2Cs investigated. Collectively, our data suggest that LRX1 acts upstream of the RALF1-FER signaling module and PP2C12 has an inhibitory activity via modulating FER activity to fine-tune CWI signaling. ### Competing Interest Statement The authors have declared no competing interest.

Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domains have been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (brassinosteroid insensitive 1-associated kinase 1/somatic embryogenesis receptor kinase 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting αC-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1.

Plant receptor kinases (RKs) function as key plasma-membrane localized receptors in the perception of molecular ligands regulating development and environmental response. Through the perception of diverse ligands, RKs regulate various aspects throughout the plant life cycle from fertilization to seed set. Thirty years of research on plant RKs has generated a wealth of knowledge on how RKs perceive ligands and activate downstream signaling. In the present review, we synthesize this body of knowledge into five central paradigms of plant RK signaling: (1) RKs are encoded by expanded gene families, largely conserved throughout land plant evolution; (2) RKs perceive many different kinds of ligands through a range of ectodomain architectures; (3) RK complexes are typically activated by co-receptor recruitment; (4) post-translational modifications fulfill central roles in both the activation and attenuation of RK-mediated signaling; and, (5) RKs activate a common set of downstream signaling processes through receptor-like cytoplasmic kinases (RLCKs). For each of these paradigms, we discuss key illustrative examples and also highlight known exceptions. We conclude by presenting five critical gaps in our understanding of RK function.

Phytocytokines regulate plant immunity via cell-surface receptors. Populus trichocarpa RUST INDUCED SECRETED PEPTIDE 1 (PtRISP1) exhibits an elicitor activity in poplar, as well as a direct antimicrobial activity against rust fungi. PtRISP1 gene directly clusters with a gene encoding a leucine-rich repeat receptor protein (LRR-RP), that we termed RISP- ASSOCIATED LRR-RP (PtRALR). In this study, we used phylogenomics to characterize the RISP and RALR gene families, and functional assays to characterize RISP/RALR pairs. Both RISP and RALR gene families specifically evolved in Salicaceae species (poplar and willow), and systematically cluster in the genomes. Two divergent RISPs, PtRISP1 and Salix purpurea RISP1 (SpRISP1), induced a reactive oxygen species (ROS) burst and mitogen- activated protein kinases (MAPKs) phosphorylation in Nicotiana benthamiana leaves expressing the respective clustered RALR. PtRISP1 triggers a rapid stomatal closure in poplar, and both PtRISP1 and SpRISP1 directly inhibit rust pathogen growth. Altogether, these results suggest that plants evolved phytocytokines with direct antimicrobial activities, and that the genes coding these phytocytokines co-evolved and physically cluster with their cognate receptors.

Plant signalling peptides are typically released from larger precursors by proteolytic cleavage to regulate plant growth, development and stress responses. Recent studies reported the characterization of a divergent family of Brassicaceae-specific peptides, SERINE RICH ENDOGENOUS PEPTIDES (SCOOPs), and their perception by the leucine-rich repeat receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2). Here, we reveal that the SCOOP family is highly expanded, containing at least 50 members in the Columbia-0 reference Arabidopsis thaliana genome. Notably, perception of these peptides is strictly MIK2-dependent. How bioactive SCOOP peptides are produced, and to what extent their perception is responsible for the multiple physiological roles associated with MIK2 are currently unclear. Using N-terminomics, we validate the N-terminal cleavage site of representative PROSCOOPs. The cleavage sites are determined by conserved motifs upstream of the minimal SCOOP bioactive epitope. We identified subtilases necessary and sufficient to process PROSCOOP peptides at conserved cleavage motifs. Mutation of these subtilases, or their recognition motifs, suppressed PROSCOOP cleavage and associated overexpression phenotypes. Furthermore, we show that higher-order mutants of these subtilases show phenotypes reminiscent of mik2 null mutant plants, consistent with impaired PROSCOOP biogenesis, and demonstrating biological relevance of SCOOP perception by MIK2. Together, this work provides insights into the molecular mechanisms underlying the functions of the recently identified SCOOP peptides and their receptor MIK2. The SCOOP signalling peptide family expands to 50 members, whose activities are strictly dependent upon the receptor kinase MIK2. Two subtilase classes process PROSCOOPs, generating bioactive SCOOP peptides. A subtilase mutant phenocopies the mik2 receptor mutant.

Calcium (Ca2+)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca2+ sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to im-mune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate path-ways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca2+ activation at physiological concentrations of Ca2+, possibly through stabilization of the Ca2+-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca2+] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific require-ments for phosphorylation-based Ca2+-sensitivity priming. We addi-tionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that en-hance resistance to microbial pathogens without consequences to yield.

Receptor kinases (RKs) are fundamental for extracellular sensing and regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) are primarily peptide receptors that regulate responses to myriad internal and external stimuli. Phosphorylation of LRR-RK cytoplasmic domains is among the earliest responses following ligand perception, and reciprocal transphosphorylation between a receptor and its coreceptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in antibacterial immunity. These results reveal a noncatalytic role for EFR in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RKs with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor, which could initiate signaling either allosterically or through driving the dissociation of negative regulators of the complex.

Reversible protein phosphorylation is a widespread post-translational modification fundamental for signaling across all domains of life. Tyrosine (Tyr) phosphorylation has recently emerged as being important for plant receptor kinase (RK)-mediated signaling, particularly during plant immunity. How Tyr phosphorylation regulates RK function is however largely unknown. Notably, the expansion of protein Tyr phosphatase and SH2 domain-containing protein families, which are the core of regulatory phospho-Tyr (pTyr) networks in choanozoans, did not occur in plants. Here, we summarize the current understanding of plant RK Tyr phosphorylation focusing on the critical role of a pTyr site ('VIa-Tyr') conserved in several plant RKs. Furthermore, we discuss the possibility of metazoan-like pTyr signaling modules in plants based on atypical components with convergent biochemical functions.