Receptor-like cytoplasmic kinases of different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Май 21, 2024

Abstract Cell-surface receptors form the front line of plant immunity. The leucine-rich repeat (LRR)-receptor-like kinases SOBIR1 and BAK1 are required for functionality tomato LRR-receptor-like protein Cf-4, which detects secreted effector Avr4 pathogenic fungus Fulvia fulva . Here, we show that kinase domains directly phosphorylate each other residues Thr522 Tyr469 domain Nicotiana benthamiana its activity interacting with signalling partners, respectively. By knocking out multiple genes belonging to different receptor-like cytoplasmic (RLCK)-VII subfamilies in N. benthamiana:Cf-4 , members RLCK-VII-6, −7, −8 differentially regulate Avr4/Cf-4-triggered biphasic burst reactive oxygen species. In addition, RLCK-VII-7 play an essential role resistance against oomycete pathogen Phytophthora palmivora Our study provides molecular evidence specific roles RLCKs downstream SOBIR1/BAK1-containing immune complexes.

Язык: Английский

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Allosteric activation of the co-receptor BAK1 by the EFR receptor kinase initiates immune signaling

eLife, Год журнала: 2023, Номер 12

Опубликована: Ноя. 23, 2023

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

Язык: Английский

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Differential contribution of Arabidopsis chitin receptor complex components to defense signaling and ubiquitination‐dependent endocytotic removal from the plasma membrane

New Phytologist, Год журнала: 2024, Номер 244(3), С. 934 - 948

Опубликована: Авг. 26, 2024

In Arabidopsis, the enzymatically active lysin motif-containing receptor-like kinase (LysM-RLK) CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) and pseudokinases LYSIN MOTIF-CONTAINING RECEPTOR-LIKE 5 (LYK5) LYK4 are core components of canonical chitin receptor complex. CERK1 dimerizes autophosphorylates upon binding, resulting in activation signaling. this study, we clarified further elucidated individual contributions LYK5 to chitin-dependent signaling using mutant (combination)s stably transformed Arabidopsis plants expressing fluorescence-tagged variants from their endogenous promoters. Our analyses revealed that interacts with treatment, independently vice versa. We show chitin-induced autophosphorylation is predominantly dependent on LYK5, whereas chitin-triggered ROS generation almost exclusively mediated by LYK4. This suggests specific functions these two co-receptor proteins apart redundant function mitogen-activated protein (MAPK) transcriptional reprogramming. Moreover, demonstrate subject CERK1-dependent ubiquitination, which serves as a signal for internalization LYK5. experiments provide evidence combination phosphorylation ubiquitination events controls removal plasma membrane via endocytosis, likely contributes complex desensitization.

Язык: Английский

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24-epibrassinolide promotes resilience against Arsenic Stress via Modulating Amino Acid Profiles and mRNA abundance of CYP450 and MRP genes in Zea mays L.

Plant Physiology and Biochemistry, Год журнала: 2025, Номер 221, С. 109631 - 109631

Опубликована: Фев. 13, 2025

Язык: Английский

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The Medicago truncatulaLYR4 intracellular domain serves as a scaffold in immunity signaling independent of its phosphorylation activity

New Phytologist, Год журнала: 2025, Номер unknown

Опубликована: Март 10, 2025

Plants engage in a wealth of interactions with beneficial and pathogenic bacteria fungi therefore need to monitor their surroundings. To this end, cell-surface receptors, such as lysin motif (LysM) perceive microbe-associated molecular patterns (MAMPs) elicit immunity responses or initiate symbiotic associations (Buendia et al., 2018). LysM receptors have an ectodomain built three interconnected domains most feature single transmembrane helix either active kinase pseudokinase intracellular domain (Gust 2012). The main ligands are carbohydrates containing N-acetylglucosamine moieties, namely chitooligosaccharides (CO; chitin), lipochitooligosaccharides (LCO), peptidoglycan (Willmann 2011; Gust 2012; Bozsoki 2017, 2020; Buendia 2018; Gysel 2021). In the model legume Lotus japonicus (Lotus), receptor CERK6 (previously called LYS6) tandem duplicated LYS13 LYS14 involved chitin-triggered (Bozsoki 2017). classified pseudokinases, expression increases roots shoots upon chitin treatment plant (Lohmann 2010; Ruman & Kawaharada, 2023). Single mutants lys13 lys14 respond similarly wild-type, suggesting functional redundancy between LYS14, whereas cerk6 cannot produce reactive oxygen species (ROS) upregulation defense-response genes phosphorylation MAPK3/6 (mitogen-activated protein kinases) impaired Further investigation signaling is difficult, double mutant not available. Medicago truncatula (Medicago), CERK1 LYK9) homolog LYR4 2017; Feng 2019). (CERK1 hereafter) (LYR4 been reported be indispensable for both cerk1 lyr4 having increased lesion size infection leaves Botrytis cinerea addition, ROS were absent MAPK3 MAPK6 decreased octamer (CO8) compared wild-type Zhang 2024). has all canonical motifs eukaryotic kinase, while due degeneration several key motifs: truncated glycine-rich loop, DFG-motif NFG, HRD-motif HKN 2023; Fig. 1a). Canonically, loop regulatory lysine on β3-strand stabilize phosphates bound ATP. Aspartate from important positioning phosphate transfer arginine aspartate contribute ordering activation incoming substrate, respectively (Taylor Kornev, Taylor study, we investigate how mediates downstream immunity. We determine crystal structure ATP-analog noncanonical manner show that it catalytically despite its lack features. However, planta experiments demonstrate ability necessary function production, but presence indispensable. Thus, serves scaffold independent catalytic activity. G. cv R108 was used wild-type. lyr4-2 (NF15280) (NF16753) previously (Tadege 2008; lyr4-1 generated by crossing (NF10265) plants. exhibit similiar CO8 described Escherichia coli TOP10 (Thermo Fisher Scientific, Waltham, MA, USA) cloning grown LB medium at 37°C. Chemically competent E. Rosetta 2 cells (Sigma-Aldrich) specified below. Agrobacterium rhizogenes strain AR1193 hairy root transformations, tumefaciens AGL1 transient transformation Nicotiana benthamiana. strains 28°C. cerk1, lyr4, seeds treated sulfuric acid 3 min, washed ddH2O, surface-sterilized 3% sodium hypochlorite, again incubated ddH2O h. Swollen transferred square plates wet filter paper 21°C under 16 h : 8 h, light dark conditions d. Seedlings then slope ¼ B&D (Broughton Dilworth, 1971) solidified 1.4% Agar Noble (Difco, Sparks, MD, USA). agar covered (AGF 651; Frisenette ApS, Knebel, Denmark) metal bar 10 holes placed top slope. Plates boxes, excluding below bars. After d plates, seedlings cut hypocotyl, one full each well white 96-well flat-bottomed polystyrene plate (Greiner Bio-One, Kremsmünster, Austria) kept overnight sterile water. water replaced reaction mixture consisting 0.5 mM L-012 (Wako Chemicals, Neuss, Germany), 5 μg ml−1 horseradish peroxidase (Sigma-Aldrich), 1 μM octa-N-acetyl-chitooctaose (CO8, obtained Sébastien Fort; Masselin 2021) 0.1 mg shrimp-cell (Sigma-Aldrich). Luminescence recorded Varioskan LUX multimode microplate reader Scientific) 30 min. For generation M. vectors, sequences variants assembled native promoter 35S terminator via Golden Gate (Weber 2011). constructs thereafter cloned into pIV10 vector alongside construct YFP fluorophores fused nuclear localization signal (tYFP-NLS), which marker. experiment N. benthamiana, sequence pICH An overview provided Supporting Information Table S1. 0.8% Gelrite (Duchefa Biochemie, Haarlem, Netherlands) supplemented ½ Gamborg's B5 nutrient solution Biochemie). Transconjugant A. carrying interest ampicillin, rifampicin, spectinomycin, final concentration 100 ml−1. Cells resuspended 4 ml YMB (5 g l−1 mannitol, yeast extract, K2HPO4, 0.2 MgSO4 × 7 H2O, NaCl; pH 6.8). Four-day-old transformed using syringe needle (Sterican Ø 0.40 20 mm), wounding hypocotyl placing droplet bacterial suspension wound. conditions. wk, primary removed emerging Magenta boxes filled clay aggregate (LECA, 2–4 mm; Saint-Gobain Weber A/S, Copenhagen, 80 KNO3. Variants tested complement mutant. wk growth expressing tYFP-NLS marker small pieces c. mm length. Pieces 1.5 cm total length Bio-One) cuttings left room temperature overnight. Afterward, Chemicals), (Sigma), added. Values derived curves without clear peak within min excluded. Agrobacterium-mediated benthamiana performed (Ochoa-Fernandez 2020). short, Lyr4-ΔIC-GFP (green fluorescent protein) driven infiltration (10 MgCl2, MES, 150 acetosyringone; 5.6) OD600 = 0.025, infiltrated abaxial side blunt end syringe. imaged after infiltration. GFP-tagged LYR4-ΔIC 488 nm excitation 491–544 emission Zeiss LSM 780 confocal microscope. (residues 298–660 residues 340–636) 307–598) codon optimized (Genscript, Leiden, N-terminally histidine tag, SUMO 3C protease cleavage site. 298–660) A377F, L392D, (307–598) K349A made site-directed mutagenesis aforementioned constructs. S2. expression, 0.6 relevant antibiotics 37°C shaking. culture chilled ice induced adding IPTG, 19°C shaking harvested 4400 15 medium, pelleted 3050 pellet lysis buffer (50 Tris–HCl 8.0, 500 NaCl, 10% v/v glycerol, imidazole, β-mercaptoethanol (BME)) lysed sonication. cleared supernatant subjected nickel-immobilized metal-affinity chromatography (Ni-IMAC) Protino Ni-NTA column (Macherey-Nagel, Düren, Germany) equilibrated buffer. sample loaded onto peristaltic pump, 20-column volumes A BME) before eluted 12 B 200 BME). added eluate 50 molar ratio digestion dialyzed against l dialysis 250 5% 4°C. product another Ni-IMAC remove digested tags purified gel filtration step Superdex75 increase 10/300 GL Superdex200 (GE Healthcare, Chicago, IL, (25 All purification steps analyzed dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) elution fractions pooled accordingly. crystallized sitting drop vapor diffusion setup 24-well (Molecular Dimensions, Rotherham, UK). Crystal formation condition M ammonium sulfate, cacodylate trihydrate 6.5 30% w/v PEG 8000 gave rise seed stock microseeding drops adenylyl-imidodiphosphate (AMP-PNP) 0.3 6.5, 25% 8000. Needle-like crystals cryoprotected supplementing crystallization glycerol fished flash-cooled liquid nitrogen. data set collected Diamond Light Source beamline I04. diffraction processed scaled XDS (Kabsch, 2010), xtriage Phenix program suite. Due high anisotropy, STARANISO server (Global Phasing Ltd (Tickle 2016)) along anisotropic resolution limit surface (CC(1/2) ≥ 0.3, I/sigI 2.0, Rpim ≤ 0.6). crystallographic phase problem solved replacement Phaser NFR5 (PDB-ID 8S79) search (McCoy 2007). Refinement (Adams manual building carried out Coot (Emsley 2010). Data collection statistics spherical ellipsoidal datasets refinement S3. atomic coordinates factors deposited Protein Bank, PDB ID: 8PS7. Structural analyses figures PyMol 2.3.2 (Schrödinger LLC, New York, NY, Binding nucleotide indirectly assayed nano differential scanning fluorimetry (nano-DSF) Tycho-NT.6 (Nanotemper, Munich, Germany). 298–660), L392D ATP, AMP-PNP, MgCl2 each. Four micrograms nCi [γ32-P] ATP (PerkinElmer, cold samples separated 15% SDS-PAGE stained InstantBlue Coomassie Stain (Abcam, Cambridge, UK) exposed Autoradiography Hypercassette (Amersham/Cytiva, Amersham, Radiographs developed Typhoon FLA 9500 (Amersham/Cytiva). individual contributions signaling, response when shrimp-shell (Fig. 1b). While see almost no mutant, CO8. production is, however, attenuated curve broadened peaks later than From conclude does fully depend can also individually signaling. understand juxtamembrane part predicted flexible C-terminal tail S1a). analog AMP-PNP. Diffraction resulted 3.1 Å anisotropically 2.1 8PS7) 1c; S3). Based amino sequence, defined pseudokinase. electron density revealed clearly AMP-PNP molecule binding site 1d), showing retained abilities. Closer examination binds way compensates traditional S1b). Pseudokinases display compensatory mechanisms retain degraded motifs, proposed (Zeqiraj Van Aalten, Murphy 2014; Sheetz Lemmon, 2022). coordinated lysine, indeed employ (K379) coordinate α-phosphate, too short reach Instead, N494 modified α- β-phosphate, R514 β- γ-phosphate. Active kinases two hydrophobic spines going through core – (R-) spine (C-) spine. cycle, assemble break these they toggle inactive states. C-spine nucleotide, R-spine αC moves in-position forms salt bridge lysine. Both able phosphorylate substrate (Kornev 2006; conformation, intact C-spine, broken being out-position S1c,d). Furthermore, shows C-terminus 1c, labeled helix). This blocks access bringing position coordinating γ-phosphate representing undescribed conformation. Guided structure, designed whether LYR4. LYR4-A377F phenylalanine site, predict will abolish ATP-binding 1e). LYR4-L392D introduces negative charge helix, make unable move R-spine, keeping retaining 1f). expressed S2) determined bind testing thermostability nano-DSF magnesium 2a). together stability 4.1°C, indicating likewise stabilized 3.7°C, present, only marginally 1.5°C, no, very weak, ATP-binding. test potential activity, vitro assays 32P-labeled observed autophosphorylate transphosphorylate myelin basic (MBP), making 2b, lanes 3, 4). As expected, neither nor showed activity (Figs had autophosphorylation lower 5). Next, investigated other. created kinase-inactive version lacked (CERK1-K349A) 9, 10, lane 11). Conversely, 12). dependent subsequent conformational changes, plants Lyr4, Lyr4-A377F (no binding), Lyr4-L392D (kinase inactive, binding). Lyr4-A377F, Lyr4-L392D, produced equal empty 2c), required all, constructed variant lacking entire domain, Lyr4-ΔIC, roots. When trafficked plasma membrane properly folded S4a). could, mediate 2c, S4b,c), crucial immune Together, our results chitin-induced comprise up 17% kinome numerous pathways, example, those (Kwon understanding remains limited. perception absence CERK1, although resulting levels attenuated. Previously, shown completely could pairing less efficient abundant partner. LYK subfamily receptor-like harboring potentially kinases, 11 members Medicago, LYR LYR7 phylogenetically closest Recently, al. LYK8 some extent functionally redundant arbuscular mycorrhizal symbiosis. Some detail yet, might overlapping capacities take over functions. better mediated pseudokinase, ATP-analog. density, prompted us relevance phosphotransfer ability. Thermostability additionally confirmed closer inspection manner. Despite pseudokinases demonstrated nucleotides various event itself lead regulation Mace Murphy, 2021; auto- transphosphorylation general, many experimentally, employing Dar, 2013). changes Complementation studies production. It intriguing apparent relevance, exclude possibility plays role other pathways. elicitation 2d). noncatalytic responsible putative CERK1–LYR4 complex. broader view, scaffolding like correctly arrange co-receptors, partners. often dynamic typical allows them act switches allosteric regulators acting scaffolds complex assembly (Sheetz 2022; Mühlenbeck Future elucidate complexes formed thank Majken Kiel Sørensen care glasshouse, Sara Bährentz help work, Leila Margot Henkes proofreading manuscript. acknowledge provision experimental facilities staff I04 assistance collection. work funded Novo Nordisk Foundation (NNF18OC0052855), Danish Council Independent Research (3103-00137B), Carlsberg (CF21-0139), Agence Nationale de la Recherche Labex ARCANE CBH-EUR-GS (ANR-17-EURE-0003), Glyco@Alps (ANR-15-IDEX-02), ICMG (UAR 2607) Grenoble Chemistry Nanobio mass spectrometry platform, USDA-NIFA grant (2022-67014-38607), project Enabling Nutrient Symbioses Agriculture (ENSA), Bill Melinda Gates Agricultural Innovations (INV-57461), Foreign, Commonwealth Development Office (INV-55767). None declared. BS, HR, MVK, MML, CK GK investigation. KG, ML, JS, SR KRA formal analysis. SF FF resources. BS HR visualization. conceptualization. supervision. administration. GEDO, funding acquisition. writing original draft preparation. authors review editing. contributed equally work. Bank (PDB code (8PS7)). S1 Purification detailed structural S2 Gel profiles corresponding SDS-PAGEs purifications recombinant proteins. S3 assay variants. S4 including versions LYR4-ΔIC. assays. Construct purification. statistics. Please note: Wiley content functionality any supplied authors. Any queries (other missing material) should directed Phytologist Central Office. publisher supporting information content) author article. neutral regard jurisdictional claims maps institutional affiliations.

Язык: Английский

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Biological functionality of non-functional protein kinases

Journal of Experimental Botany, Год журнала: 2025, Номер 76(6), С. 1478 - 1481

Опубликована: Апрель 9, 2025

This article comments on: Gonçalves Dias M, Dharmasena T, Gonzalez-Ferrer C, Maika JE, Miguel VN, Dou R, Rodriguez Gallo MC, Bredow Siegel KR, Uhrig RG, Simon Monaghan J. 2025. Catalytically inactive subgroup VIII receptor-like cytoplasmic kinases regulate the immune-triggered oxidative burst in Arabidopsis thaliana. Journal of Experimental Botany 76, 1553–1568

Язык: Английский

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Differential phosphorylation of receptor kinase SlLYK4 mediates immune responses to bacterial and fungal pathogens in tomato

Science Advances, Год журнала: 2025, Номер 11(22)

Опубликована: Май 30, 2025

Bacterial wilt caused by Ralstonia solanacearum is a devastating plant disease. Exopolysaccharide (EPS), major virulence factor of R. , elicits pattern-triggered immunity (PTI) in tomato, but the means which EPS recognized remain poorly understood. We found that tomato non-arginine-aspartate (non-RD) receptor kinase SlLYK4 mediates perception and positively regulates resistance to bacterial wilt. The RD kinases SlLYK1 SlLYK13 are required for EPS-triggered immune responses form complexes with SlLYK4. These have dual functions recognizing fungal chitin. Phosphorylation serine-320 juxtamembrane domain essential EPS- chitin-mediated signaling, whereas phosphorylation serine-334 or serine-634 C-terminal chitin respectively. Our results reveal mechanism underlying recognition provide insight into how differential antibacterial antifungal immunity.

Язык: Английский

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A large-scale screening identifies receptor-like kinases with common features in kinase domains that are potentially related to disease resistance in planta

Frontiers in Plant Science, Год журнала: 2024, Номер 15

Опубликована: Ноя. 13, 2024

The plant genome encodes a plethora of proteins with structural similarity to animal receptor protein kinases, collectively known as receptor-like kinases (RLKs), which predominantly localize the plasma membrane where they activate their kinase domains convey extracellular signals interior cell, playing crucial roles in various signaling pathways. Despite large number members within RLK family, date, only few have been identified pattern-recognition receptors (PRRs), leaving many potential RLKs that could play immunity undiscovered.

Язык: Английский

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Reverse engineering of the pattern recognition receptor FLS2 reveals key design principles of broader recognition spectra against evading flg22 epitopes

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Окт. 13, 2024

Abstract In the ongoing plant-pathogen arms race, plants employ pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs), while in successful pathogens, PAMPs can evolve evade detection. Engineering PRRs evading could potentially generate broad-spectrum and durable disease resistance. this study, we reverse-engineered two natural FLAGELLIN SENSING 2 (FLS2) variants, VrFLS2XL GmFLS2b, with extended specificities towards flg22 variants. We identified minimal gain-of-function residues enabling blind FLS2s otherwise uncovered strategies: (i) enhancing FLS2-flg22 interaction around flg22’s key evasion sites, (ii) strengthening direct between FLS2 its co-receptor BAK1 overcome weak agonistic antagonistic flg22s, respectively. Additionally, leveraged polymorphisms that enhance through unknown mechanisms engineer superior capability. These findings offer basic design principles for broader spectra, paving way PRR engineering using precise gene-editing increase resistance crops.

Язык: Английский

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A conserved juxtamembrane motif in plant NFR5 receptors is essential for root nodule symbiosis

Proceedings of the National Academy of Sciences, Год журнала: 2024, Номер 121(46)

Опубликована: Ноя. 4, 2024

Establishment of root nodule symbiosis is initiated by the perception bacterial Nod factor ligands plant LysM receptor kinases NFR1 and NFR5. Receptor signaling initiating symbiotic pathway depends on kinase activity NFR1, while mechanism catalytically inactive NFR5 pseudokinase unknown. Here, we present crystal structure signaling-competent Lotus japonicus intracellular domain, comprising juxtamembrane region domain. The structurally well defined forms two α-helices, αA αA′, which contain an exposed hydrophobic motif. We demonstrate that this “juxtamembrane motif” promotes NFR5–NFR5 NFR1–NFR5 interactions essential for signaling. Conservation analysis reveals motif throughout NFR5-type receptors required from barley RLK10, suggesting a conserved broader function in plant–microbe symbioses.

Язык: Английский

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