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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Perception of biotic and abiotic stresses often leads to stomatal closure in plants1,2. Rapid influx of calcium ions (Ca2+) across the plasma membrane has an important role in this response, but the identity of the Ca2+ channels involved has remained elusive3,4. Here we report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid-a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Imunidade Vegetal , Estômatos de Plantas/imunologia , Estômatos de Plantas/metabolismo , Ácido Abscísico/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
Perception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors activates RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) through direct phosphorylation by BOTRYTIS-INDUCED KINASE 1 (BIK1) and induces the production of reactive oxygen species (ROS). RBOHD activity must be tightly controlled to avoid the detrimental effects of ROS, but little is known about RBOHD downregulation. To understand the regulation of RBOHD, we used co-immunoprecipitation of RBOHD with mass spectrometry analysis and identified PHAGOCYTOSIS OXIDASE/BEM1P (PB1) DOMAIN-CONTAINING PROTEIN (PB1CP). PB1CP negatively regulates RBOHD and the resistance against the fungal pathogen Colletotrichum higginsianum. PB1CP competes with BIK1 for binding to RBOHD in vitro. Furthermore, PAMP treatment enhances the PB1CP-RBOHD interaction, thereby leading to the dissociation of phosphorylated BIK1 from RBOHD in vivo. PB1CP localizes at the cell periphery and PAMP treatment induces relocalization of PB1CP and RBOHD to the same small endomembrane compartments. Additionally, overexpression of PB1CP in Arabidopsis leads to a reduction in the abundance of RBOHD protein, suggesting the possible involvement of PB1CP in RBOHD endocytosis. We found PB1CP, a novel negative regulator of RBOHD, and revealed its possible regulatory mechanisms involving the removal of phosphorylated BIK1 from RBOHD and the promotion of RBOHD endocytosis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , NADPH Oxidases , Imunidade Vegetal , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , NADPH Oxidases/metabolismo , Oxirredutases/metabolismo , Fagocitose , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
In Extended Data Fig. 5d of this Letter, the blots for anti-pS612 and anti-BAK1 were inadvertently duplicated. This figure has been corrected online.
RESUMO
Multicellular organisms use cell-surface receptor kinases to sense and process extracellular signals. Many plant receptor kinases are activated by the formation of ligand-induced complexes with shape-complementary co-receptors1. The best-characterized co-receptor is BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), which associates with numerous leucine-rich repeat receptor kinases (LRR-RKs) to control immunity, growth and development2. Here we report key regulatory events that control the function of BAK1 and, more generally, LRR-RKs. Through a combination of phosphoproteomics and targeted mutagenesis, we identified conserved phosphosites that are required for the immune function of BAK1 in Arabidopsis thaliana. Notably, these phosphosites are not required for BAK1-dependent brassinosteroid-regulated growth. In addition to revealing a critical role for the phosphorylation of the BAK1 C-terminal tail, we identified a conserved tyrosine phosphosite that may be required for the function of the majority of Arabidopsis LRR-RKs, and which separates them into two distinct functional classes based on the presence or absence of this tyrosine. Our results suggest a phosphocode-based dichotomy of BAK1 function in plant signalling, and provide insights into receptor kinase activation that have broad implications for our understanding of how plants respond to their changing environment.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/imunologia , Arabidopsis/química , Arabidopsis/imunologia , Proteínas de Arabidopsis/imunologia , Ligantes , Modelos Moleculares , Fosforilação , Fosfotirosina/metabolismo , Imunidade Vegetal , Proteínas Serina-Treonina Quinases/imunologiaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Imunidade Vegetal/fisiologia , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Arabidopsis/genética , Membrana Celular/metabolismo , Expressão Gênica , Imunidade Inata/genética , Ligantes , Fator Tu de Elongação de Peptídeos/metabolismo , Fosforilação , Imunidade Vegetal/genética , Plantas Geneticamente Modificadas/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais/fisiologiaRESUMO
The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response.
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Arabidopsis/fisiologia , Quitina/metabolismo , Nicotiana/fisiologia , Plasmodesmos/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fenômenos Biomecânicos , Membrana Celular/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mecanotransdução Celular/fisiologia , Folhas de Planta/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Espécies Reativas de OxigênioRESUMO
The rapid production of reactive oxygen species (ROS) burst is a conserved signaling output in immunity across kingdoms. In plants, perception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) activates the NADPH oxidase RBOHD by hitherto unknown mechanisms. Here, we show that RBOHD exists in complex with the receptor kinases EFR and FLS2, which are the PRRs for bacterial EF-Tu and flagellin, respectively. The plasma-membrane-associated kinase BIK1, which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. BIK1 phosphorylates different residues than calcium-dependent protein kinases, and both PAMP-induced BIK1 activation and BIK1-mediated phosphorylation of RBOHD are calcium independent. Importantly, phosphorylation of these residues is critical for the PAMP-induced ROS burst and antibacterial immunity. Our study reveals a rapid regulatory mechanism of a plant RBOH, which occurs in parallel with and is essential for its paradigmatic calcium-based regulation.
Assuntos
Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Imunidade Inata , NADPH Oxidases/imunologia , Nicotiana/enzimologia , Imunidade Vegetal , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Linhagem Celular , Ativação Enzimática , Flagelina/imunologia , Flagelina/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ligantes , Dados de Sequência Molecular , Complexos Multienzimáticos , NADPH Oxidases/genética , Fator Tu de Elongação de Peptídeos/imunologia , Fator Tu de Elongação de Peptídeos/metabolismo , Fosforilação , Estômatos de Plantas/imunologia , Estômatos de Plantas/metabolismo , Proteínas Quinases/imunologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/microbiologiaRESUMO
Plant resistance inducers (PRIs) are compounds that protect plants from diseases by activating immunity responses. Exogenous treatment with glutamate (Glu), an important amino acid for all living organisms, induces resistance against fungal pathogens in rice and tomato. To understand the molecular mechanisms of Glu-induced immunity, we used the Arabidopsis model system. We found that exogenous treatment with Glu induces resistance against pathogens in Arabidopsis. Consistent with this, transcriptome analyses of Arabidopsis seedlings showed that Glu significantly induces the expression of wound-, defense-, and stress-related genes. Interestingly, Glu activates the expression of genes induced by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns at much later time points than the flg22 peptide, which is a bacterial-derived PAMP. The Glu receptor-like (GLR) proteins GLR3.3 and GLR3.6 are involved in the early expression of Glu-inducible genes; however, the sustained expression of these genes does not require the GLR proteins. Glu-inducible gene expression is also not affected by mutations in genes that encode PAMP receptors (EFR, FLS2, and CERK1), regulators of pattern-triggered immunity (BAK1, BKK1, BIK1, and PBL1), or a salicylic acid biosynthesis enzyme (SID2). The treatment of roots with Glu activates the expression of PAMP-, salicylic acid-, and jasmonic acid-inducible genes in leaves. Moreover, the treatment of roots with Glu primes chitin-induced responses in leaves, possibly through transcriptional activation of LYSIN-MOTIF RECEPTOR-LIKE KINASE 5 (LYK5), which encodes a chitin receptor. Because Glu treatment does not cause discernible growth retardation, Glu can be used as an effective PRI.
Assuntos
Arabidopsis/efeitos dos fármacos , Arabidopsis/imunologia , Ácido Glutâmico/farmacologia , Imunidade Vegetal/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Moléculas com Motivos Associados a PatógenosRESUMO
Plant immunity consists of two arms: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), induced by surface-localized receptors, and effector-triggered immunity (ETI), induced by intracellular receptors. Despite the little structural similarity, both receptor types activate similar responses with different dynamics. To better understand phosphorylation events during ETI, we employed a phosphoproteomic screen using an inducible expression system of the bacterial effector avrRpt2 in Arabidopsis thaliana, and identified 109 differentially phosphorylated residues of membrane-associated proteins on activation of the intracellular RPS2 receptor. Interestingly, several RPS2-regulated phosphosites overlap with sites that are regulated during PTI, suggesting that these phosphosites may be convergent points of both signaling arms. Moreover, some of these sites are residues of important defense components, including the NADPH oxidase RBOHD, ABC-transporter PEN3, calcium-ATPase ACA8, noncanonical Gα protein XLG2 and H+ -ATPases. In particular, we found that S343 and S347 of RBOHD are common phosphorylation targets during PTI and ETI. Our mutational analyses showed that these sites are required for the production of reactive oxygen species during both PTI and ETI, and immunity against avirulent bacteria and a virulent necrotrophic fungus. We provide, for the first time, large-scale phosphoproteomic data of ETI, thereby suggesting crucial roles of common phosphosites in plant immunity.
Assuntos
Arabidopsis/metabolismo , Moléculas com Motivos Associados a Patógenos/metabolismo , Fosfoproteínas/metabolismo , Imunidade Vegetal , Proteômica , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Autoimunidade/genética , Regulação da Expressão Gênica de Plantas , Mutação/genética , Fenótipo , Fosforilação , Imunidade Vegetal/genética , ATPases Translocadoras de Prótons/metabolismo , Pseudomonas syringae/fisiologia , Espécies Reativas de Oxigênio/metabolismo , VirulênciaRESUMO
Plasma membrane-localized pattern recognition receptors (PRRs) such as FLAGELLIN SENSING2 (FLS2), EF-TU RECEPTOR (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) recognize microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). A reverse genetics approach on genes responsive to the priming agent ß-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hypersusceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants showed defective PTI responses, notably delayed upregulation of the PTI marker gene FLG22-INDUCED RECEPTOR-LIKE KINASE1, reduced callose deposition, and mitogen-activated protein kinase activation upon MAMP treatment. Moreover, Arabidopsis lines overexpressing IOS1 were more resistant to bacteria and showed a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, coimmunoprecipitation, and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)-dependent PRRs FLS2 and EFR, as well as with the BAK1-independent PRR CERK1. IOS1 also associated with BAK1 in a ligand-independent manner and positively regulated FLS2-BAK1 complex formation upon MAMP treatment. In addition, IOS1 was critical for chitin-mediated PTI. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a novel regulatory protein of FLS2-, EFR-, and CERK1-mediated signaling pathways that primes PTI activation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Aminobutiratos/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pseudomonas syringae/patogenicidadeRESUMO
Plants use receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) that are typical of whole classes of microbes. After ligand perception, many leucine-rich repeat (LRR)-containing PRRs interact with the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1). BAK1 is thus expected to interact with unknown PRRs. Here, we used BAK1 as molecular bait to identify a previously unknown LRR-RLP required for the recognition of the csp22 peptide derived from bacterial cold shock protein. We established a method to identify proteins that interact with BAK1 only after csp22 treatment. BAK1 was expressed transiently in Nicotiana benthamiana and immunopurified after treatment with csp22. BAK1-associated proteins were identified by mass spectrometry. We identified several proteins including known BAK1 interactors and a previously uncharacterized LRR-RLP that we termed RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). This RLP associates with BAK1 upon csp22 treatment, and NbCSPR-silenced plants are impaired in csp22-induced defense responses. NbCSPR confers resistance to bacteria in an age-dependent and flagellin-induced manner. As such, it limits bacterial growth and Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transgenic expression of NbCSPR into Arabidopsis thaliana conferred responsiveness to csp22 and antibacterial resistance. Our method may be used to identify LRR-type RKs and RLPs required for PAMP perception/responsiveness, even when the active purified PAMP has not been defined.
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Proteínas de Bactérias/imunologia , Proteínas e Peptídeos de Choque Frio/fisiologia , Nicotiana/imunologia , Nicotiana/microbiologiaRESUMO
Hsp90-mediated function of NLR receptors in plant and animal innate immunity depends on the cochaperone Sgt1 and, at least in plants, on a cysteine- and histidine-rich domains (CHORD)-containing protein Rar1. Functionally, CHORD domains are associated with CS domains, either within the same protein, as in the mammalian melusin and Chp1, or in separate but interacting proteins, as in the plant Rar1 and Sgt1. Both CHORD and CS domains are independently capable of interacting with the molecular chaperone Hsp90 and can coexist in complexes with Hsp90. We have now determined the structure of an Hsp90-CS-CHORD ternary complex, providing a framework for understanding the dynamic nature of Hsp90-Rar1-Sgt1 complexes. Mutational and biochemical analyses define the architecture of the ternary complex that recruits nucleotide-binding leucine-rich repeat receptors (NLRs) by manipulating the structural elements to control the ATPase-dependent conformational cycle of the chaperone.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Glucosiltransferases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Glucosiltransferases/genética , Proteínas de Choque Térmico HSP90/genética , Peptídeos e Proteínas de Sinalização Intracelular , Complexos Multiproteicos/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Plasma membrane-localized pattern recognition receptors such as FLAGELLIN SENSING2 (FLS2) and EF-TU RECEPTOR (EFR) recognize microbe-associated molecular patterns (MAMPs) to activate the first layer of plant immunity termed pattern-triggered immunity (PTI). A reverse genetics approach with genes responsive to the priming agent ß-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hypersusceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants demonstrated defective PTI responses, notably delayed upregulation of PTI marker genes, lower callose deposition, and mitogen-activated protein kinase activities upon bacterial infection or MAMP treatment. Moreover, Arabidopsis lines overexpressing IOS1 were more resistant to P. syringae and demonstrated a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, coimmunoprecipitation, and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and FLS2 and EFR. IOS1 also associated with BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) in a ligand-independent manner and positively regulated FLS2/BAK1 complex formation upon MAMP treatment. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a regulatory protein of FLS2- and EFR-mediated signaling that primes PTI activation upon bacterial elicitation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/imunologia , Imunidade Vegetal , Proteínas Quinases/metabolismo , Transdução de Sinais , Aminobutiratos/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Expressão Gênica , Leucina/metabolismo , Mutação , Doenças das Plantas/microbiologia , Proteínas Quinases/genética , Pseudomonas syringae/fisiologia , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) that perform a wide range of functions. RbohD and RbohF, two of the 10 Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes including the response to pathogens. We hypothesized that the spatio-temporal control of RbohD and RbohF gene expression might be critical in determining their multiplicity of functions. Transgenic Arabidopsis plants with RbohD and RbohF promoter fusions to ß-glucuronidase and Luciferase reporter genes were generated. Analysis of these plants revealed a differential expression pattern for RbohD and RbohF throughout plant development and during immune responses. RbohD and RbohF gene expression was differentially modulated by pathogen-associated molecular patterns. Histochemical stains and in vivo expression analysis showed a correlation between the level of RbohD and RbohF promoter activity, H2O2 accumulation and the amount of cell death in response to the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Plectosphaerella cucumerina. A promoter-swap strategy revealed that the promoter region of RbohD was required to drive production of ROS by this gene in response to pathogens. Moreover, RbohD promoter was activated during Arabidopsis interaction with a non-virulent P. cucumerina isolate, and susceptibility tests with the double mutant rbohD rbohF uncovered a new function for these oxidases in basal resistance. Altogether, our results suggest that differential spatio-temporal expression of the Rboh genes contributes to fine-tune RBOH/NADPH oxidase-dependent ROS production and signaling in Arabidopsis immunity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , NADPH Oxidases/metabolismo , Imunidade Vegetal/genética , Arabidopsis/embriologia , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Ascomicetos/fisiologia , Genes Reporter , Teste de Complementação Genética , Mutação/genética , NADPH Oxidases/genética , Moléculas com Motivos Associados a Patógenos/metabolismo , Regiões Promotoras Genéticas/genética , Pseudomonas syringae/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Pathogen recognition induces the production of reactive oxygen species (ROS) by NADPH oxidases in both plants and animals. ROS have direct antimicrobial properties, but also serve as signaling molecules to activate further immune outputs. However, ROS production has to be tightly controlled to avoid detrimental effects on host cells, but yet must be produced in the right amount, at the right place and at the right time upon pathogen perception. Plant NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family, which contains 10 members in the model plant Arabidopsis thaliana. The perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) leads to a rapid, specific and strong production of ROS, which is dependent on RBOHD. RBOHD is mainly controlled by Ca(2+) via direct binding to EF-hand motifs and phosphorylation by Ca(2+)-dependent protein kinases. Recent studies have, however, revealed a critical role for a Ca(2+)-independent regulation of RBOHD. The plasma membrane-associated cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE1), which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. Impairment of these phosphorylation events completely abolishes the function of RBOHD in immunity. These results suggest that RBOHD activity is tightly controlled by multilayered regulations. In this review, we summarize recent advances in our understanding of the regulatory mechanisms controlling RBOHD activation.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , NADPH Oxidases/genética , Imunidade Vegetal , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , NADPH Oxidases/imunologia , NADPH Oxidases/metabolismo , Moléculas com Motivos Associados a Patógenos , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
The NLR (nucleotide-binding domain and leucine-rich repeat containing) proteins provide pathogen-sensing systems that are conserved in both plants and animals. They can be activated directly or indirectly by pathogen-derived molecules through mechanisms that remain largely elusive. Studies in plants revealed that the molecular chaperone, HSP90, and its co-chaperones, SGT1 and RAR1, are major stabilizing factors for NLR proteins. More recent work indicates that SGT1 and HSP90 are also required for the function of NLR proteins in mammals, underscoring the evolutionary conservation of innate immune system regulatory mechanisms. Comparative analyses of plant and mammalian NLR proteins, together with recent insights provided by the structure of SGT1-HSP90 complex, have begun to uncover the mechanisms by which immune NLR sensors are regulated.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Choque Térmico HSP90/imunologia , Proteínas de Choque Térmico HSP90/metabolismo , Imunidade Inata/imunologia , Proteínas Adaptadoras de Sinalização NOD/imunologia , Proteínas Adaptadoras de Sinalização NOD/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Glucosiltransferases/imunologia , Glucosiltransferases/metabolismo , Proteínas de Choque Térmico HSP90/química , Imunidade Inata/fisiologia , Mamíferos/imunologia , Mamíferos/metabolismo , Proteínas Adaptadoras de Sinalização NOD/química , Plantas/imunologia , Plantas/metabolismoRESUMO
Calcium-dependent protein kinases (CDPKs) are Ca(2+) sensors that regulate diverse biological processes in plants and apicomplexans. However, how CDPKs discriminate specific substrates in vivo is still largely unknown. Previously, we found that a potato StCDPK5 is dominantly localized to the plasma membrane and activates the plasma membrane NADPH oxidase (RBOH; for respiratory burst oxidase homolog) StRBOHB by direct phosphorylation of the N-terminal region. Here, we report the contribution of the StCDPK5 N-terminal variable (V) domain to activation of StRBOHB in vivo using heterologous expression system in Nicotiana benthamiana. Mutations of N-terminal myristoylation and palmitoylation sites in the V domain eliminated the predominantly plasma membrane localization and the capacity of StCDPK5 to activate StRBOHB in vivo. A tomato SlCDPK2, which also contains myristoylation and palmitoylation sites in its N terminus, phosphorylated StRBOHB in vitro but not in vivo. Functional domains responsible for activation and phosphorylation of StRBOHB were identified by swapping regions for each domain between StCDPK5 and SlCDPK2. The substitution of the V domain of StCDPK5 with that of SlCDPK2 abolished the activation and phosphorylation abilities of StRBOHB in vivo and relocalized the chimeric CDPK to the trans-Golgi network, as observed for SlCDPK2. Conversely, SlCDPK2 substituted with the V domain of StCDPK5 localized to the plasma membrane and activated StRBOHB. These results suggest that the V domains confer substrate specificity in vivo by dictating proper subcellular localization of CDPKs.
Assuntos
Regulação da Expressão Gênica de Plantas , Mutação , NADPH Oxidases/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Microscopia Confocal , Fosforilação , Imunidade Vegetal , Proteínas de Plantas/genética , Proteínas Quinases/genética , Espécies Reativas de Oxigênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Explosão Respiratória , Transdução de Sinais , Solanum tuberosum/enzimologia , Solanum tuberosum/genética , Especificidade por SubstratoRESUMO
Plants rely heavily on receptor-like kinases (RLKs) for perception and integration of external and internal stimuli. The Arabidopsis regulatory leucine-rich repeat RLK (LRR-RLK) BAK1 is involved in steroid hormone responses, innate immunity, and cell death control. Here, we describe the differential regulation of three different BAK1-dependent signaling pathways by a novel allele of BAK1, bak1-5. Innate immune signaling mediated by the BAK1-dependent RKs FLS2 and EFR is severely compromised in bak1-5 mutant plants. However, bak1-5 mutants are not impaired in BR signaling or cell death control. We also show that, in contrast to the RD kinase BRI1, the non-RD kinases FLS2 and EFR have very low kinase activity, and we show that neither was able to trans-phosphorylate BAK1 in vitro. Furthermore, kinase activity for all partners is completely dispensable for the ligand-induced heteromerization of FLS2 or EFR with BAK1 in planta, revealing another pathway specific mechanistic difference. The specific suppression of FLS2- and EFR-dependent signaling in bak1-5 is not due to a differential interaction of BAK1-5 with the respective ligand-binding RK but requires BAK1-5 kinase activity. Overall our results demonstrate a phosphorylation-dependent differential control of plant growth, innate immunity, and cell death by the regulatory RLK BAK1, which may reveal key differences in the molecular mechanisms underlying the regulation of ligand-binding RD and non-RD RKs.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Morte Celular , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Alelos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Hipocótilo/crescimento & desenvolvimento , Imunidade Inata , Fosforilação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Mutação Puntual , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Esteroides/metabolismoRESUMO
Cell-surface receptors play pivotal roles in many biological processes, including immunity, development, and reproduction, across diverse organisms. How cell-surface receptors evolve to become specialised in different biological processes remains elusive. To shed light on the immune-specificity of cell-surface receptors, we analyzed more than 200,000 genes encoding cell-surface receptors from 350 genomes and traced the evolutionary origin of immune-specific leucine-rich repeat receptor-like proteins (LRR-RLPs) in plants. Surprisingly, we discovered that the motifs crucial for co-receptor interaction in LRR-RLPs are closely related to those of the LRR-receptor-like kinase (RLK) subgroup Xb, which perceives phytohormones and primarily governs growth and development. Functional characterisation further reveals that LRR-RLPs initiate immune responses through their juxtamembrane and transmembrane regions, while LRR-RLK-Xb members regulate development through their cytosolic kinase domains. Our data suggest that the cell-surface receptors involved in immunity and development share a common origin. After diversification, their ectodomains, juxtamembrane, transmembrane, and cytosolic regions have either diversified or stabilised to recognise diverse ligands and activate differential downstream responses. Our work reveals a mechanism by which plants evolve to perceive diverse signals to activate the appropriate responses in a rapidly changing environment.