RESUMO
Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.
Assuntos
Proteínas Fúngicas , Oryza , Doenças das Plantas , Fosforilação , Oryza/microbiologia , Oryza/metabolismo , Doenças das Plantas/microbiologia , Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Ascomicetos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteômica , Transdução de SinaisRESUMO
Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly understood. Many effectors in the devastating plant pathogen Phytophthora contain tandem repeats of the "(L)WY" motif, which are structurally conserved but variable in sequences. Here, we discovered a functional module formed by a specific (L)WY-LWY combination in multiple Phytophthora effectors, which efficiently recruits the serine/threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Crystal structure of an effector-PP2A complex shows that the (L)WY-LWY module enables hijacking of the host PP2A core enzyme to form functional holoenzymes. While sharing the PP2A-interacting module at the amino terminus, these effectors possess divergent C-terminal LWY units and regulate distinct sets of phosphoproteins in the host. Our results highlight the appropriation of an essential host phosphatase through molecular mimicry by pathogens and diversification promoted by protein modularity in an effector repertoire.
Assuntos
Monoéster Fosfórico Hidrolases , Phytophthora , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas/metabolismo , Phytophthora/química , Phytophthora/metabolismo , Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Proteína Fosfatase 2/metabolismo , Doenças das PlantasRESUMO
Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/microbiologia , Proteínas de Arabidopsis/química , Proteínas de Bactérias/imunologia , Imunidade Inata , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Pseudomonas fluorescens/metabolismo , Pseudomonas fluorescens/patogenicidade , Pseudomonas syringae/imunologia , Pseudomonas syringae/metabolismo , Nicotiana/imunologia , Nicotiana/microbiologiaRESUMO
Plant immunity is tightly controlled by a complex and dynamic regulatory network, which ensures optimal activation upon detection of potential pathogens. Accordingly, each component of this network is a potential target for manipulation by pathogens. Here, we report that RipAC, a type III-secreted effector from the bacterial pathogen Ralstonia solanacearum, targets the plant E3 ubiquitin ligase PUB4 to inhibit pattern-triggered immunity (PTI). PUB4 plays a positive role in PTI by regulating the homeostasis of the central immune kinase BIK1. Before PAMP perception, PUB4 promotes the degradation of non-activated BIK1, while after PAMP perception, PUB4 contributes to the accumulation of activated BIK1. RipAC leads to BIK1 degradation, which correlates with its PTI-inhibitory activity. RipAC causes a reduction in pathogen-associated molecular pattern (PAMP)-induced PUB4 accumulation and phosphorylation. Our results shed light on the role played by PUB4 in immune regulation, and illustrate an indirect targeting of the immune signalling hub BIK1 by a bacterial effector.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Moléculas com Motivos Associados a Patógenos/metabolismo , Imunidade Vegetal/genética , Doenças das Plantas , Proteínas Serina-Treonina Quinases/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
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
The blast fungus Magnaporthe oryzae gains entry to its host plant by means of a specialized pressure-generating infection cell called an appressorium, which physically ruptures the leaf cuticle1,2. Turgor is applied as an enormous invasive force by septin-mediated reorganization of the cytoskeleton and actin-dependent protrusion of a rigid penetration hypha3. However, the molecular mechanisms that regulate the generation of turgor pressure during appressorium-mediated infection of plants remain poorly understood. Here we show that a turgor-sensing histidine-aspartate kinase, Sln1, enables the appressorium to sense when a critical turgor threshold has been reached and thereby facilitates host penetration. We found that the Sln1 sensor localizes to the appressorium pore in a pressure-dependent manner, which is consistent with the predictions of a mathematical model for plant infection. A Δsln1 mutant generates excess intracellular appressorium turgor, produces hyper-melanized non-functional appressoria and does not organize the septins and polarity determinants that are required for leaf infection. Sln1 acts in parallel with the protein kinase C cell-integrity pathway as a regulator of cAMP-dependent signalling by protein kinase A. Pkc1 phosphorylates the NADPH oxidase regulator NoxR and, collectively, these signalling pathways modulate appressorium turgor and trigger the generation of invasive force to cause blast disease.
Assuntos
Ascomicetos/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Proteínas Fúngicas/metabolismo , Hifas , NADPH Oxidases/metabolismo , Oryza/fisiologiaRESUMO
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
Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry. This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and colocalize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome dataset will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Phytophthora infestans/fisiologia , Proteínas/metabolismo , Vesículas Transportadoras/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas , Proteínas SNARE/metabolismo , Nicotiana/metabolismo , Nicotiana/microbiologiaRESUMO
In plants, nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NLR required for cell death (NRCs), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesised that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 165 bacterial, oomycete, nematode, and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among 5 of the identified suppressors, 1 cyst nematode protein and 1 oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network.
Assuntos
Evolução Biológica , Proteínas de Helminto/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Proteínas NLR/fisiologia , Solanaceae/microbiologia , Morte Celular , Resistência à DoençaRESUMO
Although little is known about how asymmetric cell division in plants is regulated, recent discoveries provide a starting point for exploring the mechanisms underlying this process. These studies reveal parallels with asymmetric division in yeast and animals, but also point to regulated cell expansion as a new mechanism of asymmetric division in plants.
Assuntos
Divisão Celular , Células Vegetais , Animais , Diferenciação Celular , Estômatos de Plantas , Fuso AcromáticoRESUMO
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
Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal ß-strand-and, in particular, a single amino acid polymorphism-underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein's ATG8-interacting motif (AIM). Additional proteomics experiments indicated that the N-terminal ß-strand shapes the broader ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.
Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Imunoprecipitação/métodos , Espectrometria de Massas/métodos , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas/classificação , Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Conformação Proteica em Folha beta , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteômica/métodos , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5.
Assuntos
Lipopolissacarídeos/genética , Lotus/genética , Nodulação/genética , Simbiose/genética , Citoplasma/enzimologia , Fabaceae/genética , Fabaceae/crescimento & desenvolvimento , Fabaceae/microbiologia , Regulação da Expressão Gênica de Plantas/genética , Lotus/crescimento & desenvolvimento , Lotus/microbiologia , Fosfotransferases/genética , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Rhizobium/genética , Rhizobium/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/microbiologia , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Nicotiana/microbiologiaRESUMO
Plants deploy numerous cell surface-localized pattern-recognition receptors (PRRs) to perceive host- and microbe-derived molecular patterns that are specifically released during infection and activate defense responses. The activation of the mitogen-activated protein kinases MPK3, MPK4, and MPK6 (MPK3/4/6) is a hallmark of immune system activation by all known PRRs and is crucial for establishing disease resistance. The MAP kinase kinase kinase (MAPKKK) MEKK1 controls MPK4 activation, but the MAPKKKs responsible for MPK3/6 activation downstream of diverse PRRs and how the perception of diverse molecular patterns leads to the activation of MAPKKKs remain elusive. Here, we show that two highly related MAPKKKs, MAPKKK3 and MAPKKK5, mediate MPK3/6 activation by at least four PRRs and confer resistance to bacterial and fungal pathogens in Arabidopsis thaliana The receptor-like cytoplasmic kinases VII (RLCK VII), which act downstream of PRRs, directly phosphorylate MAPKKK5 Ser-599, which is required for pattern-triggered MPK3/6 activation, defense gene expression, and disease resistance. Surprisingly, MPK6 further phosphorylates MAPKKK5 Ser-682 and Ser-692 to enhance MPK3/6 activation and disease resistance, pointing to a positive feedback mechanism. Finally, MEKK1 Ser-603 is phosphorylated by both RLCK VII and MPK4, which is required for pattern-triggered MPK4 activation. These findings illustrate central mechanisms by which multiple PRRs activate MAPK cascades and disease resistance.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/genética , Fosforilação/fisiologia , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Receptores de Reconhecimento de Padrão/genéticaRESUMO
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
Plant calcium (Ca2+)-dependent protein kinases (CPKs) represent the primary Ca2+-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca2+/CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and was inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+ activation and might also allow CPK28 to remain active when Ca2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming.
Assuntos
Arabidopsis/metabolismo , Cálcio/farmacologia , Calmodulina/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas Quinases/química , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Cinética , Fosforilação/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL-INDUCED CELL DEATH1 (RCD1). We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes. We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)-induced defense genes and alters plant growth responses to light. HaRxL106-mediated suppression of immunity is abolished in RCD1 loss-of-function mutants. We report that RCD1-type proteins are phosphorylated, and we identified Mut9-like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1-interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared with wild-type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling. Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.