RESUMO
Stomatal movement participates in plant immunity by directly affecting the invasion of bacteria, but the genes that regulate stomatal immunity have not been well identified. Here, we characterised the function of the bZIP59 transcription factor from Arabidopsis thaliana, which is constitutively expressed in guard cells. The bzip59 mutant is partially impaired in stomatal closure induced by Pseudomonas syringae pv. tomato strain (Pst) DC3000 and is more susceptible to Pst DC3000 infection. By contrast, the line overexpressing bZIP59 enhances resistance to Pst DC3000 infection. Furthermore, the bzip59 mutant is also partially impaired in stomatal closure induced by flagellin flg22 derived from Pst DC3000, and epistasis analysis revealed that bZIP59 acts upstream of reactive oxygen species (ROS) and nitric oxide (NO) and downstream of salicylic acid signalling in flg22-induced stomatal closure. In addition, the bzip59 mutant showed resistance and sensitivity to Sclerotinia sclerotiorum and Tobacco mosaic virus that do not invade through stomata, respectively. Collectively, our results demonstrate that bZIP59 plays an important role in the stomatal immunity and reveal that the same transcription factor can positively and negatively regulate disease resistance against different pathogens.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição de Zíper de Leucina Básica , Solanum lycopersicum , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Solanum lycopersicum/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal , Estômatos de Plantas/genética , Estômatos de Plantas/imunologia , Pseudomonas syringae/fisiologia , Fatores de Transcrição/genéticaRESUMO
Melatonin (MT) plays important roles in plant disease response, but the mechanisms are largely unknown. Here, we show that MT functions in stomatal immunity in Panax notoginseng and Arabidopsis thaliana. Biochemical analyses showed that MT-induced stomatal closure plays a prominent role in preventing invasion of bacteria Pseudomonas syringe pv. tomato (Pst) DC3000 via activation of mitogen-activated protein kinase (MAPK) and NADPH oxidase-mediated reactive oxygen species production in P. notoginseng. The first putative phytomelatonin receptor 1 (PMTR1) is a plasma membrane protein required for perceiving MT signaling in stomatal closure and activation of MAPK. Biochemical and genetic tests found PMTR1 is essential for flg22- and MT-induced MAPK activation in a heterotrimeric GTP-binding protein Gα subunit GPA1-independent manner. GPA1 functions in the same genetic pathways of FLS2/BAK1 (Flagellin Sensing 2/Brassinosteroid Insensitive 1-associated kinase 1)- as well as PMTR1-mediated flg22 and MT signaling in stomatal closure. The stomata in pmtr1 are insensitive to MT and flg22, but the application of MT induces stomatal closure and reduces the bacterial growth in fls2 and bak1 plants, indicating that PMTR1 might be a downstream signaling component in FLS2- and BAK1-mediated stomatal immunity. In summary, our results (i) demonstrate that phytomelatonin functions in the priming of stomatal immunity and (ii) provide insights into the phytomelatonin signaling transduction pathway.
Assuntos
Arabidopsis/imunologia , Melatonina/administração & dosagem , Panax notoginseng/imunologia , Imunidade Vegetal , Proteínas de Plantas/imunologia , Estômatos de Plantas/imunologia , Pseudomonas syringae/fisiologia , Proteínas de Arabidopsis/imunologiaRESUMO
KEY MESSAGE: We reviewed recent advances related to RIN4, including its involvement in the immune process through posttranslational modifications, PM H+-ATPase activity regulation, interaction with EXO70 and identification of RIN4-associated NLR proteins. RPM1-interacting protein 4 (RIN4) is a conserved plant immunity regulator that has been extensively studied and can be modified by pathogenic effector proteins. RIN4 plays an important role in both PTI and ETI. In this article, we review the functions of the two conserved NOI domains of RIN4, the C-terminal cysteine residues required for membrane localization and the sites targeted and modified by effector proteins during plant immunity. In addition, we discuss the effect of RIN4 on the stomatal virulence of pathogens via the regulation of PM H+-ATPase activity, which is involved in the immune process through interactions with the exocyst subunit EXO70, and progress in the identification of RIN4-related R proteins in multiple species. This review provides new insights enhancing the current understanding of the immune function of RIN4.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Peptídeos e Proteínas de Sinalização Intracelular , Imunidade Vegetal , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Imunidade Vegetal/genética , Estômatos de Plantas/imunologia , Estômatos de Plantas/microbiologiaRESUMO
Stomata regulate gas and water exchange between the plant and external atmosphere, which are vital for photosynthesis and transpiration. Stomata are also the natural entrance for pathogens invading into the apoplast. Therefore, stomata play an important role in plants against pathogens. The pattern recognition receptors (PRRs) locate in guard cells to perceive pathogen/microbe-associated molecular patterns (PAMPs) and trigger a series of plant innate immune responses, including rapid closure of stomata to limit bacterial invasion, which is termed stomatal immunity. Many PRRs involved in stomatal immunity are plasma membrane-located receptor-like protein kinases (RLKs). This review focuses on the current research progress of RLK-mediated signaling pathways involved in stomatal immunity, and discusses questions that need to be addressed in future research.
Assuntos
Imunidade Vegetal , Estômatos de Plantas/enzimologia , Estômatos de Plantas/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Quitina/metabolismo , Modelos Biológicos , Transdução de SinaisRESUMO
Chitin, a fungal microbial-associated molecular pattern, triggers various defence responses in several plant systems. Although it induces stomatal closure, the molecular mechanisms of its interactions with guard cell signalling pathways are unclear. Based on screening of public microarray data obtained from the ATH1 Affymetrix and Arabidopsis eFP browser, we isolated a cDNA encoding a Ras-related nuclear protein 1 AtRAN1. AtRAN1 expression was enriched in guard cells in a manner consistent with involvement in the control of the stomatal movement. AtRAN1 mutation impaired chitin-induced stomatal closure and accumulation of reactive oxygen species and nitric oxide in guard cells. In addition, Atran1 mutant plants exhibited compromised chitin-enhanced plant resistance to both bacterial and fungal pathogens due to changes in defence-related genes. Furthermore, Atran1 mutant plants were hypersensitive to drought stress compared to Col-0 plants, and had lower levels of stress-responsive genes. These data demonstrate a previously uncharacterized signalling role for AtRAN1, mediating chitin-induced signalling.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Quitina/metabolismo , Resistência à Doença/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Doenças das Plantas/imunologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Proteína ran de Ligação ao GTP/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Secas , Proteínas Monoméricas de Ligação ao GTP/genética , Óxido Nítrico/metabolismo , Doenças das Plantas/microbiologia , Estômatos de Plantas/genética , Estômatos de Plantas/imunologia , Estômatos de Plantas/microbiologia , Estômatos de Plantas/fisiologia , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína ran de Ligação ao GTP/genéticaRESUMO
Although untargeted metabolomic approaches hold great promise for global identification of low molecular weight metabolites in biological samples, deep coverage and confident identification of the metabolites remains challenging due to the great diversity and number of chemical structures, especially in plants. Additionally, there is a need to employ a cell-specific research approach to many physiological and biological responses to specific environmental stimuli. Here, we report an untargeted metabolomic method using Arabidopsis thaliana guard cell samples during response to systemic signals of pathogen attack. We employed a new Acquire X MSn data acquisition technology, which uses an iterative fragmentation process to increase level-2 identification of unknown metabolites. We were able to increase the number of identified metabolites and thus the metabolome coverage in Arabidopsis guard cells. This method can be applied to studying metabolomes of other cell types and tissues.
Assuntos
Arabidopsis , Metaboloma , Metabolômica , Imunidade Vegetal , Estômatos de Plantas , Arabidopsis/genética , Arabidopsis/imunologia , Metaboloma/genética , Metaboloma/imunologia , Estômatos de Plantas/genética , Estômatos de Plantas/imunologiaRESUMO
Nonspecific lipidtransfer proteins (nsLTPs), which are small, cysteine-rich proteins, belong to the pathogenesis-related protein family, and several of them act as positive regulators during plant disease resistance. However, the underlying molecular mechanisms of these proteins in plant immune responses are unclear. In this study, a typical nsLTP gene, StLTP10, was identified and functionally analysed in potato. StLTP10 expression was significantly induced by Phytophthora infestans, which causes late blight in potato, and defence-related phytohormones, including abscisic acid (ABA), salicylic acid, and jasmonic acid. Characterization of StLTP10-overexpressing and knockdown lines indicated that StLTP10 positively regulates plant resistance to P. infestans. This resistance was coupled with enhanced expression of reactive oxygen species scavenging- and defence-related genes. Furthermore, we identified that StLTP10 physically interacts with ABA receptor PYL4 and affects its subcellular localization. These two proteins work together to regulate stomatal closure during pathogen infection. Interestingly, we also found that wound-induced protein kinase interacts with StLTP10 and positively regulates its protein abundance. Taken together, our results provide insight into the role of StLTP10 in resistance to P. infestans and suggest candidates to enhance broad-spectrum resistance to pathogens in potato.
Assuntos
Proteínas de Transporte/metabolismo , Resistência à Doença/genética , Phytophthora infestans/fisiologia , Doenças das Plantas/imunologia , Solanum tuberosum/genética , Ácido Abscísico/metabolismo , Proteínas de Transporte/genética , Doenças das Plantas/parasitologia , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estômatos de Plantas/genética , Estômatos de Plantas/imunologia , Estômatos de Plantas/parasitologia , Ácido Salicílico/metabolismo , Solanum tuberosum/imunologia , Solanum tuberosum/parasitologiaRESUMO
Systemic Acquired Resistance (SAR) improves immunity of plant systemic tissue after local exposure to a pathogen. Guard cells that form stomatal pores on leaf surfaces recognize bacterial pathogens via pattern recognition receptors, such as Flagellin Sensitive 2 (FLS2). However, how SAR affects stomatal immunity is not known. In this study, we aim to reveal molecular mechanisms underlying the guard cell response to SAR using multi-omics of proteins, metabolites and lipids. Arabidopsis plants previously exposed to pathogenic bacteria Pseudomonas syringae pv. tomato DC3000 (Pst) exhibit an altered stomatal response compared to control plants when they are later exposed to the bacteria. Reduced stomatal apertures of SAR primed plants lead to decreased number of bacteria in leaves. Multi-omics has revealed molecular components of SAR response specific to guard cells functions, including potential roles of reactive oxygen species (ROS) and fatty acid signaling. Our results show an increase in palmitic acid and its derivative in the primed guard cells. Palmitic acid may play a role as an activator of FLS2, which initiates stomatal immune response. Improved understanding of how SAR signals affect stomatal immunity can aid biotechnology and marker-based breeding of crops for enhanced disease resistance.
Assuntos
Arabidopsis/imunologia , Resistência à Doença/imunologia , Lipidômica , Metabolômica , Doenças das Plantas/imunologia , Estômatos de Plantas/metabolismo , Proteoma/metabolismo , Pseudomonas syringae/crescimento & desenvolvimento , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Cromatografia Líquida , Ácidos Graxos/metabolismo , Espectrometria de Massas , Ácidos Palmíticos/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Estômatos de Plantas/imunologia , Estômatos de Plantas/microbiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pseudomonas syringae/imunologia , Pseudomonas syringae/patogenicidade , Espécies Reativas de Oxigênio/metabolismoRESUMO
MAIN CONCLUSION: Dynamic protein and phosphoprotein profiles uncovered the overall regulation of stomata movement against pathogen invasion and phosphorylation states of proteins involved in ABA, SA, calcium and ROS signaling, which may modulate the stomatal immune response. Stomatal openings represent a major route of pathogen entry into the plant, and plants have evolved mechanisms to regulate stomatal aperture as innate immune response against bacterial invasion. However, the mechanisms underlying stomatal immunity are not fully understood. Taking advantage of high-throughput liquid chromatography mass spectrometry (LC-MS), we performed label-free proteomic and phosphoproteomic analyses of enriched guard cells in response to a bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In total, 495 proteins and 1229 phosphoproteins were identified as differentially regulated. These proteins are involved in a variety of signaling pathways, including abscisic acid and salicylic acid hormone signaling, calcium and reactive oxygen species signaling. We also showed that dynamic changes of phosphoprotein WRKY transcription factors may play a crucial role in regulating stomata movement in plant immunity. The identified proteins/phosphoproteins and the pathways form interactive molecular networks to regulate stomatal immunity. This study has provided new insights into the multifaceted mechanisms of stomatal immunity. The differential proteins and phosphoproteins are potential targets for engineering or breeding of crops for enhanced pathogen defense.
Assuntos
Arabidopsis , Estômatos de Plantas , Proteômica , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Plantas/genética , Estômatos de Plantas/genética , Estômatos de Plantas/imunologia , Estômatos de Plantas/microbiologia , Pseudomonas syringae/fisiologiaRESUMO
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
Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In Arabidopsis, CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca2+, and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca2+ but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death.
Assuntos
Quitina/metabolismo , Estômatos de Plantas/imunologia , Estômatos de Plantas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Quitina/fisiologia , Quitosana/metabolismo , Fungos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Fusarium head blight (FHB) is one of the most devastating fungal diseases affecting grain crops and Fusarium graminearum is the most aggressive causal species. Several evidences shown that stomatal closure is involved in the first line of defence against plant pathogens. However, there is very little evidence to show that photosynthetic parameters change in inoculated plants. The aim of the present study was to study the role of stomatal regulation in wheat after F. graminearum inoculation and explore its possible involvement in FHB resistance. RT-qPCR revealed that genes involved in stomatal regulation are induced in the resistant Sumai3 cultivar but not in the susceptible Rebelde cultivar. Seven genes involved in the positive regulation of stomatal closure were up-regulated in Sumai3, but it is most likely, that two genes, TaBG and TaCYP450, involved in the negative regulation of stomatal closure, were strongly induced, suggesting that FHB response is linked to cross-talk between the genes promoting and inhibiting stomatal closure. Increasing temperature of spikes in the wheat genotypes and a decrease in photosynthetic efficiency in Rebelde but not in Sumai3, were observed, confirming the hypothesis that photosynthetic parameters are related to FHB resistance.
Assuntos
Resistência à Doença/genética , Fusariose/imunologia , Fotossíntese , Estômatos de Plantas , Triticum/imunologia , Fusarium/imunologia , Fusarium/patogenicidade , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Fotossíntese/genética , Fotossíntese/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Estômatos de Plantas/genética , Estômatos de Plantas/imunologia , Estômatos de Plantas/fisiologia , Triticum/genética , Triticum/microbiologiaRESUMO
Upon recognition of microbes, pattern recognition receptors (PRRs) activate pattern-triggered immunity. FLAGELLIN SENSING2 (FLS2) and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) form a typical PRR complex that senses bacteria. Here, we report that the kinase activity of the malectin-like receptor-like kinase STRESS INDUCED FACTOR 2 (SIF2) is critical for Arabidopsis (Arabidopsis thaliana) resistance to bacteria by regulating stomatal immunity. SIF2 physically associates with the FLS2-BAK1 PRR complex and interacts with and phosphorylates the guard cell SLOW ANION CHANNEL1 (SLAC1), which is necessary for abscisic acid (ABA)-mediated stomatal closure. SIF2 is also required for the activation of ABA-induced S-type anion currents in Arabidopsis protoplasts, and SIF2 is sufficient to activate SLAC1 anion channels in Xenopus oocytes. SIF2-mediated activation of SLAC1 depends on specific phosphorylation of Ser 65. This work reveals that SIF2 functions between the FLS2-BAK1 initial immunity receptor complex and the final actuator SLAC1 in stomatal immunity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Histona Desacetilases/metabolismo , Proteínas de Membrana/metabolismo , Estômatos de Plantas/imunologia , Proteínas Repressoras/metabolismo , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Animais , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Resistência à Doença/fisiologia , Feminino , Histona Desacetilases/genética , Histona Desacetilases/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Mutação , Oócitos/fisiologia , Fosforilação , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Estômatos de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Serina/metabolismo , XenopusRESUMO
KEY MESSAGE: Arabidopsis LONG-CHAIN BASE KINASE 1 (LCBK1) interacts with MEDEA, a component of PCR2 complex that negatively regulates immunity. LCBK1 phosphorylates phytosphingosine and thereby promotes stomatal immunity against bacterial pathogens. Arabidopsis polycomb-group repressor complex2 (PRC2) protein MEDEA (MEA) suppresses both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). MEA represses the expression of RPS2 and thereby attenuates AvrRpt2 effector-mediated ETI. However, the mechanism of MEA-mediated PTI diminution was not known. By screening the Arabidopsis cDNA library using yeast-2-hybrid interaction, we identified LONG-CHAIN BASE KINASE1 (LCBK1) as an MEA-interacting protein. We found that lcbk1 mutants are susceptible to virulent bacterial pathogens, such as Pseudomonas syringae pv maculicola (Psm) and P. syringae pv tomato (Pst) but not the avirulent strain of Pst that carries AvrRpt2 effector. Pathogen inoculation induces LCBK1 expression, especially in guard cells. We found that LCBK1 has a positive regulatory role in stomatal closure after pathogen inoculation. WT plants close stomata within an hour of Pst inoculation or flg22 (a 22 amino acid peptide from bacterial flagellin protein that activates PTI) treatment, but not lcbk1 mutants. LCBK1 phosphorylates phytosphingosine (PHS). Exogenous application of phosphorylated PHS (PHS-P) induces stomatal closure and rescues loss-of-PTI phenotype of lcbk1 mutant plants. MEA overexpressing (MEA-Oex) plants are defective, whereas loss-of-function mea-6 mutants are hyperactive in PTI-induced stomatal closure. Exogenous application of PHS-P rescues loss-of-PTI in MEA-Oex plants. Results altogether demonstrate that LCBK1 is an interactor of MEA that positively regulates PTI-induced stomatal closure in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Fosfotransferases/metabolismo , Estômatos de Plantas/imunologia , Arabidopsis/enzimologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Fosfotransferases/genética , Doenças das Plantas/imunologia , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Phytohormones and redox metabolites are important molecules in a number of biological processes related to plant growth, development, and stress responses. Understanding how these metabolites are involved in abiotic and biotic stress is a frequent topic of plant biology research. However, many factors, such as low physiological concentrations and the inherent complexity of plant samples, make identification and quantification of these important metabolites difficult. Here, we describe a method for metabolite extraction from whole leaves and guard cell-enriched samples and a targeted metabolomics strategy for the identification and quantification of specific hormone- and redox-related metabolites. In our experiment, we used the reference plant Arabidopsis thaliana infected with the biotrophic pathogen Pseudomonas syringe pv. tomato (Pst) DC3000, and examined the changes in hormone and redox metabolites in systemic leaves, using the targeted metabolomics strategy in order to investigate potential functions of these metabolites in systemic acquired resistance (SAR) during a plant's immune responses. The methods reported here can be expanded to other metabolites and other biological systems beyond plants and bacterial pathogens.
Assuntos
Metaboloma , Metabolômica , Oxirredução , Reguladores de Crescimento de Plantas/metabolismo , Imunidade Vegetal , Estômatos de Plantas/imunologia , Estômatos de Plantas/metabolismo , Análise de Dados , Resistência à Doença/imunologia , Espectrometria de Massas , Metabolômica/métodos , Especificidade de Órgãos , Desenvolvimento Vegetal , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologiaRESUMO
Calmodulin (CaM) regulates plant disease responses through its downstream calmodulin-binding proteins (CaMBPs) often by affecting the biosynthesis or signaling of phytohormones, such as jasmonic acid (JA) and salicylic acid. However, how these CaMBPs mediate plant hormones and other stress resistance-related signaling remains largely unknown. In this study, we conducted analyses in Arabidopsis (Arabidopsis thaliana) on the functions of AtIQM1 (IQ-Motif Containing Protein1), a Ca2+-independent CaMBP, in JA biosynthesis and defense against the necrotrophic pathogen Botrytis cinerea using molecular, biochemical, and genetic analyses. IQM1 directly interacted with and promoted CATALASE2 (CAT2) expression and CAT2 enzyme activity and indirectly increased the activity of the JA biosynthetic enzymes ACX2 and ACX3 through CAT2, thereby positively regulating JA content and B. cinerea resistance. In addition, in vitro assays showed that in the presence of CaM5, IQM1 further enhanced the activity of CAT2, suggesting that CaM5 may affect the activity of CAT2 by combining with IQM1 in the absence of Ca2+ Our data indicate that IQM1 is a key regulatory factor in signaling of plant disease responses mediated by JA. The study also provides new insights that CaMBP may play a critical role in the cross talk of multiple signaling pathways in the context of plant defense processes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Botrytis/fisiologia , Proteínas de Ligação a Calmodulina/metabolismo , Doenças das Plantas/imunologia , Reguladores de Crescimento de Plantas/metabolismo , Motivos de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Sinalização do Cálcio , Proteínas de Ligação a Calmodulina/genética , Ciclopentanos/metabolismo , Resistência à Doença , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Estômatos de Plantas/enzimologia , Estômatos de Plantas/genética , Estômatos de Plantas/imunologia , Estômatos de Plantas/microbiologia , Ácido Salicílico/metabolismoRESUMO
General Control Non-derepressible 2 (GCN2) is an evolutionarily conserved serine/threonine kinase that modulates amino acid homeostasis in response to nutrient deprivation in yeast, human and other eukaryotes. However, the GCN2 signaling pathway in plants remains largely unknown. Here, we demonstrate that in Arabidopsis, bacterial infection activates AtGCN2-mediated phosphorylation of eIF2α and promotes TBF1 translational derepression. Consequently, TBF1 regulates a subset of abscisic acid signaling components to modulate pre-invasive immunity. We show that GCN2 fine-tunes abscisic acid accumulation and signaling during both pre-invasive and post-invasive stages of an infection event. Finally, we also demonstrate that AtGCN2 participates in signaling triggered by phytotoxin coronatine secreted by P. syringae. During the preinvasive phase, AtGCN2 regulates stomatal immunity by affecting pathogen-triggered stomatal closure and coronatine-mediated stomatal reopening. Our conclusions support a conserved role of GCN2 in various forms of immune responses across kingdoms, highlighting GCN2's importance in studies on both plant and mammalian immunology.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Doenças das Plantas/microbiologia , Estômatos de Plantas/enzimologia , Proteínas Quinases/metabolismo , Pseudomonas syringae/patogenicidade , Arabidopsis/imunologia , Arabidopsis/microbiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Homeostase , Interações Hospedeiro-Patógeno , Fosforilação , Doenças das Plantas/imunologia , Estômatos de Plantas/imunologia , Estômatos de Plantas/microbiologia , Pseudomonas syringae/imunologia , Transdução de SinaisRESUMO
Plants have evolved an array of responses that provide them with protection from attack by microorganisms and other predators. Many of these mechanisms depend upon interactions between the plant hormones jasmonate (JA) and ethylene (ET). However, the molecular basis of these interactions is insufficiently understood. Gene expression and physiological assays with mutants were performed to investigate the role of Arabidopsis BIG gene in stress responses. BIG transcription is downregulated by methyl JA (MeJA), necrotrophic infection or mechanical injury. BIG deficiency promotes JA-dependent gene induction, increases JA production but restricts the accumulation of both ET and salicylic acid. JA-induced anthocyanin accumulation and chlorophyll degradation are enhanced and stomatal immunity is impaired by BIG disruption. Bacteria- and lipopolysaccaride (LPS)-induced stomatal closure is reduced in BIG gene mutants, which are hyper-susceptible to microbial pathogens with different lifestyles, but these mutants are less attractive to phytophagous insects. Our results indicate that BIG negatively and positively regulate the MYC2 and ERF1 arms of the JA signalling pathway. BIG warrants recognition as a new and distinct regulator that regulates JA responses, the synergistic interactions of JA and ET, and other hormonal interactions that reconcile the growth and defense dilemma in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Imunidade Vegetal , Estômatos de Plantas/imunologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a Calmodulina/genética , Regulação para Baixo/genética , Etilenos , Regulação da Expressão Gênica de Plantas , Mutação/genética , Ácido Salicílico/metabolismoRESUMO
The N-end rule pathway is a highly conserved constituent of the ubiquitin proteasome system, yet little is known about its biological roles. Here we explored the role of the N-end rule pathway in the plant immune response. We investigated the genetic influences of components of the pathway and known protein substrates on physiological, biochemical and metabolic responses to pathogen infection. We show that the glutamine (Gln) deamidation and cysteine (Cys) oxidation branches are both components of the plant immune system, through the E3 ligase PROTEOLYSIS (PRT)6. In Arabidopsis thaliana Gln-specific amino-terminal (Nt)-amidase (NTAQ1) controls the expression of specific defence-response genes, activates the synthesis pathway for the phytoalexin camalexin and influences basal resistance to the hemibiotroph pathogen Pseudomonas syringae pv tomato (Pst). The Nt-Cys ETHYLENE RESPONSE FACTOR VII transcription factor substrates enhance pathogen-induced stomatal closure. Transgenic barley with reduced HvPRT6 expression showed enhanced resistance to Ps. japonica and Blumeria graminis f. sp. hordei, indicating a conserved role of the pathway. We propose that that separate branches of the N-end rule pathway act as distinct components of the plant immune response in flowering plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Doenças das Plantas/imunologia , Imunidade Vegetal , Pseudomonas syringae/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Ascomicetos/fisiologia , Etilenos/metabolismo , Hordeum/genética , Hordeum/imunologia , Hordeum/microbiologia , Oxirredução , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Plantas/metabolismo , Estômatos de Plantas/genética , Estômatos de Plantas/imunologia , Estômatos de Plantas/microbiologia , Proteólise , Ubiquitina-Proteína Ligases/genéticaRESUMO
Stomatal immunity restricts bacterial entry to leaves through the recognition of microbe-associated molecular patterns (MAMPs) by pattern-recognition receptors (PRRs) and downstream abscisic acid and salicylic acid signaling. Through a reverse genetics approach, we characterized the function of the L-type lectin receptor kinase-V.2 (LecRK-V.2) and -VII.1 (LecRK-VII.1). Analyses of interactions with the PRR FLAGELLIN SENSING2 (FLS2) were performed by co-immunoprecipitation and bimolecular fluorescence complementation and whole-cell patch-clamp analyses were used to evaluate guard cell Ca2+ -permeable cation channels. The Arabidopsis thaliana LecRK-V.2 and LecRK-VII.1 and notably their kinase activities were required for full activation of stomatal immunity. Knockout lecrk-V.2 and lecrk-VII.1 mutants were hyper-susceptible to Pseudomonas syringae infection and showed defective stomatal closure in response to bacteria or to the MAMPs flagellin and EF-Tu. By contrast, Arabidopsis over-expressing LecRK-V.2 or LecRK-VII.1 demonstrated a potentiated stomatal immunity. LecRK-V.2 and LecRK-VII.1 are shown to be part of the FLS2 PRR complex. In addition, LecRK-V.2 and LecRK-VII.1 were critical for methyl jasmonate (MeJA)-mediated stomatal closure, notably for MeJA-induced activation of guard cell Ca2+ -permeable cation channels. This study highlights the role of LecRK-V.2 and LecRK-VII.1 in stomatal immunity at the FLS2 PRR complex and in MeJA-mediated stomatal closure.