RESUMO
Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.
Assuntos
Arabidopsis/imunologia , Proteínas de Bactérias/metabolismo , Imunidade Vegetal , Pseudomonas syringae/metabolismo , Transdução de Sinais , Fatores de Virulência/metabolismo , Arabidopsis/genéticaRESUMO
Multi-layered defense responses are activated in plants upon recognition of invading pathogens. Transmembrane receptors recognize conserved pathogen-associated molecular patterns (PAMPs) and activate MAP kinase cascades, which regulate changes in gene expression to produce appropriate immune responses. For example, Arabidopsis MAP kinase 4 (MPK4) regulates the expression of a subset of defense genes via at least one WRKY transcription factor. We report here that MPK4 is found in complexes in vivo with PAT1, a component of the mRNA decapping machinery. PAT1 is also phosphorylated by MPK4 and, upon flagellin PAMP treatment, PAT1 accumulates and localizes to cytoplasmic processing (P) bodies which are sites for mRNA decay. Pat1 mutants exhibit dwarfism and de-repressed immunity dependent on the immune receptor SUMM2. Since mRNA decapping is a critical step in mRNA turnover, linking MPK4 to mRNA decay via PAT1 provides another mechanism by which MPK4 may rapidly instigate immune responses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica de Plantas/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fitocromo/metabolismo , Transdução de Sinais/imunologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/imunologia , Proteínas de Transporte/imunologia , Clonagem Molecular , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica de Plantas/genética , Genótipo , Immunoblotting , Espectrometria de Massas , Microscopia Confocal , Proteínas Quinases Ativadas por Mitógeno/imunologia , Mutagênese Sítio-Dirigida , Fosforilação , Fitocromo/imunologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , LevedurasRESUMO
Plant immune responses activated through the perception of microbe-associated molecular patterns, leading to pattern-triggered immunity, are tightly regulated. This results in low immune responses in the absence of pathogens and a rapid return to the resting state following an activation event. Here, we show that two CALMODULIN-LIKE genes, CML46 and CML47, negatively regulate salicylic acid accumulation and immunity in Arabidopsis (Arabidopsis thaliana). The double mutant cml46 cml47 is highly resistant to the pathogen Pseudomonas syringae pv maculicola (Pma). The effects of cml46 cml47 on Pma growth are genetically additive to that of cbp60a, a known negative regulator in the CALMODULIN-BINDING PROTEIN60 (CBP60) family. Transcriptome profiling revealed the effects of cbp60a and cml46 cml47 on both common and separate sets of genes, with the majorities of these differentially expressed genes being Pma responsive. CBP60g, a positive regulator of immunity in the CBP60 family, was found to be transcriptionally regulated by CBP60a, CML46, and CML47 Analysis of the flg22-induced mRNA levels of CBP60g in cbp60a and cml46 cml47 revealed that cml46 cml47 plants have higher induced expression while cbp60a plants retain elevated levels longer than wild-type plants. Assays for the effect of flg22 treatment on Pma growth showed that the effect is stronger in cml46 cml47 plants and lasts longer in cbp60a plants. Thus, the expression pattern of CBP60g is reflected in flg22-induced resistance to Pma.
Assuntos
Proteínas de Arabidopsis/genética , Calmodulina/genética , Regulação da Expressão Gênica de Plantas , Mutação , Imunidade Vegetal/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Resistência à Doença/genética , Perfilação da Expressão Gênica , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Pseudomonas syringae/fisiologia , Ácido Salicílico/metabolismoRESUMO
Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-D-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-D-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Pectinas/metabolismo , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Transdução de Sinais , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Botrytis/fisiologia , Parede Celular/metabolismo , Ácidos Hexurônicos/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Pseudomonas syringae/fisiologiaRESUMO
SARD1 is an activator of plant immunity that promotes production of the hormone salicylic acid (SA) and activation of defense gene expression. SARD1 itself is strongly inducible by infection. Here, we investigated the transcriptional control of SARD1. We used yeast one-hybrid assays to identify WRKY70. The WRKY70 binding site was defined using electrophoretic mobility shift assays, and its importance was investigated using an Arabidopsis thaliana protoplast system. The effect of wrky70 mutations was studied by measurements of pathogen growth, SA concentrations, and gene expression by RNA-seq. WRKY70 binds to a GACTTTT motif in the SARD1 promoter in yeast and Arabidopsis protoplasts. Plants with wrky70 mutations have elevated expression of SARD1 in the absence of pathogens, but not when infected. Expression profiling revealed that WRKY70 represses many pathogen-inducible genes in the absence of pathogens, yet is required for activation of many other pathogen-inducible genes in infected plants. The GACTTTT motif is enriched in the promoters of both these gene sets, and conserved in SARD1 orthologs within the Brassicaceae. WRKY70 represses SARD1 by binding the motif GACTTTT in the absence of pathogens. Conservation of the WRKY70 binding among the Brassicaceae suggests that WRKY70 repression of SARD1 is important for fitness.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Cultura Axênica , Imunidade Vegetal , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Sequência de Bases , Sítios de Ligação , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Modelos Biológicos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Pseudomonas syringae/fisiologiaRESUMO
Plant biology is rapidly entering an era where we have the ability to conduct intricate studies that investigate how a plant interacts with the entirety of its environment. This requires complex, large studies to measure how plant genotypes simultaneously interact with a diverse array of environmental stimuli. Successful interpretation of the results from these studies requires us to transition away from the traditional standard of conducting an array of pairwise t tests toward more general linear modeling structures, such as those provided by the extendable ANOVA framework. In this Perspective, we present arguments for making this transition and illustrate how it will help to avoid incorrect conclusions in factorial interaction studies (genotype × genotype, genotype × treatment, and treatment × treatment, or higher levels of interaction) that are becoming more prevalent in this new era of plant biology.
Assuntos
Análise de Variância , Epistasia Genética , Interação Gene-Ambiente , Plantas/genética , Genótipo , Glucosinolatos/metabolismo , Modelos Genéticos , Mutação , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas/metabolismoRESUMO
Clavibacter michiganensis is the most economically important gram-positive bacterial plant pathogen, with subspecies that cause serious diseases of maize, wheat, tomato, potato, and alfalfa. Much less is known about pathogenesis involving gram-positive plant pathogens than is known for gram-negative bacteria. Comparative genome analyses of C. michiganensis subspecies affecting tomato, potato, and maize have provided insights on pathogenicity. In this study, we identified strains of C. michiganensis subsp. insidiosus with contrasting pathogenicity on three accessions of the model legume Medicago truncatula. We generated complete genome sequences for two strains and compared these to a previously sequenced strain and genome sequences of four other subspecies. The three C. michiganensis subsp. insidiosus strains varied in gene content due to genome rearrangements, most likely facilitated by insertion elements, and plasmid number, which varied from one to three depending on strain. The core C. michiganensis genome consisted of 1,917 genes, with 379 genes unique to C. michiganensis subsp. insidiosus. An operon for synthesis of the extracellular blue pigment indigoidine, enzymes for pectin degradation, and an operon for inositol metabolism are among the unique features. Secreted serine proteases belonging to both the pat-1 and ppa families were present but highly diverged from those in other subspecies.
Assuntos
Genoma Bacteriano/genética , Genômica , Medicago truncatula/microbiologia , Micrococcaceae/genética , Doenças das Plantas/microbiologia , Micrococcaceae/isolamento & purificação , Micrococcaceae/patogenicidade , Anotação de Sequência Molecular , Óperon/genética , Filogenia , Plasmídeos/genética , VirulênciaRESUMO
Network robustness is a crucial property of the plant immune signaling network because pathogens are under a strong selection pressure to perturb plant network components to dampen plant immune responses. Nevertheless, modulation of network robustness is an area of network biology that has rarely been explored. While two modes of plant immunity, Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), extensively share signaling machinery, the network output is much more robust against perturbations during ETI than PTI, suggesting modulation of network robustness. Here, we report a molecular mechanism underlying the modulation of the network robustness in Arabidopsis thaliana. The salicylic acid (SA) signaling sector regulates a major portion of the plant immune response and is important in immunity against biotrophic and hemibiotrophic pathogens. In Arabidopsis, SA signaling was required for the proper regulation of the vast majority of SA-responsive genes during PTI. However, during ETI, regulation of most SA-responsive genes, including the canonical SA marker gene PR1, could be controlled by SA-independent mechanisms as well as by SA. The activation of the two immune-related MAPKs, MPK3 and MPK6, persisted for several hours during ETI but less than one hour during PTI. Sustained MAPK activation was sufficient to confer SA-independent regulation of most SA-responsive genes. Furthermore, the MPK3 and SA signaling sectors were compensatory to each other for inhibition of bacterial growth as well as for PR1 expression during ETI. These results indicate that the duration of the MAPK activation is a critical determinant for modulation of robustness of the immune signaling network. Our findings with the plant immune signaling network imply that the robustness level of a biological network can be modulated by the activities of network components.
Assuntos
Proteínas de Arabidopsis/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Imunidade Vegetal/genética , Ácido Salicílico/metabolismo , Transdução de Sinais/genética , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/imunologia , Redes Reguladoras de Genes/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/genética , Fatores de Transcrição/metabolismoRESUMO
Clavibacter michiganensis subspp. michiganensis and sepedonicus cause diseases on solanaceous crops. The genomes of both subspecies encode members of the pat-1 family of putative serine proteases known to function in virulence on host plants and induction of hypersensitive responses (HR) on nonhosts. One gene of this family in C. michiganensis subsp. sepedonicus, chp-7, is required for triggering HR in Nicotiana tabacum. Here, further investigation revealed that mutation of the putative catalytic serine residue at position 232 to threonine abolished the HR induction activity of Chp-7, suggesting that enzymatic activity is required. Purified Chp-7 triggered an HR in N. tabacum leaves in the absence of the pathogen, indicating Chp-7 itself is the HR elicitor from C. michiganensis subsp. sepedonicus. Ectopic expression of chp-7 constructs in N. tabacum leaves revealed that Chp-7 targeted to the apoplast triggered an HR while cytoplasmic Chp-7 did not, indicating that Chp-7 induces the HR in the apoplast of N. tabacum leaves. Chp-7 also induced HR in N. sylvestris, a progenitor of N. tabacum, but not in other Nicotiana species tested. ChpG, a related protein from C. michiganensis subsp. michiganensis, also triggered HR in N. tabacum and N. sylvestris. Unlike Chp-7, ChpG triggered HR in N. clevelandii and N. glutinosa.
Assuntos
Actinobacteria/imunologia , Nicotiana/imunologia , Doenças das Plantas/imunologia , Proteínas/imunologia , Serina Proteases/imunologia , Actinobacteria/genética , Actinobacteria/patogenicidade , Sequência de Aminoácidos , Parede Celular/genética , Parede Celular/imunologia , Interações Hospedeiro-Patógeno/imunologia , Immunoblotting , Dados de Sequência Molecular , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Mutação Puntual , Proteínas/genética , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Serina Proteases/genética , Serina Proteases/metabolismo , Especificidade da Espécie , Nicotiana/classificação , Nicotiana/genética , Nicotiana/microbiologia , Virulência/genética , Virulência/imunologiaRESUMO
In this paper we describe PATTERN-TRIGGERED IMMUNITY (PTI) COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (PCRK1) of Arabidopsis thaliana, an RLCK that is important for defense against the pathogen Pseudomonas syringae pv. maculicola ES4326 (Pma ES4326). We examined defense responses such as bacterial growth, production of reactive oxygen species (ROS) and callose deposition in pcrk1 mutant plants to determine the role of PCRK1 during pathogen infection. Expression of PCRK1 was induced following pathogen infection. Pathogen growth was significantly higher in pcrk1 mutant lines than in wild-type Col-0. Mutant pcrk1 plants showed reduced pattern-triggered immunity (PTI) against Pma ES4326 after pretreatment with peptides derived from flagellin (flg22), elongation factor-Tu (elf18), or an endogenous protein (pep1). Deposition of callose was reduced in pcrk1 plants, indicating a role of PCRK1 in activation of early immune responses. A PCRK1 transgene containing a mutation in a conserved lysine residue important for phosphorylation activity of kinases (K118E) failed to complement a pcrk1 mutant for the Pma ES4326 growth phenotype. Our study shows that PCRK1 plays an important role during PTI and that a conserved lysine residue in the putative kinase domain is important for PCRK1 function.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Imunidade Vegetal , Proteínas Serina-Treonina Quinases/metabolismo , Pseudomonas syringae/fisiologia , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência Conservada , Flagelina/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucanos/metabolismo , Lisina/metabolismo , Dados de Sequência Molecular , Mutação/genética , Imunidade Vegetal/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Pseudomonas syringae/efeitos dos fármacos , Pseudomonas syringae/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/metabolismoRESUMO
Pectins, major components of dicot cell walls, are synthesized in a heavily methylesterified form in the Golgi and are partially deesterified by pectin methylesterases (PMEs) upon export to the cell wall. PME activity is important for the virulence of the necrotrophic fungal pathogen Botrytis cinerea. Here, the roles of Arabidopsis PMEs in pattern-triggered immunity and immune responses to the necrotrophic fungus Alternaria brassicicola and the bacterial hemibiotroph Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) were studied. Plant PME activity increased during pattern-triggered immunity and after inoculation with either pathogen. The increase of PME activity in response to pathogen treatment was concomitant with a decrease in pectin methylesterification. The pathogen-induced PME activity did not require salicylic acid or ethylene signaling, but was dependent on jasmonic acid signaling. In the case of induction by A. brassicicola, the ethylene response factor, but not the MYC2 branch of jasmonic acid signaling, contributed to induction of PME activity, whereas in the case of induction by Pma ES4326, both branches contributed. There are 66 PME genes in Arabidopsis, suggesting extensive genetic redundancy. Nevertheless, selected pme single, double, triple and quadruple mutants allowed significantly more growth of Pma ES4326 than wild-type plants, indicating a role of PMEs in resistance to this pathogen. No decreases in total PME activity were detected in these pme mutants, suggesting that the determinant of immunity is not total PME activity; rather, it is some specific effect of PMEs such as changes in the pattern of pectin methylesterification.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/imunologia , Hidrolases de Éster Carboxílico/metabolismo , Imunidade Vegetal/imunologia , Pseudomonas syringae/fisiologia , Alternaria/patogenicidade , Alternaria/fisiologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Ciclopentanos/metabolismo , Esterificação , Regulação da Expressão Gênica de Plantas , Mutação/genética , Oxilipinas/metabolismo , Pectinas/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Pseudomonas syringae/patogenicidade , Receptores de Reconhecimento de Padrão/metabolismo , Regulação para Cima/genéticaRESUMO
Two members of the eight-member CALMODULIN-BINDING PROTEIN60 (CBP60) gene family, CBP60g and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1 (SARD1), encode positive regulators of plant immunity that promote the production of salicylic acid (SA) and affect the expression of SA-dependent and SA-independent defense genes. Here, we investigated the other six family members in Arabidopsis (Arabidopsis thaliana). Only cbp60a mutations affected growth of the bacterial pathogen Pseudomonas syringae pv maculicola ES4326. In contrast to cbp60g and sard1 mutations, cbp60a mutations reduced pathogen growth, indicating that CBP60a is a negative regulator of immunity. Bacterial growth was increased by cbp60g only in the presence of CBP60a, while the increase in growth due to sard1 was independent of CBP60a, suggesting that the primary function of CBP60g may be to counter the repressive effect of CBP60a. In the absence of pathogen, levels of SA as well as of several SA-dependent and SA-independent pathogen-inducible genes were higher in cbp60a plants than in the wild type, suggesting that the enhanced resistance of cbp60a plants may result from the activation of immune responses prior to pathogen attack. CBP60a bound calmodulin, and the calmodulin-binding domain was defined at the C-terminal end of the protein. Transgenes encoding mutant versions of CBP60a lacking the ability to bind calmodulin failed to complement null cbp60a mutations, indicating that calmodulin-binding ability is required for the immunity-repressing function of CBP60a. Regulation at the CBP60 node involves negative regulation by CBP60a as well as positive regulation by CBP60g and SARD1, providing multiple levels of control over the activation of immune responses.
Assuntos
Arabidopsis/imunologia , Proteínas de Ligação a Calmodulina/metabolismo , Família Multigênica , Imunidade Vegetal , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Epistasia Genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Teste de Complementação Genética , Modelos Biológicos , Mutação/genética , Imunidade Vegetal/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Estrutura Terciária de Proteína , Pseudomonas syringae/crescimento & desenvolvimento , Ácido Salicílico/metabolismo , TransgenesRESUMO
Agrobacterium tumefaciens-mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein-protein interactions. Agrobacterium-mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium-mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)-inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a ß-glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock-pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein-protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium-mediated transient transformation in Arabidopsis thaliana.
Assuntos
Agrobacterium tumefaciens/fisiologia , Arabidopsis/genética , Proteínas de Bactérias/genética , Plantas Geneticamente Modificadas , Transformação Genética/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/microbiologia , Arabidopsis/fisiologia , DNA Bacteriano/genética , Dexametasona/farmacologia , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , Regiões Promotoras Genéticas/genética , Mapeamento de Interação de Proteínas , Pseudomonas syringae/genéticaRESUMO
Resistance gene-mediated immunity confers protection against pathogen infection in a wide range of plants. A genetic screen for Arabidopsis thaliana mutants compromised for recognition of turnip crinkle virus previously identified CRT1, a member of the GHKL ATPase/kinase superfamily. Here, we demonstrate that CRT1 interacts with various resistance proteins from different structural classes, and this interaction is disrupted when these resistance proteins are activated. The Arabidopsis mutant crt1-2 crh1-1, which lacks CRT1 and its closest homolog, displayed compromised resistance to avirulent Pseudomonas syringae and Hyaloperonospora arabidopsidis. Additionally, resistance-associated hypersensitive cell death was suppressed in Nicotiana benthamiana silenced for expression of CRT1 homolog(s). Thus, CRT1 appears to be a general factor for resistance gene-mediated immunity. Since elevation of cytosolic calcium triggered by avirulent P. syringae was compromised in crt1-2 crh1-1 plants, but cell death triggered by Nt MEK2(DD) was unaffected in CRT1-silenced N. benthamiana, CRT1 likely functions at an early step in this pathway. Genome-wide transcriptome analysis led to identification of CRT1-Associated genes, many of which are associated with transport processes, responses to (a)biotic stress, and the endomembrane system. Confocal microscopy and subcellular fractionation revealed that CRT1 localizes to endosome-like vesicles, suggesting a key process in resistance protein activation/signaling occurs in this subcellular compartment.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Endossomos/metabolismo , Doenças das Plantas/genética , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Morte Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Imunidade Inata , Mutagênese Insercional , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Pseudomonas syringae/fisiologia , RNA de Plantas/genética , Nicotiana/genética , Nicotiana/imunologiaRESUMO
Arabidopsis RPS2 is a typical nucleotide-binding leucine-rich repeat resistance protein, which indirectly recognizes the bacterial effector protein AvrRpt2 and thereby activates effector-triggered immunity (ETI). Previously, we identified two hypersensitive induced reaction (AtHIR) proteins, AtHIR1 (At1g09840) and AtHIR2 (At3g01290), as potential RPS2 complex components. AtHIR proteins contain the stomatin/prohibitin/flotillin/HflK/C domain (also known as the prohibitin domain or band 7 domain). In this study, we confirmed that AtHIR1 and AtHIR2 form complexes with RPS2 in Arabidopsis and Nicotiana benthamiana using a pulldown assay and fluorescence resonance energy transfer (FRET) analysis. Arabidopsis has four HIR family genes (AtHIR1-4). All AtHIR proteins could form homo- and hetero-oligomers in vivo and were enriched in membrane microdomains of the plasma membrane. The mRNA levels of all except AtHIR4 were significantly induced by microbe-associated molecular patterns, such as the bacterial flagellin fragment flg22. Athir2-1 and Athir3-1 mutants allowed more growth of Pto DC3000 AvrRpt2, but not Pto DC3000, indicating that these mutations reduce RPS2-mediated ETI but do not affect basal resistance to the virulent strain. Overexpression of AtHIR1 and AtHIR2 reduced growth of Pto DC3000. Taken together, the results show that the AtHIR proteins are physically associated with RPS2, are localized in membrane microdomains, and quantitatively contribute to RPS2-mediated ETI.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Imunidade Inata , Receptores Imunológicos/metabolismo , Agrobacterium tumefaciens , Proteínas de Arabidopsis/imunologia , Microdomínios da Membrana , Ligação Proteica , NicotianaRESUMO
Arabidopsis thaliana calmodulin binding protein 60g (CBP60g) contributes to production of salicylic acid (SA) in response to recognition of microbe-associated molecular patterns (MAMPs) such as flg22, a fragment of bacterial flagellin. Calmodulin binding is required for the function of CBP60g in limiting growth of the bacterial pathogen Pseudomonas syringae pv. maculicola (Pma) ES4326 and activation of SA synthesis. Here, we describe a closely related protein, SARD1. Unlike CBP60g, SARD1 does not bind calmodulin. Growth of Pma ES4326 is enhanced in sard1 mutants. In cbp60g sard1 double mutants, growth of Pma ES4326 is greatly enhanced, and SA levels and expression of PR-1 and SID2 are dramatically reduced. Expression profiling placed the CBP60g/SARD1 node between the PAD4/EDS1 and SA nodes in the defense signaling network, and indicated that CBP60g and SARD1 affect defense responses in addition to SA production. A DNA motif bound by CBP60g and SARD1, GAAATTT, was significantly over-represented in promoters of CBP60g/SARD1-dependent genes, suggesting that expression of these genes is modulated by CBP60g/SARD1 binding. Gene expression patterns showed a stronger effect of cbp60g mutations soon after activation of a defense response, and a stronger effect of sard1 mutations at later times. The results are consistent with a model in which CBP60g and SARD1 comprise a partially redundant protein pair that is required for activation of SA production as well as other defense responses, with CBP60g playing a more important role early during the defense response, and SARD1 to playing a more important role later.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Ácido Salicílico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/genética , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Resistência à Doença , Flagelina/metabolismo , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Interações Hospedeiro-Patógeno , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Motivos de Nucleotídeos , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas , Pseudomonas syringae/patogenicidade , Transdução de SinaisRESUMO
The interaction between the pathogenic ascomycete Alternaria brassicicola and Arabidopsis was investigated by metabolite profiling. The effect of A. brassicicola challenge on metabolite levels was substantial, with nearly 50% of detected compounds undergoing significant changes. Mutations blocking ethylene, jasmonic acid, or ethylene signaling had little effect on metabolite levels. The effects of altering levels of some metabolites were tested by exogenous application during A. brassicicola inoculation. Gamma amino-butyric acid (GABA) or xylitol promoted, while trehalose and ascorbate inhibited, disease severity. GABA promoted, and ascorbate strongly inhibited, fungal growth in culture. Arabidopsis vtc1 and vtc2 mutants, that have low levels of ascorbate, were more susceptible to A. brassicicola. Ascorbate levels declined following A. brassicicola inoculation while levels of dehydroascorbate increased, resulting in a shift of the redox balance between these compounds in the direction of oxidation. These results demonstrate that ascorbate is an important component of resistance to this pathogen.
Assuntos
Alternaria/efeitos dos fármacos , Antioxidantes/farmacologia , Arabidopsis/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Metabolômica , Doenças das Plantas/imunologia , Alternaria/crescimento & desenvolvimento , Alternaria/patogenicidade , Antioxidantes/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácido Ascórbico/metabolismo , Resistência à Doença/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Genótipo , Interações Hospedeiro-Patógeno , Manitol/metabolismo , Mutação , Oxirredução , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Ácido gama-Aminobutírico/farmacologiaRESUMO
Mitogen-activated protein kinases (MAPK) mediate cellular signal transduction during stress responses, as well as diverse growth and developmental processes in eukaryotes. Pathogen infection or treatments with conserved pathogen-associated molecular patterns (PAMPs) such as the bacterial flagellin-derived flg22 peptide are known to activate three Arabidopsis thaliana MAPK: MPK3, MPK4, and MPK6. Several stresses, including flg22 treatment, are known to increase MPK11 expression but activation of MPK11 has not been shown. Here, we show that MPK11 activity can, indeed, be increased through flg22 elicitation. A small-scale microarray for profiling defense-related genes revealed that cinnamyl alcohol dehyrogenase 5 requires MPK11 for full flg22-induced expression. An mpk11 mutant showed increased flg22-mediated growth inhibition but no altered susceptibility to Pseudomonas syringae, Botrytis cinerea, or Alternaria brassicicola. In mpk3, mpk6, or mpk4 backgrounds, MPK11 is required for embryo or seed development or general viability. Although this developmental deficiency in double mutants and the lack of or only subtle mpk11 phenotypes suggest functional MAPK redundancies, comparison with the paralogous MPK4 reveals distinct functions. Taken together, future investigations of MAPK roles in stress signaling should include MPK11 as a fourth PAMP-activated MAPK.