RESUMO
Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions.
Assuntos
Arabidopsis/imunologia , Bactérias/metabolismo , Membrana Celular/fisiologia , Doenças das Plantas/imunologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Bactérias/classificação , Regulação da Expressão Gênica de Plantas/imunologiaRESUMO
The membrane-bound Brassinosteroid insensitive1-associated receptor kinase1 (BAK1) is a common coreceptor in plants and regulates distinct cellular programs ranging from growth and development to defense against pathogens. BAK1 functions through binding to ligand-stimulated transmembrane receptors and activating their kinase domains via transphosphorylation. In the absence of microbes, BAK1 activity may be suppressed by different mechanisms, like interaction with the regulatory BIR (for BAK1-interacting receptor-like kinase) proteins. Here, we demonstrated that BAK1 overexpression in Arabidopsis (Arabidopsis thaliana) could cause detrimental effects on plant development, including growth arrest, leaf necrosis, and reduced seed production. Further analysis using an inducible expression system showed that BAK1 accumulation quickly stimulated immune responses, even under axenic conditions, and led to increased resistance to pathogenic Pseudomonas syringae pv tomato DC3000. Intriguingly, our study also revealed that the plasma membrane-associated BAK1 ectodomain was sufficient to induce autoimmunity, indicating a novel mode of action for BAK1 in immunity control. We postulate that an excess of BAK1 or its ectodomain could trigger immune receptor activation in the absence of microbes through unbalancing regulatory interactions, including those with BIRs. Consistently, mutation of suppressor of BIR1-1, which encodes an emerging positive regulator of transmembrane receptors in plants, suppressed the effects of BAK1 overexpression. In conclusion, our findings unravel a new role for the BAK1 ectodomain in the tight regulation of Arabidopsis immune receptors necessary to avoid inappropriate activation of immunity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Autoimunidade , Imunidade Vegetal , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/microbiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Autoimunidade/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Resistência à Doença/efeitos dos fármacos , Flagelina/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Dominantes , Genes de Plantas , Células do Mesofilo/citologia , Células do Mesofilo/efeitos dos fármacos , Células do Mesofilo/metabolismo , Mutação/genética , Moléculas com Motivos Associados a Patógenos/metabolismo , Fenótipo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Pseudomonas syringae/crescimento & desenvolvimento , Pseudomonas syringae/fisiologia , Plântula/citologia , Plântula/efeitos dos fármacos , Plântula/metabolismoRESUMO
Peptide signals have emerged as an important class of regulators in cell-to-cell communication in plants. Several families of small, secreted proteins with a conserved C-terminal Pro-rich motif have been identified as functional peptide signals in Arabidopsis thaliana. These proteins are presumed to be trimmed proteolytically and undergo posttranslational modifications, such as hydroxylation of Pro residues and glycosylation, to form mature, bioactive signals. Identification and matching of such ligands with their respective receptors remains a major challenge since the genes encoding them often show redundancy and low expression restricted to a few cells or particular developmental stages. To overcome these difficulties, we propose the use of ectopic expression of receptor genes in suitable plant cells like Nicotiana benthamiana for testing ligand candidates in receptor output assays and in binding studies. As an example, we used the IDA peptide HAE/HSL2 receptor signaling system known to regulate floral organ abscission. We demonstrate that the oxidative burst response can be employed as readout for receptor activation by synthetic peptides and that a new, highly sensitive, nonradioactive labeling approach can be used to reveal a direct correlation between peptide activity and receptor affinity. We suggest that these approaches will be of broad value for the field of ligand-receptor studies in plants.
RESUMO
We characterized the molecular function of the Pseudomonas syringae pv. tomato DC3000 (Pto) effector HopQ1. In silico studies suggest that HopQ1 might possess nucleoside hydrolase activity based on the presence of a characteristic aspartate motif. Transgenic Arabidopsis lines expressing HopQ1 or HopQ1 aspartate mutant variants were characterized with respect to flagellin triggered immunity, phenotype and changes in phytohormone content by high-performance liquid chromatography-MS (HPLC-MS). We found that HopQ1, but not its aspartate mutants, suppressed all tested immunity marker assays. Suppression of immunity was the result of a lack of the flagellin receptor FLS2, whose gene expression was abolished by HopQ1 in a promoter-dependent manner. Furthermore, HopQ1 induced cytokinin signaling in Arabidopsis and the elevation in cytokinin signaling appears to be responsible for the attenuation of FLS2 expression. We conclude that HopQ1 can activate cytokinin signaling and that moderate activation of cytokinin signaling leads to suppression of FLS2 accumulation and thus defense signaling.
Assuntos
Arabidopsis/imunologia , Proteínas de Bactérias/fisiologia , Citocininas/metabolismo , Resistência à Doença , Pseudomonas syringae/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Citocininas/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Quinases/metabolismo , Pseudomonas syringae/genética , Transdução de SinaisRESUMO
Receptor kinases sense extracellular signals and trigger intracellular signaling and physiological responses. However, how does signal binding to the extracellular domain activate the cytoplasmic kinase domain? Activation of the plant immunoreceptor Flagellin sensing2 (FLS2) by its bacterial ligand flagellin or the peptide-epitope flg22 coincides with rapid complex formation with a second receptor kinase termed brassinosteroid receptor1 associated kinase1 (BAK1). Here, we show that the receptor pair of FLS2 and BAK1 is also functional when the roles of the complex partners are reversed by swapping their cytosolic domains. This reciprocal constellation prevents interference by redundant partners that can partially substitute for BAK1 and demonstrates that formation of the heteromeric complex is the molecular switch for transmembrane signaling. A similar approach with swaps between the Elongation factor-Tu receptor and BAK1 also resulted in a functional receptor/coreceptor pair, suggesting that a "two-hybrid-receptor assay" is of more general use for studying heteromeric receptor complexes.
Assuntos
Plantas/metabolismo , Multimerização Proteica , Receptores Imunológicos/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Flagelina/metabolismo , Ligantes , Fator Tu de Elongação de Peptídeos/metabolismo , Plantas/imunologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Receptores Imunológicos/imunologia , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
As part of their immune system, plants have pattern recognition receptors (PRRs) that can detect a broad range of microbe-associated molecular patterns (MAMPs). Here, we identified a PRR of Arabidopsis thaliana with specificity for the bacterial MAMP eMax from xanthomonads. Response to eMax seems to be restricted to the Brassicaceae family and also varied among different accessions of Arabidopsis. In crosses between sensitive accessions and the insensitive accession Shakhdara, eMax perception mapped to receptor-like protein1 (RLP1). Functional complementation of rlp1 mutants required gene constructs that code for a longer version of RLP1 that we termed ReMAX (for receptor of eMax). ReMAX/RLP1 is a typical RLP with structural similarity to the tomato (Solanum lycopersicum) RLP Eix2, which detects fungal xylanase as a MAMP. Attempts to demonstrate receptor function by interfamily transfer of ReMAX to Nicotiana benthamiana were successful after using hybrid receptors with the C-terminal part of ReMAX replaced by that of Eix2. These results show that ReMAX determines specificity for eMax. They also demonstrate hybrid receptor technology as a promising tool to overcome problems that impede interfamily transfer of PRRs to enhance pathogen detection in crop plants.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Xanthomonas/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte/metabolismo , Interações Hospedeiro-Patógeno/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Protoplastos/metabolismo , Homologia de Sequência de Aminoácidos , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologia , Xanthomonas/metabolismo , Xanthomonas/fisiologiaRESUMO
As part of their innate immune system plants carry a number of pattern recognition receptors (PRRs) that can detect a broad range of microbe-associated molecular patterns (MAMPs). In a recently published article (1) we described a novel, proteinaceous MAMP termed eMax (enigmatic MAMP of Xanthomonas) that derives from Xanthomonas and gets recognized by the receptor-like protein ReMAX (RECEPTOR OF eMax) of Arabidopsis thaliana. ReMAX has no ortholog in Nicotiana benthamiana and this species does not respond to eMax even when transformed with ReMAX. However, interfamily transfer of eMax perception was successful with a chimeric form of ReMAX where the C-terminal part of the protein was replaced by the corresponding part of the tomato RLP EIX2 (ETHYLENE INDUCING XYLANASE2). In this addendum we describe the difficulties with the purification and identification of the MAMP eMax and we present data demonstrating that functionality of ReMAX, much like that of related RLPs, depends on the presence of the receptor kinase SOBIR (SUPPRESSOR OF BIR1-1).
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Transporte/metabolismo , Proteínas Quinases/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Xanthomonas/fisiologia , Proteínas de Bactérias/metabolismo , Etilenos/biossíntese , Flagelina/metabolismo , Mutação/genéticaRESUMO
The pattern recognition receptor FLAGELLIN SENSING2 (FLS2) renders plant cells responsive to subnanomolar concentrations of flg22, the active epitope of bacterial flagellin. We recently observed that a preparation of the peptide IDL1, a signal known to regulate abscission processes via the receptor kinases HAESA and HAESA-like2, apparently triggered Arabidopsis thaliana cells in an FLS2-dependent manner. However, closer investigation revealed that this activity was due to contamination by a flg22-type peptide, and newly synthesized IDL1 peptide was completely inactive in FLS2 signaling. This raised alert over contamination events occurring in the process of synthesis or handling of peptides. Two recent reports have suggested that FLS2 has further specificities for structurally unrelated peptides derived from CLV3 and from Ax21. We thus scrutinized these peptides for activity in Arabidopsis cells as well. While responding to <1 nM flg22, Arabidopsis cells proved blind even to 100 µM concentrations of CLV3p and axY(s)22. Our results confirm the exquisite sensitivity and selectivity of FLS2 for flg22. They also show that inadvertent contaminations with flg22-type peptides do occur and can be detected even in trace amounts by FLS2.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Flagelina/química , Peptídeos/análise , Proteínas Quinases/química , Bactérias/química , Ligantes , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica , Protoplastos/química , Transdução de Sinais , Especificidade por SubstratoRESUMO
The flagellin receptor of Arabidopsis thaliana, At-FLAGELLIN SENSING2 (FLS2), has become a model for mechanistic and functional studies on plant immune receptors. Here, we started out with a comparison of At-FLS2 and the orthologous tomato (Solanum lycopersicum) receptor Sl-FLS2. Both receptors specifically responded to picomolar concentrations of the genuine flg22 ligand but proved insensitive to >10(6)-fold higher concentrations of CLV3 peptides that have recently been reported as a second type of ligand for At-FLS2. At-FLS2 and Sl-FLS2 exhibit species-specific differences in the recognition of shortened or sequence-modified flg22 ligands. To map the sites responsible for these species-specific traits on the FLS2 receptors, we performed domain swaps, substituting subsets of the 28 leucine-rich repeats (LRRs) in At-FLS2 with the corresponding LRRs from Sl-FLS2. We found that the LRRs 7 to 10 of Sl-FLS2 determine the high affinity of Sl-FLS2 for the core part RINSAKDD of flg22. In addition, we discovered importance of the LRRs 19 to 24 for the responsiveness to C-terminally modified flagellin peptides. These results indicate that ligand perception in FLS2 is a complex molecular process that involves LRRs from both the outermost and innermost LRRs of the FLS2 ectodomain.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flagelina/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Solanum lycopersicum/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flagelina/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Proteínas Quinases/genéticaRESUMO
Recognition of microbial patterns by host pattern recognition receptors is a key step in immune activation in multicellular eukaryotes. Peptidoglycans (PGNs) are major components of bacterial cell walls that possess immunity-stimulating activities in metazoans and plants. Here we show that PGN sensing and immunity to bacterial infection in Arabidopsis thaliana requires three lysin-motif (LysM) domain proteins. LYM1 and LYM3 are plasma membrane proteins that physically interact with PGNs and mediate Arabidopsis sensitivity to structurally different PGNs from gram-negative and gram-positive bacteria. lym1 and lym3 mutants lack PGN-induced changes in transcriptome activity patterns, but respond to fungus-derived chitin, a pattern structurally related to PGNs, in a wild-type manner. Notably, lym1, lym3, and lym3 lym1 mutant genotypes exhibit supersusceptibility to infection with virulent Pseudomonas syringae pathovar tomato DC3000. Defects in basal immunity in lym3 lym1 double mutants resemble those observed in lym1 and lym3 single mutants, suggesting that both proteins are part of the same recognition system. We further show that deletion of CERK1, a LysM receptor kinase that had previously been implicated in chitin perception and immunity to fungal infection in Arabidopsis, phenocopies defects observed in lym1 and lym3 mutants, such as peptidoglycan insensitivity and enhanced susceptibility to bacterial infection. Altogether, our findings suggest that plants share with metazoans the ability to recognize bacterial PGNs. However, as Arabidopsis LysM domain proteins LYM1, LYM3, and CERK1 form a PGN recognition system that is unrelated to metazoan PGN receptors, we propose that lineage-specific PGN perception systems have arisen through convergent evolution.
Assuntos
Proteínas de Arabidopsis/metabolismo , Bactérias/metabolismo , Peptidoglicano/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Bactérias/crescimento & desenvolvimento , Bactérias/imunologia , Resistência à Doença/genética , Resistência à Doença/imunologia , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Immunoblotting , Microscopia Confocal , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Peptidoglicano/imunologia , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/genética , Pseudomonas syringae/imunologia , Pseudomonas syringae/metabolismo , Pseudomonas syringae/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo , Staphylococcus aureus/fisiologia , TranscriptomaRESUMO
The receptor kinase EFR of Arabidopsis thaliana detects the microbe-associated molecular pattern elf18, a peptide that represents the N terminus of bacterial elongation factor Tu. Here, we tested subdomains of EFR for their importance in receptor function. Transient expression of tagged versions of EFR and EFR lacking its cytoplasmic domain in leaves of Nicotiana benthamiana resulted in functional binding sites for elf18. No binding of ligand was found with the ectodomain lacking the transmembrane domain or with EFR lacking the first 5 of its 21 leucine-rich repeats (LRRs). EFR is structurally related to the receptor kinase flagellin-sensing 2 (FLS2) that detects bacterial flagellin. Chimeric receptors with subdomains of FLS2 substituting for corresponding parts of EFR were tested for functionality in ligand binding and receptor activation assays. Substituting the transmembrane domain and the cytoplasmic domain resulted in a fully functional receptor for elf18. Replacing also the outer juxtamembrane domain with that of FLS2 led to a receptor with full affinity for elf18 but with a lower efficiency in response activation. Extending the substitution to encompass also the last two of the LRRs abolished binding and receptor activation. Substitution of the N terminus by the first six LRRs from FLS2 reduced binding affinity and strongly affected receptor activation. In summary, chimeric receptors allow mapping of subdomains relevant for ligand binding and receptor activation. The results also show that modular assembly of chimeras from different receptors can be used to form functional receptors.
Assuntos
Arabidopsis/metabolismo , Flagelina/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Tu de Elongação de Peptídeos/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Sequência de Aminoácidos , Arabidopsis/microbiologia , Bioquímica/métodos , Regulação da Expressão Gênica de Plantas , Ligantes , Modelos Biológicos , Dados de Sequência Molecular , Estresse Oxidativo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Reprodutibilidade dos TestesRESUMO
In plants leucine-rich repeat receptor kinases (LRR-RKs) located at the plasma membrane play a pivotal role in the perception of extracellular signals. For two of these LRR-RKs, the brassinosteroid receptor BRI1 and the flagellin receptor FLS2, interaction with the LRR receptor-like kinase BAK1 (BRI1-associated receptor kinase 1) was shown to be required for signal transduction. Here we report that FLS2.BAK1 heteromerization occurs almost instantaneously after perception of the ligand, the flagellin-derived peptide flg22. Flg22 can induce formation of a stable FLS2.BAK1 complex in microsomal membrane preparations in vitro, and the kinase inhibitor K-252a does not prevent complex formation. A kinase dead version of BAK1 associates with FLS2 in a flg22-dependent manner but does not restore responsiveness to flg22 in cells of bak1 plants, demonstrating that kinase activity of BAK1 is essential for FLS2 signaling. Furthermore, using in vivo phospholabeling, we are able to detect de novo phosphorylation of both FLS2 and BAK1 within 15 s of stimulation with flg22. Similarly, brassinolide induces BAK1 phosphorylation within seconds. Other triggers of plant defense, such as bacterial EF-Tu and the endogenous AtPep1 likewise induce rapid formation of heterocomplexes consisting of de novo phosphorylated BAK1 and proteins representing the ligand-specific binding receptors EF-Tu receptor and Pep1 receptor 1, respectively. Thus, we propose that several LRR-RKs form tight complexes with BAK1 almost instantaneously after ligand binding and that the subsequent phosphorylation events are key initial steps in signal transduction.
Assuntos
Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Fosforilação , Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Dimerização , Cinética , Ligantes , Microssomos/metabolismo , Fator Tu de Elongação de Peptídeos/química , Plantas Geneticamente Modificadas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Transativadores/químicaRESUMO
In this review we focus on pattern recognition receptors in plants that detect extracellular signals indicative for pathogen attack and injury. We start out with a discussion on FLS2, which binds and responds to bacterial flagellin, and then concentrate on ligand-receptor interactions as initial steps in the molecular receptor activation process. Comparison with other receptor kinases, whether involved in plant immunity or regulation of other cellular programs, might indicate common principles of receptor activation.