RESUMEN
In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.
Asunto(s)
Ancirinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ácido Salicílico/metabolismo , Ancirinas/genética , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Flagelina/farmacología , Regulación de la Expresión Génica de las Plantas , Glucanos/metabolismo , Interacciones Huésped-Patógeno , Immunoblotting , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Unión Proteica/efectos de los fármacos , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Pseudomonas syringae/fisiología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ácido Salicílico/agonistas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tiadiazoles/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Autoinmunidad , Inmunidad de la Planta , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/microbiología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Autoinmunidad/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Resistencia a la Enfermedad/efectos de los fármacos , Flagelina/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes Dominantes , Genes de Plantas , Células del Mesófilo/citología , Células del Mesófilo/efectos de los fármacos , Células del Mesófilo/metabolismo , Mutación/genética , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Fenotipo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/efectos de los fármacos , Plantas Modificadas Genéticamente , Proteínas Quinasas/genética , Estructura Terciaria de Proteína , Pseudomonas syringae/crecimiento & desarrollo , Pseudomonas syringae/fisiología , Plantones/citología , Plantones/efectos de los fármacos , Plantones/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flagelina/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Solanum lycopersicum/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flagelina/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Proteínas Quinasas/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Flagelina/química , Péptidos/análisis , Proteínas Quinasas/química , Bacterias/química , Ligandos , Péptidos/síntesis química , Péptidos/química , Unión Proteica , Protoplastos/química , Transducción de Señal , Especificidad por SustratoRESUMEN
Plant cells can be sensitized toward a subsequent pathogen attack by avirulent pathogens or by chemicals such as ß-aminobutyric acid (BABA). This process is called priming. Using a reverse genetic approach in Arabidopsis thaliana, we demonstrate that the BABA-responsive L-type lectin receptor kinase-VI.2 (LecRK-VI.2) contributes to disease resistance against the hemibiotrophic Pseudomonas syringae and the necrotrophic Pectobacterium carotovorum bacteria. Accordingly, LecRK-VI.2 mRNA levels increased after bacterial inoculation or treatments with microbe-associated molecular patterns (MAMPs). We also show that LecRK-VI.2 is required for full activation of pattern-triggered immunity (PTI); notably, lecrk-VI.2-1 mutants show reduced upregulation of PTI marker genes, impaired callose deposition, and defective stomatal closure. Overexpression studies combined with genome-wide microarray analyses indicate that LecRK-VI.2 positively regulates the PTI response. LecRK-VI.2 is demonstrated to act upstream of mitogen-activated protein kinase signaling, but independently of reactive oxygen production and Botrytis-induced kinase1 phosphorylation. In addition, complex formation between the MAMP receptor flagellin sensing2 and its signaling partner brassinosteroid insensitive1-associated kinase1 is observed in flg22-treated lecrk-VI.2-1 mutants. LecRK-VI.2 is also required for full BABA-induced resistance and priming of PTI. Our work identifies LecRK-VI.2 as a novel mediator of the Arabidopsis PTI response and provides insight into molecular mechanisms governing priming.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/genética , Inmunidad de la Planta , Proteínas Serina-Treonina Quinasas/inmunología , Aminobutiratos/farmacología , Arabidopsis/enzimología , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ADN Bacteriano/genética , Resistencia a la Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos , Pectobacterium carotovorum/patogenicidad , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Estomas de Plantas/inmunología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pseudomonas syringae/patogenicidad , ARN de Planta/genéticaRESUMEN
Plants and animals use innate immunity as a first defense against pathogens, a costly yet necessary tradeoff between growth and immunity. In Arabidopsis, the regulatory leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1 combines with the LRR-RLKs FLS2 and EFR in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and the LRR-RLK BRI1 in brassinosteroid (BR)-mediated growth. Therefore, a potential tradeoff between these pathways mediated by BAK1 is often postulated. Here, we show a unidirectional inhibition of FLS2-mediated immune signaling by BR perception. Unexpectedly, this effect occurred downstream or independently of complex formation with BAK1 and associated downstream phosphorylation. Thus, BAK1 is not rate-limiting in these pathways. BRs also inhibited signaling triggered by the BAK1-independent recognition of the fungal PAMP chitin. Our results suggest a general mechanism operative in plants in which BR-mediated growth directly antagonizes innate immune signaling.
Asunto(s)
Arabidopsis/inmunología , Arabidopsis/microbiología , Brasinoesteroides/farmacología , Inmunidad de la Planta/efectos de los fármacos , Pseudomonas/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal/efectos de los fármacos , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Activación Enzimática/efectos de los fármacos , Flagelina/farmacología , Inmunidad de la Planta/inmunología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Pseudomonas/efectos de los fármacos , Transducción de Señal/inmunología , Esteroides Heterocíclicos/farmacologíaRESUMEN
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.
Asunto(s)
Arabidopsis/inmunología , Proteínas Bacterianas/fisiología , Citocininas/metabolismo , Resistencia a la Enfermedad , Pseudomonas syringae/fisiología , Arabidopsis/efectos de los fármacos , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/genética , Cromatografía Líquida de Alta Presión , Citocininas/farmacología , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Proteínas Quinasas/metabolismo , Pseudomonas syringae/genética , Transducción de SeñalRESUMEN
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.
Asunto(s)
Plantas/metabolismo , Multimerización de Proteína , Receptores Inmunológicos/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Flagelina/metabolismo , Ligandos , Factor Tu de Elongación Peptídica/metabolismo , Plantas/inmunología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Receptores Inmunológicos/inmunología , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs for the peptide PAMPs elf18 and flg22, which are derived from bacterial EF-Tu and flagellin, respectively. Using coimmunoprecipitation and mass spectrometry analyses, we demonstrated that EFR and FLS2 undergo ligand-induced heteromerization in planta with several LRR receptor-like kinases that belong to the SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1/SERK3 (BAK1/SERK3) and BAK1-LIKE1/SERK4 (BKK1/SERK4). Using a novel bak1 allele that does not exhibit pleiotropic defects in brassinosteroid and cell death responses, we determined that BAK1 and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important layer of plant innate immunity.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/enzimología , Arabidopsis/inmunología , Inmunidad Innata , Oomicetos/patogenicidad , Enfermedades de las Plantas/inmunología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/inmunología , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ligandos , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Oomicetos/inmunología , Péptidos/genética , Péptidos/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Pseudomonas syringae/inmunología , Pseudomonas syringae/patogenicidad , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
The plant's innate immune system detects potential biotic threats through recognition of microbe-associated molecular patterns (MAMPs) or danger-associated molecular patterns (DAMPs) by pattern recognition receptors (PRR). A central regulator of pattern-triggered immunity (PTI) is the BRI1-associated kinase 1 (BAK1), which undergoes complex formation with PRR upon ligand binding. Although viral patterns inducing PTI are well known from animal systems, nothing similar has been reported for plants. Rather, antiviral defense in plants is thought to be mediated by post-transcriptional gene silencing of viral RNA or through effector-triggered immunity, i.e., recognition of virus-specific effectors by resistance proteins. Nevertheless, infection by compatible viruses can also lead to the induction of defense gene expression, indicating that plants may also recognize viruses through PTI. Here, we show that PTI, or at least the presence of the regulator BAK1, is important for antiviral defense of Arabidopsis plants. Arabidopsis bak1 mutants show increased susceptibility to three different RNA viruses during compatible interactions. Furthermore, crude viral extracts but not purified virions induce several PTI marker responses in a BAK1-dependent manner. Overall, we conclude that BAK1-dependent PTI contributes to antiviral resistance in plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/enzimología , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta , Proteínas Serina-Treonina Quinasas/genética , Virus ARN/fisiología , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Etilenos/metabolismo , Interacciones Huésped-Patógeno , Mutación , Enfermedades de las Plantas/virología , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas , Virus de Plantas/aislamiento & purificación , Virus de Plantas/fisiología , Unión Proteica , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Virus ARN/aislamiento & purificación , ARN Viral/genética , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Plantones , Transducción de Señal , Virión/aislamiento & purificación , Virión/fisiologíaRESUMEN
In plant innate immunity, the surface-exposed leucine-rich repeat receptor kinases EFR and FLS2 mediate recognition of the bacterial pathogen-associated molecular patterns EF-Tu and flagellin, respectively. We identified the Arabidopsis stromal-derived factor-2 (SDF2) as being required for EFR function, and to a lesser extent FLS2 function. SDF2 resides in an endoplasmic reticulum (ER) protein complex with the Hsp40 ERdj3B and the Hsp70 BiP, which are components of the ER-quality control (ER-QC). Loss of SDF2 results in ER retention and degradation of EFR. The differential requirement for ER-QC components by EFR and FLS2 could be linked to N-glycosylation mediated by STT3a, a catalytic subunit of the oligosaccharyltransferase complex involved in co-translational N-glycosylation. Our results show that the plasma membrane EFR requires the ER complex SDF2-ERdj3B-BiP for its proper accumulation, and provide a demonstration of a physiological requirement for ER-QC in transmembrane receptor function in plants. They also provide an unexpected differential requirement for ER-QC and N-glycosylation components by two closely related receptors.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Retículo Endoplásmico/metabolismo , Enfermedades de las Plantas/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Inmunidad Innata , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/metabolismoRESUMEN
The bacterial flagellin (FliC) epitopes flg22 and flgII-28 are microbe-associated molecular patterns (MAMPs). Although flg22 is recognized by many plant species via the pattern recognition receptor FLS2, neither the flgII-28 receptor nor the extent of flgII-28 recognition by different plant families is known. Here, we tested the significance of flgII-28 as a MAMP and the importance of allelic diversity in flg22 and flgII-28 in plant-pathogen interactions using purified peptides and a Pseudomonas syringae ∆fliC mutant complemented with different fliC alleles. The plant genotype and allelic diversity in flg22 and flgII-28 were found to significantly affect the plant immune response, but not bacterial motility. The recognition of flgII-28 is restricted to a number of solanaceous species. Although the flgII-28 peptide does not trigger any immune response in Arabidopsis, mutations in both flg22 and flgII-28 have FLS2-dependent effects on virulence. However, the expression of a tomato allele of FLS2 does not confer to Nicotiana benthamiana the ability to detect flgII-28, and tomato plants silenced for FLS2 are not altered in flgII-28 recognition. Therefore, MAMP diversification is an effective pathogen virulence strategy, and flgII-28 appears to be perceived by an as yet unidentified receptor in the Solanaceae, although it has an FLS2-dependent virulence effect in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/microbiología , Flagelina/genética , Genotipo , Inmunidad de la Planta/genética , Proteínas Quinasas/metabolismo , Pseudomonas syringae/patogenicidad , Solanaceae/microbiología , Alelos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiología , Mutación , Enfermedades de las Plantas/genética , Proteínas Quinasas/genética , Pseudomonas syringae/genética , Pseudomonas syringae/fisiología , Solanaceae/genética , Solanaceae/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologíaRESUMEN
Plants sense potential microbial invaders by using pattern-recognition receptors to recognize pathogen-associated molecular patterns (PAMPs). In Arabidopsis thaliana, the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) (ref. 2) and elongation factor Tu receptor (EFR) (ref. 3) act as pattern-recognition receptors for the bacterial PAMPs flagellin and elongation factor Tu (EF-Tu) (ref. 5) and contribute to resistance against bacterial pathogens. Little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. Here we show that BAK1 (BRI1-associated receptor kinase 1), a leucine-rich repeat receptor-like kinase that has been reported to regulate the brassinosteroid receptor BRI1 (refs 6,7), is involved in signalling by FLS2 and EFR. Plants carrying bak1 mutations show normal flagellin binding but abnormal early and late flagellin-triggered responses, indicating that BAK1 acts as a positive regulator in signalling. The bak1-mutant plants also show a reduction in early, but not late, EF-Tu-triggered responses. The decrease in responses to PAMPs is not due to reduced sensitivity to brassinosteroids. We provide evidence that FLS2 and BAK1 form a complex in vivo, in a specific ligand-dependent manner, within the first minutes of stimulation with flagellin. Thus, BAK1 is not only associated with developmental regulation through the plant hormone receptor BRI1 (refs 6,7), but also has a functional role in PRR-dependent signalling, which initiates innate immunity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Flagelina/inmunología , Enfermedades de las Plantas/inmunología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Sitios de Unión , Flagelina/química , Flagelina/metabolismo , Ligandos , Mutación/genética , Factor Tu de Elongación Peptídica/metabolismo , Unión Proteica , Proteínas Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunologíaRESUMEN
Plant innate immunity depends in part on recognition of pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin, EF-Tu, and fungal chitin. Recognition is mediated by pattern-recognition receptors (PRRs) and results in PAMP-triggered immunity. EF-Tu and flagellin, and the derived peptides elf18 and flg22, are recognized in Arabidopsis by the leucine-rich repeat receptor kinases (LRR-RK), EFR and FLS2, respectively. To gain insights into the molecular mechanisms underlying PTI, we investigated EFR-mediated PTI using genetics. A forward-genetic screen for Arabidopsis elf18-insensitive (elfin) mutants revealed multiple alleles of calreticulin3 (CRT3), UDP-glucose glycoprotein glucosyl transferase (UGGT), and an HDEL receptor family member (ERD2b), potentially involved in endoplasmic reticulum quality control (ER-QC). Strikingly, FLS2-mediated responses were not impaired in crt3, uggt, and erd2b null mutants, revealing that the identified mutations are specific to EFR. A crt3 null mutant did not accumulate EFR protein, suggesting that EFR is a substrate for CRT3. Interestingly, Erd2b did not accumulate CRT3 protein, although they accumulate wild-type levels of other ER proteins. ERD2B seems therefore to be a specific HDEL receptor for CRT3 that allows its retro-translocation from the Golgi to the ER. These data reveal a previously unsuspected role of a specific subset of ER-QC machinery components for PRR accumulation in plant innate immunity.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Retículo Endoplásmico/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/fisiología , Alelos , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Secuencia de Bases , Calreticulina/genética , Calreticulina/inmunología , Calreticulina/fisiología , Cartilla de ADN/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/fisiología , Genes de Plantas , Glucosiltransferasas/genética , Glucosiltransferasas/inmunología , Glucosiltransferasas/fisiología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Inmunidad Innata/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/fisiología , Mutación , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Plantas Modificadas Genéticamente , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteínas Quinasas/fisiología , Pseudomonas syringae/inmunología , Pseudomonas syringae/patogenicidad , Transducción de SeñalRESUMEN
N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR(N143Q) lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR(N143Q) in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Inmunidad Innata/fisiología , Receptores de Reconocimiento de Patrones/química , Receptores de Reconocimiento de Patrones/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Biología Computacional , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Glicosilación/efectos de los fármacos , Inmunidad Innata/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Mutación , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/inmunología , Plantas Modificadas Genéticamente/metabolismo , Receptores de Reconocimiento de Patrones/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tunicamicina/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Fosforilación , Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Dimerización , Cinética , Ligandos , Microsomas/metabolismo , Factor Tu de Elongación Peptídica/química , Plantas Modificadas Genéticamente/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal , Transactivadores/químicaRESUMEN
Plasma membrane-borne pattern recognition receptors, which recognize microbe-associated molecular patterns and endogenous damage-associated molecular patterns, provide the first line of defense in innate immunity. In plants, leucine-rich repeat receptor kinases fulfill this role, as exemplified by FLS2 and EFR, the receptors for the microbe-associated molecular patterns flagellin and elongation factor Tu. Here we examined the perception of the damage-associated molecular pattern peptide 1 (AtPep1), an endogenous peptide of Arabidopsis identified earlier and shown to be perceived by the leucine-rich repeat protein kinase PEPR1. Using seedling growth inhibition, elicitation of an oxidative burst and induction of ethylene biosynthesis, we show that wild type plants and the pepr1 and pepr2 mutants, affected in PEPR1 and in its homologue PEPR2, are sensitive to AtPep1, but that the double mutant pepr1/pepr2 is completely insensitive. As a central body of our study, we provide electrophysiological evidence that at the level of the plasma membrane, AtPep1 triggers a receptor-dependent transient depolarization through activation of plasma membrane anion channels, and that this effect is absent in the double mutant pepr1/pepr2. The double mutant also fails to respond to AtPep2 and AtPep3, two distant homologues of AtPep1 on the basis of homology screening, implying that the PEPR1 and PEPR2 are responsible for their perception too. Our findings provide a basic framework to study the biological role of AtPep1-related danger signals and their cognate receptors.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Plantones/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Potenciales de la Membrana/fisiología , Mutación , Receptores de Reconocimiento de Patrones/genética , Plantones/genética , Homología de Secuencia de Aminoácido , Transactivadores/genéticaRESUMEN
Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.
Asunto(s)
Plantas/inmunología , Plantas/microbiología , Polisacáridos Bacterianos/toxicidad , Arabidopsis/inmunología , Arabidopsis/microbiología , Bacterias/patogenicidad , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/inmunología , Inmunidad Innata/efectos de los fármacos , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Plantas/efectos de los fármacos , Virulencia/inmunología , Xanthomonas campestris/patogenicidadRESUMEN
Plants face many biotic and abiotic challenges in nature; one of them is attack by disease-causing microbes. Phytophthora infestans, the causal agent of late blight is one of the most prominent pathogens of the potato responsible for multi-billion-dollar losses every year. We have previously reported that potato-associated Pseudomonas strains inhibited P. infestans at various developmental stages. A comparative genomics approach identified several factors putatively involved in this anti-oomycete activity, among which was the production of hydrogen cyanide (HCN). Here, we report the relative contribution of HCN emission to the overall anti-Phytophthora activity of two cyanogenic Pseudomonas strains, P. putida R32 and P. chlororaphis R47. To quantify this contribution, we generated HCN-negative mutants (Δhcn) and compared their activities to those of their respective wild types in different experiments assessing P. infestans mycelial growth, zoospore germination, and infection of potato leaf disks. Using in vitro experiments allowing only volatile-mediated interactions, we observed that HCN accounted for most of the mycelial growth inhibition (57% in R47 and 80% in R32). However, when allowing both volatile and diffusible compound-mediated interactions, HCN only accounted for 1% (R47) and 18% (R32) of mycelial growth inhibition. Likewise, both mutants inhibited zoospore germination in a similar way as their respective wild types. More importantly, leaf disk experiments showed that both wild-type and Δhcn strains of R47 and R32 were able to limit P. infestans infection to a similar extent. Our results suggest that while HCN is a major contributor to the in vitro volatile-mediated restriction of P. infestans mycelial growth, it does not play a major role in the inhibition of other disease-related features such as zoospore germination or infection of plant tissues.
RESUMEN
(1) Background: S-methyl methanethiosulfonate (MMTS), a sulfur containing volatile organic compound produced by plants and bacterial species, has recently been described to be an efficient anti-oomycete agent with promising perspectives for the control of the devastating potato late blight disease caused by Phytophthora infestans. However, earlier work raised questions regarding the putative toxicity of this compound. To assess the suitability of MMTS for late blight control in the field, the present study thus aimed at evaluating the effect of MMTS on a wide range of non-target organisms in comparison to P. infestans. (2) Methods: To this end, we exposed P. infestans, as well as different pathogenic and non-pathogenic fungi, bacteria, the nematode Caenorhabditis elegans as well as the plant Arabidopsis thaliana to MMTS treatment and evaluated their response by means of in vitro assays. (3) Results: Our results showed that fungi (both mycelium and spores) tolerated MMTS better than the oomycete P. infestans, but that the compound nevertheless exhibited non-negligible toxic effects on bacteria, nematodes and plants. (4) Conclusions: We discuss the mode of action of MMTS and conclude that even though this compound might be too toxic for chemical application in the field, its strong anti-oomycete activity could still be exploited when naturally released at the site of infection by plant-associated microbes inoculated as biocontrol agents.