RESUMEN
Perception of microbes by plants leads to dynamic reprogramming of the transcriptome, which is essential for plant health. The appropriate amplitude of this transcriptional response can be regulated at multiple levels, including chromatin. However, the mechanisms underlying the interplay between chromatin remodeling and transcription dynamics upon activation of plant immunity remain poorly understood. Here, we present evidence that activation of plant immunity by bacteria leads to nucleosome repositioning, which correlates with altered transcription. Nucleosome remodeling follows distinct patterns of nucleosome repositioning at different loci. Using a reverse genetic screen, we identify multiple chromatin remodeling ATPases with previously undescribed roles in immunity, including EMBRYO SAC DEVELOPMENT ARREST 16, EDA16. Functional characterization of the immune-inducible chromatin remodeling ATPase EDA16 revealed a mechanism to negatively regulate immunity activation and limit changes in redox homeostasis. Our transcriptomic data combined with MNase-seq data for EDA16 functional knock-out and over-expressor mutants show that EDA16 selectively regulates a defined subset of genes involved in redox signaling through nucleosome repositioning. Thus, collectively, chromatin remodeling ATPases fine-tune immune responses and provide a previously uncharacterized mechanism of immune regulation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Ensamble y Desensamble de Cromatina/inmunología , ADN Helicasas/metabolismo , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/genética , Pseudomonas syringae/inmunología , Adenosina Trifosfatasas/genética , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Cromatina/genética , ADN Helicasas/genética , Homeostasis , Nucleosomas/genética , Oxidación-Reducción , Estrés Oxidativo , Enfermedades de las Plantas/microbiología , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/fisiologíaRESUMEN
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, At-FLAGELLIN SENSING 2 (FLS2), has become a model for mechanistic and functional studies on plant immune receptors. Responses to flagellin or its active epitope flagellin 22 (flg22) have been extensively studied in Arabidopsis leaves. However, the perception of microbe-associated molecular patterns (MAMPs) and the immune responses in roots are poorly understood. Here, we show that isolated root tissue is able to induce pattern-triggered immunity (PTI) responses upon flg22 perception, in contrast to elf18 (the active epitope of elongation factor thermo unstable (EF-Tu)). Making use of fls2 mutant plants and tissue-specific promoters, we generated transgenic Arabidopsis lines expressing FLS2 only in certain root tissues. This allowed us to study the spatial requirements for flg22 responses in the root. Remarkably, the intensity of the immune responses did not always correlate with the expression level of the FLS2 receptor, but depended on the expressing tissue, supporting the idea that MAMP perception and sensitivity in different tissues contribute to a proper balance of defense responses according to the expected exposure to elicitors. In summary, we conclude that each investigated root tissue is able to perceive flg22 if FLS2 is present and that tissue identity is a major element of MAMP perception in roots.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/inmunología , Enfermedades de las Plantas/inmunología , Raíces de Plantas/inmunología , Proteínas Quinasas/genética , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Epítopos/inmunología , Flagelina/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Especificidad de Órganos , Fosforilación , Enfermedades de las Plantas/microbiología , Raíces de Plantas/citología , Raíces de Plantas/genética , Plantas Modificadas Genéticamente , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
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
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
Plants have benefited from interactions with symbionts for coping with challenging environments since the colonisation of land. The mechanisms of symbiont-mediated beneficial effects and similarities and differences to pathogen strategies are mostly unknown. Here, we use 106 (effector-) proteins, secreted by the symbiont Serendipita indica (Si) to modulate host physiology, to map interactions with Arabidopsis thaliana host proteins. Using integrative network analysis, we show significant convergence on target-proteins shared with pathogens and exclusive targeting of Arabidopsis proteins in the phytohormone signalling network. Functional in planta screening and phenotyping of Si effectors and interacting proteins reveals previously unknown hormone functions of Arabidopsis proteins and direct beneficial activities mediated by effectors in Arabidopsis. Thus, symbionts and pathogens target a shared molecular microbe-host interface. At the same time Si effectors specifically target the plant hormone network and constitute a powerful resource for elucidating the signalling network function and boosting plant productivity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Reguladores del Crecimiento de las Plantas , Transducción de Señal , Proteínas de Arabidopsis/genética , HormonasRESUMEN
In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen recognition and activation of innate immunity. In plants, NLRs recognise pathogen-derived effector proteins and initiate effector-triggered immunity (ETI). However, the molecular mechanisms that link NLR-mediated effector recognition and downstream signalling are not fully understood. By exploiting the well-characterised tomato Prf/Pto NLR resistance complex, we identified the 14-3-3 proteins TFT1 and TFT3 as interacting partners of both the NLR complex and the protein kinase MAPKKKα. Moreover, we identified the helper NRC proteins (NLR-required for cell death) as integral components of the Prf /Pto NLR recognition complex. Notably our studies revealed that TFTs and NRCs interact with distinct modules of the NLR complex and, following effector recognition, dissociate facilitating downstream signalling. Thus, our data provide a mechanistic link between activation of immune receptors and initiation of downstream signalling cascades.
Asunto(s)
Solanum lycopersicum , Animales , Proteínas , Transducción de Señal , Inmunidad Innata , Plantas/metabolismo , Receptores Inmunológicos , Inmunidad de la Planta , Proteínas de Plantas/metabolismo , Enfermedades de las PlantasRESUMEN
The wide host range of Xylella fastidiosa (Xf) indicates the existence of yet uncharacterized virulence mechanisms that help pathogens to overcome host defences. Various bioinformatics tools combined with prediction of the functions of putative virulence proteins are valuable approaches to study microbial pathogenicity. We collected a number of putative effectors from three Xf strains belonging to different subspecies: Temecula-1 (subsp. fastidiosa), CoDiRO (subsp. pauca), and Ann-1 (subsp. sandyi). We designed an in planta Agrobacterium-based expression system that drives the expressed proteins to the cell apoplast, in order to investigate their ability to activate defence in Nicotiana model plants. Multiple Xf proteins differentially elicited cell death-like phenotypes in different Nicotiana species. These proteins are members of different enzymatic groups: (a) hydrolases/hydrolase inhibitors, (b) serine proteases, and (c) metal transferases. We also classified the Xf proteins according to their sequential and structural similarities via the I-TASSER online tool. Interestingly, we identified similar proteins that were able to differentially elicit cell death in different cultivars of the same species. Our findings provide a basis for further studies on the mechanisms that underlie both defence activation in Xf resistant hosts and pathogen adaptation in susceptible hosts.
Asunto(s)
Nicotiana , Xylella , Muerte Celular , Enfermedades de las Plantas , PlantasRESUMEN
BACKGROUND: Swarming is a multicellular phenomenom characterized by the coordinated and rapid movement of bacteria across semisolid surfaces. In Sinorhizobium meliloti this type of motility has been described in a fadD mutant. To gain insights into the mechanisms underlying the process of swarming in rhizobia, we compared the transcriptome of a S. meliloti fadD mutant grown under swarming inducing conditions (semisolid medium) to those of cells grown under non-swarming conditions (broth and solid medium). RESULTS: More than a thousand genes were identified as differentially expressed in response to growth on agar surfaces including genes for several metabolic activities, iron uptake, chemotaxis, motility and stress-related genes. Under swarming-specific conditions, the most remarkable response was the up-regulation of iron-related genes. We demonstrate that the pSymA plasmid and specifically genes required for the biosynthesis of the siderophore rhizobactin 1021 are essential for swarming of a S. meliloti wild-type strain but not in a fadD mutant. Moreover, high iron conditions inhibit swarming of the wild-type strain but not in mutants lacking either the iron limitation response regulator RirA or FadD. CONCLUSIONS: The present work represents the first transcriptomic study of rhizobium growth on surfaces including swarming inducing conditions. The results have revealed major changes in the physiology of S. meliloti cells grown on a surface relative to liquid cultures. Moreover, analysis of genes responding to swarming inducing conditions led to the demonstration that iron and genes involved in rhizobactin 1021 synthesis play a role in the surface motility shown by S. meliloti which can be circumvented in a fadD mutant. This work opens a way to the identification of new traits and regulatory networks involved in swarming by rhizobia.
Asunto(s)
Citratos/biosíntesis , Perfilación de la Expresión Génica , Sinorhizobium meliloti/crecimiento & desarrollo , Sinorhizobium meliloti/genética , Alquenos , Proteínas Bacterianas/genética , Medios de Cultivo , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Genes Bacterianos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Plásmidos , ARN Bacteriano/genéticaRESUMEN
Pathogenic bacteria and mutualistic rhizobia are able to invade and establish chronic infections within their host plants. The success of these plant-bacteria interactions requires evasion of the plant innate immunity by either avoiding recognition or by suppressing host defences. The primary plant innate immunity is triggered upon recognition of common microbe-associated molecular patterns. Different studies reveal striking similarities between the molecular bases underlying the perception of rhizobial nodulation factors and microbe-associated molecular patterns from plant pathogens. However, in contrast to general elicitors, nodulation factors can control plant defences when recognized by their cognate legumes. Nevertheless, in response to rhizobial infection, legumes show transient or local defence-like responses suggesting that Rhizobium is perceived as an intruder although the plant immunity is controlled. Whether these responses are involved in limiting the number of infections or whether they are required for the progression of the interaction is not yet clear. Further similarities in both plant-pathogen and Rhizobium-legume associations are factors such as surface polysaccharides, quorum sensing signals and secreted proteins, which play important roles in modulating plant defence responses and determining the outcome of the interactions.
Asunto(s)
Bacterias/patogenicidad , Fenómenos Fisiológicos Bacterianos , Enfermedades de las Plantas/microbiología , Plantas/inmunología , Plantas/microbiología , SimbiosisRESUMEN
Phytohormones mediate most diverse processes in plants, ranging from organ development to immune responses. Receptor protein complexes perceive changes in intracellular phytohormone levels and trigger a signaling cascade to effectuate downstream responses. The in planta analysis of elements involved in phytohormone signaling can be achieved through transient expression in mesophyll protoplasts, which are a fast and versatile alternative to generating plant lines that stably express a transgene. While promoter-reporter constructs have been used successfully to identify internal or external factors that change phytohormone signaling, the range of available marker constructs does not meet the potential of the protoplast technique for large scale approaches. The aim of our study was to provide novel markers for phytohormone signaling in the Arabidopsis mesophyll protoplast system. We validated 18 promoter::luciferase constructs towards their phytohormone responsiveness and specificity and suggest an experimental setup for high-throughput analyses. We recommend novel markers for the analysis of auxin, abscisic acid, cytokinin, salicylic acid and jasmonic acid responses that will facilitate future screens for biological elements and environmental stimuli affecting phytohormone signaling.
Asunto(s)
Reguladores del Crecimiento de las Plantas/metabolismo , Protoplastos/citología , Transducción de Señal , Arabidopsis/citología , Biomarcadores/metabolismoRESUMEN
The disaccharide trehalose is a well-known osmoprotectant, and trehalose accumulation through de novo biosynthesis is a common response of bacteria to abiotic stress. In this study, we have investigated the role of endogenous trehalose synthesis in the osmotolerance of Sinorhizobium meliloti. Genes coding for three possible trehalose synthesis pathways are present in the genome of S. meliloti 1021: OtsA, TreYZ, and TreS. Among these, OtsA has a major role in trehalose accumulation under all of the conditions tested and is the main system involved in osmoadaptation. Nevertheless, the other two systems are also important for growth in hyperosmotic medium. Genes for the three pathways are transcriptionally responsive to osmotic stress. The presence of at least one functional trehalose biosynthesis pathway is required for optimal competitiveness of S. meliloti to nodulate alfalfa roots.
Asunto(s)
Medicago sativa/microbiología , Presión Osmótica , Nódulos de las Raíces de las Plantas/crecimiento & desarrollo , Nódulos de las Raíces de las Plantas/microbiología , Sinorhizobium meliloti/fisiología , Estrés Fisiológico , Trehalosa/biosíntesis , Fusión Artificial Génica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Perfilación de la Expresión Génica , Genes Reporteros , Sinorhizobium meliloti/crecimiento & desarrollo , Sinorhizobium meliloti/metabolismo , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
Stimulation of potassium uptake is the most rapid response to an osmotic upshock in bacteria. This cation accumulates by a number of different transport systems whose importance has not been previously addressed for rhizobia. In silico analyses reveal the presence of genes encoding four possible potassium uptake systems in the genome of Sinorhizobium meliloti 1021: Kup1, Kup2, Trk, and Kdp. The study of the relevance of these systems under a number of different growth conditions and in symbiosis showed that the integrity of Kup1 or Trk is essential for growth under laboratory conditions even in osmotically balanced media and the absence of both systems leads to a reduced infectivity and competitiveness of the bacteria in alfalfa roots. Trk is the main system involved in the accumulation of potassium after an osmotic upshift and the most important system for growth of S. meliloti under hyperosmotic conditions. The other three systems, especially Kup1, are also relevant during the osmotic adaptation of the cell, and the relative importance of the Kdp system increases at low potassium concentrations.
Asunto(s)
Proteínas Bacterianas/metabolismo , Potasio/metabolismo , Sinorhizobium meliloti/fisiología , Simbiosis , Adaptación Fisiológica , Proteínas Bacterianas/genética , Transporte Biológico , Medicago sativa/microbiología , Medicago sativa/fisiología , Ósmosis , Raíces de Plantas/microbiología , Raíces de Plantas/fisiología , Sinorhizobium meliloti/genéticaRESUMEN
The discovery of insecticidal activity in root-colonizing pseudomonads, best-known for their plant-beneficial effects, raised fundamental questions about the ecological relevance of insects as alternative hosts for these bacteria. Since soil bacteria are limited in their inherent abilities of dispersal, insects as vectors might be welcome vehicles to overcome large distances. Here, we report on the transmission of the root-colonizing, plant-beneficial and insecticidal bacterium Pseudomonas protegens CHA0 from root to root by the cabbage root fly, Delia radicum. Following ingestion by root-feeding D. radicum larvae, CHA0 persisted inside the insect until the pupal and adult stages. The emerging flies were then able to transmit CHA0 to a new plant host initiating bacterial colonization of the roots. CHA0 did not reduce root damages caused by D. radicum and had only small effects on Delia development suggesting a rather commensal than pathogenic relationship. Interestingly, when the bacterium was fed to two highly susceptible lepidopteran species, most of the insects died, but CHA0 could persist throughout different life stages in surviving individuals. In summary, this study investigated for the first time the interaction of P. protegens CHA0 and related strains with an insect present in their rhizosphere habitat. Our results suggest that plant-colonizing pseudomonads have different strategies for interaction with insects. They either cause lethal infections and use insects as food source or they live inside insect hosts without causing obvious damages and might use insects as vectors for dispersal, which implies a greater ecological versatility of these bacteria than previously thought.
Asunto(s)
Brassica/microbiología , Dípteros/microbiología , Raíces de Plantas/microbiología , Pseudomonas/fisiología , Microbiología del Suelo , Animales , Antibiosis , Dípteros/crecimiento & desarrollo , Dípteros/fisiología , Herbivoria , Larva/microbiología , Larva/fisiología , Pseudomonas/química , Pseudomonas/clasificación , Pupa/microbiología , Pupa/fisiología , Rizosfera , SimbiosisRESUMEN
Bacteria of the genus Pseudomonas occupy diverse environments. The Pseudomonas fluorescens group is particularly well-known for its plant-beneficial properties including pathogen suppression. Recent observations that some strains of this group also cause lethal infections in insect larvae, however, point to a more versatile ecology of these bacteria. We show that 26 P. fluorescens group strains, isolated from three continents and covering three phylogenetically distinct sub-clades, exhibited different activities toward lepidopteran larvae, ranging from lethal to avirulent. All strains of sub-clade 1, which includes Pseudomonas chlororaphis and Pseudomonas protegens, were highly insecticidal regardless of their origin (animals, plants). Comparative genomics revealed that strains in this sub-clade possess specific traits allowing a switch between plant- and insect-associated lifestyles. We identified 90 genes unique to all highly insecticidal strains (sub-clade 1) and 117 genes common to all strains of sub-clade 1 and present in some moderately insecticidal strains of sub-clade 3. Mutational analysis of selected genes revealed the importance of chitinase C and phospholipase C in insect pathogenicity. The study provides insight into the genetic basis and phylogenetic distribution of traits defining insecticidal activity in plant-beneficial pseudomonads. Strains with potent dual activity against plant pathogens and herbivorous insects have great potential for use in integrated pest management for crops.
Asunto(s)
Lepidópteros/microbiología , Plantas/microbiología , Pseudomonas/genética , Pseudomonas/patogenicidad , Animales , Genómica , Especificidad del Huésped , Larva/microbiología , Filogenia , Pseudomonas/aislamiento & purificación , Pseudomonas/fisiología , VirulenciaRESUMEN
In this work, DNA microarrays were used to investigate genome-wide transcriptional responses of Sinorhizobium meliloti to a sudden increase in external osmolarity elicited by addition of either NaCl or sucrose to exponentially growing cultures. A time course of the response within the first 4 h after the osmotic shock was established. We found that there was a general redundancy in the differentially expressed genes after NaCl or sucrose addition. Both kinds of stress resulted in induction of a large number of genes having unknown functions and in repression of many genes coding for proteins with known functions. There was a strong replicon bias in the pattern of the osmotic stress response; whereas 64% of the upregulated genes had a plasmid localization, 85% of the downregulated genes were chromosomal. Among the pSymB osmoresponsive genes, 83% were upregulated, suggesting the importance of this plasmid for S. meliloti osmoadaptation. Indeed, we identified a 200-kb region in pSymB needed for adaptation to saline shock which has a high density of osmoregulated genes.
Asunto(s)
Adaptación Fisiológica , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Presión Osmótica , Plásmidos/genética , Sinorhizobium meliloti/fisiología , Cromosomas Bacterianos/genética , Soluciones Hipertónicas , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/crecimiento & desarrollo , Cloruro de Sodio , SacarosaRESUMEN
An approach originally designed to identify functional origins of conjugative transfer (oriT or mob) in a bacterial genome (J. A. Herrera-Cervera, J. M. Sanjuán-Pinilla, J. Olivares, and J. Sanjuán, J. Bacteriol. 180:4583-4590, 1998) was modified to improve its reliability and prevent selection of undesired false mob clones. By following this modified approach, we were able to identify two functional mob regions in the genome of Rhizobium etli CFN42. One corresponds to the recently characterized transfer region of the nonsymbiotic, self-transmissible plasmid pRetCFN42a (C. Tun-Garrido, P. Bustos, V. González, and S. Brom, J. Bacteriol. 185:1681-1692, 2003), whereas the second mob region belongs to the symbiotic plasmid pRetCFN42d. The new transfer region identified contains a putative oriT and a typical conjugative (tra) gene cluster organization. Although pRetCFN42d had not previously been shown to be self-transmissible, mobilization of cosmids containing this tra region required the presence of a wild-type pRetCFN42d in the donor cell; the presence of multiple copies of this mob region in CFN42 also promoted conjugal transfer of the Sym plasmid pRetCFN42d. The overexpression of a small open reading frame, named yp028, located downstream of the putative relaxase gene traA, appeared to be responsible for promoting the conjugal transfer of the R. etli pSym under laboratory conditions. This yp028-dependent conjugal transfer required a wild-type pRetCFN42d traA gene. Our results suggest for the first time that the R. etli symbiotic plasmid is self-transmissible and that its transfer is subject to regulation. In wild-type CFN42, pRetCFN42d tra gene expression appears to be insufficient to promote plasmid transfer under standard laboratory conditions; gene yp028 may play some role in the activation of conjugal transfer in response to as-yet-unknown environmental conditions.