RESUMEN
As the gall-inducing smut fungus Ustilago maydis colonizes maize (Zea mays) plants, it secretes a complex effector blend that suppresses host defense responses, including production of reactive oxygen species (ROS) and redirects host metabolism to facilitate colonization. We show that the U. maydis effector ROS burst interfering protein 1 (Rip1), which is involved in pathogen-associated molecular pattern (PAMP)-triggered suppression of host immunity, is functionally conserved in several other monocot-infecting smut fungi. We also have identified a conserved C-terminal motif essential for Rip1-mediated PAMP-triggered suppression of the ROS burst. The maize susceptibility factor lipoxygenase 3 (Zmlox3) bound by Rip1 was relocalized to the nucleus, leading to partial suppression of the ROS burst. Relocalization was independent of its enzymatic activity, revealing a distinct function for ZmLox3. Most importantly, whereas Zmlox3 maize mutant plants showed increased resistance to U. maydis wild-type strains, rip1 deletion strains infecting the Zmlox3 mutant overcame this effect. This could indicate that Rip1-triggered host resistance depends on ZmLox3 to be suppressed and that lox3 mutation-based resistance of maize to U. maydis requires functional Rip1. Together, our results reveal that Rip1 acts in several cellular compartments to suppress immunity and that targeting of ZmLox3 by Rip1 is responsible for the suppression of Rip1-dependent reduced susceptibility of maize to U. maydis.
Asunto(s)
Ustilago , Zea mays , Basidiomycota , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Enfermedades de las Plantas/microbiología , Especies Reactivas de Oxígeno/metabolismo , Ustilago/genéticaRESUMEN
Elongation factor TFIIS (transcription factor IIS) is structurally and biochemically probably the best characterized elongation cofactor of RNA polymerase II. However, little is known about TFIIS regulation or its roles during stress responses. Here, we show that, although TFIIS seems unnecessary under optimal conditions in Arabidopsis, its absence renders plants supersensitive to heat; tfIIs mutants die even when exposed to sublethal high temperature. TFIIS activity is required for thermal adaptation throughout the whole life cycle of plants, ensuring both survival and reproductive success. By employing a transcriptome analysis, we unravel that the absence of TFIIS makes transcriptional reprogramming sluggish, and affects expression and alternative splicing pattern of hundreds of heat-regulated transcripts. Transcriptome changes indirectly cause proteotoxic stress and deterioration of cellular pathways, including photosynthesis, which finally leads to lethality. Contrary to expectations of being constantly present to support transcription, we show that TFIIS is dynamically regulated. TFIIS accumulation during heat occurs in evolutionary distant species, including the unicellular alga Chlamydomonas reinhardtii, dicot Brassica napus and monocot Hordeum vulgare, suggesting that the vital role of TFIIS in stress adaptation of plants is conserved.
Asunto(s)
Arabidopsis , Factores Generales de Transcripción , Arabidopsis/genética , Arabidopsis/fisiología , Respuesta al Choque Térmico , ARN Polimerasa II/metabolismo , Factores Generales de Transcripción/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
ARHGAP25, a RAC-specific GTPase activating protein (GAP), is an essential regulator of phagocyte effector functions such as phagocytosis, superoxide production, and transendothelial migration. Furthermore, its complex role in tumor behavior has recently been recognized. We previously demonstrated that phosphorylation of serine 363 in ARHGAP25 regulates hematopoietic stem cells and progenitor cells in mouse bone marrow. However, the significance of other potential phosphorylation sites of ARHGAP25 remained unknown. Now, we developed a novel, real-time bioluminescence resonance energy transfer (BRET) assay to monitor the GAP activity of ARHGAP25 in vitro. Using this approach, we revealed that phosphorylation of S363 and S488, but not that of S379-380, controls ARHGAP25's RACGAP activity. On the other hand, we found in granulocyte-differentiated human PLB-985 cells that superoxide production and actin depolymerization are regulated by residues S363 and S379-380. The present data demonstrate the value of our BRET-GAP assay and show that different phosphorylation patterns regulate ARHGAP25's GAP activity and its effect on superoxide production and phagocytosis.
Asunto(s)
Proteínas Activadoras de GTPasa , Superóxidos , Animales , Transferencia de Energía , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Fosforilación , Serina/metabolismo , Superóxidos/metabolismoRESUMEN
The human proteome is more complex than the genetic code predicts it to be. Epitomics, or protein epitome profiling, is a tool for understanding sub-protein level variation. With the ultimate goal to explore C9 proteoforms and their relevance to lung cancer, here we report plasma C9 epitope-associated molecular heterogeneity in plasma samples of lung cancer patients and control subjects. We show three C9 epitopes (BSI0449, BSI0581, BSI0639) with markedly different association with lung cancer ("unaltered", "upregulated" and "downregulated"). In order to exclude confounding effects, we show first that the three epitope-defining mAbs recognize C9 in purified form and in the natural context, in the human plasma. Then, we present data demonstrating the lack of major epitope interdependence or overlap. The next experiments represent a quest toward the understanding of the molecular basis of apparent disparate association with lung cancer. Using immunochemistry, SDS PAGE and LC-MS/MS technologies, we demonstrate that epitope-specific immunoprecipitates of plasma C9 seem identical regarding peptide sequence. However, we found epitope-specific posttranslational modification and coprecipitated protein composition differences with respect to control and lung cancer plasma. Epitope profiling enabled the classification of hypothetical C9 proteoforms through differential association with lung cancer.
Asunto(s)
Complemento C9 , Neoplasias Pulmonares , Humanos , Epítopos/genética , Complemento C9/análisis , Cromatografía Liquida , Espectrometría de Masas en Tándem , Neoplasias Pulmonares/genéticaRESUMEN
Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated virus (AAV) vectors emerged as safe and efficient gene delivery tools, over-coming setbacks experienced with other viral gene delivery systems in early gene therapy trials. Among AAVs, AAV9 can translocate through the blood-brain barrier (BBB), making it a promising gene delivery tool for transducing the central nervous system (CNS) via systemic administration. Recent reports on the shortcomings of AAV9-mediated gene delivery into the CNS require reviewing the molecular base of AAV9 cellular biology. A more detailed understanding of AAV9's cellular entry would eradicate current hurdles and enable more efficient AAV9-based gene therapy approaches. Syndecans, the transmembrane family of heparan-sulfate proteoglycans, facilitate the cellular uptake of various viruses and drug delivery systems. Utilizing human cell lines and syndecan-specific cellular assays, we assessed the involvement of syndecans in AAV9's cellular entry. The ubiquitously expressed isoform, syndecan-4 proved its superiority in facilitating AAV9 internalization among syndecans. Introducing syndecan-4 into poorly transducible cell lines enabled robust AAV9-dependent gene transduction, while its knockdown reduced AAV9's cellular entry. Attachment of AAV9 to syndecan-4 is mediated not just by the polyanionic heparan-sulfate chains but also by the cell-binding domain of the extracellular syndecan-4 core protein. Co-immunoprecipitation assays and affinity proteomics also confirmed the role of syndecan-4 in the cellular entry of AAV9. Overall, our findings highlight the universally expressed syndecan-4 as a significant contributor to the cellular internalization of AAV9 and provide a molecular-based, rational explanation for the low gene delivery potential of AAV9 into the CNS.
Asunto(s)
Dependovirus , Sindecano-4 , Humanos , Dependovirus/metabolismo , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/metabolismo , Sulfatos , Sindecano-1 , Sindecanos/metabolismoRESUMEN
Despite the growing list of identified SARS-CoV-2 receptors, the human angiotensin-converting enzyme 2 (ACE2) is still viewed as the main cell entry receptor mediating SARS-CoV-2 internalization. It has been reported that wild-type mice, like other rodent species of the Muridae family, cannot be infected with SARS-CoV-2 due to differences in their ACE2 receptors. On the other hand, the consensus heparin-binding motif of SARS-CoV-2's spike protein, PRRAR, enables the attachment to rodent heparan sulfate proteoglycans (HSPGs), including syndecans, a transmembrane HSPG family with a well-established role in clathrin- and caveolin-independent endocytosis. As mammalian syndecans possess a relatively conserved structure, we analyzed the cellular uptake of inactivated SARS-CoV-2 particles in in vitro and in vivo mice models. Cellular studies revealed efficient uptake into murine cell lines with established syndecan-4 expression. After intravenous administration, inactivated SARS-CoV-2 was taken up by several organs in vivo and could also be detected in the brain. Internalized by various tissues, inactivated SARS-CoV-2 raised tissue TNF-α levels, especially in the heart, reflecting the onset of inflammation. Our studies on in vitro and in vivo mice models thus shed light on unknown details of SARS-CoV-2 internalization and help broaden the understanding of the molecular interactions of SARS-CoV-2.
Asunto(s)
COVID-19 , SARS-CoV-2 , Distribución Tisular , Internalización del Virus , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/metabolismo , COVID-19/virología , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Mamíferos/metabolismo , Ratones , SARS-CoV-2/metabolismo , Sindecanos/metabolismo , Distribución Tisular/fisiologíaRESUMEN
The two paralogous Arabidopsis genes MAINTENANCE OF MERISTEMS (MAIN) and MAINTENANCE OF MERISTEMS LIKE1 (MAIL1) encode a conserved retrotransposon-related plant mobile domain and are known to be required for silencing of transposable elements (TE) and for primary root development. Loss of function of either MAIN or MAIL1 leads to release of heterochromatic TEs, reduced condensation of pericentromeric heterochromatin, cell death of meristem cells and growth arrest of the primary root soon after germination. Here, we show that they act in one protein complex that also contains the inactive isoform of PROTEIN PHOSPHATASE 7 (PP7), which is named PROTEIN PHOSPHATASE 7-LIKE (PP7L). PP7L was previously shown to be important for chloroplast biogenesis and efficient chloroplast protein synthesis. We show that loss of PP7L function leads to the same root growth phenotype as loss of MAIL1 or MAIN. In addition, pp7l mutants show similar silencing defects. Double mutant analyses confirmed that the three proteins act in the same molecular pathway. The primary root growth arrest, which is associated with cell death of stem cells and their daughter cells, is a consequence of genome instability. Our data demonstrate so far unrecognized functions of an inactive phosphatase isoform in a protein complex that is essential for silencing of heterochromatic elements and for maintenance of genome stability in dividing cells.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Elementos Transponibles de ADN/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Silenciador del Gen , Germinación , Heterocromatina/genética , Isoenzimas , Meristema/genética , Meristema/crecimiento & desarrollo , Meristema/fisiología , Mutación , Proteínas Nucleares/genética , Fenotipo , Fosfoproteínas Fosfatasas/genética , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , Retroelementos/genéticaRESUMEN
The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Puntos de Control del Ciclo Celular , Daño del ADN , Reparación del ADN , Factores de Transcripción E2F/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , ADN de Plantas/metabolismoRESUMEN
Self-renewal and differentiation of stem cells is one of the fundamental biological phenomena relying on proper chromatin organization. In our study, we describe a novel chromatin regulator encoded by the Drosophila small ovary (sov) gene. We demonstrate that sov is required in both the germline stem cells (GSCs) and the surrounding somatic niche cells to ensure GSC survival and differentiation. sov maintains niche integrity and function by repressing transposon mobility, not only in the germline, but also in the soma. Protein interactome analysis of Sov revealed an interaction between Sov and HP1a. In the germ cell nuclei, Sov colocalizes with HP1a, suggesting that Sov affects transposon repression as a component of the heterochromatin. In a position-effect variegation assay, we found a dominant genetic interaction between sov and HP1a, indicating their functional cooperation in promoting the spread of heterochromatin. An in vivo tethering assay and FRAP analysis revealed that Sov enhances heterochromatin formation by supporting the recruitment of HP1a to the chromatin. We propose a model in which sov maintains GSC niche integrity by regulating transposon silencing and heterochromatin formation.
Asunto(s)
Diferenciación Celular , Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Silenciador del Gen , Células Germinativas/citología , Heterocromatina/metabolismo , Células Madre/citología , Animales , Apoptosis , Supervivencia Celular , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Genoma , Células Germinativas/metabolismo , Mutación/genética , Transducción de Señal , Nicho de Células Madre , Células Madre/metabolismo , Transcripción GenéticaRESUMEN
In Arabidopsis thaliana, phytochrome B (phyB) is the dominant receptor of photomorphogenic development under red light. Phytochrome B interacts with a set of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR 3 (PIF3). The interaction between PIF3 and photoactivated phyB leads to the rapid phosphorylation and degradation of PIF3 and also to the degradation of phyB, events which are required for proper photomorphogenesis. Here we report that PIF3 is SUMOylated at the Lys13 (K13) residue and that we could detect this posttranslational modification in a heterologous experimental system and also in planta. We also found that the SUMO acceptor site mutant PIF3(K13R) binds more strongly to the target promoters than its SUMOylated, wild-type counterpart. Seedlings expressing PIF3(K13R) show an elongated hypocotyl response, elevated photoprotection and higher transcriptional induction of red-light responsive genes compared with plantlets expressing wild-type PIF3. These observations are supported by the lower level of phyB in plants which possess only PIF3(K13R), indicating that SUMOylation of PIF3 also alters photomorphogenesis via the regulation of phyB levels. In conclusion, whereas SUMOylation is generally connected to different stress responses, it also fine-tunes light signalling by reducing the biological activity of PIF3, thus promoting photomorphogenesis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Fitocromo B , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Luz , Fitocromo B/genética , Fitocromo B/metabolismo , SumoilaciónRESUMEN
The ErbB-3 BINDING PROTEIN 1 (EBP1) drives growth, but the mechanism of how it acts in plants is little understood. Here, we show that EBP1 expression and protein abundance in Arabidopsis (Arabidopsis thaliana) are predominantly confined to meristematic cells and are induced by sucrose and partially dependent on TARGET OF RAPAMYCIN (TOR) kinase activity. Consistent with being downstream of TOR, silencing of EBP1 restrains, while overexpression promotes, root growth, mostly under sucrose-limiting conditions. Inducible overexpression of RETINOBLASTOMA RELATED (RBR), a sugar-dependent transcriptional repressor of cell proliferation, depletes meristematic activity and causes precocious differentiation, which is attenuated by EBP1. To understand the molecular mechanism, we searched for EBP1- and RBR-interacting proteins by affinity purification and mass spectrometry. In line with the double-stranded RNA-binding activity of EBP1 in human (Homo sapiens) cells, the overwhelming majority of EBP1 interactors are part of ribonucleoprotein complexes regulating many aspects of protein synthesis, including ribosome biogenesis and mRNA translation. We confirmed that EBP1 associates with ribosomes and that EBP1 silencing hinders ribosomal RNA processing. We revealed that RBR also interacts with a set of EBP1-associated nucleolar proteins as well as factors that function in protein translation. This suggests EBP1 and RBR act antagonistically on common processes that determine the capacity for translation to tune meristematic activity in relation to available resources.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Meristema/metabolismo , Raíces de Plantas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Diferenciación Celular/genética , Cromatografía de Afinidad , Espectrometría de Masas , Meristema/genética , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Raíces de Plantas/genética , Unión Proteica , Biosíntesis de Proteínas/genética , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Ribosomas/metabolismo , Sacarosa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Plants heat shock factors (HSFs) are encoded by large gene families with variable structure, expression, and function. HSFs are components of complex signaling systems that control responses not only to high temperatures but also to a number of abiotic stresses such as cold, drought, hypoxic conditions, soil salinity, toxic minerals, strong irradiation, and to pathogen threats. Here we provide an overview of the diverse world of plant HSFs through compilation and analysis of their functional versatility, diverse regulation, and interactions. Bioinformatic data on gene expression profiles of Arabidopsis HSF genes were re-analyzed to reveal their characteristic transcript patterns. While HSFs are regulated primarily at the transcript level, alternative splicing and post-translational modifications such as phosphorylation and sumoylation provides further variability. Plant HSFs are involved in an intricate web of protein-protein interactions which adds considerable complexity to their biological function. A list of such interactions was compiled from public databases and published data, and discussed to pinpoint their relevance in transcription control. Although most fundamental studies of plant HSFs have been conducted in the model plant, Arabidopsis, information on HSFs is accumulating in other plants such as tomato, rice, wheat, and sunflower. Understanding the function, interactions, and regulation of HSFs will facilitate the design of novel strategies to use engineered proteins to improve tolerance and adaptation of crops to adverse environmental conditions.
Asunto(s)
Arabidopsis , Regulación de la Expresión Génica de las Plantas , Arabidopsis/genética , Arabidopsis/metabolismo , Sequías , Factores de Transcripción del Choque Térmico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estrés FisiológicoRESUMEN
In multicellular organisms, temporal and spatial regulation of cell proliferation is central for generating organs with defined sizes and morphologies. For establishing and maintaining the post-mitotic quiescent state during cell differentiation, it is important to repress genes with mitotic functions. We found that three of the Arabidopsis MYB3R transcription factors synergistically maintain G2/M-specific genes repressed in post-mitotic cells and restrict the time window of mitotic gene expression in proliferating cells. The combined mutants of the three repressor-type MYB3R genes displayed long roots, enlarged leaves, embryos, and seeds. Genome-wide chromatin immunoprecipitation revealed that MYB3R3 binds to the promoters of G2/M-specific genes and to E2F target genes. MYB3R3 associates with the repressor-type E2F, E2FC, and the RETINOBLASTOMA RELATED proteins. In contrast, the activator MYB3R4 was in complex with E2FB in proliferating cells. With mass spectrometry and pairwise interaction assays, we identified some of the other conserved components of the multiprotein complexes, known as DREAM/dREAM in human and flies. In plants, these repressor complexes are important for periodic expression during cell cycle and to establish a post-mitotic quiescent state determining organ size.
Asunto(s)
Arabidopsis/fisiología , Ciclo Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Organogénesis/fisiología , Factores de Transcripción/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Inmunoprecipitación de Cromatina , Regulación del Desarrollo de la Expresión Génica/genética , Regulación de la Expresión Génica de las Plantas/genética , Espectrometría de Masas , Análisis por Micromatrices , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARNRESUMEN
Heat shock factors regulate responses to high temperature, salinity, water deprivation, or heavy metals. Their function in combinations of stresses is, however, not known. Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature, and a combination of these conditions. Fast translocation of HSFA4A tagged with yellow fluorescent protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by mitogen-activated protein (MAP) kinases MPK3 and MPK6 but also by MPK4, and Ser309 is the dominant MAP kinase phosphorylation site. In vivo data suggest that HSFA4A can be the substrate of other kinases as well. Changing Ser309 to Asp or Ala alters intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes.
Asunto(s)
Arabidopsis/genética , Respuesta al Choque Térmico/genética , Estrés Salino/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Cloruro de Sodio/efectos adversos , Factores de TranscripciónRESUMEN
Symbiotic nitrogen fixation by Rhizobium bacteria in the cells of legume root nodules alleviates the need for nitrogen fertilizers. Nitrogen fixation requires the endosymbionts to differentiate into bacteroids which can be reversible or terminal. The latter is controlled by the plant, it is more beneficial and has evolved in multiple clades of the Leguminosae family. The plant effectors of terminal differentiation in inverted repeat-lacking clade legumes (IRLC) are nodule-specific cysteine-rich (NCR) peptides, which are absent in legumes such as soybean where there is no terminal differentiation of rhizobia. It was assumed that NCRs co-evolved with specific transcription factors, but our work demonstrates that expression of NCR genes does not require NCR-specific transcription factors. Introduction of the Medicago truncatula NCR169 gene under its own promoter into soybean roots resulted in its nodule-specific expression, leading to bacteroid changes associated with terminal differentiation. We identified two AT-Hook Motif Nuclear Localized (AHL) transcription factors from both M. truncatula and soybean nodules that bound to AT-rich sequences in the NCR169 promoter inducing its expression. Whereas mutation of NCR169 arrested bacteroid development at a late stage, the absence of MtAHL1 or MtAHL2 completely blocked bacteroid differentiation indicating that they also regulate other NCR genes required for the development of nitrogen-fixing nodules. Regulation of NCRs by orthologous transcription factors in non-IRLC legumes opens up the possibility of increasing the efficiency of nitrogen fixation in legumes lacking NCRs.
Asunto(s)
Medicago truncatula , Rhizobium , Nódulos de las Raíces de las Plantas/genética , Medicago truncatula/genética , Péptidos/metabolismo , Rhizobium/fisiología , Glycine max/genética , Expresión Génica , Simbiosis/fisiología , Regulación de la Expresión Génica de las PlantasRESUMEN
The growth of bacterial populations has been described as a dynamic process of continuous reproduction and cell death. However, this is far from the reality. In a well fed, growing bacterial population, the stationary phase inevitably occurs, and it is not due to accumulated toxins or cell death. A population spends the most time in the stationary phase, where the phenotype of the cells alters from the proliferating ones, and only the colony forming unit (CFU) decreases after a while, not the total cell concentration. A bacterial population can be considered as a virtual tissue as a result of a specific differentiation process, in which the exponential-phase cells develop to stationary-phase cells and eventually reach the unculturable form. The richness of the nutrient had no effect on growth rate or on stationary cell density. The generation time seems not to be a constant value, but it depended on the concentration of the starter cultures. Inoculations with serial dilutions of stationary populations reveal a so-called minimal stationary cell concentration (MSCC) point, up to which the cell concentrations remain constant upon dilutions; that seems to be universal among unicellular organisms.
Asunto(s)
División Celular , Citocinesis , Synechococcus , Synechococcus/crecimiento & desarrollo , Synechococcus/metabolismo , Técnicas de Cultivo Celular por Lotes , Proteómica , Medios de Cultivo/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
Endoplasmic reticulum (ER) stress-dependent accumulation of incorrectly folded proteins leads to activation of the unfolded protein response. The role of the unfolded protein response (UPR) is to avoid cell damage and restore the homeostatic state by autophagy; however, excessive ER stress results in apoptosis. Here we investigated the ER stress-dependent feedback loops inside one of the UPR branches by focusing on PERK-induced ATF4 and its two targets, called CHOP and GADD34. Our goal was to qualitatively describe the dynamic behavior of the system by exploring the key regulatory motifs using both molecular and theoretical biological techniques. Using the HEK293T cell line as a model system, we confirmed that the life-or-death decision is strictly regulated. We investigated the dynamic characteristics of the crucial elements of the PERK pathway at both the RNA and protein level upon tolerable and excessive levels of ER stress. Of particular note, inhibition of GADD34 or CHOP resulted in various phenotypes upon high levels of ER stress. Our computer simulations suggest the existence of two new feedback loops inside the UPR. First, GADD34 seems to have a positive effect on ATF4 activity, while CHOP inhibits it. We claim that these newly described feedback loops ensure the fine-tuning of the ATF4-dependent stress response mechanism of the cell.
Asunto(s)
Factor de Transcripción Activador 4 , Estrés del Retículo Endoplásmico , Humanos , Factor de Transcripción Activador 4/metabolismo , Células HEK293 , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
The DNA of all organisms is constantly damaged by physiological processes and environmental conditions. Upon persistent damage, plant growth and cell proliferation are reduced. Based on previous findings that RBR1, the only Arabidopsis homolog of the mammalian tumor suppressor gene retinoblastoma, plays a key role in the DNA damage response in plants, we unravel here the network of RBR1 interactors under DNA stress conditions. This led to the identification of homologs of every DREAM component in Arabidopsis, including previously not recognized homologs of LIN52. Interestingly, we also discovered NAC044, a mediator of DNA damage response in plants and close homolog of the major DNA damage regulator SOG1, to directly interact with RBR1 and the DREAM component LIN37B. Consistently, not only mutants in NAC044 but also the double mutant of the two LIN37 homologs and mutants for the DREAM component E2FB showed reduced sensitivities to DNA-damaging conditions. Our work indicates the existence of multiple DREAM complexes that work in conjunction with NAC044 to mediate growth arrest after DNA damage.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Daño del ADN/genética , Factores de Transcripción E2F/metabolismo , Proteínas Mutantes/metabolismo , Transducción de Señal/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/genética , Puntos de Control del Ciclo Celular/genética , Reparación del ADN/genética , Factores de Transcripción E2F/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Mutantes/genética , Mutación , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Transactivadores/genéticaRESUMEN
Necroptosis is a regulated necrotic-like cell death modality which has come into the focus of attention since it is known to contribute to the pathogenesis of many inflammatory and degenerative diseases as well as to tumor regulation. Based on current data, necroptosis serves as a backup mechanism when death receptor-induced apoptosis is inhibited or absent. However, the necroptotic role of the proteins involved in mitochondrial apoptosis has not been investigated. Here, we demonstrated that the stimulation of several death and pattern recognition receptors induced necroptosis under caspase-compromised conditions in wild-type, but not in caspase-9-negative human Jurkat and murine MEF cells. Cerulein-induced pancreatitis was significantly reduced in mice with acinar cell-restricted caspase-9 gene knockout. The absence of caspase-9 led to impaired association of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 and resulted in decreased phosphorylation of RIP kinases, but the overexpression of RIPK1 or RIPK3 rescued the effect of caspase-9 deficiency. Inhibition of either Aurora kinase A (AURKA) or its known substrate, glycogen synthase kinase 3ß (GSK3ß) restored necroptosis sensitivity of caspase-9-deficient cells, indicating an interplay between caspase-9 and AURKA-mediated pathways to regulate necroptosis. Our findings suggest that caspase-9 acts as a newly identified regulator of necroptosis, and thus, caspase-9 provides a promising therapeutic target to manipulate the immunological outcome of cell death.