RESUMO
There is an urgent need for novel protection strategies to sustainably secure crop production under changing climates. Studying microbial effectors, defined as microbe-derived proteins that alter signalling inside plant cells, has advanced our understanding of plant immunity and microbial plant colonisation strategies. Our understanding of effectors in the establishment and beneficial outcome of plant symbioses is less well known. Combining functional and comparative interaction assays uncovered specific symbiont effector targets in highly interconnected plant signalling networks and revealed the potential of effectors in beneficially modulating plant traits. The diverse functionality of symbiont effectors differs from the paradigmatic immuno-suppressive function of pathogen effectors. These effectors provide solutions for improving crop resilience against climate stress by their evolution-driven specification in host protein targeting and modulation. Symbiont effectors represent stringent tools not only to identify genetic targets for crop breeding, but to serve as applicable agents in crop management strategies under changing environments.
Assuntos
Proteínas de Plantas , Resiliência Psicológica , Proteínas de Plantas/metabolismo , Melhoramento Vegetal , Plantas/metabolismo , SimbioseRESUMO
Mitochondrial function depends on the RNA processing of mitochondrial gene transcripts by nucleus-encoded proteins. This posttranscriptional processing involves the large group of nuclear-encoded pentatricopeptide repeat (PPR) proteins. Mitochondrial processes represent a crucial part in animal immunity, but whether mitochondria play similar roles in plants remains unclear. Here, we report the identification of RESISTANCE TO PHYTOPHTHORA PARASITICA 7 (AtRTP7), a P-type PPR protein, in Arabidopsis thaliana and its conserved function in immunity to diverse pathogens across distantly related plant species. RTP7 affects the levels of mitochondrial reactive oxygen species (mROS) by participating in RNA splicing of nad7, which encodes a critical subunit of the mitochondrial respiratory chain Complex I, the largest of the four major components of the mitochondrial oxidative phosphorylation system. The enhanced resistance of rtp7 plants to Phytophthora parasitica is dependent on an elevated mROS burst, but might be independent from the ROS burst associated with plasma membrane-localized NADPH oxidases. Our study reveals the immune function of RTP7 and the defective processing of Complex I subunits in rtp7 plants resulted in enhanced resistance to both biotrophic and necrotrophic pathogens without affecting overall plant development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Imunidade Vegetal/genética , Processamento Pós-Transcricional do RNA , Splicing de RNA , RNA Mitocondrial/metabolismo , Explosão RespiratóriaRESUMO
Roots are a highly organised plant tissue consisting of different cell types with distinct developmental functions defined by cell identity networks. Roots are the target of some of the most devastating diseases and possess a highly effective immune system. The recognition of microbe- or plant-derived molecules released in response to microbial attack is highly important in the activation of complex immunity gene networks. Development and immunity are intertwined, and immunity activation can result in growth inhibition. In turn, by connecting immunity and cell identity regulators, cell types are able to launch a cell type-specific immunity based on the developmental function of each cell type. By this strategy, fundamental developmental processes of each cell type contribute their most basic functions to drive cost-effective but highly diverse and, thus, efficient immune responses. This review highlights the interdependence of root development and immunity and how the developmental age of root cells contributes to positive and negative outcomes of development-immunity cross-talk.
RESUMO
While root diseases are among the most devastating stresses in global crop production, our understanding of root immunity is still limited relative to our knowledge of immune responses in leaves. Considering that root performance is based on the concerted functions of its different cell types, we undertook a cell type-specific transcriptome analysis to identify gene networks activated in epidermis, cortex, and pericycle cells of Arabidopsis (Arabidopsis thaliana) roots challenged with two immunity elicitors, the bacterial flagellin-derived flg22 and the endogenous Pep1 peptide. Our analyses revealed distinct immunity gene networks in each cell type. To further substantiate our understanding of regulatory patterns underlying these cell type-specific immunity networks, we developed a tool to analyze paired transcription factor binding motifs in the promoters of cell type-specific genes. Our study points toward a connection between cell identity and cell type-specific immunity networks that might guide cell types in launching immune response according to the functional capabilities of each cell type.
Assuntos
Arabidopsis/citologia , Arabidopsis/imunologia , Redes Reguladoras de Genes/imunologia , Raízes de Plantas/imunologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis , Basidiomycota , Sítios de Ligação , Regulação da Expressão Gênica de Plantas , Células Vegetais/imunologia , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Raízes de Plantas/citologia , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Transdução de Sinais , TransativadoresRESUMO
The interaction of plants with complex microbial communities is the result of co-evolution over millions of years and contributed to plant transition and adaptation to land. The ability of plants to be an essential part of complex and highly dynamic ecosystems is dependent on their interaction with diverse microbial communities. Plant microbiota can support, and even enable, the diverse functions of plants and are crucial in sustaining plant fitness under often rapidly changing environments. The composition and diversity of microbiota differs between plant and soil compartments. It indicates that microbial communities in these compartments are not static but are adjusted by the environment as well as inter-microbial and plant-microbe communication. Hormones take a crucial role in contributing to the assembly of plant microbiomes, and plants and microbes often employ the same hormones with completely different intentions. Here, the function of hormones as go-betweens between plants and microbes to influence the shape of plant microbial communities is discussed. The versatility of plant and microbe-derived hormones essentially contributes to the creation of habitats that are the origin of diversity and, thus, multifunctionality of plants, their microbiota and ultimately ecosystems.
Assuntos
Microbiota , Reguladores de Crescimento de Plantas/fisiologia , Plantas/microbiologia , Ácido Abscísico/metabolismo , Ciclopentanos/metabolismo , Etilenos/metabolismo , Ácidos Indolacéticos/metabolismo , Microbiota/fisiologia , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Rizosfera , Ácido Salicílico/metabolismo , Transdução de SinaisRESUMO
In more than 30 years of aptamer research, it has become widely accepted that aptamers are fascinating binding molecules for a vast variety of applications. However, the majority of targets have been proteins, although special variants of the so-called SELEX process for the molecular evolution of specific aptamers have also been developed, allowing for the targeting of small molecules as well as larger structures such as cells and even cellular networks of human (tumor) tissues. Although the provocative thesis is widely accepted in the field, that is, in principle, any level of complexity for SELEX targets is possible, the number of studies on whole organs or at least parts of them is limited. To pioneer this thesis, and based on our FluCell-SELEX process, here, we have developed polyclonal aptamer libraries against apices and the elongation/differentiation zones of plant roots as examples of organs. We show that dedicated libraries can specifically label the respective parts of the root, allowing us to distinguish them in fluorescence microscopy. We consider this achievement to be an initial but important evidence for the robustness of this SELEX variant. These libraries may be valuable tools for plant research and a promising starting point for the isolation of more specific individual aptamers directed against root-specific epitopes.
Assuntos
Aptâmeros de Nucleotídeos , Arabidopsis , Humanos , Aptâmeros de Nucleotídeos/química , Arabidopsis/genética , Arabidopsis/metabolismo , Epitopos , Técnica de Seleção de Aptâmeros , Raízes de Plantas/metabolismoRESUMO
OBJECTIVE: To assess the effect of a routine medication review service in German community pharmacies (ATHINA) on drug-related problems (DRPs) and patient-related outcomes. MATERIALS AND METHODS: From 2015 to 2017, ATHINA patients were invited by their pharmacists to participate in a prospective, observational trial, meaning that they needed to attend to a follow-up visit (T2) 3 - 6 months after the routine ATHINA baseline (T0) and concluding visit (T1) to assess implementation rates of the pharmacists' interventions. Moreover, they were asked to fill in 2 surveys on drug treatment-related quality of life and satisfaction with the amount of information received about medicines at T0, T1, and T2. RESULTS: Of 132 recruited patients, 115 completed T2. At T0, pharmacists documented a DRP or information need for 114 of 115 patients. About half of these issues were resolved leading to 43/115 patients without any DRP or information need at T1 and 50/115 patients without any DRP or information need at T2 (i.e., absolute reduction by 42.6%, p < 0.001). Also, the number of patients who felt that their daily life was not impaired at all or only very slightly by their drug treatment increased from 54.7% (58/106) at T0 to 67.6% (73/108, p = 0.011) at T2. While the overall satisfaction score with the amount of information on medicines increased from 10.2 ± 5.5 at T0 over 14.6 ± 3.8 (T1) to 15.4 ± 3.1 (T2, p < 0.001), this increase did not correlate with reduced information needs. CONCLUSION: The results suggest that the intervention improves medication- and patient-related outcomes. However, causal relationships are still questionable.
Assuntos
Serviços Comunitários de Farmácia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Farmácias , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Alemanha , Humanos , Farmacêuticos , Estudos Prospectivos , Qualidade de VidaRESUMO
Background and Objectives: Producing consistent measures of femoral version amongst observers are necessary to allow for an assessment of version for possible corrective procedures. The purpose of this study was to compare two computed tomography (CT)-based techniques for the reliability of measuring femoral version amongst observers. Materials and Methods: Review was performed for 15 patients post-femoral nailing for comminuted (Winquist III and IV) femoral shaft fractures where CT scanograms were obtained. Two CT-based techniques were utilized to measure femoral version by five observers. Results: The mean femoral version, when utilizing a proximal line drawn down the center of the femoral head-neck through CT, was 9.50 ± 4.82°, while the method utilizing the head and shaft at lesser trochanter centers produced a mean version of 18.73 ± 2.69°. A significant difference was noted between these two (p ≤ 0.001). The method of measuring in the center of the femoral head and neck produced an intraclass correlation coefficient (ICC) of 0.960 with a 95% confidence interval lower bound of 0.909 and upper bound of 0.982. For the method assessing version via the center of the head and shaft at the lesser trochanter region, the ICC was 0.993 with a 95% confidence interval lower bound of 0.987 and an upper bound of 0.996. Conclusions: The method of measuring version proximally through a CT image of the femoral head-neck versus overlaying the femoral head with the femoral shaft at the most prominent aspect of the lesser trochanter produces differing version measurements by roughly 10° while yielding an almost perfect interobserver reliability in the new technique. Both techniques result in significantly high interobserver reliability.
Assuntos
Colo do Fêmur , Fêmur , Fêmur/diagnóstico por imagem , Fêmur/cirurgia , Colo do Fêmur/diagnóstico por imagem , Humanos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Tomografia Computadorizada por Raios XRESUMO
The interest in dry electroencephalography (EEG) electrodes has increased in recent years, especially as everyday suitability earplugs for measuring drowsiness or focus of auditory attention. However, the challenge is still the need for a good electrode material, which is reliable and can be easily processed for highly personalized applications. Laser processing, as used here, is a fast and very precise method to produce personalized electrode configurations that meet the high requirements of in-ear EEG electrodes. The arrangement of the electrodes on the flexible and compressible mats allows an exact alignment to the ear mold and contributes to high wearing comfort, as no edges or metal protrusions are present. For better transmission properties, an adapted coating process for surface enlargement of platinum electrodes is used, which can be controlled precisely. The resulting porous platinum-copper alloy is chemically very stable, shows no exposed copper residues, and enlarges the effective surface area by 40. In a proof-of-principle experiment, these porous platinum electrodes could be used to measure the Berger effect in a dry state using just one ear of a test person. Their signal-to-noise ratio and the frequency transfer function is comparable to gel-based silver/silver chloride electrodes.
Assuntos
Eletrodos , Eletroencefalografia , Platina , Orelha , Humanos , Porosidade , Razão Sinal-RuídoRESUMO
Oomycete pathogens secrete numerous effectors to manipulate host immunity. While some effectors share a conserved structural fold, it remains unclear if any have conserved host targets. Avr3a-like family effectors, which are related to Phytophthora infestans effector PiAvr3a and are widely distributed across diverse clades of Phytophthora species, were used to study this question. By using yeast-two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays, we identified members of the plant cinnamyl alcohol dehydrogenase 7 (CAD7) subfamily as targets of multiple Avr3a-like effectors from Phytophthora pathogens. The CAD7 subfamily has expanded in plant genomes but lost the lignin biosynthetic activity of canonical CAD subfamilies. In turn, we identified CAD7s as negative regulators of plant immunity that are induced by Phytophthora infection. Moreover, AtCAD7 was stabilized by Avr3a-like effectors and involved in suppression of pathogen-associated molecular pattern-triggered immunity, including callose deposition, reactive oxygen species burst and WRKY33 expression. Our results reveal CAD7 subfamily proteins as negative regulators of plant immunity that are exploited by multiple Avr3a-like effectors to promote infection in different host plants.
RESUMO
BACKGROUND: Renal dysfunction following intraoperative arterial hypotension is mainly caused by an insufficient renal blood flow. It is associated with higher mortality and morbidity rates. We hypothesised that the intraoperative haemodynamics are more stable during xenon anaesthesia than during isoflurane anaesthesia in patients undergoing partial nephrectomy. METHODS: We performed a secondary analysis of the haemodynamic variables collected during the randomised, single-blinded, single-centre PaNeX study, which analysed the postoperative renal function in 46 patients who underwent partial nephrectomy. The patients received either xenon or isoflurane anaesthesia with 1:1 allocation ratio. We analysed the duration of the intraoperative systolic blood pressure decrease by > 40% from baseline values and the cumulative duration of a mean arterial blood pressure (MAP) of < 65 mmHg as primary outcomes. The secondary outcomes were related to other blood pressure thresholds, the amount of administered norepinephrine, and the analysis of confounding factors on the haemodynamic stability. RESULTS: The periods of an MAP of < 65 mmHg were significantly shorter in the xenon group than in the isoflurane group. The medians [interquartile range] were 0 [0-10.0] and 25.0 [10.0-47.5] minutes, for the xenon and isoflurane group, respectively (P = 0.002). However, the cumulative duration of a systolic blood pressure decrease by > 40% did not significantly differ between the groups (P = 0.51). The periods with a systolic blood pressure decrease by 20% from baseline, MAP decrease to values < 60 mmHg, and the need for norepinephrine, as well as the cumulative dose of norepinephrine were significantly shorter and lower, respectively, in the xenon group. The confounding factors, such as demographic data, surgical technique, or anaesthesia data, were similar in the two groups. CONCLUSION: The patients undergoing xenon anaesthesia showed a better haemodynamic stability, which might be attributed to the xenon properties. The indirect effect of xenon anaesthesia might be of importance for the preservation of renal function during renal surgery and needs further elaboration. TRIAL REGISTRATION: ClinicalTrials.gov : NCT01839084. Registered 24 April 2013.
Assuntos
Anestésicos Inalatórios/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Isoflurano/farmacologia , Monitorização Intraoperatória , Xenônio/farmacologia , Relação Dose-Resposta a Droga , Feminino , Frequência Cardíaca/efeitos dos fármacos , Hemodinâmica , Humanos , Masculino , Pessoa de Meia-Idade , Nefrectomia , Norepinefrina/administração & dosagem , Ensaios Clínicos Controlados Aleatórios como Assunto , Sístole/efeitos dos fármacos , Vasoconstritores/administração & dosagemRESUMO
Reactive gliosis is an umbrella term for various glia functions in neurodegenerative diseases and upon injury. Specifically, Müller glia (MG) in some species readily regenerate retinal neurons to restore vision loss after insult, whereas mammalian MG respond by reactive gliosis-a heterogeneous response which frequently includes cell hypertrophy and proliferation. Limited regeneration has been stimulated in mammals, with a higher propensity in young MG, and in vitro compared to in vivo, but the underlying processes are unknown. To facilitate studies on the mechanisms regulating and limiting glia functions, we developed a strategy to purify glia and their progeny by fluorescence-activated cell sorting. Dual-transgenic nuclear reporter mice, which label neurons and glia with red and green fluorescent proteins, respectively, have enabled MG enrichment up to 93% purity. We applied this approach to MG in a mouse retina regeneration ex vivo assay. Combined cell size and cell cycle analysis indicates that most MG hypertrophy and a subpopulation proliferates which, over time, become even larger in cell size than the ones that do not proliferate. MG undergo timed differential genomic changes in genes controlling stemness and neurogenic competence; and glial markers are downregulated. Genes that are potentially required for, or associated with, regeneration and reactive gliosis are differentially regulated by retina explant culture time, epidermal growth factor stimulation, and animal age. Thus, MG enrichment facilitates cellular and molecular studies which, in combination with the mouse retina regeneration assay, provide an experimental approach for deciphering mechanisms that possibly regulate reactive gliosis and limit regeneration in mammals.
Assuntos
Diferenciação Celular/fisiologia , Regeneração Nervosa/fisiologia , Neuroglia/citologia , Retina/citologia , Neurônios Retinianos/citologia , Animais , Proliferação de Células/fisiologia , Células Ependimogliais/fisiologia , Camundongos Transgênicos , Neurogênese/fisiologia , Células-Tronco/fisiologiaRESUMO
A growing body of research is highlighting the impacts root-associated microbial communities can have on plant health and development. These impacts can include changes in yield quantity and quality, timing of key developmental stages and tolerance of biotic and abiotic stresses. With such a range of effects it is clear that understanding the factors that contribute to a plant-beneficial root microbiome may prove advantageous. Increasing demands for food by a growing human population increases the importance and urgency of understanding how microbiomes may be exploited to increase crop yields and reduce losses caused by disease. In addition, climate change effects may require novel approaches to overcoming abiotic stresses such as drought and salinity as well as new emerging diseases. This review discusses current knowledge on the formation and maintenance of root-associated microbial communities and plant-microbe interactions with a particular emphasis on the effect of microbe-microbe interactions on the shape of microbial communities at the root surface. Further, we discuss the potential for root microbiome modification to benefit agriculture and food production.
Assuntos
Raízes de Plantas/microbiologia , Microbiologia do Solo , Agricultura , Produtos Agrícolas/microbiologia , Microbiota , Rizosfera , Transdução de SinaisRESUMO
The mechanisms limiting neuronal regeneration in mammals and their relationship with reactive gliosis are unknown. Müller glia (MG), common to all vertebrate retinas, readily regenerate neuron loss in some species, but normally not in mammals. However, experimental stimulation of limited mammalian retina regeneration has been reported. Here, we use a mouse retina organ culture approach to investigate the MG responses at different mouse ages. We found that MG undergo defined spatio-temporal changes upon stimulation. In EGF-stimulated juvenile postmitotic retinas, most MG upregulate cell-cycle regulators (Mcm6, Pcna, Ki67, Ccnd1) within 48 h ex vivo; some also express the neurogenic factors Ascl1, Pax6, and Vsx2; up to 60% re-enter the cell cycle, some of which delaminate to divide mostly apically; and the majority cease to proliferate after stimulation. A subpopulation of MG progeny starts to express transcription factors (Ptf1a, Nr4a2) and neuronal (Calb1, Calb2, Rbfox3), but not glial, markers, indicating neurogenesis. BrdU-tracking, genetic lineage-tracing, and transgenic-reporter experiments suggest that MG reprogram to a neurogenic stage and proliferate; and that some MG progeny differentiate into neuronal-like cells, most likely amacrines, no photoreceptors; most others remain in a de-differentiated state. The mouse MG regeneration potential becomes restricted, dependent on the age of the animal, as observed by limited activation of the cell cycle and neurogenic factors. The stage-dependent analysis of mouse MG revealed similarities and differences when compared with MG-derived regeneration in fish and chicks. Therefore, the mouse retina ex vivo approach is a potential assay for understanding and overcoming the limitations of mammalian MG-derived neuronal regeneration. Postmitotic MG in mouse retina ex vivo can be stimulated to proliferate, express neurogenic factors, and generate progeny expressing neuronal or glial markers. This potential regenerative competence becomes limited with increasing mouse age.
Assuntos
Envelhecimento/fisiologia , Células Ependimogliais/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Retina/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bromodesoxiuridina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Ependimogliais/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Gliose/patologia , Técnicas In Vitro , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
In Arabidopsis thaliana roots, the mutualistic fungus Piriformospora indica initially colonizes living cells, which die as the colonization proceeds. We aimed to clarify the molecular basis of this colonization-associated cell death. Our cytological analyses revealed endoplasmic reticulum (ER) swelling and vacuolar collapse in invaded cells, indicative of ER stress and cell death during root colonization. Consistent with this, P. indica-colonized plants were hypersensitive to the ER stress inducer tunicamycin. By clear contrast, ER stress sensors bZIP60 and bZIP28 as well as canonical markers for the ER stress response pathway, termed the unfolded protein response (UPR), were suppressed at the same time. Arabidopsis mutants compromised in caspase 1-like activity, mediated by cell death-regulating vacuolar processing enzymes (VPEs), showed reduced colonization and decreased cell death incidence. We propose a previously unreported microbial invasion strategy during which P. indica induces ER stress but inhibits the adaptive UPR. This disturbance results in a VPE/caspase 1-like-mediated cell death, which is required for the establishment of the symbiosis. Our results suggest the presence of an at least partially conserved ER stress-induced caspase-dependent cell death pathway in plants as has been reported for metazoans.
Assuntos
Arabidopsis/citologia , Basidiomycota/fisiologia , Morte Celular , Estresse do Retículo Endoplasmático , Raízes de Plantas/microbiologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Caspases/metabolismo , Retículo Endoplasmático/microbiologia , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Simbiose , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas , Vacúolos/metabolismoRESUMO
The root endophytic fungus Serendipita indica establishes beneficial symbioses with a broad spectrum of plants and enhances host resilience against biotic and abiotic stresses. However, little is known about the mechanisms underlying S. indica-mediated plant protection. Here, we report S. indica effector (SIE) 141 and its host target CDSP32, a conserved thioredoxin-like protein, and underlying mechanisms for enhancing pathogen resistance and abiotic salt tolerance in Arabidopsis thaliana. SIE141 binding interfered with canonical targeting of CDSP32 to chloroplasts, leading to its re-location into the plant nucleus. This nuclear translocation is essential for both their interaction and resistance function. Furthermore, SIE141 enhanced oxidoreductase activity of CDSP32, leading to CDSP32-mediated monomerization and activation of NON-EXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1), a key regulator of systemic resistance. Our findings provide functional insights on how S. indica transfers well-known beneficial effects to host plants and indicate CDSP32 as a genetic resource to improve plant resilience to abiotic and biotic stresses.
Assuntos
Arabidopsis , Estresse Salino , Simbiose , Arabidopsis/microbiologia , Arabidopsis/fisiologia , Arabidopsis/genética , Basidiomycota/fisiologia , Oxirredutases/metabolismo , Oxirredutases/genética , Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Plastídeos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Doenças das Plantas/microbiologiaRESUMO
Tubby and Tubby-like proteins (TLPs) were first discovered in mammals, where they are involved in the development and function of neuronal cells. Due to their importance as plasma membrane (PM)-tethered transcription factors or mediators of vesicle trafficking, their lack causes obesity and other disease syndromes. Phosphatidylinositol 4,5-bisphosphate binding of the carboxyl-terminal Tubby domain attaches these proteins to the PM and vesicles and is essential for function. TLPs are conserved across eukaryotic kingdoms including plants, suggesting fundamental biological functions of TLPs. Plant TLPs possess an amino-terminal F-box domain that distinguishes them from other eukaryotic TLPs. Arabidopsis (Arabidopsis thaliana) encodes 11 AtTLPs that fall into six phylogenetic clades. We identified the significance of AtTLPs for root colonization of Arabidopsis by the mutualistic fungus Piriformospora indica. Our results further indicate conserved phosphatidylinositol 4,5-bisphosphate-binding sites in the Tubby domains that are required for PM anchoring of AtTLPs. More detailed studies revealed phospholipase C-triggered release of AtTLP3 from the PM, indicating a conserved mechanism as reported for mammalian Tubby and TLP3. We further show that hydrogen peroxide stimulates the release of AtTLP3 from the PM, presumably for activating downstream events. Different from mammalian homologs, the amino-terminal part of almost all AtTLPs has nucleocytosolic and plastidial localization patterns. Thus, it is tempting to assume that TLPs translate reactive oxygen species currents into signaling not only for transcriptional regulation in the nucleus but also affect plastid-associated functions after release from the PM.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/microbiologia , Basidiomycota/crescimento & desenvolvimento , Proteínas F-Box/metabolismo , Raízes de Plantas/microbiologia , Estresse Fisiológico , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Peróxido de Hidrogênio/farmacologia , Dados de Sequência Molecular , Mutagênese Insercional , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Plastídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Simbiose , Transformação Genética , Fosfolipases Tipo C/metabolismoRESUMO
EndA is a membrane-attached surface-exposed DNA-entry nuclease previously known to be required for genetic transformation of Streptococcus pneumoniae. More recent studies have shown that the enzyme also plays an important role during the establishment of invasive infections by degrading extracellular chromatin in the form of neutrophil extracellular traps (NETs), enabling streptococci to overcome the innate immune system in mammals. As a virulence factor, EndA has become an interesting target for future drug design. Here we present the first mutational and biochemical analysis of recombinant forms of EndA produced either in a cell-free expression system or in Escherichia coli. We identify His160 and Asn191 to be essential for catalysis and Asn182 to be required for stability of EndA. The role of His160 as the putative general base in the catalytic mechanism is supported by chemical rescue of the H160A variant of EndA with imidazole added in excess. Our study paves the way for the identification and development of protein or low-molecular-weight inhibitors for EndA in future high-throughput screening assays.
Assuntos
Proteínas de Bactérias/química , Endodesoxirribonucleases/química , Proteínas de Membrana/química , Streptococcus pneumoniae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Biossíntese de Proteínas , Espalhamento a Baixo Ângulo , Transcrição Gênica , Difração de Raios XRESUMO
The aim of the present study was to assess possible adverse effects of transgene expression in leaves of field-grown barley relative to the influence of genetic background and the effect of plant interaction with arbuscular mycorrhizal fungi. We conducted transcript profiling, metabolome profiling, and metabolic fingerprinting of wild-type accessions and barley transgenics with seed-specific expression of (1,3-1, 4)-beta-glucanase (GluB) in Baronesse (B) as well as of transgenics in Golden Promise (GP) background with ubiquitous expression of codon-optimized Trichoderma harzianum endochitinase (ChGP). We found more than 1,600 differential transcripts between varieties GP and B, with defense genes being strongly overrepresented in B, indicating a divergent response to subclinical pathogen challenge in the field. In contrast, no statistically significant differences between ChGP and GP could be detected based on transcriptome or metabolome analysis, although 22 genes and 4 metabolites were differentially abundant when comparing GluB and B, leading to the distinction of these two genotypes in principle component analysis. The coregulation of most of these genes in GluB and GP, as well as simple sequence repeat-marker analysis, suggests that the distinctive alleles in GluB are inherited from GP. Thus, the effect of the two investigated transgenes on the global transcript profile is substantially lower than the effect of a minor number of alleles that differ as a consequence of crop breeding. Exposing roots to the spores of the mycorrhizal Glomus sp. had little effect on the leaf transcriptome, but central leaf metabolism was consistently altered in all genotypes.
Assuntos
Regulação da Expressão Gênica de Plantas , Hordeum/genética , Hordeum/metabolismo , Perfilação da Expressão Gênica , Glucana 1,3-beta-Glucosidase/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Metaboloma , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente ModificadasRESUMO
Matteson homologations of chiral boronic esters with the aid of unsaturated nucleophiles are powerful for gaining access to a range of different O-heterocycles via subsequent ring-closing metatheses. Using this protocol, six- to eight-membered rings become available and almost any position of the ring can be substituted and/or functionalized.