RESUMO
MAIN CONCLUSION: PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis, which rely on the phosphorylation status of T589, the endocytic YXXØ motif and the kinase activity of the receptor. Legume-rhizobia nodulation is a complex developmental process. It initiates when the rhizobia-produced Nod factors are perceived by specific LysM receptors present in the root hair apical membrane. Consequently, SYMRK (Symbiosis Receptor-like Kinase) becomes active in the root hair and triggers an extensive signaling network essential for the infection process and nodule organogenesis. Despite its relevant functions, the underlying cellular mechanisms involved in SYMRK signaling activity remain poorly characterized. In this study, we demonstrated that PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis. We found that in uninoculated roots, PvSYMRK-EGFP is mainly associated with the plasma membrane, although intracellular puncta labelled with PvSymRK-EGFP were also observed in root hair and nonhair-epidermal cells. Inoculation with Rhizobium etli producing Nod factors induces in the root hair a redistribution of PvSYMRK-EGFP from the plasma membrane to intracellular puncta. In accordance, deletion of the endocytic motif YXXØ (YKTL) and treatment with the endocytosis inhibitors ikarugamycin (IKA) and tyrphostin A23 (TyrA23), as well as brefeldin A (BFA), drastically reduced the density of intracellular PvSYMRK-EGFP puncta. A similar effect was observed in the phosphorylation-deficient (T589A) and kinase-dead (K618E) mutants of PvSYMRK-EGFP, implying these structural features are positive regulators of PvSYMRK-EGFP endocytosis. Our findings lead us to postulate that rhizobia-induced endocytosis of SYMRK modulates the duration and amplitude of the SYMRK-dependent signaling pathway.
Assuntos
Phaseolus , Rhizobium , Nódulos Radiculares de Plantas/metabolismo , Phaseolus/metabolismo , Nodulação , Rhizobium/fisiologia , Simbiose , Proteínas de Transporte/metabolismo , Endocitose , Raízes de Plantas/metabolismo , Proteínas de Plantas/metabolismoRESUMO
The development of a symbiotic nitrogen-fixing nodule in legumes involves infection and organogenesis. Infection begins when rhizobia enter a root hair through an inward structure, the infection thread (IT), which guides the bacteria towards the cortical tissue. Concurrently, organogenesis takes place by inducing cortical cell division (CCD) at the infection site. Genetic analysis showed that both events are well-coordinated; however, the dynamics connecting them remain to be elucidated. To visualize the crossroads between IT and CCD, we benefited from the fact that, in Phaseolus vulgaris nodulation, where the first division occurs in subepidermal cortical cells located underneath the infection site, we traced a Rhizobium etli strain expressing DsRed, the plant cytokinesis marker YFP-PvKNOLLE, a nuclear stain and cell wall auto-fluorescence. We found that the IT exits the root hair to penetrate an underlying subepidermal cortical (S-E) cell when it is concluding cytokinesis.
Assuntos
Phaseolus , Rhizobium , Divisão Celular , Phaseolus/microbiologia , Proteínas de Plantas/genética , Nodulação , Raízes de Plantas/genética , Rhizobium/genética , Nódulos Radiculares de Plantas/microbiologia , Simbiose/genéticaRESUMO
MAIN CONCLUSION: Rhizobium etli CE3-DsRed pMP604 drives infection 12-24 h earlier than R. etli CE3-DsRed and it is an excellent tool in live-cell imaging studies of IT developement in P. vulgaris roots. The study of the cellular dynamics of nodulation has frequently been limited by the difficulty of performing live-cell imaging in nodule primordia and legume roots, which are constituted by multiple cell layers, such is the case of Phaseolus vulgaris. Seeking conditions to reduce the time it takes for rhizobia to infect P. vulgaris root, we decided to explore the nodulation properties of Rhizobium etli CE3 pMP604, a strain that constitutively produces Nod factors through a flavonoids-independent transcriptional activation which is often used to purify Nod factors. Even though the strain infects 12-24 h earlier than the parental R. etli CE3 strain, infection thread (IT) formation, nodule organogenesis processes and N2-fixation activity are similar for both strains. Additionally, we have confirmed that R. etli CE3-DsRed pMP604 is an excellent tool to trace IT development in P. vulgaris roots.
Assuntos
Phaseolus , Nodulação , Rhizobium etli , Rhizobium , Botânica/métodos , Proteínas Luminescentes/metabolismo , Imagem Óptica , Phaseolus/microbiologia , Rhizobium etli/genética , SimbioseRESUMO
Solvent conditions modulate the expression of the amyloidogenic potential of proteins. In this work the effect of pH on the fibrillogenic behavior and the conformational properties of 6aJL2, a model protein of the highly amyloidogenic variable light chain λ6a gene segment, was examined. Ordered aggregates showing the ultrastructural and spectroscopic properties observed in amyloid fibrils were formed in the 2.0-8.0â¯pH range. At pH <3.0 a drastic decrease in lag time and an increase in fibril formation rate were found. In the 4.0-8.0â¯pH range there was no spectroscopic evidence for significant conformational changes in the native state. Likewise, heat capacity measurements showed no evidence for residual structure in the unfolded state. However, at pH <3.0 stability is severely decreased and the protein suffers conformational changes as detected by circular dichroism, tryptophan and ANS fluorescence, as well as by NMR spectroscopy. Molecular dynamics simulations indicate that acid-induced conformational changes involve the exposure of the loop connecting strands E and F. These results are compatible with pH-induced changes in the NMR spectra. Overall, the results indicate that the mechanism involved in the acid-induced increase in the fibrillogenic potential of 6aJL2 is profoundly different to that observed in κ light chains, and is promoted by localized conformational changes in a region of the protein that was previously not known to be involved in acid-induced light chain fibril formation. The identification of this region opens the potential for the design of specific inhibitors.
Assuntos
Amiloide/química , Cadeias lambda de Imunoglobulina/química , Agregados Proteicos , Ácidos/farmacologia , Varredura Diferencial de Calorimetria , Humanos , Concentração de Íons de Hidrogênio , Cadeias lambda de Imunoglobulina/genética , Microscopia Eletrônica , Modelos Moleculares , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/química , Espectrometria de Fluorescência , Ureia/farmacologiaRESUMO
Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40-60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL.
Assuntos
Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/metabolismo , Cadeias lambda de Imunoglobulina/química , Cadeias lambda de Imunoglobulina/metabolismo , Mutagênese Sítio-Dirigida/métodos , Sequência de Aminoácidos , Amiloidose/metabolismo , Cristalografia por Raios X , Humanos , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Emerging evidence indicates that some reactive oxygen species (ROS), such as the superoxide anion radical and hydrogen peroxide (H2O2), are central regulators of plant responses to biotic and abiotic stresses. Thus, the cellular levels of ROS are thought to be tightly regulated by an efficient and elaborate pro- and antioxidant system that modulates the production and scavenging of ROS. Until recently, studies of ROS in plant cells have been limited to biochemical assays and the use of fluorescent probes; however, the irreversible oxidation of these fluorescent probes makes it impossible to visualize dynamic changes in ROS levels. In this work, we describe the use of Hyper, a recently developed live cell probe for H2O2 measurements in living cells, to monitor oxidative stress in Arabidopsis roots subjected to aluminum treatment. Hyper consists of a circularly permuted YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide-binding protein (OxyR), and is a H2O2-specific ratiometric, and therefore quantitative, probe that can be expressed in plant and animal cells. Now we demonstrate that H2O2 levels drop sharply in the elongation zone of roots treated with aluminum. This response could contribute to root growth arrest and provides evidence that H2O2 is involved in early Al sensing.
Assuntos
Alumínio/toxicidade , Arabidopsis/crescimento & desenvolvimento , Técnicas Biossensoriais , Peróxido de Hidrogênio/análise , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/efeitos dos fármacos , Espaço Intracelular/metabolismo , Raízes de Plantas/efeitos dos fármacos , Plantas Geneticamente ModificadasRESUMO
Legume plants secrete signaling molecules called flavonoids into the rhizosphere. These molecules activate the transcription of rhizobial nod genes, which encode proteins involved in the synthesis of signaling compounds named Nod factors (NFs). NFs, in turn, trigger changes in plant gene expression, cortical cell dedifferentiation and mitosis, depolarization of the root hair cell membrane potential and rearrangement of the actin cytoskeleton. Actin polymerization plays an important role in apical growth in hyphae and pollen tubes. Using sublethal concentrations of fluorescently labeled cytochalasin D (Cyt-Fl), we visualized the distribution of filamentous actin (F-actin) plus ends in living Phaseolus vulgaris and Arabidopsis root hairs during apical growth. We demonstrated that Cyt-Fl specifically labeled the newly available plus ends of actin microfilaments, which probably represent sites of polymerization. The addition of unlabeled competing cytochalasin reduced the signal, suggesting that the labeled and unlabeled forms of the drug bind to the same site on F-actin. Exposure to Rhizobium etli NFs resulted in a rapid increase in the number of F-actin plus ends in P. vulgaris root hairs and in the re-localization of F-actin plus ends to infection thread initiation sites. These data suggest that NFs promote the formation of F-actin plus ends, which results in actin cytoskeleton rearrangements that facilitate infection thread formation.
Assuntos
Actinas/metabolismo , Lipopolissacarídeos/farmacologia , Phaseolus/efeitos dos fármacos , Phaseolus/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismoRESUMO
It has been suggested that the N-terminal strand of the light chain variable domain (V(L)) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V(L) protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.
Assuntos
Amiloide/química , Cadeias Leves de Imunoglobulina/química , Cadeias lambda de Imunoglobulina/química , Amiloide/genética , Sítios de Ligação , Cadeias Leves de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The reactive oxygen species (ROS) generated by respiratory burst oxidative homologs (Rbohs) are involved in numerous plant cell signaling processes, and have critical roles in the symbiosis between legumes and nitrogen-fixing bacteria. Previously, down-regulation of RbohB in Phaseolus vulgaris was shown to suppress ROS production and abolish Rhizobium infection thread (IT) progression, but also to enhance arbuscular mycorrhizal fungal (AMF) colonization. Thus, Rbohs function both as positive and negative regulators. Here, we assessed the effect of enhancing ROS concentrations, by overexpressing PvRbohB, on the P. vulgaris--rhizobia and P. vulgaris--AMF symbioses. We estimated superoxide concentrations in hairy roots overexpressing PvRbohB, determined the status of early and late events of both Rhizobium and AMF interactions in symbiont-inoculated roots, and analyzed the nodule ultrastructure of transgenic plants overexpressing PvRbohB. Overexpression of PvRbohB significantly enhanced ROS production, the formation of ITs, nodule biomass, and nitrogen-fixing activity, and increased the density of symbiosomes in nodules, and the density and size of bacteroides in symbiosomes. Furthermore, PvCAT, early nodulin, PvSS1, and PvGOGAT transcript abundances were elevated in these nodules. By contrast, mycorrhizal colonization was reduced in roots that overexpressed RbohB. Overexpression of PvRbohB augmented nodule efficiency by enhancing nitrogen fixation and delaying nodule senescence, but impaired AMF colonization.
Assuntos
Genes de Plantas , Micorrizas/crescimento & desenvolvimento , NADPH Oxidases/genética , Fixação de Nitrogênio/genética , Phaseolus/enzimologia , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/microbiologia , Simbiose/genética , Biomassa , Clonagem Molecular , Contagem de Colônia Microbiana , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , NADPH Oxidases/metabolismo , Phaseolus/genética , Phaseolus/microbiologia , Phaseolus/ultraestrutura , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/ultraestruturaRESUMO
Rotavirus VP6 nanotubes are an attractive option for a recombinant vaccine against rotavirus disease. Protection against rotavirus infection and an adjuvant effect have been observed upon immunization with VP6 nanotubes. However, little information exists on how VP6 nanotubes interact with cells and trigger an immune response. In this work, the interaction between VP6 nanotubes and different cell lines was characterized. VP6 nanotubes were not cytotoxic to any of the animal or human cell lines tested. Uptake of nanotubes into cells was cell-line-dependent, as only THP1 and J774 macrophage cells internalized them. Moreover, the size and spatial arrangement of VP6 assembled into nanotubes allowed their uptake by macrophages, as double-layered rotavirus-like particles also displaying VP6 in their surface were not taken up. The internalization of VP6 nanotubes was inhibited by methyl-ß-cyclodextrin, but not by genistein, indicating that nanotube entry is specific, depends on the presence of cholesterol in the plasma membrane, and does not require the activity of tyrosine kinases. The information generated here expands our understanding of the interaction of protein nanotubes with cells, which is useful for the application of VP6 nanotubes as a vaccine.
Assuntos
Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Nanotubos/virologia , Rotavirus/fisiologia , Vacinas Sintéticas , Internalização do Vírus , Animais , Células COS , Células CACO-2 , Chlorocebus aethiops , Colesterol , Endocitose/efeitos dos fármacos , Genisteína/farmacologia , Células HEK293 , Humanos , Nanotubos/química , Proteínas Tirosina Quinases , Rotavirus/imunologia , Vacinas Virais/imunologia , beta-Ciclodextrinas/farmacologiaRESUMO
Proteins are biological units whose essence is defined by their functional relationships with other proteins or biomolecules such as RNA, DNA, lipids, or carbohydrates. These functions encompass enzymatic, structural, regulatory, or physical interaction roles. The STRING database (Nucleic Acids Research, 8 Jan 2021;49(D1): D605-12) provides an index that defines the functional interaction networks between proteins in model organisms. To facilitate the identification, visualization, and evaluation of potential functional networks across organisms from different phylogenetic lineages, we have developed PhyloString (https://biocomputo.ibt.unam.mx/phylostring/), a web server that utilizes the indices of the STRING database. PhyloString decomposes these functional networks into modules, representing cohesive units of proteins grouped based on their similarity of STRING values and the phylogenetic origins of their respective organisms. This study presents and thoroughly discusses examples of such functional networks and their modules identified using PhyloString.
Assuntos
Proteínas , Software , Filogenia , Proteínas/química , Computadores , RNA/química , InternetRESUMO
Plant NADPH oxidases (RBOHs) regulate the early stages of rhizobial infection in Phaseolus vulgaris and affect nodule function in Medicago truncatula. In contrast, the role of RBOHs in the plant-arbuscular mycorrhizal (AM) symbiosis and in the regulation of reactive oxygen species (ROS) production during the establishment of the AM interaction is largely unknown. In this study, we assessed the role of P. vulgaris Rboh (PvRbohB) during the symbiosis with the AM fungus, Rhizophagus irregularis. Our results indicate that the PvRbohB transcript is significantly up-regulated in the mycorrhized roots of P. vulgaris. Further, the PvRbohB promoter was found to be active during the invasion of R. irregularis. Down-regulation of PvRbohB transcription by RNAi (RNA interference) silencing resulted in diminished ROS levels in the transgenic mycorrhized roots and induced early hyphal root colonization. Interestingly, the size of appressoria increased in PvRbohB-RNAi roots (760 ± 70.1 µm) relative to controls (251 ± 73.2 µm). Finally, the overall level of mycorrhizal colonization significantly increased in PvRbohB-RNAi roots [48.1 ± 3.3% root length colonization (RLC)] compared with controls (29.4 ± 1.9% RLC). We propose that PvRbohB negatively regulates AM colonization in P. vulgaris.
Assuntos
Regulação da Expressão Gênica de Plantas , Glomeromycota/fisiologia , Micorrizas/genética , NADPH Oxidases/genética , Phaseolus/genética , Espécies Reativas de Oxigênio/metabolismo , Regulação para Baixo , Genes Reporter , Micorrizas/citologia , Micorrizas/fisiologia , NADPH Oxidases/metabolismo , Phaseolus/citologia , Phaseolus/microbiologia , Phaseolus/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Interferência de RNA , Espécies Reativas de Oxigênio/análise , SimbioseRESUMO
Plant NADPH oxidases [respiratory burst oxidase homologs (RBOHs)] have emerged as key players in the regulation of plant-pathogen interactions. Nonetheless, their role in mutualistic associations, such as the rhizobia-legume symbiosis, is poorly understood. In this work, nine members of the Phaseolus vulgaris Rboh gene family were identified. The transcript of one of these, PvRbohB, accumulated abundantly in shoots, roots and nodules. PvRbohB promoter activity was detected in meristematic regions of P. vulgaris roots, as well as during infection thread (IT) progression and nodule development. RNA interference (RNAi)-mediated PvRbohB down-regulation in transgenic roots reduced reactive oxygen species (ROS) production and lateral root density, and greatly impaired nodulation. Microscopy analysis revealed that progression of the ITs was impeded at the base of root hairs in PvRbohB-RNAi roots. Furthermore, the few nodules that formed in PvRbohB-down-regulated roots displayed abnormally wide ITs and reduced nitrogen fixation. These findings indicate that this common bean NADPH oxidase is crucial for successful rhizobial colonization and probably maintains proper IT growth and shape.
Assuntos
Regulação da Expressão Gênica de Plantas , NADPH Oxidases/genética , Phaseolus/genética , Phaseolus/microbiologia , Proteínas de Plantas/genética , Raízes de Plantas/genética , Rhizobium/fisiologia , Sequência de Bases , Dados de Sequência Molecular , Família Multigênica , NADPH Oxidases/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/microbiologia , Simbiose/genéticaRESUMO
The symbiotic interaction of legumes and rhizobia results in the formation of nitrogen-fixing nodules. Nodulation depends on the finely coordinated expression of a battery of genes involved in the infection and the organogenesis processes. After Nod factor perception, symbiosis receptor kinase (SymRK) receptor triggers a signal transduction cascade essential for nodulation leading to cortical cell divisions, infection thread (IT) formation and final release of rhizobia to the intracellular space, forming the symbiosome. Herein, the participation of SymRK receptor during the nodule organogenesis in Phaseolus vulgaris is addressed. Our findings indicate that besides its expression in the nodule epidermis, in IT, and in uninfected cells of the infection zone, PvSymRK immunolocalizes in the root and nodule vascular system. On the other hand, knockdown expression of PvSymRK led to the formation of scarce and defective nodules, which presented alterations in both IT/symbiosome formation and vascular system.
Assuntos
Phaseolus/genética , Proteínas de Plantas/metabolismo , Nodulação , Proteínas Serina-Treonina Quinases/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Regulação para Baixo , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Phaseolus/enzimologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Proteínas Serina-Treonina Quinases/genética , Nódulos Radiculares de Plantas/enzimologia , Transdução de Sinais , SimbioseRESUMO
Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix alpha-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix alpha-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Endotoxinas/genética , Endotoxinas/metabolismo , Engenharia Genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Insetos/efeitos dos fármacos , Larva/efeitos dos fármacos , Aedes/efeitos dos fármacos , Animais , Anopheles/efeitos dos fármacos , Toxinas de Bacillus thuringiensis , Bioensaio , Western Blotting , Antígenos CD13/metabolismo , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Insetos/metabolismo , Inseticidas/farmacologia , Larva/metabolismo , Larva/microbiologia , Bicamadas Lipídicas , Manduca/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Mutação/genética , Controle Biológico de Vetores , Multimerização Proteica , TriptofanoRESUMO
Conjugative transfer is one of the mechanisms allowing diversification and evolution of bacteria. Rhizobium etli CFN42 is a bacterial strain whose habitat is the rhizosphere and is able to form nodules as a result of the nitrogen-fixing symbiotic relationship it may establish with the roots of Phaseolus vulgaris. R. etli CFN42 contains one chromosome and six large plasmids (pRet42a - pRet42f). Most of the genetic information involved in the establishment of the symbiosis is localized on plasmid pRet42d, named as the symbiotic plasmid (pSym). This plasmid is able to perform conjugation, using pSym encoded transfer genes controlled by the RctA/RctB system. Another plasmid of CFN42, pRet42a, has been shown to perform conjugative transfer not only in vitro, but also on the surface of roots and inside nodules, using other rhizobia as recipients. In addition to the rhizobia involved in the formation of nodules, these structures have been shown to contain endophytic bacteria from different genera and species. In this work, we have explored the conjugative transfer of the pSym (pRet42d) from R. etli CFN42 to endophytic bacteria as putative recipients, using as donor a CFN42 derivative labeled with GFP in the pRet42d and RFP in the chromosome. We were able to isolate some transconjugants, which inherit the GFP, but not the RFP marker. Some of them were identified, analyzed and evaluated for their ability to nodulate. We found transconjugants from genera such as Stenotrophomonas, Achromobacter, and Bacillus, among others. Although all the transconjugants carried the GFP marker, and nod, fix, and nif genes from pRet42d, not all were able to nodulate. Ultrastructure microscopy analysis showed some differences in the structure of the nodules of one of the transconjugants. A replicon of the size of pRet42d (371 Kb) could not be visualized in the transconjugants, suggesting that the pSym or a segment of the plasmid is integrated in the chromosome of the recipients. These findings strengthen the proposal that nodules constitute a propitious environment for exchange of genetic information among bacteria, in addition to their function as structures where nitrogen fixation and assimilation takes place.
RESUMO
Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.
Assuntos
Células Germinativas , Cadeias lambda de Imunoglobulina/química , Termodinâmica , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Humanos , Cadeias lambda de Imunoglobulina/genética , Mutagênese Sítio-Dirigida , Conformação Proteica , Análise Espectral/métodosRESUMO
Reactive oxygen species (ROS) produced by respiratory burst oxidase homologs (RBOHs) regulate numerous plant cell processes, including the symbiosis between legumes and nitrogen-fixing bacteria. Rapid and transient ROS production was reported after Phaseolus vulgaris root hairs were treated with Nod factors, indicating the presence of a ROS-associated molecular signature in the symbiosis signaling pathway. Rboh is a multigene family containing nine members (RbohA-I) in P. vulgaris. RNA interference of RbohB suppresses ROS production and attenuates rhizobial infection thread (IT) progression in P. vulgaris root hairs. However, the roles of other Rboh members in symbiotic interactions are largely unknown. In this study, we characterized the role of the NADPH oxidase-encoding gene RbohA (Phvulv091020621) in the P. vulgaris-Rhizobium tropici symbiosis. The spatiotemporal activity of the RbohA promoter colocalized with growing ITs and was associated with vascular bundles in developing nodules. Subcellular localization studies indicated that RBOHA was localized in the plasma membrane of P. vulgaris root hairs. After rhizobial inoculation, PvRBOHA was mainly distributed in the infection pocket and, to a lesser extent, throughout the IT. In PvRbohA RNAi lines, the rhizobial infection events were significantly reduced and, in successful infections, IT progression was arrested within the root hair, but did not impede cortical cell division. PvRbohA-RNAi nodules failed to fix nitrogen, since the infected cells in the few nodules formed were empty. RbohA-dependent ROS production and upregulation of several antioxidant enzymes was attenuated in rhizobia-inoculated PvRbohA-RNAi roots. These combined results indicate that PvRbohA is crucial for effective Rhizobium infection and its release into the nodule cells. This oxidase is partially or indirectly required to promote nodule organogenesis, altering the expression of auxin- and cyclin-related genes and genes involved in cell growth and division.
RESUMO
Approximately 25% of the λ6 light chains have glycine rather than arginine at position 25, which is an allelic variant of the IGLV6-57 (6a) locus. The Gly25 variant has been shown to decrease the folding stability of the germline λ6 V(L) protein 6aJL2 by 1.7 kcal·mol(-1). In this work, we compared the thermodynamic and fibrillogenic properties of the amyloidosis (AL) derived recombinant (r) V(L) protein AR, which contains the allelic variant Gly25, with those of germline rV(L) 6aJL2-R25G and the λ6 disease-associated V(L) proteins Wil (AL) and Jto (myeloma). Our experiments show that of the four proteins AR is the least stable; forms amyloid fibrils at physiological temperature, pH and ionic strength; has the shortest lag time; and elongates homologous seeds most efficiently. We conclude that the Gly25 allelic variant, together with the somatic mutations, contributes importantly to the extremely low stability and high amyloidogenicity of the AL-derived protein AR.