Leguminous plants have established mutualistic endosymbiotic interactions with nitrogen-fixing rhizobia to secure nitrogen sources in root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule, and shoot formation, raising the question of how SIMK modulates these processes. In particular, detailed subcellular spatial distribution, activation, and developmental relocation of SIMK during early stages of alfalfa nodulation remain unclear. Here, we characterized SIMK distribution in Ensifer meliloti-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In the SIMKK-RNAi line, showing down-regulation of SIMKK and SIMK, we found considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads. However, this was strongly increased in the GFP-SIMK line, constitutively overexpressing green fluorescent protein (GFP)-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. Advanced light-sheet fluorescence microscopy on intact plants allowed non-invasive imaging of spatiotemporal interactions between root hairs and symbiotic E. meliloti, while immunofluorescence detection confirmed that SIMK was activated in these locations. Our results shed new light on SIMK spatiotemporal participation in early interactions between alfalfa and E. meliloti, and its internalization into root hairs, showing that local accumulation of active SIMK modulates early nodulation in alfalfa.

• Klíčová slova
• Ensifer meliloti, Alfalfa, MAPKs, SIMK, immunolocalization, infection pocket, infection thread, light-sheet fluorescence microscopy, root hairs, subcellular localization,
• MeSH
• Medicago sativa genetika   metabolismus   MeSH
• mikroskopie MeSH
• mitogenem aktivované proteinkinasy * metabolismus   MeSH
• rostliny metabolismus   MeSH
• Sinorhizobium meliloti * metabolismus   MeSH
• symbióza fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitogenem aktivované proteinkinasy * MeSH

In nature, proteins have evolved sophisticated cavities tailored for capturing target guests selectively among competitors of similar size, shape, and charge. The fundamental principles guiding the molecular recognition, such as self-assembly and complementarity, have inspired the development of biomimetic receptors. In the current work, we report a self-assembled triple anion helicate (host 2) featuring a cavity resembling that of the choline-binding protein ChoX, as revealed by crystal and density functional theory (DFT)-optimized structures, which binds choline in a unique dual-site-binding mode. This similarity in structure leads to a similarly high selectivity of host 2 for choline over its derivatives, as demonstrated by the NMR and fluorescence competition experiments. Furthermore, host 2 is able to act as a fluorescence displacement sensor for discriminating choline, acetylcholine, L-carnitine, and glycine betaine effectively.The choline-binding protein ChoX exhibits a synergistic dual-site binding mode that allows it to discriminate choline over structural analogues. Here, the authors design a biomimetic triple anion helicate receptor whose selectivity for choline arises from a similar binding mechanism.

• MeSH
• acetylcholin chemie   metabolismus   MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• cholin chemie   metabolismus   MeSH
• fosfáty chemie   metabolismus   MeSH
• kinetika MeSH
• kompetitivní vazba MeSH
• krystalografie rentgenová MeSH
• membránové transportní proteiny chemie   metabolismus   MeSH
• molekulární modely MeSH
• proteinové domény * MeSH
• protonová magnetická rezonanční spektroskopie MeSH
• Sinorhizobium meliloti metabolismus   MeSH
• transportní proteiny chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• acetylcholin MeSH
• bakteriální proteiny MeSH
• cholin MeSH
• choline transporter MeSH Prohlížeč
• fosfáty MeSH
• membránové transportní proteiny MeSH
• transportní proteiny MeSH

Quorum sensing is a cell density-dependent communication system of bacteria relying on autoinducer molecules. During the analysis of the post-transcriptional regulation of quorum sensing in the nitrogen fixing plant symbiont Sinorhizobium meliloti, we predicted and verified a direct interaction between the 5'-UTR of sinI mRNA encoding the autoinducer synthase and a small RNA (sRNA), which we named RcsR1. In vitro, RcsR1 prevented cleavage in the 5'-UTR of sinI by RNase E and impaired sinI translation. In line with low ribosomal occupancy and transcript destabilization upon binding of RcsR1 to sinI, overproduction of RcsR1 in S. meliloti resulted in lower level and shorter half-life of sinI mRNA, and in decreased autoinducer amount. Although RcsR1 can influence quorum sensing via sinI, its level did not vary at different cell densities, but decreased under salt stress and increased at low temperature. We found that RcsR1 and its stress-related expression pattern, but not the interaction with sinI homologs, are conserved in Sinorhizobium, Rhizobium and Agrobacterium. Consistently, overproduction of RcsR1 in S. meliloti and Agrobacterium tumefaciens inhibited growth at high salinity. We identified conserved targets of RcsR1 and showed that most conserved interactions and the effect on growth under salt stress are mediated by the first stem-loop of RcsR1, while its central part is responsible for the species-specific interaction with sinI. We conclude that RcsR1 is an ancient, stress-related riboregulator in rhizobia and propose that it links stress responses to quorum sensing in S. meliloti.

• Klíčová slova
• Agrobacterium, RNA degradation, RNase E, Sinorhizobium, autoinducer synthase, degradosome, quorum sensing, small RNA, stress response,
• MeSH
• 5' nepřekládaná oblast MeSH
• bakteriální proteiny genetika   MeSH
• bakteriální RNA genetika   MeSH
• fyziologický stres * MeSH
• konzervovaná sekvence MeSH
• malá nekódující RNA genetika   MeSH
• messenger RNA genetika   MeSH
• quorum sensing MeSH
• regulace genové exprese u bakterií MeSH
• salinita MeSH
• sekvence nukleotidů MeSH
• Sinorhizobium meliloti genetika   fyziologie   MeSH
• transkripční faktory genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• bakteriální proteiny MeSH
• bakteriální RNA MeSH
• LuxI protein, Bacteria MeSH Prohlížeč
• malá nekódující RNA MeSH
• messenger RNA MeSH
• transkripční faktory MeSH

Medicago truncatula represents a model plant species for understanding legume-bacteria interactions. M. truncatula roots form a specific root-nodule symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti. Symbiotic nitrogen fixation generates high iron (Fe) demands for bacterial nitrogenase holoenzyme and plant leghemoglobin proteins. Leguminous plants acquire Fe via "Strategy I," which includes mechanisms such as rhizosphere acidification and enhanced ferric reductase activity. In the present work, we analyzed the effect of S. meliloti volatile organic compounds (VOCs) on the Fe-uptake mechanisms of M. truncatula seedlings under Fe-deficient and Fe-rich conditions. Axenic cultures showed that both plant and bacterium modified VOC synthesis in the presence of the respective symbiotic partner. Importantly, in both Fe-rich and -deficient experiments, bacterial VOCs increased the generation of plant biomass, rhizosphere acidification, ferric reductase activity, and chlorophyll content in plants. On the basis of our results, we propose that M. truncatula perceives its symbiont through VOC emissions, and in response, increases Fe-uptake mechanisms to facilitate symbiosis.

• MeSH
• biomasa MeSH
• chlorofyl analýza   MeSH
• FMN-reduktasa metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• kořenové hlízky rostlin mikrobiologie   MeSH
• Medicago truncatula chemie   růst a vývoj   metabolismus   mikrobiologie   MeSH
• půda chemie   MeSH
• Sinorhizobium meliloti metabolismus   MeSH
• těkavé organické sloučeniny metabolismus   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl MeSH
• ferric citrate iron reductase MeSH Prohlížeč
• FMN-reduktasa MeSH
• půda MeSH
• těkavé organické sloučeniny MeSH
• železo MeSH

In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and "washed pellets") were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of "washed pellets" and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI-TOF MS of "washed pellets" enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI-TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.

• MeSH
• elektroforéza kapilární metody   MeSH
• isoelektrická fokusace metody   MeSH
• mikrobiální viabilita MeSH
• Rhizobium chemie   klasifikace   izolace a purifikace   MeSH
• Sinorhizobium meliloti chemie   klasifikace   izolace a purifikace   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.

• MeSH
• DNA bakterií chemie   genetika   MeSH
• genom bakteriální MeSH
• hybridizace nukleových kyselin MeSH
• Klebsiella pneumoniae enzymologie   genetika   MeSH
• oxidoreduktasy genetika   MeSH
• Sinorhizobium meliloti genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• nitrogenase reductase MeSH Prohlížeč
• oxidoreduktasy MeSH

The root exudates of alfalfa (Medicago sativa) and mungbean (Vigna radiata) induced the Tsr (thick and short roots) factor production in Rhizobium meliloti. The factor caused a 30-40% reduction of root length in alfalfa seedlings. Pea root exudate had no Tsr induction activity. The flavonoid naringenin could replace the roots in inducing Tsr production. Naringenin-induced Tsr factor caused 70% shortening of main roots. The Tsr inducing property of naringenin was specific since quercetin and syringaldehyde had no such effect.

• MeSH
• bakteriální proteiny biosyntéza   MeSH
• flavanony * MeSH
• flavonoidy farmakologie   MeSH
• Medicago sativa metabolismus   mikrobiologie   MeSH
• membránové proteiny biosyntéza   MeSH
• Sinorhizobium meliloti účinky léků   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• flavanony * MeSH
• flavonoidy MeSH
• membránové proteiny MeSH
• naringenin MeSH Prohlížeč
• Tsr protein, Bacteria MeSH Prohlížeč