RESUMO
Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants, which has subsequently been lost by species scattered throughout the radiation of plant diversity to the present day, including the model Arabidopsis thaliana. To explore if elements of this apparently beneficial trait are still present and could be reactivated we generated Arabidopsis plants expressing a constitutively active form of Interacting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost from Arabidopsis along with the AM host trait. We characterize the transcriptomic effect of expressing IPD3 in Arabidopsis with and without exposure to the AM fungus (AMF) Rhizophagus irregularis, and compare these results to the AM model Lotus japonicus and its ipd3 knockout mutant cyclops-4. Despite its long history as a non-AM species, restoring IPD3 in the form of its constitutively active DNA-binding domain to Arabidopsis altered expression of specific gene networks. Surprisingly, the effect of expressing IPD3 in Arabidopsis and knocking it out in Lotus was strongest in plants not exposed to AMF, which is revealed to be due to changes in IPD3 genotype causing a transcriptional state, which partially mimics AMF exposure in non-inoculated plants. Our results indicate that molecular connections to symbiosis machinery remain in place in this nonAM species, with implications for both basic science and the prospect of engineering this trait for agriculture.
Assuntos
Arabidopsis , Lotus , Arabidopsis/genética , Simbiose/genética , Genótipo , Agricultura , Evolução Biológica , Lotus/genéticaRESUMO
Hybrids account for nearly all commercially planted varieties of maize and many other crop plants because crosses between inbred lines of these species produce first-generation [F1] offspring that greatly outperform their parents. The mechanisms underlying this phenomenon, called heterosis or hybrid vigor, are not well understood despite over a century of intensive research. The leading hypotheses-which focus on quantitative genetic mechanisms (dominance, overdominance, and epistasis) and molecular mechanisms (gene dosage and transcriptional regulation)-have been able to explain some but not all of the observed patterns of heterosis. Abiotic stressors are known to impact the expression of heterosis; however, the potential role of microbes in heterosis has largely been ignored. Here, we show that heterosis of root biomass and other traits in maize is strongly dependent on the belowground microbial environment. We found that, in some cases, inbred lines perform as well by these criteria as their F1 offspring under sterile conditions but that heterosis can be restored by inoculation with a simple community of seven bacterial strains. We observed the same pattern for seedlings inoculated with autoclaved versus live soil slurries in a growth chamber and for plants grown in steamed or fumigated versus untreated soil in the field. In a different field site, however, soil steaming increased rather than decreased heterosis, indicating that the direction of the effect depends on community composition, environment, or both. Together, our results demonstrate an ecological phenomenon whereby soil microbes differentially impact the early growth of inbred and hybrid maize.
Assuntos
Bactérias/metabolismo , Fungos/fisiologia , Vigor Híbrido , Plântula/crescimento & desenvolvimento , Microbiologia do Solo , Zea mays/crescimento & desenvolvimento , Plântula/microbiologia , Zea mays/microbiologiaRESUMO
Metaproteomics is a powerful tool for the characterization of metabolism, physiology, and functional interactions in microbial communities, including plant-associated microbiota. However, the metaproteomic methods that have been used to study plant-associated microbiota are very laborious and require large amounts of plant tissue, hindering wider application of these methods. We optimized and evaluated different protein extraction methods for metaproteomics of plant-associated microbiota in two different plant species (Arabidopsis and maize). Our main goal was to identify a method that would work with low amounts of input material (40 to 70 mg) and that would maximize the number of identified microbial proteins. We tested eight protocols, each comprising a different combination of physical lysis method, extraction buffer, and cell-enrichment method on roots from plants grown with synthetic microbial communities. We assessed the performance of the extraction protocols by liquid chromatography-tandem mass spectrometry-based metaproteomics and found that the optimal extraction method differed between the two species. For Arabidopsis roots, protein extraction by beating whole roots with small beads provided the greatest number of identified microbial proteins and improved the identification of proteins from gram-positive bacteria. For maize, vortexing root pieces in the presence of large glass beads yielded the greatest number of microbial proteins identified. Based on these data, we recommend the use of these two methods for metaproteomics with Arabidopsis and maize. Furthermore, detailed descriptions of the eight tested protocols will enable future optimization of protein extraction for metaproteomics in other dicot and monocot plants. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Assuntos
Arabidopsis , Microbiota , Cromatografia Líquida , Proteoma , Espectrometria de Massas , PlantasRESUMO
IMPORTANCE: Synthetic communities (SynComs) are an invaluable tool to characterize and model plant-microbe interactions. Multimember SynComs approximate intricate real-world interactions between plants and their microbiome, but the complexity and time required for their construction increase enormously for each additional member added to the SynCom. Therefore, researchers who study a diversity of microbiomes using SynComs are looking for ways to simplify the use of SynComs. In this manuscript, we evaluate the feasibility of creating ready-to-use freezer stocks of a well-studied seven-member SynCom for maize roots. The frozen ready-to-use SynCom stocks work according to the principle of "just add buffer and apply to sterilized seeds or seedlings" and thus can save time applied in multiple days of laborious growing and combining of multiple microorganisms. We show that ready-to-use SynCom stocks provide comparable results to those of freshly constructed SynComs and thus allow for significant time savings when working with SynComs.
Assuntos
Microbiota , Zea mays , Raízes de Plantas , Bactérias , Plantas , Microbiologia do SoloRESUMO
Lucinid clams are one of the most diverse and widespread symbiont-bearing animal groups in both shallow and deep-sea chemosynthetic habitats. Lucinids harbor Ca. Thiodiazotropha symbionts that can oxidize inorganic and organic substrates such as hydrogen sulfide and formate to gain energy. The interplay between these key metabolic functions, nutrient uptake and biotic interactions in Ca. Thiodiazotropha is not fully understood. We collected Lucinoma kazani individuals from next to a deep-sea brine pool in the eastern Mediterranean Sea, at a depth of 1150 m and used Oxford Nanopore and Illumina sequencing to obtain high-quality genomes of their Ca. Thiodiazotropha gloverae symbiont. The genomes served as the basis for transcriptomic and proteomic analyses to characterize the in situ gene expression, metabolism and physiology of the symbionts. We found genes needed for N2 fixation in the deep-sea symbiont's genome, which, to date, were only found in shallow-water Ca. Thiodiazotropha. However, we did not detect the expression of these genes and thus the potential role of nitrogen fixation in this symbiosis remains to be determined. We also found the high expression of carbon fixation and sulfur oxidation genes, which indicate chemolithoautotrophy as the key physiology of Ca. Thiodiazotropha. However, we also detected the expression of pathways for using methanol and formate as energy sources. Our findings highlight the key traits these microbes maintain to support the nutrition of their hosts and interact with them.
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Objective biomarkers of food intake are a sought-after goal in nutrition research. Most biomarker development to date has focused on metabolites detected in blood, urine, skin or hair, but detection of consumed foods in stool has also been shown to be possible via DNA sequencing. An additional food macromolecule in stool that harbors sequence information is protein. However, the use of protein as an intake biomarker has only been explored to a very limited extent. Here, we evaluate and compare measurement of residual food-derived DNA and protein in stool as potential biomarkers of intake. We performed a pilot study of DNA sequencing-based metabarcoding (FoodSeq) and mass spectrometry-based metaproteomics in five individuals' stool sampled in short, longitudinal bursts accompanied by detailed diet records (n=27 total samples). Dietary data provided by stool DNA, stool protein, and written diet record independently identified a strong within-person dietary signature, identified similar food taxa, and had significantly similar global structure in two of the three pairwise comparisons between measurement techniques (DNA-to-protein and DNA-to-diet record). Metaproteomics identified proteins including myosin, ovalbumin, and beta-lactoglobulin that differentiated food tissue types like beef from dairy and chicken from egg, distinctions that were not possible by DNA alone. Overall, our results lay the groundwork for development of targeted metaproteomic assays for dietary assessment and demonstrate that diverse molecular components of food can be leveraged to study food intake using stool samples.
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The source of protein in a persons diet affects their total life expectancy. However, the mechanisms by which dietary protein sources differentially impact human health and life expectancy are poorly understood. Dietary choices have major impacts on the composition and function of the intestinal microbiota that ultimately mediate host health. This raises the possibility that health outcomes based on dietary protein sources might be driven by interactions between dietary protein and the gut microbiota. In this study, we determine the effects of seven different sources of dietary protein on the gut microbiota in mice. We apply an integrated metagenomics-metaproteomics approach to simultaneously investigate the effects of these dietary protein sources on the gut microbiotas composition and function. The protein abundances measured by metaproteomics can provide microbial species abundances, and evidence for the phenotype of microbiota members on the molecular level because measured proteins allow us to infer the metabolic and physiological processes used by a microbial community. We showed that dietary protein source significantly altered the species composition and overall function of the gut microbiota. Different dietary protein sources led to changes in the abundance of microbial amino acid degrading proteins and proteins involved in the degradation of glycosylations on dietary protein. In particular, brown rice and egg white protein increased the abundance of amino acid degrading enzymes and egg white protein increased the abundance of bacteria and proteins usually associated with the degradation of the intestinal mucus barrier. These results show that dietary protein source can change the gut microbiotas metabolism, which could have major implications in the context of gut microbiota mediated diseases.
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Bathymodioline mussels rely on thiotrophic and/or methanotrophic chemosynthetic symbionts for nutrition, yet, secondary heterotrophic symbionts are often present and play an unknown role in the fitness of the organism. The bathymodioline Idas mussels that thrive in gas seeps and on sunken wood in the Mediterranean Sea and the Atlantic Ocean, host at least six symbiont lineages that often co-occur. These lineages include the primary symbionts chemosynthetic methane- and sulfur-oxidizing gammaproteobacteria, and the secondary symbionts, Methylophagaceae, Nitrincolaceae and Flavobacteriaceae, whose physiology and metabolism are obscure. Little is known about if and how these symbionts interact or exchange metabolites. Here we curated metagenome-assembled genomes of Idas modiolaeformis symbionts and used genome-centered metatranscriptomics and metaproteomics to assess key symbiont functions. The Methylophagaceae symbiont is a methylotrophic autotroph, as it encoded and expressed the ribulose monophosphate and Calvin-Benson-Bassham cycle enzymes, particularly RuBisCO. The Nitrincolaceae ASP10-02a symbiont likely fuels its metabolism with nitrogen-rich macromolecules and may provide the holobiont with vitamin B12. The Urechidicola (Flavobacteriaceae) symbionts likely degrade glycans and may remove NO. Our findings indicate that these flexible associations allow for expanding the range of substrates and environmental niches, via new metabolic functions and handoffs.
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Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants, which has subsequently been lost by species scattered throughout the radiation of plant diversity to the present day, including the model Arabidopsis thaliana. To explore why an apparently beneficial trait would be repeatedly lost, we generated Arabidopsis plants expressing a constitutively active form of Interacting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost from Arabidopsis along with the AM host trait. We characterize the transcriptomic effect of expressing IPD3 in Arabidopsis with and without exposure to the AM fungus (AMF) Rhizophagus irregularis, and compare these results to the AM model Lotus japonicus and its ipd3 knockout mutant cyclops-4. Despite its long history as a non-AM species, restoring IPD3 in the form of its constitutively active DNA-binding domain to Arabidopsis altered expression of specific gene networks. Surprisingly, the effect of expressing IPD3 in Arabidopsis and knocking it out in Lotus was strongest in plants not exposed to AMF, which is revealed to be due to changes in IPD3 genotype causing a transcriptional state which partially mimics AMF exposure in non-inoculated plants. Our results indicate that despite the long interval since loss of AM and IPD3 in Arabidopsis, molecular connections to symbiosis machinery remain in place in this nonAM species, with implications for both basic science and the prospect of engineering this trait for agriculture.
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Pathogenic isolates of Pyrenochaeta lycopersici, the causal agent of corky root rot of tomato, secrete cell death in tomato 1 (CDiT1), a homodimeric protein of 35 kDa inducing cell death after infiltration into the leaf apoplast of tomato. CDiT1 was purified by fast protein liquid chromatography, characterized by mass spectrometry and cDNA cloning. Its activity was confirmed after infiltration of an affinity-purified recombinant fusion of the protein with a C-terminal polyhistidine tag. CDiT1 is highly expressed during tomato root infection compared with axenic culture, and has a putative ortholog in other pathogenic Pleosporales species producing proteinaceous toxins that contribute to virulence. Infiltration of CDiT1 into leaves of other plants susceptible to P. lycopersici revealed that the protein affects them differentially. All varieties of cultivated tomato (Solanum lycopersicum) tested were more sensitive to CDiT1 than those of currant tomato (S. pimpinellifolium). Root infection assays showed that varieties of currant tomato are also significantly less prone to intracellular colonization of their root cells by hyphae of P. lycopersici than varieties of cultivated tomato. Therefore, secretion of this novel type of inducer of cell death during penetration of the fungus inside root cells might favor infection of host species that are highly sensitive to this molecule.
Assuntos
Ascomicetos/metabolismo , Cucumis/microbiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Doenças das Plantas/microbiologia , Solanaceae/microbiologia , Sequência de Aminoácidos , Ascomicetos/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologiaRESUMO
Nodularia spumigena is a filamentous cyanobacterium that is commonly found in brackish water bodies. The species is capable of fixing atmospheric nitrogen in specialized cells termed heterocysts. N. spumigena dominates the annual toxic summer blooms in the Baltic Sea causing environmental and economical problems. We have previously demonstrated that N. spumigena strain AV1 exhibits a different response to the presence of combined nitrogen as compared to model cyanobacteria such as Nostoc PCC7120 and Nostoc punctiforme by uncoupling between nitrogen fixation and heterocyst differentiation.In order to assess whether or not the behaviour of N. spumigena strain AV1 is characteristic of N. spumigena populations, especially in the Baltic Sea, we have investigated the effect of nitrate and ammonium ions on growth, nitrogen fixation activity and presence of heterocysts in eight different Nodularia strains. Our results show that most of the strains retain heterocysts in the absence of nitrogenase activity indicating that uncoupling between nitrogen fixation and heterocyst differentiation is most likely a common behaviour among N. spumigena strains, especially in the Baltic Sea. In addition, there are variations between strains in regard to nitrate uptake.
Assuntos
Fixação de Nitrogênio , Nitrogênio/metabolismo , Nodularia/metabolismo , Geografia , Nitratos/farmacologia , Nodularia/genética , Oceanos e Mares , Compostos de Amônio Quaternário/farmacologia , Especificidade da EspécieRESUMO
Cyanobacterial blooms in the Baltic Sea are a common phenomenon and are formed by the heterocystous, filamentous species Nodularia spumigena. The toxicity of these blooms is attributed to the hepatotoxin nodularin, produced by N. spumigena. Little is known regarding the regulatory mechanisms or environmental signaling that control nodularin production. Here we report the characterization of the transcriptional expression pattern of the nodularin synthetase gene cluster (nda) during phosphate depletion, and nitrogen supplementation. Real-time PCR analysis of these genes revealed that while cells continuously expressed the nda cluster, the expression of all nda genes increased when cells were subjected to phosphate depletion, and decreased in the presence of ammonium. In contrast to the shifts in expression, the intracellular and extracellular nodularin concentrations did not vary significantly during the treatments.
Assuntos
Regulação Bacteriana da Expressão Gênica , Ligases/genética , Família Multigênica , Nodularia/enzimologia , Peptídeos Cíclicos/metabolismo , Água do Mar/microbiologia , Ligases/metabolismo , Nitrogênio/metabolismo , Nodularia/classificação , Nodularia/genética , Nodularia/crescimento & desenvolvimento , Fosfatos/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuéciaRESUMO
The annually reoccurring blooms that characterize the surface waters of the Baltic Sea are dominated by filamentous, heterocystous cyanobacteria such as Nodularia spumigena. In a previous study, we have demonstrated that N. spumigena strain AV1 differentiates heterocysts in the absence of detectable nitrogen fixation activity, an unusual physiological trait that is clearly distinct from other well-studied cyanobacteria. To further analyze the uncoupling between these two processes, we analyzed the gene expression and modification of the nitrogenase enzyme (the enzyme responsible for nitrogen fixation) in N. spumigena AV1, as well as in several other N. spumigena strains. Here, we demonstrate the occurrence of two nifH gene copies in N. spumigena strain AV1, only one of which is located in a complete nifHDK cluster and several NifH protein forms. Furthermore, we demonstrate the occurrence of a DNA rearrangement mechanism acting within the nifH gene copy located in the nifHDK cluster and present only in the strains exhibiting the previously reported uncoupling between heterocyst differentiation and nitrogen fixation processes. These data stress the existence of a distinct and complex regulatory circuit related to nitrogen fixation in this ecologically significant bloom-forming cyanobacterium.
Assuntos
Proteínas de Bactérias/genética , Fixação de Nitrogênio , Nodularia/genética , Oxirredutases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Rearranjo Gênico , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Insercional , Nodularia/enzimologia , Oxirredutases/metabolismo , Fotoperíodo , Água do Mar/microbiologia , Alinhamento de Sequência , Microbiologia da ÁguaRESUMO
The cyanobacterium Nodularia spumigena dominates the annual, toxic summer blooms in the Baltic Sea. Although Nodularia has been receiving attention due to its production of the hepatotoxin nodularin, molecular data regarding the regulation of nitrogen fixation is lacking. We have previously reported that N. spumigena strain AV1, unlike model filamentous cyanobacteria, differentiates heterocysts in the absence of detectable nitrogen fixation activity. To further analyze the uncoupling between these two linked processes, we assessed the impact of ammonium ions on the N. spumigena metabolism using a proteomic approach. Proteomic profiling was performed at three different times during ammonium supplementation using quantitative 2-dimensional gel electrophoresis followed by MS/MS analysis. Using this approach, we identified 34 proteins, 28 of which were unique proteins that changed successively in abundance during growth on ammonium. Our results indicate that N. spumigena generally exhibits lower energy production and carbon fixation in the presence of ammonium and seems to be inefficient in utilizing ammonium as an external nitrogen source. The possibility of ammonium toxicity due to PSII damage was investigated and the results are discussed. Our findings have implications in regard to the strategies considered to manage the cyanobacterial blooms in the Baltic Sea.
Assuntos
Nodularia/metabolismo , Compostos de Amônio Quaternário/metabolismo , Perfilação da Expressão Gênica , Fixação de Nitrogênio , Nodularia/efeitos dos fármacos , Nodularia/crescimento & desenvolvimento , Proteômica , Compostos de Amônio Quaternário/farmacologiaRESUMO
In the presence of ammonium ion, Nodularia spumigena strain AV1, a filamentous, heterocystous cyanobacterium isolated from the Baltic Sea, lost aerobic nitrogen-fixation activity while maintaining heterocyst frequency along the filaments. Real-time RT-PCR showed that the expression of nifH (encoding the dinitrogenase reductase component of the nitrogenase enzyme) was suppressed and the levels of NifH protein decreased dramatically in response to treatment with ammonium. On the other hand, ntcA (encoding the global nitrogen regulator in cyanobacteria) and hetR (the key regulatory gene in heterocyst differentiation) were expressed and their expression patterns were not affected by the treatment with ammonium. These data demonstrate that N. spumigena strain AV1 maintains heterocyst frequency along the filaments in the presence of ammonium and in the absence of detectable N2-fixation activity.