RESUMO
Mycorrhizal fungi form mutualistic associations with the roots of most land plants and provide them with mineral nutrients from the soil in exchange for fixed carbon derived from photosynthesis. The common symbiosis pathway (CSP) is a conserved molecular signaling pathway in all plants capable of associating with arbuscular mycorrhizal fungi. It is required not only for arbuscular mycorrhizal symbiosis but also for rhizobia-legume and actinorhizal symbioses. Given its role in such diverse symbiotic associations, we hypothesized that the CSP also plays a role in ectomycorrhizal associations. We showed that the ectomycorrhizal fungus Laccaria bicolor produces an array of lipochitooligosaccharides (LCOs) that can trigger both root hair branching in legumes and, most importantly, calcium spiking in the host plant Populus in a CASTOR/POLLUX-dependent manner. Nonsulfated LCOs enhanced lateral root development in Populus in a calcium/calmodulin-dependent protein kinase (CCaMK)-dependent manner, and sulfated LCOs enhanced the colonization of Populus by L. bicolor Compared with the wild-type Populus, the colonization of CASTOR/POLLUX and CCaMK RNA interference lines by L. bicolor was reduced. Our work demonstrates that similar to other root symbioses, L. bicolor uses the CSP for the full establishment of its mutualistic association with Populus.
Assuntos
Canais de Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Laccaria/metabolismo , Lipopolissacarídeos/metabolismo , Raízes de Plantas/microbiologia , Simbiose/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Regulação da Expressão Gênica de Plantas , Lipopolissacarídeos/química , Micorrizas/crescimento & desenvolvimento , Micorrizas/metabolismo , Micorrizas/fisiologia , Raízes de Plantas/química , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Populus/genética , Populus/metabolismo , Transdução de SinaisRESUMO
Signals and signaling pathways underlying the symbiosis between legumes and rhizobia have been studied extensively over the past decades. In a previous phosphoproteomic study on the Medicago truncatula-Sinorhizobium meliloti symbiosis, we identified plant proteins that are differentially phosphorylated upon the perception of rhizobial signals, called Nod factors. In this study, we provide experimental evidence that one of these proteins, Early Phosphorylated Protein 1 (EPP1), is required for the initiation of this symbiosis. Upon inoculation with rhizobia, MtEPP1 expression was induced in curled root hairs. Down-regulation of MtEPP1 in M. truncatula roots almost abolished calcium spiking, reduced the expression of essential symbiosis-related genes (MtNIN, MtNF-YB1, MtERN1 and MtENOD40) and strongly decreased nodule development. Phylogenetic analyses revealed that orthologs of MtEPP1 are present in legumes and specifically in plant species able to host arbuscular mycorrhizal fungi, suggesting a possible role in this association too. Short chitin oligomers induced the phosphorylation of MtEPP1 like Nod factors. However, the down-regulation of MtEPP1 affected the colonization of M. truncatula roots by arbuscular mycorrhizal fungi only moderately. Altogether, these findings indicate that MtEPP1 is essential for the establishment of the legume-rhizobia symbiosis but might plays a limited role in the arbuscular mycorrhizal symbiosis.
Assuntos
Medicago truncatula/metabolismo , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/genética , Nódulos Radiculares de Plantas/genética , Simbiose/genética , Simbiose/fisiologiaRESUMO
Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.
Assuntos
Lotus/microbiologia , Medicago truncatula/microbiologia , Micorrizas/fisiologia , Oryza/microbiologia , Transdução de Sinais , Simbiose , Sinalização do Cálcio/efeitos dos fármacos , Quitina/análogos & derivados , Quitina/farmacologia , Quitosana , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucuronidase/metabolismo , Lipopolissacarídeos/farmacologia , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/genética , Dados de Sequência Molecular , Micorrizas/efeitos dos fármacos , Oligossacarídeos/farmacologia , Oryza/efeitos dos fármacos , Oryza/genética , Plântula/efeitos dos fármacos , Plântula/microbiologia , Transdução de Sinais/efeitos dos fármacos , Simbiose/efeitos dos fármacosRESUMO
Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca(2+) concentration (Ca(2+) spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca(2+) spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume-rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca(2+) spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca(2+) spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca(2+) spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca(2+) spiking in this heterologous system.
Assuntos
Redes e Vias Metabólicas , Ácido Mevalônico/metabolismo , Transdução de Sinais , Simbiose , Arabidopsis/genética , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/genética , Medicago truncatula/microbiologia , Redes e Vias Metabólicas/efeitos dos fármacos , Ácido Mevalônico/farmacologia , Mutação/genética , Micorrizas/efeitos dos fármacos , Micorrizas/fisiologia , Epiderme Vegetal/citologia , Epiderme Vegetal/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transdução de Sinais/efeitos dos fármacos , Simbiose/efeitos dos fármacos , Simbiose/genéticaRESUMO
Arbuscular mycorrhiza and the rhizobia-legume symbiosis are two major root endosymbioses that facilitate plant nutrition. In Lotus japonicus, two symbiotic cation channels, CASTOR and POLLUX, are indispensable for the induction of nuclear calcium spiking, one of the earliest plant responses to symbiotic partner recognition. During recent evolution, a single amino acid substitution in DOES NOT MAKE INFECTIONS1 (DMI1), the POLLUX putative ortholog in the closely related Medicago truncatula, rendered the channel solo sufficient for symbiosis; castor, pollux, and castor pollux double mutants of L. japonicus were rescued by DMI1 alone, while both Lj-CASTOR and Lj-POLLUX were required for rescuing a dmi1 mutant of M. truncatula. Experimental replacement of the critical serine by an alanine in the selectivity filter of Lj-POLLUX conferred a symbiotic performance indistinguishable from DMI1. Electrophysiological characterization of DMI1 and Lj-CASTOR (wild-type and mutants) by planar lipid bilayer experiments combined with calcium imaging in Human Embryonic Kidney-293 cells expressing DMI1 (the wild type and mutants) suggest that the serine-to-alanine substitution conferred reduced conductance with a long open state to DMI1 and improved its efficiency in mediating calcium oscillations. We propose that this single amino acid replacement in the selectivity filter made DMI1 solo sufficient for symbiosis, thus explaining the selective advantage of this allele at the mechanistic level.
Assuntos
Sinalização do Cálcio/fisiologia , Evolução Molecular , Canais Iônicos/metabolismo , Lotus/fisiologia , Medicago truncatula/fisiologia , Proteínas de Plantas/genética , Alanina/genética , Substituição de Aminoácidos , Evolução Biológica , Linhagem Celular , Fenômenos Eletrofisiológicos , Fabaceae/fisiologia , Teste de Complementação Genética , Humanos , Canais Iônicos/genética , Dados de Sequência Molecular , Mutação , Micorrizas/fisiologia , Filogenia , Proteínas de Plantas/metabolismo , Potássio/metabolismo , Serina/genética , Simbiose/fisiologiaRESUMO
Symbiotic associations between leguminous plants and nitrogen-fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula-Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen-fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique - matrix-assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) - to study metabolite distribution in roots and root nodules of M. truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8-bis(dimethyl-amino) naphthalene, DMAN] with a conventional matrix 2,5-dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non-fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.
Assuntos
Medicago truncatula/metabolismo , Metaboloma , Imagem Molecular/métodos , Sinorhizobium meliloti/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Regulação da Expressão Gênica de Plantas , Gentisatos/química , Medicago truncatula/microbiologia , Naftóis/química , Nitrogênio/metabolismo , Fixação de Nitrogênio , Fenótipo , Nodulação , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Nódulos Radiculares de Plantas , SimbioseRESUMO
Symbiotic associations between legumes and rhizobia usually commence with the perception of bacterial lipochitooligosaccharides, known as Nod factors (NF), which triggers rapid cellular and molecular responses in host plants. We report here deep untargeted tandem mass spectrometry-based measurements of rapid NF-induced changes in the phosphorylation status of 13,506 phosphosites in 7739 proteins from the model legume Medicago truncatula. To place these phosphorylation changes within a biological context, quantitative phosphoproteomic and RNA measurements in wild-type plants were compared with those observed in mutants, one defective in NF perception (nfp) and one defective in downstream signal transduction events (dmi3). Our study quantified the early phosphorylation and transcription dynamics that are specifically associated with NF-signaling, confirmed a dmi3-mediated feedback loop in the pathway, and suggested "cryptic" NF-signaling pathways, some of them being also involved in the response to symbiotic arbuscular mycorrhizal fungi.
Assuntos
Lipopolissacarídeos/metabolismo , Medicago truncatula/metabolismo , Medicago truncatula/microbiologia , Micorrizas/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Sinorhizobium meliloti/metabolismo , Simbiose , Medicago truncatula/genética , Fosforilação , Rhizobium/metabolismo , Transdução de Sinais/genética , Sinorhizobium meliloti/genética , Espectrometria de Massas em Tandem , TranscriptomaRESUMO
Peptide sequencing by computational assignment of tandem mass spectra to a database of putative protein sequences provides an independent approach to confirming or refuting protein predictions based on large-scale DNA and RNA sequencing efforts. This use of mass spectrometrically-derived sequence data for testing and refining predicted gene models has been termed proteogenomics. We report herein the application of proteogenomic methodology to a database of 10.9 million tandem mass spectra collected over a period of two years from proteolytically generated peptides isolated from the model legume Medicago truncatula. These spectra were searched against a database of predicted M. truncatula protein sequences generated from public databases, in silico gene model predictions, and a whole-genome six-frame translation. This search identified 78,647 distinct peptide sequences, and a comparison with the publicly available proteome from the recently published M. truncatula genome supported translation of 9,843 existing gene models and identified 1,568 novel peptides suggesting corrections or additions to the current annotations. Each supporting and novel peptide was independently validated using mRNA-derived deep sequencing coverage and an overall correlation of 93% between the two data types was observed. We have additionally highlighted examples of several aspects of structural annotation for which tandem MS provides unique evidence not easily obtainable through typical DNA or RNA sequencing. Proteogenomic analysis is a valuable and unique source of information for the structural annotation of genomes and should be included in such efforts to ensure that the genome models used by biologists mirror as accurately as possible what is present in the cell.
Assuntos
Genoma de Planta , Medicago truncatula/genética , Peptídeos/genética , Proteínas de Plantas/genética , Proteômica , Algoritmos , Sequência de Aminoácidos , Bases de Dados de Proteínas/normas , Disseminação de Informação , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/análise , Proteínas de Plantas/análise , Proteoma , Análise de Sequência de DNARESUMO
Nuclear-associated oscillations in calcium act as a secondary messenger in the symbiotic signaling pathway of legumes. These are decoded by a nuclear-localized calcium and calmodulin-dependent protein kinase, the activation of which is sufficient to drive downstream responses. This implies that the calcium oscillations within the nucleus are the predominant signals for legume symbiosis. However, the mechanisms that allow targeted release of calcium in the nuclear region have not been defined. Here we show that symbiosis-induced calcium changes occur in both the nucleoplasm and the perinuclear cytoplasm and seem to originate from the nuclear membranes. Reaction diffusion simulations suggest that spike generation within the nucleoplasm is not possible through transmission of a calcium wave from the cytoplasm alone and that calcium is likely to be released across the inner nuclear membrane to allow nuclear calcium changes. In agreement with this, we found that the cation channel DMI1, which is essential for symbiotic calcium oscillations, is preferentially located on the inner nuclear membrane, implying an essential function for the inner nuclear membrane in symbiotic calcium signaling. Furthermore, a sarco/endoplasmic reticulum calcium ATPase (SERCA) essential for symbiotic calcium oscillations is targeted to the inner nuclear membrane, as well as the outer nuclear membrane and endoplasmic reticulum (ER). We propose that release of calcium across the inner nuclear membrane allows targeted release of the ER calcium store, and efficient reloading of this calcium store necessitates the capture of calcium from the nucleoplasm and nuclear-associated cytoplasm.
Assuntos
Sinalização do Cálcio , Medicago truncatula/citologia , Medicago truncatula/metabolismo , Membrana Nuclear/metabolismo , Simbiose/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Difusão/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inativação Gênica/efeitos dos fármacos , Canais Iônicos/metabolismo , Lipopolissacarídeos/farmacologia , Medicago truncatula/enzimologia , Medicago truncatula/ultraestrutura , Modelos Biológicos , Dados de Sequência Molecular , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/ultraestrutura , Epiderme Vegetal/citologia , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/ultraestrutura , Transporte Proteico/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Simbiose/efeitos dos fármacosRESUMO
Root hairs play important roles in the interaction of plants with their environment. Root hairs anchor the plant in the soil, facilitate nutrient uptake from the rhizosphere, and participate in symbiotic plant-microbe interactions. These specialized cells grow in a polar fashion which gives rise to their elongated shape, a process mediated in part by a family of small GTPases known as Rops. RopGEFs (GEF, guanine nucleotide exchange factor) activate Rops to effect tip growth in Arabidopsis pollen and root hairs, but the genes mediating tip growth in legumes have not yet been characterized. In this report we describe the Rop and RopGEF gene families from the model legume Medicago truncatula and from the crop legume soybean. We find that one member of the M. truncatula gene family, MtRopGEF2, is required for root hair development because silencing this gene by RNA interference affects the cytosolic Ca2+ gradient and subcellular structure of root hairs, and reduces root hair growth. Consistent with its role in polar growth, we find that a GFP::MtRopGEF2 fusion protein localizes in the apex of emerging and actively growing root hairs. The amino terminus of MtRopGEF2 regulates its ability to interact with MtRops in yeast, and regulates its biological activity in vivo.
Assuntos
Regulação da Expressão Gênica de Plantas , Glycine max/crescimento & desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/genética , Raízes de Plantas/crescimento & desenvolvimento , Sequência de Aminoácidos , Cálcio/metabolismo , Regulação Enzimológica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Medicago truncatula/metabolismo , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Glycine max/genética , Glycine max/metabolismo , Leveduras/metabolismoRESUMO
Symbioses between angiosperms and rhizobia or arbuscular mycorrhizal fungi are controlled through a conserved signaling pathway. Microbe-derived, chitin-based elicitors activate plant cell surface receptors and trigger nuclear calcium oscillations, which are decoded by a calcium/calmodulin-dependent protein kinase (CCaMK) and its target transcription factor interacting protein of DMI3 (IPD3). Genes encoding CCaMK and IPD3 have been lost in multiple non-mycorrhizal plant lineages yet retained among non-mycorrhizal mosses. Here, we demonstrated that the moss Physcomitrium is equipped with a bona fide CCaMK that can functionally complement a Medicago loss-of-function mutant. Conservation of regulatory phosphosites allowed us to generate predicted hyperactive forms of Physcomitrium CCaMK and IPD3. Overexpression of synthetically activated CCaMK or IPD3 in Physcomitrium led to abscisic acid (ABA) accumulation and ectopic development of brood cells, which are asexual propagules that facilitate escape from local abiotic stresses. We therefore propose a functional role for Physcomitrium CCaMK-IPD3 in stress-associated developmental reprogramming.
RESUMO
A family of plant nuclear ion channels, including DMI1 (Does not Make Infections 1) and its homologs CASTOR and POLLUX, are required for the establishment of legume-microbe symbioses by generating nuclear and perinuclear Ca2+ spiking. Here we show that CASTOR from Lotus japonicus is a highly selective Ca2+ channel whose activation requires cytosolic/nucleosolic Ca2+, contrary to the previous suggestion of it being a K+ channel. Structurally, the cytosolic/nucleosolic ligand-binding soluble region of CASTOR contains two tandem RCK (Regulator of Conductance for K+) domains, and four subunits assemble into the gating ring architecture, similar to that of large conductance, Ca2+-gated K+ (BK) channels despite the lack of sequence similarity. Multiple ion binding sites are clustered at two locations within each subunit, and three of them are identified to be Ca2+ sites. Our in vitro and in vivo assays also demonstrate the importance of these gating-ring Ca2+ binding sites to the physiological function of CASTOR as well as DMI1.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Lotus , Micorrizas , Rhizobium , Simbiose , Ativação do Canal Iônico , Membrana Nuclear/metabolismo , Proteínas de Plantas/metabolismo , Domínios ProteicosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
In plants and fungi the plasma membrane proton pump generates a large proton-motive force that performs essential functions in many processes, including solute transport and the control of cell elongation. Previous studies in yeast and higher plants have indicated that phosphorylation of an auto-inhibitory domain is involved in regulating pump activity. In this report we examine the Medicago truncatula plasma membrane proton pump gene family, and in particular MtAHA5. Yeast complementation assays with phosphomimetic mutations at six candidate sites support a phosphoregulatory role for two residues, suggesting a molecular model to explain early Nod factor-induced changes in the plasma membrane proton-motive force of legume root cells.
Assuntos
Membrana Celular/enzimologia , Medicago truncatula/enzimologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Análise por Conglomerados , Regulação Enzimológica da Expressão Gênica , Teste de Complementação Genética , Interações Hospedeiro-Patógeno , Medicago truncatula/genética , Medicago truncatula/microbiologia , Dados de Sequência Molecular , Família Multigênica , Mutação , Fosforilação , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , ATPases Translocadoras de Prótons/classificação , ATPases Translocadoras de Prótons/genética , Rhizobium/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Serina/genética , Serina/metabolismo , Transdução de Sinais , Simbiose , Treonina/genética , Treonina/metabolismoRESUMO
In the Internet era, communicating with friends and colleagues via social networks constitutes a significant proportion of our daily activities. Similarly animals and plants also interact with many organisms, some of which are pathogens and do no good for the plant, while others are beneficial symbionts. Almost all plants indulge in developing social networks with microbes, in particular with arbuscular mycorrhizal fungi, and emerging evidence indicates that most employ an ancient and widespread central 'social media' pathway made of signaling molecules within what is called the SYM pathway. Some plants, like legumes, are particularly active recruiters of friends, as they have established very sophisticated and beneficial interactions with nitrogen-fixing bacteria, also via the SYM pathway. Interestingly, many members of the Brassicaceae, including the model plant Arabidopsis thaliana, seem to have removed themselves from this ancestral social network and lost the ability to engage in mutually favorable interactions with arbuscular mycorrhizal fungi. Despite these generalizations, recent studies exploring the root microbiota of A. thaliana have found that in natural conditions, A. thaliana roots are colonized by many different bacterial species and therefore may be using different and probably more recent 'social media' for these interactions. In general, recent advances in the understanding of such molecular machinery required for plant-symbiont associations are being obtained using high throughput genomic profiling strategies including transcriptomics, proteomics and metabolomics. The crucial mechanistic understanding that such data reveal may provide the infrastructure for future efforts to genetically manipulate crop social networks for our own food and fiber needs.
Assuntos
Fungos/fisiologia , Micorrizas/fisiologia , Fenômenos Fisiológicos Vegetais , Plantas/microbiologia , Simbiose/fisiologia , Bactérias , Fenômenos Fisiológicos Bacterianos , Brassicaceae/metabolismo , Brassicaceae/microbiologia , Brassicaceae/fisiologia , Fabaceae/metabolismo , Fabaceae/microbiologia , Fabaceae/fisiologia , Redes e Vias Metabólicas , Fixação de Nitrogênio , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Plantas/metabolismoRESUMO
The ability of legume crops to fix atmospheric nitrogen via a symbiotic association with soil rhizobia makes them an essential component of many agricultural systems. Initiation of this symbiosis requires protein phosphorylation-mediated signaling in response to rhizobial signals named Nod factors. Medicago truncatula (Medicago) is the model system for studying legume biology, making the study of its phosphoproteome essential. Here, we describe the Medicago PhosphoProtein Database (MPPD; http://phospho.medicago.wisc.edu), a repository built to house phosphoprotein, phosphopeptide, and phosphosite data specific to Medicago. Currently, the MPPD holds 3,457 unique phosphopeptides that contain 3,404 non-redundant sites of phosphorylation on 829 proteins. Through the web-based interface, users are allowed to browse identified proteins or search for proteins of interest. Furthermore, we allow users to conduct BLAST searches of the database using both peptide sequences and phosphorylation motifs as queries. The data contained within the database are available for download to be investigated at the user's discretion. The MPPD will be updated continually with novel phosphoprotein and phosphopeptide identifications, with the intent of constructing an unparalleled compendium of large-scale Medicago phosphorylation data.
RESUMO
The establishment of symbiosis between leguminous plants and rhizobial bacteria requires rapid metabolic changes in both partners. We utilized untargeted quantitative mass spectrometry to perform metabolomic profiling of small molecules in extracts of the model legume Medicago truncatula treated with rhizobial Nod factors. One metabolite closely resembling the 9(R)-HODE class of oxylipins reproducibly showed a decrease in concentration within the first hour of in planta nod factor treatment. Oxylipins are precursors of the jasmonic acid biosynthetic pathway and we showed that both this metabolite and jasmonic acid inhibit Nod factor signaling. Since, oxylipins have been implicated as antimicrobial compounds produced by plants, these observations suggest that the oxylipin pathway may play multiple roles in facilitating Nod factor signaling during the early stages of symbiosis.
Assuntos
Medicago truncatula/metabolismo , Oxilipinas/metabolismo , Simbiose/fisiologia , Sequência de Bases , DNA de Plantas/genética , Genes de Plantas , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/genética , Medicago truncatula/microbiologia , Metaboloma , Metabolômica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Rhizobium/metabolismo , Transdução de Sinais , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Potato is the third most important food crop worldwide. However, genetic and genomic research of potato has lagged behind other major crops due to the autopolyploidy and highly heterozygous nature associated with the potato genome. Reliable and technically undemanding techniques are not available for functional gene assays in potato. Here we report the development of a transient gene expression and silencing system in potato. Gene expression or RNAi-based gene silencing constructs were delivered into potato leaf cells using Agrobacterium-mediated infiltration. Agroinfiltration of various gene constructs consistently resulted in potato cell transformation and spread of the transgenic cells around infiltration zones. The efficiency of agroinfiltration was affected by potato genotypes, concentration of Agrobacterium, and plant growth conditions. We demonstrated that the agroinfiltration-based transient gene expression can be used to detect potato proteins in sub-cellular compartments in living cells. We established a double agroinfiltration procedure that allows to test whether a specific gene is associated with potato late blight resistance pathway mediated by the resistance gene RB. This procedure provides a powerful approach for high throughput functional assay for a large number of candidate genes in potato late blight resistance.
Assuntos
Regulação da Expressão Gênica , Inativação Gênica , Rhizobium/genética , Solanum tuberosum/genética , Regulação da Expressão Gênica de Plantas , Técnicas Genéticas , Genoma de Planta , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Phytophthora infestans/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Plasmídeos/metabolismo , Interferência de RNA , Solanum tuberosum/microbiologiaRESUMO
In addition to establishing symbiotic relationships with arbuscular mycorrhizal fungi, legumes also enter into a nitrogen-fixing symbiosis with rhizobial bacteria that results in the formation of root nodules. Several genes involved in the development of both arbuscular mycorrhiza and legume nodulation have been cloned in model legumes. Among them, Medicago truncatula DMI1 (DOESN'T MAKE INFECTIONS1) is required for the generation of nucleus-associated calcium spikes in response to the rhizobial signaling molecule Nod factor. DMI1 encodes a membrane protein with striking similarities to the Methanobacterium thermoautotrophicum potassium channel (MthK). The cytosolic C terminus of DMI1 contains a RCK (regulator of the conductance of K(+)) domain that in MthK acts as a calcium-regulated gating ring controlling the activity of the channel. Here we show that a dmi1 mutant lacking the entire C terminus acts as a dominant-negative allele interfering with the formation of nitrogen-fixing nodules and abolishing the induction of calcium spikes by the G-protein agonist Mastoparan. Using both the full-length DMI1 and this dominant-negative mutant protein we show that DMI1 increases the sensitivity of a sodium- and lithium-hypersensitive yeast (Saccharomyces cerevisiae) mutant toward those ions and that the C-terminal domain plays a central role in regulating this response. We also show that DMI1 greatly reduces the release of calcium from internal stores in yeast, while the dominant-negative allele appears to have the opposite effect. This work suggests that DMI1 is not directly responsible for Nod factor-induced calcium changes, but does have the capacity to regulate calcium channels in both yeast and plants.