RESUMO
The Arabidopsis (Arabidopsis thaliana) calmodulin-binding transcription activator3 (CAMTA3) is a repressor of immunity-related genes but an activator of cold-induced or general stress-responsive genes in plants. Post-transcriptional or posttranslational mechanisms have been proposed to control CAMTA3 functions in different stress responses. Here, we show that treatment with the bacterial flg22 elicitor induces CAMTA3 phosphorylation, which is accompanied by its destabilization and nuclear export. Two flg22-responsive mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, directly phosphorylate CAMTA3, with the phospho-sites contributing to CAMTA3 degradation and suppression of downstream target gene expression. However, the flg22-induced nuclear export and phospho-mobility shift can still be observed for the CAMTA3 phospho-null variant of the MAPK-modified sites, suggesting additional flg22-responsive kinases might be involved. Taken together, we propose that flg22-induced CAMTA3 depletion facilitates de-repression of downstream defense target genes, which involves phosphorylation, increased protein turnover, and nucleo-cytoplasmic trafficking.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Calmodulina/metabolismo , Fosforilação/fisiologia , Fatores de Transcrição/metabolismo , Calmodulina/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Fosforilação/genética , Fatores de Transcrição/genéticaRESUMO
Plants exude a diverse cocktail of metabolites into the soil as response to exogenous and endogenous factors. So far, root exudates have mainly been studied under artificial conditions due to methodological difficulties. In this study, each five perennial grass and forb species were investigated for polar and semi-polar metabolites in exudates under field conditions. Metabolite collection and untargeted profiling approaches combined with a novel classification method allowed the designation of 182 metabolites. The composition of exuded polar metabolites depended mainly on the local environment, especially soil conditions, whereas the pattern of semi-polar metabolites was primarily affected by the species identity. The profiles of both polar and semi-polar metabolites differed between growth forms, with grass species being generally more similar to each other and more responsive to the abiotic environment than forb species. This study demonstrated the feasibility of investigating exudates under field conditions and to identify the driving factors of exudate composition.
Assuntos
Exsudatos de Plantas/metabolismo , Raízes de Plantas/metabolismo , Poaceae/metabolismo , Asteraceae/classificação , Asteraceae/metabolismo , Ecossistema , Cromatografia Gasosa-Espectrometria de Massas , Pradaria , Fenômenos Fisiológicos Vegetais , Plantaginaceae/classificação , Plantaginaceae/metabolismo , Poaceae/classificação , Ranunculaceae/classificação , Ranunculaceae/metabolismo , Rizosfera , Rubiaceae/classificação , Rubiaceae/metabolismoRESUMO
Changes in cellular calcium levels are one of the earliest signalling events in plants exposed to pathogens or other exogenous factors. In a genetic screen, we identified an Arabidopsis thaliana 'changed calcium elevation 1' (cce1) mutant with attenuated calcium response to the bacterial flagellin flg22 peptide and several other elicitors. Whole-genome resequencing revealed a mutation in asparagine-linked glycosylation 12 that encodes the mannosyltransferase responsible for adding the eighth mannose residue in an α-1,6 linkage to the dolichol-PP-oligosaccharide N-glycosylation glycan tree precursors. While properly targeted to the plasma membrane, misglycosylation of several receptors in the cce1 background suggests that N-glycosylation is required for proper functioning of client proteins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Manosiltransferases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Glicosilação , Manosiltransferases/genéticaRESUMO
Mitogen-activated protein kinase (MAPK) cascades are key signalling modules of plant defence responses to pathogen-associated molecular patterns [PAMPs; e.g. the bacterial peptide flagellin (flg22)]. Tandem zinc finger protein 9 (TZF9) is a RNA-binding protein that is phosphorylated by two PAMP-responsive MAPKs, MPK3 and MPK6. We mapped the major phosphosites in TZF9 and showed their importance for controlling in vitro RNA-binding activity, in vivo flg22-induced rapid disappearance of TZF9-labelled processing body-like structures and TZF9 protein turnover. Microarray analysis showed a strong discordance between transcriptome (total mRNA) and translatome (polysome-associated mRNA) in the tzf9 mutant, with more mRNAs associated with ribosomes in the absence of TZF9. This suggests that TZF9 may sequester and inhibit the translation of subsets of mRNAs. Fittingly, TZF9 physically interacts with poly(A)-binding protein 2 (PAB2), a hallmark constituent of stress granules - sites for stress-induced translational stalling/arrest. TZF9 even promotes the assembly of stress granules in the absence of stress. Hence, MAPKs may control defence gene expression post-transcriptionally through release from translation arrest within TZF9-PAB2-containing RNA granules or by perturbing the function of PAB2 in translation control (e.g. in the mRNA closed-loop model of translation).
Assuntos
Arabidopsis/genética , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Arabidopsis/fisiologia , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Fosforilação , Doenças das Plantas/microbiologia , Transporte Proteico , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genéticaRESUMO
The active bacterial rhizobiomes and root exudate profiles of phytometers of six plant species growing in central European temperate grassland communities were investigated in three regions located up to 700 km apart, across diverse edaphic conditions and along a strong land use gradient. The recruitment process from bulk soil communities was identified as the major direct driver of the composition of active rhizosphere bacterial communities. Unexpectedly, the effect of soil properties, particularly soil texture, water content, and soil type, strongly dominated over plant properties and the composition of polar root exudates of the primary metabolism. While plant species-specific selection of bacteria was minor, the RNA-based composition of active rhizosphere bacteria substantially differed between rhizosphere and bulk soil. Although other variables could additionally be responsible for the consistent enrichment of particular bacteria in the rhizosphere, distinct bacterial OTUs were linked to the presence of specific polar root exudates independent of individual plant species. Our study also identified numerous previously unknown taxa that are correlated with rhizosphere dynamics and hence represent suitable targets for future manipulations of the plant rhizobiome.
Assuntos
Bactérias/isolamento & purificação , Pradaria , Rizosfera , Microbiologia do Solo , Bactérias/classificação , Raízes de Plantas/microbiologia , Solo/químicaRESUMO
Potato plants treated with the pathogen-associated molecular pattern Pep-13 mount salicylic acid- and jasmonic acid-dependent defense responses, leading to enhanced resistance against Phytophthora infestans, the causal agent of late blight disease. Recognition of Pep-13 is assumed to occur by binding to a yet unknown plasma membrane-localized receptor kinase. The potato genes annotated to encode the co-receptor BAK1, StSERK3A and StSERK3B, are activated in response to Pep-13 treatment. Transgenic RNAi-potato plants with reduced expression of both SERK3A and SERK3B were generated. In response to Pep-13 treatment, the formation of reactive oxygen species and MAP kinase activation, observed in wild type plants, is highly reduced in StSERK3A/B-RNAi plants, suggesting that StSERK3A/B are required for perception of Pep-13 in potato. In contrast, defense gene expression is induced by Pep-13 in both control and StSERK3A/B-depleted plants. Altered morphology of StSERK3A/B-RNAi plants correlates with major shifts in metabolism, as determined by untargeted metabolite profiling. Enhanced levels of hydroxycinnamic acid amides, typical phytoalexins of potato, in StSERK3A/B-RNAi plants are accompanied by significantly decreased levels of flavonoids and steroidal glycoalkaloids. Thus, altered metabolism in StSERK3A/B-RNAi plants correlates with the ability of StSERK3A/B-depleted plants to mount defense, despite highly decreased early immune responses.
Assuntos
Regulação da Expressão Gênica de Plantas/imunologia , Phytophthora infestans/patogenicidade , Doenças das Plantas/imunologia , Proteínas de Plantas/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Solanum tuberosum/imunologia , Alcaloides/imunologia , Alcaloides/metabolismo , Amidas/imunologia , Amidas/metabolismo , Ácidos Cumáricos/imunologia , Ácidos Cumáricos/metabolismo , Ciclopentanos/imunologia , Ciclopentanos/metabolismo , Resistência à Doença/genética , Flavonoides/imunologia , Flavonoides/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/imunologia , Metaboloma/genética , Metaboloma/imunologia , Oxilipinas/imunologia , Oxilipinas/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Phytophthora infestans/fisiologia , Doenças das Plantas/genética , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Reconhecimento de Padrão/antagonistas & inibidores , Receptores de Reconhecimento de Padrão/genética , Ácido Salicílico/imunologia , Ácido Salicílico/metabolismo , Sesquiterpenos/imunologia , Sesquiterpenos/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/parasitologia , FitoalexinasRESUMO
In the rhizosphere, plants are exposed to a multitude of different biotic and abiotic factors, to which they respond by exuding a wide range of secondary root metabolites. So far, it has been unknown to which degree root exudate composition is species-specific and is affected by land use, the local impact and local neighborhood under field conditions. In this study, root exudates of 10 common grassland species were analyzed, each five of forbs and grasses, in the German Biodiversity Exploratories using a combined phytometer and untargeted liquid chromatography-mass spectrometry (LC-MS) approach. Redundancy analysis and hierarchical clustering revealed a large set of semi-polar metabolites common to all species in addition to species-specific metabolites. Chemical richness and exudate composition revealed that forbs, such as Plantago lanceolata and Galium species, exuded more species-specific metabolites than grasses. Grasses instead were primarily affected by environmental conditions. In both forbs and grasses, plant functional traits had only a minor impact on plant root exudation patterns. Overall, our results demonstrate the feasibility of obtaining and untargeted profiling of semi-polar metabolites under field condition and allow a deeper view in the exudation of plants in a natural grassland community.
RESUMO
Cytosolic Ca2+ ([Ca2+]cyt) elevation is an early signaling response upon exposure to pathogen-derived molecules (so-called microbe-associated molecular patterns, MAMPs) and has been successfully used as a quantitative read-out in genetic screens to identify MAMP receptors or their associated components. Here, we isolated and identified by mass spectrometry the dipeptide γ-Glu-Leu as a component of a Phytophthora infestans mycelium extract that induces [Ca2+]cyt elevation. Treatment of Arabidopsis seedlings with synthetic γ-Glu-Leu revealed stimulatory effects on defense signaling, including a weak enhancement of the expression of some MAMP-inducible genes or affecting the refractory period to a second MAMP elicitation. However, γ-Glu-Leu is not a classical MAMP since pH adjustment abolished these activities and importantly, the observed effects of γ-Glu-Leu could be recapitulated by mimicking extracellular acidification. Thus, although γ-Glu-Leu can act as a direct agonist of calcium sensing receptors in animal systems, the Ca2+-mobilizing activity in plants reported here is due to acidification. Low pH also shapes the Ca2+ signature of well-studied MAMPs (e.g. flg22) or excitatory amino acids such as glutamate. Overall, this work serves as a cautionary reminder that in defense signaling studies where Ca2+ flux measurements are concerned, it is important to monitor and consider the effects of pH.
Assuntos
Cálcio/metabolismo , Dipeptídeos/fisiologia , Concentração de Íons de Hidrogênio , Phytophthora infestans/química , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Arabidopsis/parasitologia , Sinalização do Cálcio , Espectrometria de Massas , Phytophthora infestans/patogenicidade , Plântula/efeitos dos fármacosRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0204128.].
RESUMO
Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system. Arabidopsis thaliana controls pathogen entry through the penetration-resistance genes PEN2 and PEN3, encoding an atypical myrosinase and an ABC transporter, respectively, required for synthesis and export of unknown indole compounds. To identify pathogen-elicited leaf surface metabolites and further unravel nonhost resistance in Arabidopsis, we performed untargeted metabolite profiling by incubating a P. infestans zoospore suspension on leaves of WT or pen3 mutant Arabidopsis plants. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-yl-methyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen3 mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. The syntheses of both compounds were dependent on functional PEN2 and phytochelatin synthase 1. None of these compounds inhibited mycelial growth of P. infestans in vitro Of note, exogenous application of 4-methoxyindol-3-yl methanol slightly elevated cytosolic Ca2+ levels and enhanced callose deposition in hydathodes of seedlings treated with a bacterial pathogen-associated molecular pattern (PAMP), flagellin (flg22). Loss of flg22-induced callose deposition in leaves of pen3 seedlings was partially reverted by the addition of 4-methoxyindol-3-yl methanol. In conclusion, we have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Arabidopsis/metabolismo , Flagelina/metabolismo , Glucanos/metabolismo , Arabidopsis/microbiologia , Cálcio/metabolismo , Citosol/metabolismo , Indóis/metabolismo , Phytophthora infestans/isolamento & purificação , Folhas de Planta/metabolismo , Especificidade por SubstratoRESUMO
Primary and secondary metabolites exuded by plant roots have mainly been studied under laboratory conditions, while knowledge of root exudate patterns of plants growing in natural communities is very limited. Focusing on ten common European grassland plant species, we asked to which degree exuded metabolite compositions are specific to species or growth forms (forbs and grasses), depend on environments and local neighbourhoods, and reflect traditional plant functional traits. Root exudates were collected under field conditions and analysed using a non-targeted gas chromatography coupled mass spectrometry (GC-MS) approach. In total, we annotated 153 compounds of which 36 were identified by structure and name as metabolites mainly derived from the primary metabolism. Here we show by using variance partitioning, that the composition of exuded polar metabolites was mostly explained by plot identity, followed by plant species identity while plant species composition of the local neighbourhood played no role. Total and root dry biomass explained the largest proportion of variance in exudate composition, with additional variance explained by traditional plant traits. Although the exudate composition was quite similar between the two growth forms, we found some metabolites that occurred only in one of the two growth forms. Our study demonstrated the feasibility of measuring polar exudates under non-sterile field conditions by mass spectrometry, which opens new avenues of research for functional plant ecology.
Assuntos
Exsudatos de Plantas/química , Raízes de Plantas/metabolismo , Plantas/metabolismo , Poaceae/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Pradaria , Plântula/crescimento & desenvolvimento , Sementes/crescimento & desenvolvimento , Água/químicaRESUMO
Mitogen-activated protein kinases (MAPKs) cascades play essential roles in plants by transducing developmental cues and environmental signals into cellular responses. Among the latter are microbe-associated molecular patterns perceived by pattern recognition receptors (PRRs), which trigger immunity. We found that YODA (YDA) - a MAPK kinase kinase regulating several Arabidopsis developmental processes, like stomatal patterning - also modulates immune responses. Resistance to pathogens is compromised in yda alleles, whereas plants expressing the constitutively active YDA (CA-YDA) protein show broad-spectrum resistance to fungi, bacteria, and oomycetes with different colonization modes. YDA functions in the same pathway as ERECTA (ER) Receptor-Like Kinase, regulating both immunity and stomatal patterning. ER-YDA-mediated immune responses act in parallel to canonical disease resistance pathways regulated by phytohormones and PRRs. CA-YDA plants exhibit altered cell-wall integrity and constitutively express defense-associated genes, including some encoding putative small secreted peptides and PRRs whose impairment resulted in enhanced susceptibility phenotypes. CA-YDA plants show strong reprogramming of their phosphoproteome, which contains protein targets distinct from described MAPKs substrates. Our results suggest that, in addition to stomata development, the ER-YDA pathway regulates an immune surveillance system conferring broad-spectrum disease resistance that is distinct from the canonical pathways mediated by described PRRs and defense hormones.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Resistência à Doença , MAP Quinase Quinase Quinases/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal , Padronização Corporal , Parede Celular/metabolismo , Flagelina/farmacologia , Fungos/fisiologia , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , Mutação/genética , Moléculas com Motivos Associados a Patógenos/metabolismo , Estômatos de Plantas/crescimento & desenvolvimento , Transdução de Sinais , Regulação para Cima/genéticaRESUMO
Plant functional traits are widely used to predict community productivity. However, they are rarely used to predict individual plant performance in grasslands. To assess the relative importance of traits compared to environment, we planted seedlings of 20 common grassland species as phytometers into existing grassland communities varying in land-use intensity. After 1 year, we dug out the plants and assessed root, leaf, and aboveground biomass, to measure plant performance. Furthermore, we determined the functional traits of the phytometers and of all plants growing in their local neighborhood. Neighborhood impacts were analyzed by calculating community-weighted means (CWM) and functional diversity (FD) of every measured trait. We used model selection to identify the most important predictors of individual plant performance, which included phytometer traits, environmental conditions (climate, soil conditions, and land-use intensity), as well as CWM and FD of the local neighborhood. Using variance partitioning, we found that most variation in individual plant performance was explained by the traits of the individual phytometer plant, ranging between 19.30% and 44.73% for leaf and aboveground dry mass, respectively. Similarly, in a linear mixed effects model across all species, performance was best predicted by phytometer traits. Among all environmental variables, only including land-use intensity improved model quality. The models were also improved by functional characteristics of the local neighborhood, such as CWM of leaf dry matter content, root calcium concentration, and root mass per volume as well as FD of leaf potassium and root magnesium concentration and shoot dry matter content. However, their relative effect sizes were much lower than those of the phytometer traits. Our study clearly showed that under realistic field conditions, the performance of an individual plant can be predicted satisfyingly by its functional traits, presumably because traits also capture most of environmental and neighborhood conditions.
RESUMO
A delicate balance in cellular signaling is required for plants to respond to microorganisms or to changes in their environment. Mitogen-activated protein kinase (MAPK) cascades are one of the signaling modules that mediate transduction of extracellular microbial signals into appropriate cellular responses. Here, we employ a transgenic system that simulates activation of two pathogen/stress-responsive MAPKs to study release of metabolites and proteins into root exudates. The premise is based on our previous proteomics study that suggests upregulation of secretory processes in this transgenic system. An advantage of this experimental set-up is the direct focus on MAPK-regulated processes without the confounding complications of other signaling pathways activated by exposure to microbes or microbial molecules. Using non-targeted metabolomics and proteomics studies, we show that MAPK activation can indeed drive the appearance of dipeptides, defense-related metabolites and proteins in root apoplastic fluid. However, the relative levels of other compounds in the exudates were decreased. This points to a bidirectional control of metabolite and protein release into the apoplast. The putative roles for some of the identified apoplastic metabolites and proteins are discussed with respect to possible antimicrobial/defense or allelopathic properties. Overall, our findings demonstrate that sustained activation of MAPKs alters the composition of apoplastic root metabolites and proteins, presumably to influence the plant-microbe interactions in the rhizosphere. The reported metabolomics and proteomics data are available via Metabolights (Identifier: MTBLS441) and ProteomeXchange (Identifier: PXD006328), respectively.
RESUMO
The molecular actions of mitogen-activated protein kinases (MAPKs) are ultimately accomplished by the substrate proteins where phosphorylation affects their molecular properties and function(s), but knowledge regarding plant MAPK substrates is currently still fragmentary. Here, we uncovered a previously uncharacterized protein family consisting of three proline/serine-rich proteins (PRPs) that are substrates of stress-related MAPKs. We demonstrated the importance of a MAPK docking domain necessary for protein-protein interaction with MAPKs and consequently also for phosphorylation. The main phosphorylated site was mapped to a residue conserved between all three proteins, which when mutated to a non-phosphorylatable form, differentially affected their protein stability. Together with their distinct gene expression patterns, this differential accumulation of the three proteins upon phosphorylation probably contributes to their distinct function(s). Transgenic over-expression of PRP, the founding member, led to plants with enhanced resistance to Pseudomonas syringae pv. tomato DC3000. Older plants of the over-expressing lines have curly leaves and were generally smaller in stature. This growth phenotype was lost in plants expressing the phosphosite variant, suggesting a phosphorylation-dependent effect. Thus, this novel family of PRPs may be involved in MAPK regulation of plant development and / or pathogen resistance responses. As datamining associates PRP expression profiles with hypoxia or oxidative stress and PRP-overexpressing plants have elevated levels of reactive oxygen species, PRP may connect MAPK and oxidative stress signaling.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Família Multigênica , Prolina/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/química , Sítios de Ligação , Resistência à Doença/efeitos dos fármacos , Flagelina/farmacologia , Homeostase/efeitos dos fármacos , Mutação/genética , Fosforilação/efeitos dos fármacos , Filogenia , Desenvolvimento Vegetal/efeitos dos fármacos , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Pseudomonas syringae/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Especificidade por Substrato/efeitos dos fármacosRESUMO
The chemical composition of root exudates strongly impacts the interactions of plants with microorganisms in the rhizosphere and the efficiency of nutrient acquisition. Exudation of metabolites is in part mediated by ATP-binding cassette (ABC) transporters. In order to assess the contribution of individual ABC transporters to root exudation, we performed an LC-MS based non-targeted metabolite profiling of semi-polar metabolites accumulating in root exudates of Arabidopsis thaliana plants and mutants deficient in the expression of ABCG36 (PDR8/PEN3), ABCG37 (PDR9) or both transporters. Comparison of the metabolite profiles indicated distinct roles for each ABC transporter in root exudation. Thymidine exudation could be attributed to ABCG36 function, whereas coumarin exudation was strongly reduced only in ABCG37 deficient plants. However, coumarin exudation was compromised in abcg37 mutants only with respect to certain metabolites of this substance class. The specificity of ABCG37 for individual coumarins was further verified by a targeted LC-MS based coumarin profiling method. The response to iron deficiency, which is known to strongly induce coumarin exudation, was also investigated. In either treatment, the distribution of individual coumarins between roots and exudates in the investigated genotypes suggested the involvement of ABCG37 in the exudation specifically of highly oxygenated rather than monohydroxylated coumarins.
Assuntos
Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Arabidopsis/metabolismo , Cumarínicos/metabolismo , Oxigênio/metabolismo , Exsudatos de Plantas/metabolismo , Raízes de Plantas/metabolismo , Metaboloma , Metabolômica/métodosRESUMO
AvrRpt2 is one of the first Pseudomonas syringae effector proteins demonstrated to be delivered into host cells. It suppresses plant immunity by modulating auxin signaling and cleavage of the membrane-localized defense regulator RIN4. We recently uncovered a novel potential virulence function of AvrRpt2, where it specifically blocked activation of mitogen-activated protein kinases, MPK4 and MPK11, but not of MPK3 and MPK6. Putative AvrRpt2 homologs from different phytopathogens and plant-associated bacteria showed distinct activities with respect to MPK4/11 activation suppression and RIN4 cleavage. Apart from differences in sequence similarity, 3 of the analyzed homologs were apparently "truncated." To examine the role of the AvrRpt2 N-terminus, we modeled the structures of these AvrRpt2 homologs and performed deletion and domain swap experiments. Our results strengthen the finding that RIN4 cleavage is irrelevant for the ability to suppress defense-related MPK4/11 activation and indicate that full protease activity or cleavage specificity is affected by the N-terminus.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Pseudomonas syringae/patogenicidade , Virulência/genética , Virulência/fisiologiaRESUMO
Natural variation of secondary metabolism between different accessions of Arabidopsis thaliana (A. thaliana) has been studied extensively. In this study, we extended the natural variation approach by including biological variability (plant-to-plant variability) and analysed root metabolic patterns as well as their variability between plants and naturally occurring accessions. To screen 19 accessions of A. thaliana, comprehensive non-targeted metabolite profiling of single plant root extracts was performed using ultra performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS). Linear mixed models were applied to dissect the total observed variance. All metabolic profiles pointed towards a larger plant-to-plant variability than natural variation between accessions and variance of experimental batches. Ratios of plant-to-plant to total variability were high and distinct for certain secondary metabolites. None of the investigated accessions displayed a specifically high or low biological variability for these substance classes. This study provides recommendations for future natural variation analyses of glucosinolates, flavonoids, and phenylpropanoids and also reference data for additional substance classes.
Assuntos
Arabidopsis/metabolismo , Extratos Vegetais/química , Arabidopsis/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Metabolômica , Raízes de Plantas/metabolismo , Metabolismo Secundário , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Piriformospora indica is a root-colonizing fungus, which interacts with a variety of plants including Arabidopsis thaliana. This interaction has been considered as mutualistic leading to growth promotion of the host. So far, only indolic glucosinolates and phytohormones have been identified as key players. In a comprehensive non-targeted metabolite profiling study, we analyzed Arabidopsis thaliana's roots, root exudates, and leaves of inoculated and non-inoculated plants by ultra performance liquid chromatography/electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLC/(ESI)-QTOFMS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS), and identified further biomarkers. Among them, the concentration of nucleosides, dipeptides, oligolignols, and glucosinolate degradation products was affected in the exudates. In the root profiles, nearly all metabolite levels increased upon co-cultivation, like carbohydrates, organic acids, amino acids, glucosinolates, oligolignols, and flavonoids. In the leaf profiles, we detected by far less significant changes. We only observed an increased concentration of organic acids, carbohydrates, ascorbate, glucosinolates and hydroxycinnamic acids, and a decreased concentration of nitrogen-rich amino acids in inoculated plants. These findings contribute to the understanding of symbiotic interactions between plant roots and fungi of the order of Sebacinales and are a valid source for follow-up mechanistic studies, because these symbioses are particular and clearly different from interactions of roots with mycorrhizal fungi or dark septate endophytes.