RESUMO
The Lotus japonicus SYMBIOSIS RECEPTOR-LIKE KINASE (SYMRK) is required for symbiotic signal transduction upon stimulation of root cells by microbial signaling molecules. Here, we identified members of the SEVEN IN ABSENTIA (SINA) E3 ubiquitin-ligase family as SYMRK interactors and confirmed their predicted ubiquitin-ligase activity. In Nicotiana benthamiana leaves, SYMRK-yellow fluorescent protein was localized at the plasma membrane, and interaction with SINAs, as determined by bimolecular fluorescence complementation, was observed in small punctae at the cytosolic interface of the plasma membrane. Moreover, fluorescence-tagged SINA4 partially colocalized with SYMRK and caused SYMRK relocalization as well as disappearance of SYMRK from the plasma membrane. Neither the localization nor the abundance of Nod-factor receptor1 was altered by the presence of SINA4. SINA4 was transcriptionally upregulated during root symbiosis, and rhizobia inoculated roots ectopically expressing SINA4 showed reduced SYMRK protein levels. In accordance with a negative regulatory role in symbiosis, infection thread development was impaired upon ectopic expression of SINA4. Our results implicate SINA4 E3 ubiquitin ligase in the turnover of SYMRK and provide a conceptual mechanism for its symbiosis-appropriate spatio-temporal containment.
Assuntos
Lotus/enzimologia , Lotus/microbiologia , Proteínas Nucleares/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Rhizobium/fisiologia , Simbiose , Ubiquitina-Proteína Ligases/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Dominantes/genética , Lotus/genética , Proteínas Nucleares/genética , Nodulação/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Nódulos Radiculares de Plantas/citologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/microbiologia , Saccharomyces cerevisiae/metabolismo , Frações Subcelulares/enzimologia , Nicotiana/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+) -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+) fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+) imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+) dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+) spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+) dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures.
Assuntos
Arabidopsis/genética , Cálcio/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Lotus/citologia , Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais/métodos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/análise , Caulimovirus/genética , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Cotilédone/genética , Cotilédone/metabolismo , Citosol/metabolismo , Vetores Genéticos , Lotus/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , TransgenesRESUMO
⢠Legume roots develop two types of lateral organs, lateral roots and nodules. Nodules develop as a result of a symbiotic interaction with rhizobia and provide a niche for the bacteria to fix atmospheric nitrogen for the plant. ⢠The Arabidopsis NAC1 transcription factor is involved in lateral root formation, and is regulated post-transcriptionally by miRNA164 and by SINAT5-dependent ubiquitination. We analyzed in Medicago truncatula the role of the closest NAC1 homolog in lateral root formation and in nodulation. ⢠MtNAC1 shows a different expression pattern in response to auxin than its Arabidopsis homolog and no changes in lateral root number or nodulation were observed in plants affected in MtNAC1 expression. In addition, no interaction was found with SINA E3 ligases, suggesting that post-translational regulation of MtNAC1 does not occur in M. truncatula. Similar to what was found in Arabidopsis, a conserved miR164 target site was retrieved in MtNAC1, which reduced protein accumulation of a GFP-miR164 sensor. Furthermore, miR164 and MtNAC1 show an overlapping expression pattern in symbiotic nodules, and overexpression of this miRNA led to a reduction in nodule number. ⢠This work suggests that regulatory pathways controlling a conserved transcription factor are complex and divergent between M. truncatula and Arabidopsis.
Assuntos
Medicago truncatula/fisiologia , Proteínas de Plantas/metabolismo , Sinorhizobium meliloti/fisiologia , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Flores/efeitos dos fármacos , Flores/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/farmacologia , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/genética , MicroRNAs/genética , Dados de Sequência Molecular , Mutação , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Nodulação/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/genética , Brotos de Planta/fisiologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Estrutura Terciária de Proteína , RNA de Plantas/genética , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The CLAVATA3/embryo-surrounding region (CLE) peptides control the fine balance between proliferation and differentiation in plant development. We studied the role of CLE peptides during indeterminate nodule development and identified 25 MtCLE peptide genes in the Medicago truncatula genome, of which two genes, MtCLE12 and MtCLE13, had nodulation-related expression patterns that were linked to proliferation and differentiation. MtCLE13 expression was up-regulated early in nodule development. A high-to-low expression gradient radiated from the inner toward the outer cortical cell layers in a region defining the incipient nodule. At later stages, MtCLE12 and MtCLE13 were expressed in differentiating nodules and in the apical part of mature, elongated nodules. Functional analysis revealed a putative role for MtCLE12 and MtCLE13 in autoregulation of nodulation, a mechanism that controls the number of nodules and involves systemic signals mediated by a leucine-rich repeat receptor-like kinase, SUNN, which is active in the shoot. When MtCLE12 and MtCLE13 were ectopically expressed in transgenic roots, nodulation was abolished at the level of the nodulation factor signal transduction, and this inhibition involved long-distance signaling. In addition, composite plants with roots ectopically expressing MtCLE12 or MtCLE13 had elongated petioles. This systemic effect was not observed in transgenic roots ectopically expressing MtCLE12 and MtCLE13 in a sunn-1 mutant background, although nodulation was still strongly reduced. These results suggest multiple roles for CLE signaling in nodulation.