RESUMO
A wide variety of intrinsic and extrinsic cues lead to cell death with unclear mechanisms. The infertility of some death mutants often hurdles the classical suppressor screens for death regulators. We have developed a transient RNA interference (RNAi)-based screen using a virus-induced gene silencing approach to understand diverse cell death pathways in Arabidopsis (Arabidopsis thaliana). One death pathway is due to the depletion of a MAP kinase (MAPK) cascade, consisting of MAPK kinase kinase 1 (MEKK1), MKK1/2, and MPK4, which depends on a nucleotide-binding site Leu-rich repeat (NLR) protein SUMM2. Silencing of MEKK1 by virus-induced gene silencing resembles the mekk1 mutant with autoimmunity and defense activation. The RNAi-based screen toward Arabidopsis T-DNA insertion lines identified SUMM2, MEKK2, and Calmodulin-binding receptor-like cytoplasmic kinase 3 (CRCK3) to be vital regulators of RNAi MEKK1-induced cell death, consistent with the reports of their requirement in the mekk1-mkk1/2-mpk4 death pathway. Similar with MEKK2, overexpression of CRCK3 caused dosage- and SUMM2-dependent cell death, and the transcripts of CRCK3 were up-regulated in mekk1, mkk1/2, and mpk4 MEKK2-induced cell death depends on CRCK3. Interestingly, CRCK3-induced cell death also depends on MEKK2, consistent with the biochemical data that MEKK2 complexes with CRCK3. Furthermore, the kinase activity of CRCK3 is essential, whereas the kinase activity of MEKK2 is dispensable, for triggering cell death. Our studies suggest that MEKK2 and CRCK3 exert concerted functions in the control of NLR SUMM2 activation and MEKK2 may play a structural role, rather than function as a kinase, in regulating CRCK3 protein stability.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 1/metabolismo , MAP Quinase Quinase Quinase 2/genética , MAP Quinase Quinase Quinase 2/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estabilidade Proteica , Interferência de RNA/fisiologiaRESUMO
Sugar-rich environments represent an important challenge for microorganisms. The osmotic and molecular imbalances resulting from this condition severely limit microbial metabolism and growth. Gluconacetobacter diazotrophicus is one of the most sugar-tolerant prokaryotes, able to grow in the presence of sucrose concentrations up to 30%. However, the mechanisms that control its tolerance to such conditions remain poorly exploited. The present work investigated the key mechanisms of tolerance to high sugar in G. diazotrophicus. Comparative proteomics was applied to investigate the main functional pathways regulated in G. diazotrophicus when cultivated in the presence of high sucrose. Among 191 proteins regulated by high sucrose, regulatory pathways related to sugar metabolism, nutrient uptake, compatible solute synthesis, amino acid metabolism, and proteolytic system were highlighted. The role of these pathways on high-sucrose tolerance was investigated by mutagenesis analysis, which revealed that the knockout mutants zwf::Tn5 (sugar metabolism), tbdr::Tn5 (nutrient uptake), mtlK::Tn5 (compatible solute synthesis), pepN::Tn5 (proteolytic system), metH::Tn5 (amino acid metabolism), and ilvD::Tn5 (amino acid metabolism) became more sensitive to high sucrose. Together, our results identified mechanisms involved in response to high sugar in G. diazotrophicus, shedding light on the combination of osmotolerance and sugar-tolerance mechanisms. KEY POINTS: ⢠G. diazotrophicus intensifies glycolysis to metabolize the excess of sugar. ⢠G. diazotrophicus turns down the uptake of nutrients in response to high sugar. ⢠G. diazotrophicus requires amino acid availability to resist high sugar.
Assuntos
Sacarose , Açúcares , Gluconacetobacter , Pressão OsmóticaRESUMO
Gluconacetobacter diazotrophicus is a diazotrophic endophytic bacterium that promotes the growth and development of several plant species. However, the molecular mechanisms activated during plant response to this bacterium remain unclear. Here, we used the RNA-seq approach to understand better the effect of G. diazotrophicus PAL5 on the transcriptome of shoot and root tissues of Arabidopsis thaliana. G. diazotrophicus colonized A. thaliana roots and promoted growth, increasing leaf area and biomass. The transcriptomic analysis revealed several differentially expressed genes (DEGs) between inoculated and non-inoculated plants in the shoot and root tissues. A higher number of DEGs were up-regulated in roots compared to shoots. Genes up-regulated in both shoot and root tissues were associated with nitrogen metabolism, production of glucosinolates and flavonoids, receptor kinases, and transcription factors. In contrast, the main groups of down-regulated genes were associated with pathogenesis-related proteins and heat-shock proteins in both shoot and root tissues. Genes encoding enzymes involved in cell wall biogenesis and modification were down-regulated in shoots and up-regulated in roots. In contrast, genes associated with ROS detoxification were up-regulated in shoots and down-regulated in roots. These results highlight the fine-tuning of the transcriptional regulation of A. thaliana in response to colonization by G. diazotrophicus PAL5.
RESUMO
Cell death is a vital and ubiquitous process that is tightly controlled in all organisms. However, the mechanisms underlying precise cell death control remain fragmented. As an important shared module in plant growth, development, and immunity, Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate plant cell death. By deploying an RNAi-based genetic screen for bak1/serk4 cell death suppressors, we revealed that cyclic nucleotide-gated channel 20 (CNGC20) functions as a hyperpolarization-activated Ca2+-permeable channel specifically regulating bak1/serk4 cell death. BAK1 directly interacts with and phosphorylates CNGC20 at specific sites in the C-terminal cytosolic domain, which in turn regulates CNGC20 stability. CNGC19, the closest homolog of CNGC20 with a low abundance compared with CNGC20, makes a quantitative genetic contribution to bak1/serk4 cell death only in the absence of CNGC20, supporting the biochemical data showing homo- and heteromeric assembly of the CNGC20 and CNGC19 channel complexes. Transcripts of CNGC20 and CNGC19 are elevated in bak1/serk4 compared with wild-type plants, further substantiating a critical role of homeostasis of CNGC20 and CNGC19 in cell death control. Our studies not only uncover a unique regulation of ion channel stability by cell-surface-resident receptor kinase-mediated phosphorylation but also provide evidence for fine-tuning Ca2+ channel functions in maintaining cellular homeostasis by the formation of homo- and heterotetrameric complexes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Morte Celular/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Homeostase , Fosforilação , Células Vegetais/metabolismo , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de SinaisRESUMO
Precise control of cell death is essential for the survival of all organisms. Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate cell death through elusive mechanisms. By deploying a genetic screen for suppressors of cell death triggered by virus-induced gene silencing of BAK1/SERK4 on Arabidopsis knockout collections, we identified STT3a, a protein involved in N-glycosylation modification, as an important regulator of bak1/serk4 cell death. Systematic investigation of glycosylation pathway and endoplasmic reticulum (ER) quality control (ERQC) components revealed distinct and overlapping mechanisms of cell death regulated by BAK1/SERK4 and their interacting protein BIR1. Genome-wide transcriptional analysis revealed the activation of members of cysteine-rich receptor-like kinase (CRK) genes in the bak1/serk4 mutant. Ectopic expression of CRK4 induced STT3a/N-glycosylation-dependent cell death in Arabidopsis and Nicotiana benthamiana. Therefore, N-glycosylation and specific ERQC components are essential to activate bak1/serk4 cell death, and CRK4 is likely to be among client proteins of protein glycosylation involved in BAK1/SERK4-regulated cell death.