Project description:The target of rapamycin (TOR) kinase is a central regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. Although the TOR pathway is conserved among eukaryotes, plants developed unique adaptations to this pathway to cope with their autotrophic and sessile nature. Here, we captured for the first time a proteome-wide view of the plant TOR phosphorylation and interaction landscape. After sampling four biological repeats, proteins were extracted, digested and phosphopeptides were enriched. In total, 8302 phosphopeptides on 2558 proteins were identified from all samples. After label free quantitative analysis, a filtered dataset of 5500 phosphopeptides on 2056 proteins was retained. To identify TOR-dependent sites, the control-, AZD8055- and rapamycin-treated samples were statistically analyzed using a linear mixed model to evaluate the effect of the treatment, time and the interaction between both. For T10, T20 and T40, the sucrose control samples were compared to the AZD8055 or rapamycin samples. In addition, the AZD8055 samples were compared to the rapamycin samples, and the sucrose control samples (T10, T20, T40) were compared to T0. Overall, the strongest effect occurred with AZD8055. Therefore, an additional filter was applied retrieving only phosphopeptides that changed at least two-fold upon AZD8055 treatment. In total, 96 unambiguous TOR-dependent phosphosites were detected on 66 proteins, linking TOR to a plethora of biological processes.
Project description:Colletotrichum orbiculare Whi2, yeast stress response Whi2 homolog, is involved in switch from biotrophic to necrotrophic stage. To elucidate downstream genes regulated by Co Whi2, we have conducted DNA microarray. About 3100 genes were up or down regulated in the Co whi2Δ mutant compared with the wild-type. In particularly, 44 genes among up-regulated 58 genes in the Co whi2Δ mutant are ribosomal protein related gene. Eukaryote is widely conserved TOR (Target Of Rapamycin) which is known to regulator of ribosomal gene expression. To elucidate whether up-regulated ribosomal genes in the Co whi2Δ mutant are regulated by TOR activity, we have conducted DNA microarray in the Co whi2Δ mutant treated with rapamycin inhibiting TOR activity. The enormous ribosomal gene expression in the Co whi2Δ mutant treated with the rapamycin is lower than that without rapamycin treatment.
Project description:Rapamycin-sensitive transgenic Arabidopsis lines (BP12) expressing yeast FK506 Binding Protein12 (FKBP12) were developed. Inhibition of TOR in BP12 plants by rapamycin resulted in slower overall root, leaf and shoot growth and development leading to poor nutrient uptake and light energy utilization. Genetic and physiological studies together with RNA-Seq and metabolite analysis of TOR-suppressed lines revealed that TOR regulates development and lifespan in Arabidopsis by restructuring cell growth, carbon and nitrogen metabolism, gene expression, ribosomal RNA and protein synthesis.
Project description:Colletotrichum orbiculare Whi2, yeast stress response Whi2 homolog, is involved in switch from biotrophic to necrotrophic stage. To elucidate downstream genes regulated by Co Whi2, we have conducted DNA microarray. About 3100 genes were up or down regulated in the Co whi2Î mutant compared with the wild-type. In particularly, 44 genes among up-regulated 58 genes in the Co whi2Î mutant are ribosomal protein related gene. Eukaryote is widely conserved TOR (Target Of Rapamycin) which is known to regulator of ribosomal gene expression. To elucidate whether up-regulated ribosomal genes in the Co whi2Î mutant are regulated by TOR activity, we have conducted DNA microarray in the Co whi2Î mutant treated with rapamycin inhibiting TOR activity. The enormous ribosomal gene expression in the Co whi2Î mutant treated with the rapamycin is lower than that without rapamycin treatment. In gene expression of the Co whi2Î mutant, the wild-type and the Co whi2Î mutant infecting on cucumber cotyledons were assessed at 4 hours post-inoculation. In gene expression of the Co whi2Î mutant with rapamycin treatment, Co whi2Î mutant treated with 100nM rapamycin and Co whi2Î mutant without rapamycin treatment infecting on cucumber cotyledons were assesed at 4 hours post-inoculation. Four replication were performed for each experiments.
Project description:Rapamycin-sensitive transgenic Arabidopsis lines (BP12) expressing yeast FK506 Binding Protein12 (FKBP12) were developed. Inhibition of TOR in BP12 plants by rapamycin resulted in slower overall root, leaf and shoot growth and development leading to poor nutrient uptake and light energy utilization. Genetic and physiological studies together with RNA-Seq and metabolite analysis of TOR-suppressed lines revealed that TOR regulates development and lifespan in Arabidopsis by restructuring cell growth, carbon and nitrogen metabolism, gene expression, ribosomal RNA and protein synthesis. Arabidopsis WT (Col)and BP12-2 (TOR knockdown line) seedlings at 15 DAG were treated with rapamycin for 3 days by transferring from 0.5 MS medium to 0.5 MS+10 ug/ml rapamycin. Triplicate samples of rapamycin treated WT and BP12-2 seedlings were used for RNA-Seq analysis (Illumina Hiseq 2000). Paired-end alignments were obtained through aligning short reads onto the reference Arabidopsis Genome (TAIR9) using Bowtie. More than 80% of the reads mapped onto the genome. Htseq-count was used to count the reads from the Bowtie derived output files. Differential expressed genes were identified using edgeR. The FDR-corrected P value for differential expression was set to be <=0.05.
Project description:The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyper-activation of the mTOR pathway in human cancers, novel strategies to enhance TOR pathway inhibition are highly desirable. We used a yeast-based high-throughput chemical genetic screen to identify small-molecule enhancers of rapamycin (SMERs) and used whole genome expression analysis to identify their mechanisms of action.
Project description:The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyper-activation of the mTOR pathway in human cancers, novel strategies to enhance TOR pathway inhibition are highly desirable. We used a yeast-based high-throughput chemical genetic screen to identify small-molecule enhancers of rapamycin (SMERs) and used whole genome expression analysis to identify their mechanisms of action.
Project description:The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyper-activation of the mTOR pathway in human cancers, novel strategies to enhance TOR pathway inhibition are highly desirable. We used a yeast-based high-throughput chemical genetic screen to identify small-molecule enhancers of rapamycin (SMERs) and used whole genome expression analysis to identify their mechanisms of action.
Project description:The eukaryote-specific ribosomal protein of the small subunit eS6 is a target of phosphorylation for the Target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been known and studied for about 50 years, its biochemical and physiological significance remains controversial and poorly understood. To explore the role of eS6 and its phosphorylation we performed RNA-seq on the shoots of 12-day-old seedlings in five Arabidopsis thaliana genotypes. eS6 protein is encoded by two paralogous genes, RPS6A and RPS6B. Our data describe mRNA transcript levels in wild type (ecotype Columbia), two single mutants (rps6a and rps6b), an HA-epitope-tagged transgenic complementation line (rps6a rps6b; eS6A-HA) and a 'phosphonull' transgenic complementation line (rps6a rps6b; eS6A-Δ6SA) with six phosphorylatable residues mutated to alanine. Single mutants showed broad, but moderate changes to mRNA levels with the primary effects encompassing increases in translation and ribosome biogenesis genes and down regulation of photosynthesis. The rps6a rps6b; eS6A-HA plant almost fully complemented defects observed in the single mutants highlighting that the eS6 paralogs are functionally equivalent. Residual mRNA expression defects of the phospho-deficient seedlings could be understood as an incomplete rescue of the rps6 mutant defects, with little or no evidence for gain-of function defects. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.
Project description:Diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEF2), is the human host cell-sensitizing target of diphtheria toxin. Diphthamide biosynthesis depends on the 4Fe-4S-cluster protein Dph1 catalyzing the first committed step, as well as Dph2 to Dph7, in yeast and mammals. Here we show that diphthamide modification of eEF2 is conserved in Arabidopsis thaliana and requires AtDPH1. Ribosomal -1 frameshifting-error rates are increased in Arabidopsis dph1 mutants, similar to yeast and mice. Compared to the wild type, shorter roots and smaller rosettes of dph1 mutants result from fewer formed cells. TARGET OF RAPAMYCIN (TOR) kinase activity is attenuated, and autophagy is activated, in dph1 mutants. Under abiotic stress diphthamide-unmodified eEF2 accumulates in wild-type seedlings, most strongly upon heavy metal excess, which is conserved in human cells. In summary, our results suggest that diphthamide contributes to the functionality of the translational machinery monitored by plants to regulate growth.