The hot issue: TOR signalling network in plants.

Plant diseases caused by fungal phytopathogens have led to significant economic losses in agriculture worldwide. The management of fungal diseases is mainly dependent on the application of fungicides, which are not suitable for sustainable agriculture, human health, and environmental safety. Thus, it is necessary to develop novel targets and green strategies to mitigate the losses caused by these pathogens. The target of rapamycin (TOR) complexes and key components of the TOR signalling pathway are evolutionally conserved in pathogens and closely related to the vegetative growth and pathogenicity. As indicated in recent systems, chemical, genetic, and genomic studies on the TOR signalling pathway, phytopathogens with TOR dysfunctions show severe growth defects and nonpathogenicity, which makes the TOR signalling pathway to be developed into an ideal candidate target for controlling plant disease. In this review, we comprehensively discuss the current knowledge on components of the TOR signalling pathway in microorganisms and the diverse roles of various plant TOR in response to plant pathogens. Furthermore, we analyse a range of disease management strategies that rely on the TOR signalling pathway, including genetic modification technologies and chemical controls. In the future, disease control strategies based on the TOR signalling network are expected to become a highly effective weapon for crop protection.

Target of rapamycin (TOR) functions within the cell as a transducer of information from various sources, including growth factors, energy sensors, and hypoxia sensors, as well as components of the cell regulating growth and division. Blocking TOR function mimics amino acid, and to some extent, growth factor deprivation and has a cytostatic effect on proliferating cells in vivo. Inhibition of TOR in vivo, utilising its namesake rapamycin, leads to immunosuppression. This property has been exploited successfully with the use of rapamycin and its derivatives as a therapeutic agent in the prevention of organ rejection after transplantation with relatively mild side effects when compared to other immunosuppressive agents. The cytostatic effect of TOR on vascular smooth muscle cell proliferation has also recently been exploited in the therapeutic application of rapamycin to drug eluting stents for angioplasty. These stents significantly reduce the amount of arterial reblockage that results from proliferating vascular smooth muscle cells. In cancer, the effect of blocking TOR function on tumour growth and disease progression is currently of major interest and is the basis for a number of ongoing clinical trials. However, different cell types and tumours respond differently to TOR inhibition, and TOR is clearly not cytostatic for all types of cancer cells in vitro or in vivo. As the molecular details of how TOR functions and the targets of TOR activity are further elucidated, tumour and tissue specific functions are being identified that implicate TOR in angiogenesis, apoptosis, and the reversal of some forms of cellular transformation. This review will describe our current understanding of TOR function, describe the current strategies for employing TOR inhibitors in clinical and preclinical development, and outline future strategies for appropriate targets of TOR inhibitors in the treatment of disease.

TOR (Target Of Rapamycin) is a highly conserved protein kinase that is important in both fundamental and clinical biology. In fundamental biology, TOR is a nutrient-sensitive, central controller of cell growth and aging. In clinical biology, TOR is implicated in many diseases and is the target of the drug rapamycin used in three different therapeutic areas. The yeast Saccharomyces cerevisiae has played a prominent role in both the discovery of TOR and the elucidation of its function. Here we review the TOR signaling network in S. cerevisiae.

The TOR (target of rapamycin) signalling network plays a pivotal role in sugar metabolism and plant growth. In this study, we used grape (Vitis vinifera L.) calli to explore the function of the kinase and FATC domains (C-terminal of FAT (FRAP-ATM-TTRAP) of VvTOR (Vitis vinifera target of rapamycin). We found that the activity of VvTOR affected sugar-related gene expression. VvTOR-VvS6K pathway potentially participated in regulating sugar gene expression. We obtained the over-expression of kinase and FATC domains in transgenic calli by Agrobacterium -mediated transformation. Even though the kinase and FATC domains all belong to VvTOR protein, their functions were different in the regulating sugar accumulation and sugar-related gene expression. We speculated that the kinase domain positively regulated sugar accumulation and FATC domain may negatively influenced sugar accumulation. FATC and kinase domains of VvTOR co-regulated sugar accumulation in grape. These observations will provide framework for future investigations to address other functions of TOR signalling in plant development and signalling pathways.

Mutations of the AtYAK1 Kinase Suppress TOR Deficiency in Arabidopsis

16 - Systems Biology and TOR: Past, Present, and Future

All organisms need to sense and process information about the availability of nutrients, energy status, and environmental cues to determine the best time for growth and development. The conserved target of rapamycin (TOR) protein kinase has a central role in sensing and perceiving nutritional information. TOR connects environmental information about nutrient availability to developmental and metabolic processes to maintain cellular homeostasis. Under favourable energy conditions, TOR is activated and promotes anabolic processes such as cell division, while suppressing catabolic processes. Conversely, when nutrients are limited or environmental stresses are present, TOR is inactivated, and catabolic processes are promoted. Given the central role of TOR in regulating metabolism, several previous works have examined whether TOR is wired to plant defence. To date, the mechanisms by which TOR influences plant defence are not entirely clear. Here, we addressed this question by testing the effect of inhibiting TOR on immunity and pathogen resistance in tomato. Examining which hormonal defence pathways are influenced by TOR, we show that tomato immune responses and disease resistance to several pathogens increase on TOR inhibition, and that TOR inhibition‐mediated resistance probably requires a functional salicylic acid, but not jasmonic acid, pathway. Our results support the notion that TOR is a master regulator of the development–defence switch in plants.

The target of rapamycin (TOR) kinase is a conserved regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. Despite the increased appreciation of the essential role of the TOR complex in plants, no large-scale phosphoproteomics or interactomics studies have been performed to map TOR signalling events in plants. To fill this gap, we combined a systematic phosphoproteomics screen with a targeted protein complex analysis in the model plant Arabidopsis thaliana. Integration of the phosphoproteome and protein complex data on the one hand shows that both methods reveal complementary subspaces of the plant TOR signalling network, enabling proteome-wide discovery of both upstream and downstream network components. On the other hand, the overlap between both data sets reveals a set of candidate direct TOR substrates. The integrated network embeds both evolutionarily-conserved and plant-specific TOR signalling components, uncovering an intriguing complex interplay with protein synthesis. Overall, the network provides a rich data set to start addressing fundamental questions about how TOR controls key processes in plants, such as autophagy, auxin signalling, chloroplast development, lipid metabolism, nucleotide biosynthesis, protein translation or senescence.

ABSTRACTThe Ser/Thr protein kinase MTOR (mechanistic target of rapamycin kinase) regulates cellular metabolism and controls macroautophagy/autophagy. Autophagy has both metabolic and quality control functions, including recycling nutrients at times of starvation and removing dysfunctional intracellular organelles. Lysosomal damage is one of the strongest inducers of autophagy, and yet mechanisms of its activation in response to lysosomal membrane damage are not fully understood. Our recent study has uncovered a new signal transduction system based on cytosolic galectins that elicits autophagy by controlling master regulators of metabolism and autophagy, MTOR and AMPK, in response to lysosomal damage. Thus, intracellular galectins are not, as previously thought, passive tags recognizing damage to guide selective autophagy receptors, but control the activation state of AMPK and MTOR in response to endomembrane damage.Abbreviations: MTOR: mechanistic target of rapamycin kinase; AMPK: AMP-activated protein kinase / Protein Kinase AMP-Activated; SLC38A9: Solute Carrier Family 38 Member 9; APEX2: engineered ascorbate peroxidase 2; RRAGA/B: Ras Related GTP Binding A or B; LAMTOR1: Late Endosomal/Lysosomal Adaptor, MAPK and MTOR Activator 1; LGALS8: Lectin, Galactoside-Binding, Soluble, 8 / Galectin 8; LGALS9: Lectin, Galactoside-Binding, Soluble, 9 / Galectin 9; TAK1: TGF-Beta Activated Kinase 1 / Mitogen-Activated Protein Kinase Kinase Kinase 7 (MAP3K7); STK11/LKB1: Serine/Threonine Kinase 11 / Liver Kinase B1; ULK1: Unc-51 Like Autophagy Activating Kinase 1.

A complex network of endocrine/paracrine signals regulates granulosa-cell function in ovarian follicles. Mechanistic target of rapamycin (MTOR) has recently emerged as a master intracellular integrator of extracellular signals and nutrient availability. The objectives of the present study were to characterize the expression pattern and kinase activity of MTOR during follicular and corpus luteum development, and to examine how inhibition of MTOR kinase activity affects preovulatory maturation of ovarian follicles. MTOR expression was constitutive throughout follicular and corpus luteum development. Gonadotropins induced MTOR kinase activity in the ovary, which was inhibited by rapamycin treatment (10 µg/g body weight, intraperitoneal injection). Inhibition of human chorionic gonadotropin (hCG)-induced MTOR activity during preovulatory follicle maturation did not change key events of ovulation. Granulosa cells of rapamycin-treated mice showed reduced MTOR kinase activity at 1 and 4 hr post-hCG and overexpression of hCG-induced ovulation genes at 4 hr post-hCG. Overexpression of these ovulatory genes was associated with hyper-activation of extracellular signal-regulated kinase 1/2 (ERK1/2), which occurred in response to inhibition of MTOR with rapamycin and suggested that MTOR may function as a negative regulator of the mitogen-activated protein kinase (MAPK) pathway. Indeed, simultaneous inhibition of MTOR and ERK1/2 activities during preovulatory follicle maturation caused anovulation. Inhibition of hCG-induced ERK1/2 activity alone suppressed MTOR kinase activity, indicating that MAPK pathway is upstream of MTOR. Thus, normal ovulation appears to be a result of complex interactions between MTOR and MAPK signaling pathways in granulosa cells of ovulating follicles in mice.

Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae

Introduction: The PI3K/AKT/TOR signaling network has been the focus of intense research in the cancer field as its activity is commonly elevated across cancers including leukemia and lymphoma. As an integral effector of the network, the target of rapamycin (TOR) functions as the catalytic subunit of two distinct protein complexes, TORC1 and TORC2. Both complexes promote survival of hematological malignancies through protein translation (TORC1) and AKT activation (TORC2). Unlike the classical allosteric TOR inhibitors (e.g. rapamycin), a new class of active-site TOR inhibitors (asTORi) more completely inhibits TOR enzymatic function in both TORC −1 and −2 complexes. TORC2 inhibition correlates with accumulation of FOXO transcription factors in the nucleus. Our lab showed that asTORi kill mouse leukemia and inhibit proliferation of human leukemia cells in vitro and in vivo. Here we investigated mechanisms of resistance to asTORi-mediated cell death in a human leukemia as well as a panel of human lymphoma cell lines. We show that asTORi induce nuclear localization of FOXO and transcription of cytostatic (i.e. p27) but not pro-apoptotic (ex. TRAIL) FOXO target genes. Combining asTORi with pharmacologically achievable concentrations of the histone deacetylase inhibitor (HDACi) upregulates transcription of TRAIL and dramatically enhances death of human leukemia cells in vitro. Similarly, asTORi potentiate a small molecule antagonist of the anti-apoptotic Bcl-2 family proteins whose expression is commonly elevated in lymphoma. Material and Methods: Cell Lines and reagents: The mouse p190 and human SUP-B15 cell lines are Ph+ B precursor acute lymphoblastic leukemia cells. The human lymphoma cell lines OCI-LY1, SUDHL-4, SUDHL-6 and RCK8 are germinal-center diffuse large-B-cell non-Hodgkins lymphoma (DLBCL). The HDACi (Vorinostat) and BCL-2 antagonist (ABT-263) were purchased from Merck & Co and Selleck. The active-site TOR inhibitors were generously provided by Intellikine Inc. Cell cycle and death analysis: Flow cytometry was used in conjunction with 7-amino actinomycin D or annexin V staining along with forward scatter profile to assay cell viability. Flow cytometry and propidium iodide staining was used to measure DNA content for cell cycle analysis. qPCR: RNA was isolated using trizol method and analyzed by real time PCR. Subcellular localization: SUP-B15 and OCI-LY1 cell lines were fractionated into cytosolic and nuclear fraction then probed with anti-FOXO1 antibody. Anti -actin and -histone antibodies were used to confirm the purity of the fractions. Conclusion:The human leukemia cell line SUP-B15 and DLBCL cell lines survive inhibition of TOR by asTORi.asTORi promotes nuclear localization of FOXO and transcription of the cytostatic effector and FOXO target gene, p27.Combining HDACi with asTORi promotes transcription of TRAIL, a pro-apoptotic FOXO target gene, and markedly enhances apoptosis in SUP-B15 cells.asTORi potentiates cytotoxic effects of the BCL-2 inhibitor in DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C71.

Identification of TOR Signaling Complexes: More TORC for the Cell Growth Engine

TARGET OF RAPAMYCIN signaling plays a role in Arabidopsis growth promotion by Azospirillum brasilense Sp245

Azospirillum brasilense Sp245 lipopolysaccharides induce target of rapamycin signaling and growth in Arabidopsis thaliana