Target of Rapamycin (TOR) Negatively Regulates Ethylene Signals in Arabidopsis.

Target of rapamycin (TOR) acts as a master regulator in coordination of cell growth with energy and nutrient availability. Despite the increased appreciation of the essential role of the TOR complex in interaction with phytohormone signaling, little is known about its function on ethylene signaling. Here, through expression analysis, genetic and biochemical approaches, we reveal that TOR functions in the regulation of ethylene signals. Transcriptional analysis indicates that TOR inhibition by AZD8055 upregulated senescence- and ethylene-related genes expression. Furthermore, ethylene insensitive mutants like etr1-1, ein2-5 and ein3 eil1, showed more hyposensitivity to AZD8055 than that of WT in hypocotyl growth inhibition. Similarly, blocking ethylene signals by ethylene action inhibitor Ag+ or biosynthesis inhibitor aminoethoxyvinylglycine (AVG) largely rescued hypocotyl growth even in presence of AZD8055. In addition, we also demonstrated that Type 2A phosphatase-associated protein of 46 kDa (TAP46), a downstream component of TOR signaling, physically interacts with 1-aminocy-clopropane-1-carboxylate (ACC) synthase ACS2 and ACS6. Arabidopsis overexpressing ACS2 or ACS6 showed more hypersensitivity to AZD8055 than WT in hypocotyl growth inhibition. Moreover, ACS2/ACS6 protein was accumulated under TOR suppression, implying TOR modulates ACC synthase protein levels. Taken together, our results indicate that TOR participates in negatively modulating ethylene signals and the molecular mechanism is likely involved in the regulation of ethylene biosynthesis by affecting ACSs in transcription and protein levels.

Host-induced gene silencing of BcTOR in Botrytis cinerea enhances plant resistance to grey mould.

Botrytis cinerea is the causal agent of grey mould for more than 200 plant species, including economically important vegetables, fruits and crops, which leads to economic losses worldwide. Target of rapamycin (TOR) acts a master regulator to control cell growth and proliferation by integrating nutrient, energy and growth factors in eukaryotic species, but little is known about whether TOR can function as a practicable target in the control of plant fungal pathogens. Here, we characterize TOR signalling of B. cinerea in the regulation of growth and pathogenicity as well as its potential value in genetic engineering for crop protection by bioinformatics analysis, pharmacological assays, biochemistry and genetics approaches. The results show that conserved TOR signalling occurs, and a functional FK506-binding protein 12 kD (FKBP12) mediates the interaction between rapamycin and B. cinerea TOR (BcTOR). RNA sequencing (RNA-Seq) analysis revealed that BcTOR displayed conserved functions, particularly in controlling growth and metabolism. Furthermore, pathogenicity assay showed that BcTOR inhibition efficiently reduces the infection of B. cinerea in plant leaves of Arabidopsis and potato or tomato fruits. Additionally, transgenic plants expressing double-stranded RNA of BcTOR through the host-induced gene silencing method could produce abundant small RNAs targeting BcTOR, and significantly block the occurrence of grey mould in potato and tomato. Taken together, our results suggest that BcTOR is an efficient target for genetic engineering in control of grey mould, and also a potential and promising target applied in the biocontrol of plant fungal pathogens.

Hypocotyl Elongation Inhibition of Melatonin Is Involved in Repressing Brassinosteroid Biosynthesis in Arabidopsis.

Melatonin functions as a plant hormone/regulator in the regulation of growth and development. However, the underlying mechanisms are still unclear. In this study, we found that a high dose of melatonin inhibited hypocotyl elongation in a dose-dependent manner in Arabidopsis. An expression profile analysis showed that hypocotyl growth inhibition by melatonin was involved in reprograming the expression of cell elongation genes and brassinosteroid (BRs) biosynthetic genes. Furthermore, similar to BR biosynthetic inhibitor brassinazole (BRZ), a high concentration of melatonin upregulated BR-biosynthetic genes and downregulated BR-induced genes involved in cell elongation, while melatonin was inefficient in brassinazole-resistant mutants like the bzr1-1D and bes1-D in hypocotyl inhibition. The comparative expression profile analysis showed an opposite expression mode in the co-regulated genes between melatonin and BZR1 or melatonin and brassinolide (BL). Additionally, exogenous BL rescued the repressive phenotype of BR biosynthesis-deficient mutant like det2-1 even in the presence of high-dose melatonin, but not BR receptor mutant bri1-5 or signal transduction mutant bin2-1. A biochemical analysis further confirmed that melatonin reduced endogenous BR levels in a dose-dependent manner in Arabidopsis. Taken together, these results indicate that melatonin inhibits BR biosynthesis but does not block BR signaling in the inhibition of hypocotyl elongation and extends insights on the role of melatonin in cross-talking with plant hormone signaling.

Functional Characterization of Target of Rapamycin Signaling in Verticillium dahliae.

More than 200 plants have been suffering from Verticillium wilt caused by Verticillium dahliae (V. dahliae) across the world. The target of rapamycin (TOR) is a lethal gene and controls cell growth and development in various eukaryotes, but little is known about TOR signaling in V. dahliae. Here, we found that V. dahliae strain is hypersensitive to rapamycin in the presence of rapamycin binding protein VdFKBP12 while the deletion mutant aaavdfkbp12 is insensitive to rapamycin. Heterologous expressing VdFKBP12 in Arabidopsis conferred rapamycin sensitivity, indicating that VdFKBP12 can bridge the interaction between rapamycin and TOR across species. The key across species of TOR complex 1 (TORC1) and TORC2 have been identified in V. dahliae, suggesting that TOR signaling pathway is evolutionarily conserved in eukaryotic species. Furthermore, the RNA-seq analysis showed that ribosomal biogenesis, RNA polymerase II transcription factors and many metabolic processes were significantly suppressed in rapamycin treated cells of V. dahliae. Importantly, transcript levels of genes associated with cell wall degrading enzymes (CWEDs) were dramatically down-regulated in TOR-inhibited cells. Further infection assay showed that the pathogenicity of V. dahliae and occurrence of Verticillium wilt can be blocked in the presence of rapamycin. These observations suggested that VdTOR is a key target of V. dahliae for controlling and preventing Verticillium wilt in plants.

Target of Rapamycin (TOR) Regulates the Expression of lncRNAs in Response to Abiotic Stresses in Cotton.

TOR (Target of Rapamycin) kinase is an evolutionarily conserved protein kinase, which integrates stress-related cues with growth and metabolic outputs. Long non-coding RNAs (lncRNAs) play a vital role in the regulation of eukaryotic genes. However, little is known about TOR's function in regulating the expression of lncRNAs in plants. In this study, four putative homologous genes encoding the TOR protein were identified by utilizing the recently completed cotton genome. Pharmacological experiments with TOR inhibitor AZD8055 and on silencing GhTOR genes resulted in obvious cotton growth retardation, indicating the conserved role of TOR in plant growth. The expression pattern analyses in different tissues reveal that TOR may play a role in root development, and the transcript levels of TOR genes were changed under different stress conditions. Importantly, we found TOR may be a key player in regulating the expression of long non-coding RNAs (lncRNAs). A total of 10,315 lncRNAs were discovered in cotton seedlings, 90.7% of which were long intergenic ncRNAs. Moreover, we identified the differentially expressed lncRNAs, of which 296 were significantly upregulated and 105 were downregulated in TOR inactivated plants. GO and KEGG analyses of differentially expressed lncRNA neighboring genes reveal that these differentially expressed lncRNA-targeted genes are involved in many life processes, including stress response, glutathione, and ribosomes in cotton. A series of differentially expressed lncRNAs potentially involved in plant stress response was identified under TOR inhibition. Collectively, these results suggest that cotton TOR proteins may directly modulate the expression of putative stress-related lncRNAs and eventually play a potential role in the cotton stress response.

The crosstalk between Target of Rapamycin (TOR) and Jasmonic Acid (JA) signaling existing in Arabidopsis and cotton.

Target of rapamycin (TOR) acts as an important regulator of cell growth, development and stress responses in most examined diploid eukaryotes. However, little is known about TOR in tetraploid species such as cotton. Here, we show that TORC1-S6K-RPS6, the major signaling components, are conserved and further expanded in cotton genome. Though the cotton seedlings are insensitive to rapamycin, AZD8055, the second-generation inhibitor of TOR, can significantly suppress the growth in cotton. Global transcriptome analysis revealed that genes associated with jasmonic acid (JA) biosynthesis and transduction were significantly altered in AZD8055 treated cotton seedlings, suggesting the potential crosstalk between TOR and JA signaling. Pharmacological and genetic approaches have been employed to get further insights into the molecular mechanism of the crosstalk between TOR and JA. Combination of AZD8055 with methyl jasmonate can synergistically inhibit cotton growth, and additionally JA levels were significantly increased when cotton seedlings were subjected to AZD8055. JA biosynthetic and signaling mutants including jar1, coi1-2 and myc2-2 displayed TOR inhibitor-resistant phenotypes, whereas COI1 overexpression transgenic lines and jaz10 exhibited sensitivity to AZD8055. Consistently, cotton JAZ can partially rescue TOR-suppressed phenotypes in Arabidopsis. These evidences revealed that the crosstalk between TOR and JA pathway operates in cotton and Arabidopsis.

Brassinosteriod Insensitive 2 (BIN2) acts as a downstream effector of the Target of Rapamycin (TOR) signaling pathway to regulate photoautotrophic growth in Arabidopsis.

The components of the target of rapamycin (TOR) signaling pathway have been well characterized in heterotrophic organisms from yeast to humans. However, because of rapamycin insensitivity, embryonic lethality in tor null mutants and a lack of reliable ways of detecting TOR protein kinase in higher plants, the key players upstream and downstream of TOR remain largely unknown in plants. Using engineered rapamycin-sensitive Binding Protein 12-2 (BP12-2) plants, the present study showed that combined treatment with rapamycin and active-site TOR inhibitors (asTORis) results in synergistic inhibition of TOR activity and plant growth in Arabidopsis. Based on this system, we revealed that TOR signaling plays a crucial role in modulating the transition from heterotrophic to photoautotrophic growth in Arabidopsis. Ribosomal protein S6 kinase 2 (S6K2) was identified as a direct downstream target of TOR, and the growth of TOR-suppressed plants could be rescued by up-regulating S6K2. Systems, genetic, and biochemical analyses revealed that Brassinosteriod Insensitive 2 (BIN2) acts as a novel downstream effector of S6K2, and the phosphorylation of BIN2 depends on TOR-S6K2 signaling in Arabidopsis. By combining pharmacological with genetic and biochemical approaches, we determined that the TOR-S6K2-BIN2 signaling pathway plays important roles in regulating the photoautotrophic growth of Arabidopsis.

Tomato FK506 Binding Protein 12KD (FKBP12) Mediates the Interaction between Rapamycin and Target of Rapamycin (TOR).

Target of Rapamycin (TOR) signaling is an important regulator in multiple organisms including yeast, plants, and animals. However, the TOR signaling in plants is much less understood as compared to that in yeast and animals. TOR kinase can be efficiently suppressed by rapamycin in the presence of functional FK506 Binding Protein 12 KD (FKBP12) in yeast and animals. In most examined higher plants rapamycin fails to inhibit TOR kinase due to the non-functional FKBP12. Here we find that tomato plants showed obvious growth inhibition when treated with rapamycin and the inhibitory phenotype is similar to suppression of TOR causing by active-site TOR inhibitors (asTORis) such as KU63794, AZD8055, and Torin1. The chemical genetic assays using TOR inhibitors and heterologous expressing SlFKBP12 in Arabidopsis indicated that the TOR signaling is functional in tomato. The protein gel shifting and TOR inhibitors combination assays showed that SlFKBP12 can mediate the interaction between rapamycin and TOR. Furthermore, comparative expression profile analysis between treatments with rapamycin and KU63794 identified highly overlapped Differentially Expressed Genes (DEGs) which are involved in many anabolic and catabolic processes, such as photosynthesis, cell wall restructuring, and senescence in tomato. These observations suggest that SlFFBP12 is functional in tomato. The results provided basic information of TOR signaling in tomato, and also some new insights into how TOR controls plant growth and development through reprogramming the transcription profiles.