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.

Target of Rapamycin (TOR) is an eukaryotic protein kinase and evolutionally conserved from the last eukaryotic common ancestor (LECA) to humans. The growing evidences have shown that TOR signaling acts as a central controller of cell growth and development. The downstream effectors of TOR have been well-identified in yeast and animals by using the immunosuppression agent rapamycin. However, less is known about TOR in plants. This is largely due to the fact that plants are insensitive to rapamycin. In this study, AZD8055 (AZD), the novel ATP-competitive inhibitor of TOR, was employed to decipher the downstream effectors of TOR in Arabidopsis. One AZD insensitive mutant, TOR-inhibitor insensitive-1 (trin1), was screened from 10,000 EMS-induced mutation seeds. The cotyledons of trin1 can turn green when its seeds were germinated on ½ MS medium supplemented with 2 µM AZD, whereas the cotyledons greening of wild-type (WT) can be completely blocked at this concentration. In addition, trin1 also showed a longer hypocotyl than that observed in WT in the presence of 2 µM AZD. Through genetic mapping, TRIN1 was mapped onto the long arm of chromosome 2, between markers SGCSNP26 and MI277. Positional cloning revealed that TRIN1 was an allele of ABI4, which encoded an ABA-regulated AP2 domain transcription factor. Plants containing P35S::TRIN1 or P35S::TRIN1-GUS were hypersensitive to AZD treatment and displayed the opposite phenotype observed in trin1. Importantly, GUS signaling was significantly enhanced in P35S::TRIN1-GUS transgenic plants in response to AZD treatment, indicating that suppression of TOR resulted in the accumulation of TRIN1. These observations revealed that TOR controlled seed-to-seedling transition by negatively regulating the stability of TRIN1 in Arabidopsis. For the first time, TRIN1, the downstream effector of TOR signaling, was identified through a chemical genetics approach.

Verticillium wilt is caused by the soil‐borne vascular pathogen Verticillium dahliae, and affects a wide range of economically important crops, including upland cotton (Gossypium hirsutum). Previous studies showed that expression levels of BIN2 were significantly down‐regulated during infestation with V. dahliae. However, the underlying molecular mechanism of BIN2 in plant regulation against V. dahliae remains enigmatic. Here, we characterized a protein kinase GhBIN2 from Gossypium hirsutum, and identified GhBIN2 as a negative regulator of resistance to V. dahliae. The Verticillium wilt resistance of Arabidopsis and cotton were significantly enhanced when BIN2 was knocked down. Constitutive expression of BIN2 attenuated plant resistance to V. dahliae. We found that BIN2 regulated plant endogenous JA content and influenced the expression of JA‐responsive marker genes. Further analysis revealed that BIN2 interacted with and phosphorylated JAZ family proteins, key repressors of the JA signalling pathway in both Arabidopsis and cotton. Spectrometric analysis and site‐directed mutagenesis showed that BIN2 phosphorylated AtJAZ1 at T196, resulting in the degradation of JAZ proteins. Collectively, these results show that BIN2 interacts with JAZ proteins and plays a negative role in plant resistance to V. dahliae. Thus, BIN2 may be a potential target gene for genetic engineering against Verticillium wilt in crops.

During the arms race between plants and pathogens, pathogenesis-related proteins (PR) in host plants play a crucial role in disease resistance, especially PR1. PR1 constitute a secretory peptide family, and their role in plant defense has been widely demonstrated in both hosts and in vitro. However, the mechanisms by which they control host-pathogen interactions and the nature of their targets within the pathogen remain poorly understood. The present study was aimed to investigate the anti-oomycete activity of secretory PR1 proteins and elaborate their underlying mechanisms. This study was conducted in the potato-Phytophthora infestans pathosystem. After being induced by the pathogen infection, the cross-kingdom translocation of secretory PR1 was demonstrated by histochemical assays and western blot, and their targets in P. infestans were identified by yeast-two-hybrid assays, bimolecular fluorescence complementation assays, and co-immunoprecipitation assay. The results showed that the expression of secretory PR1-encoding genes was induced during pathogen infection, and the host could deliver PR1 into P. infestans to inhibit its vegetative growth and pathogenicity. The translocated secretory PR1 targeted the subunits of the AMPK kinase complex in P. infestans, thus affecting the AMPK-driven phosphorylation of downstream target proteins, preventing ROS homeostasis, and down-regulating the expression of RxLR effectors. The results provide novel insights into the molecular function of PR1 in protecting plants against pathogen infection, and uncover a potential target for preventing pre- and post-harvest late blight.

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 △vdfkbp12 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.

DNA methylation is an indispensable epigenetic modification that dynamically regulates gene expression and genome stability during cell growth and development processes. The target of rapamycin (TOR) has emerged as a central regulator to regulate many fundamental cellular metabolic processes from protein synthesis to autophagy in all eukaryotic species. However, little is known about the functions of TOR in DNA methylation. In this study, the synergistic growth inhibition of Arabidopsis seedlings can be observed when DNA methylation inhibitor azacitidine was combined with TOR inhibitors. Global DNA methylation level was evaluated using whole-genome bisulfite sequencing (WGBS) under TOR inhibition. Hypomethylation level of whole genome DNA was observed in AZD-8055 (AZD), rapamycin (RAP) and AZD + RAP treated Arabidopsis seedlings. Based on functional annotation and KEGG pathway analysis of differentially methylated genes (DMGs), most of DMGs were enriched in carbon metabolism, biosynthesis of amino acids and other metabolic processes. Importantly, the suppression of TOR caused the change in DNA methylation of the genes associated with plant hormone signal transduction, indicating that TOR played an important role in modulating phytohormone signals in Arabidopsis. These observations are expected to shed light on the novel functions of TOR in DNA methylation and provide some new insights into how TOR regulates genome DNA methylation to control plant growth.