Autophagy is a critical process for recycling of cytoplasmic materials during environmental stress, senescence and cellular remodeling. It is upregulated under a wide range of abiotic stress conditions and is important for stress tolerance. Autophagy is repressed by the protein kinase target of rapamycin (TOR), which is activated in response to nutrients and in turn upregulates cell growth and translation and inhibits autophagy. Down-regulation of TOR in Arabidopsis thaliana leads to constitutive autophagy and to decreased growth, but the relationship to stress conditions is unclear. Here, we assess the extent to which TOR controls autophagy activation by abiotic stress. Overexpression of TOR inhibited autophagy activation by nutrient starvation, salt and osmotic stress, indicating that activation of autophagy under these conditions requires down-regulation of TOR activity. In contrast, TOR overexpression had no effect on autophagy induced by oxidative stress or ER stress, suggesting that activation of autophagy by these conditions is independent of TOR function. The plant hormone auxin has been shown previously to up-regulate TOR activity. To confirm the existence of two pathways for activation of autophagy, dependent on the stress conditions, auxin was added exogenously to activate TOR, and the effect on autophagy under different conditions was assessed. Consistent with the effect of TOR overexpression, the addition of the auxin NAA inhibited autophagy during nutrient deficiency, salt and osmotic stress, but not during oxidative or ER stress. NAA treatment was unable to block autophagy induced by a TOR inhibitor or by a mutation in the TOR complex component RAPTOR1B, indicating that auxin is upstream of TOR in the regulation of autophagy. We conclude that repression of auxin-regulated TOR activity is required for autophagy activation in response to a subset of abiotic stress conditions.

The pseudokinase mixed lineage kinase domain-like protein (MLKL) is a key component of tumor necrosis factor (TNF)-induced necroptosis and plays a crucial role in necroptosis execution. However, the mechanisms that control MLKL activity are not completely understood. Here, we identify the molecular chaperone Hsp90 as a novel MLKL-interacting protein. We show that Hsp90 associates with MLKL and is required for MLKL stability. Moreover, we find that Hsp90 also regulates the stability of the upstream RIP3 kinase. Interference with Hsp90 function with the 17AAG inhibitor destabilizes MLKL and RIP3, resulting in their degradation by the proteasome pathway. Furthermore, we find that Hsp90 is required for TNF-stimulated necrosome assembly. Disruption of Hsp90 function prevents necrosome formation and strongly reduces MLKL phosphorylation and inhibits TNF-induced necroptosis. Consistent with a positive role of Hsp90 in necroptosis, coexpression of Hsp90 increases MLKL oligomerization and plasma membrane translocation and enhances MLKL-mediated necroptosis. Our findings demonstrate that an efficient necrotic response requires a functional Hsp90.

ACT2: actin 2; ATG: autophagy-related; BGLU21: β-glucosidase 21; BIP3: binding protein 3; BZIP: basic leucine zipper; DAPI: 4', 6-diamidino-2-phenylindole; DTT: dithiothreitol; ER: endoplasmic reticulum; ERN1: endoplasmic reticulum to nucleus signaling 1; IRE1: inositol requiring 1; GFP: green fluorescent protein; MAP3K5/ASK1: mitogen-activated protein kinase kinase kinase 5; MAPK8/JNK1: mitogen-activated protein kinase 8/c-Jun N-terminal kinase 1; MDC: monodansylcadaverine; PR-14: pathogenesis-related protein 14; RIDD: Regulated IRE1-Dependent Decay of Messenger RNA; ROSY1/ML: interactor of synaptotagmin1/MD2-related lipid recognition protein; Tm: tunicamycin; UPR: unfolded protein response; WT: wild-type.

The endoplasmic reticulum (ER) is responsible for the synthesis of one third of the cellular proteome and is constantly challenged by physiological and environmental situations that can perturb its homeostasis and lead to the accumulation of misfolded secretory proteins, a condition referred to as ER stress. In response, the ER evokes a set of intracellular signaling processes, collectively known as the unfolded protein response (UPR), which are designed to restore biosynthetic capacity of the ER. As single cell organisms evolved into multicellular life, the UPR complexity has increased to suit their growth and development. In this review, we discuss recent advances in the understanding in the UPR, emphasizing conserved UPR elements between plants and metazoans, and highlighting unique plant-specific features.

BackgroundThermophilic, Gram-positive, anaerobic bacteria (TGPAs) are generally recalcitrant to chemical and electrotransformation due to their special cell-wall structure and the low intrinsic permeability of plasma membranes.Methodology/Principal FindingsHere we established for any Gram-positive or thermophiles an ultrasound-based sonoporation as a simple, rapid, and minimally invasive method to genetically transform TGPAs. We showed that by applying a 40 kHz ultrasound frequency over a 20-second exposure, Texas red-conjugated dextran was delivered with 27% efficiency into Thermoanaerobacter sp. X514, a TGPA that can utilize both pentose and hexose for ethanol production. Experiments that delivered plasmids showed that host-cell viability and plasmid DNA integrity were not compromised. Via sonoporation, shuttle vectors pHL015 harboring a jellyfish gfp gene and pIKM2 encoding a Clostridium thermocellum β-1,4-glucanase gene were delivered into X514 with an efficiency of 6×102 transformants/µg of methylated DNA. Delivery into X514 cells was confirmed via detecting the kanamycin-resistance gene for pIKM2, while confirmation of pHL015 was detected by visualization of fluorescence signals of secondary host-cells following a plasmid-rescue experiment. Furthermore, the foreign β-1,4-glucanase gene was functionally expressed in X514, converting the host into a prototypic thermophilic consolidated bioprocessing organism that is not only ethanologenic but cellulolytic.Conclusions/SignificanceIn this study, we developed an ultrasound-based sonoporation method in TGPAs. This new DNA-delivery method could significantly improve the throughput in developing genetic systems for TGPAs, many of which are of industrial interest yet remain difficult to manipulate genetically.