Macroautophagy/autophagy is a strategy cells use to cope with detrimental conditions, e.g. nutrient deficiency. Phagophores, the precursors to autophagosomes, are initiated and expanded on the endoplasmic reticulum (ER). However, how phagophores and completed autophagosomes are linked to the ER remains incompletely understood. We recently unveiled a RAB GTPase-based linkage between the two structures. RABC1 is a plant member of RABC/RAB18 GTPases. Our biochemical and microscopy data indicated that RABC1 promotes autophagy in response to nutrient starvation, but not under ER stress. Under nutrient-starvation conditions, active RABC1 interacts with ATG18a on the ER, controlling the association of ATG18a to the ER. Subsequently, active RABC1 is turned off allowing expanded phagophores or autophagosomes to detach from the ER. Our work identifies a RAB GTPase-mediated autophagy process in plant cells, opening a door for improving crop productivity in the changing environment.

Rice production accounts for approximately half of the freshwater resources utilized in agriculture, resulting in greenhouse gas emissions such as methane (CH4) from flooded paddy fields. To address this challenge, environmentally friendly and cost-effective water-saving techniques have become widely adopted in rice cultivation. However, the implementation of water-saving treatments (WSTs) in paddy-field rice has been associated with a substantial yield loss of up to 50% as well as a reduction in nitrogen use efficiency (NUE). In this study, we discovered that the target of rapamycin (TOR) signaling pathway is compromised in rice under WST. Polysome profiling-coupled transcriptome sequencing (polysome-seq) analysis unveiled a substantial reduction in global translation in response to WST associated with the downregulation of TOR activity. Molecular, biochemical, and genetic analyses revealed new insights into the impact of the positive TOR-S6K-RPS6 and negative TOR-MAF1 modules on translation repression under WST. Intriguingly, ammonium exhibited a greater ability to alleviate growth constraints under WST by enhancing TOR signaling, which simultaneously promoted uptake and utilization of ammonium and nitrogen allocation. We further demonstrated that TOR modulates the ammonium transporter AMT1;1 as well as the amino acid permease APP1 and dipeptide transporter NPF7.3 at the translational level through the 5\' untranslated region. Collectively, these findings reveal that enhancing TOR signaling could mitigate rice yield penalty due to WST by regulating the processes involved in protein synthesis and NUE. Our study will contribute to the breeding of new rice varieties with increased water and fertilizer utilization efficiency.

With the rise of the internet, there has been an increasing focus on user anonymity. Anonymous communication networks (ACNs) aim to protect the identity privacy of users in the network. As a typical ACN, Tor achieves user anonymity by relaying user data through a series of relay nodes. However, this results in higher latency due to the transmission of network traffic between multiple nodes. This paper proposes a port-based anonymous communication network (PBACN) to address this issue. First, we propose a path construction algorithm. This algorithm describes constructing paths by partitioning the communication path information, which can reduce the probability of being discovered by adversaries. Secondly, we design a port-based source routing addressing method. During data transmission from the source to the destination, each node can directly forward the data by resolving the address into the port of each node. This method eliminates the need for table lookups, reducing the complexity of routing. Lastly, we propose an entropy-based metric to measure the anonymity of different ACNs. In terms of experimental evaluation, we quantitatively analyze the anonymity and end-to-end delay of various ACNs. The experimental results show that our proposed method reduces end-to-end delay by approximately 25% compared to Tor. When the adversary fraction is 20%, PBACN can improve the anonymity degree by approximately 4%.

The regulation of autophagy initiation is a key step in autophagosome biogenesis. However, our understanding of the molecular mechanisms underlying the stepwise assembly of ATG proteins during this process remains incomplete. The Rab GTPase Ypt1/Rab1 is recognized as an essential autophagy regulator. Here, we identify Atg23 and Atg17 as binding partners of Ypt1, with their direct interaction proving crucial for the stepwise assembly of autophagy initiation complexes. Disruption of Ypt1-Atg23 binding results in significantly reduced Atg9 interactions with Atg11, Atg13, and Atg17, thus preventing the recruitment of Atg9 vesicles to the phagophore assembly site (PAS). Likewise, Ypt1-Atg17 binding contributes to the PAS recruitment of Ypt1 and Atg1. Importantly, we found that Ypt1 is phosphorylated by TOR at the Ser174 residue. Converting this residue to alanine blocks Ypt1 phosphorylation by TOR and enhances autophagy. Conversely, the Ypt1S174D phosphorylation mimic impairs both PAS recruitment and activation of Atg1, thus inhibiting subsequent autophagy. Thus, we propose TOR-mediated Ypt1 as a multifunctional assembly factor that controls autophagy initiation via its regulation of the stepwise assembly of ATG proteins.

Aedes aegypti female mosquitoes require vertebrate blood for their egg production and consequently they become vectors of devastating human diseases. Amino acids (AAs) and nutrients originating from a blood meal activate vitellogenesis and fuel embryo development of anautogenous mosquitoes. Insulin-like peptides (ILPs) are indispensable in reproducing female mosquitoes, regulating glycogen and lipid metabolism, and other essential functions. However, how ILPs coordinate their action in response to the AA influx in mosquito reproduction was unknown. We report here that the AA/Target of Rapamycin (TOR) signaling pathway regulates ILPs through GATA transcription factors (TFs). AA infusion combined with RNA-interference TOR silencing of revealed their differential action on ILPs, elevating circulating levels of several ILPs but inhibiting others, in the female mosquito. Experiments involving isoform-specific CRISPR-Cas9 genomic editing and chromatin immunoprecipitation assays showed that the expression of ilp4, ilp6, and ilp7 genes was inhibited by the GATA repressor (GATAr) isoform in response to low AA-TOR signaling, while the expression of ilp1, ilp2, ilp3, ilp5, and ilp8 genes was activated by the GATA activator isoform after a blood meal in response to the increased AA-TOR signaling. FoxO, a downstream TF in the insulin pathway, was involved in the TOR-GATAr-mediated repression of ilp4, ilp6, and ilp7 genes. This work uncovered how AA/TOR signaling controls the ILP pathway in modulation of metabolic requirements of reproducing female mosquitoes.

Sclerotinia sclerotiorum is a necrotrophic phytopathogenic fungus that cross-talks with its hosts for control of cell-death pathways for colonization. Target of rapamycin (TOR) is a central regulator that controls cell growth, intracellular metabolism, and stress responses in a variety of eukaryotes, but little is known about TOR signaling in S. sclerotiorum. In this study, we identified a conserved TOR signaling pathway and characterized SsTOR as a critical component of this pathway. Hyphal growth of S. sclerotiorum was retarded by silencing SsTOR, moreover, sclerotia and compound appressoria formation were severely disrupted. Notably, pathogenicity assays of strains shows that the virulence of the SsTOR-silenced strains were dramatically decreased. SsTOR was determined to participate in cell wall integrity (CWI) by regulating the phosphorylation level of SsSmk3, a core MAP kinase in the CWI pathway. Importantly, the inactivation of SsTOR induced autophagy in S. sclerotiorum potentially through SsAtg1 and SsAtg13. Taken together, our results suggest that SsTOR is a global regulator controlling cell growth, stress responses, cell wall integrity, autophagy, and virulence of S. sclerotiorum. IMPORTANCE TOR is a conserved protein kinase that regulates cell growth and metabolism in response to growth factors and nutrient abundance. Here, we used gene silencing to characterize SsTOR, which is a critical component of TOR signaling pathway. SsTOR-silenced strains have limited mycelium growth, and the virulence of the SsTOR-silenced strains was decreased. Phosphorylation analysis indicated that SsTOR influenced CWI by regulating the phosphorylation level of SsSmk3. Autophagy is essential to preserve cellular homeostasis in response to cellular and environmental stresses. Inactivation of SsTOR induced autophagy in S. sclerotiorum potentially through SsAtg1 and SsAtg13. These findings further indicated that SsTOR is a global regulator of the growth, development, and pathogenicity of S. sclerotiorum in multiple ways.

Wing dimorphism in insects is an evolutionarily adaptive trait to maximize insect fitness under various environments, by which the population could be balanced between dispersing and reproduction. Most studies concern the regulatory mechanisms underlying the stimulation of wing morph in aphids, but relatively little research addresses the molecular basis of wing loss. Here, we found that, while developing normally in winged-destined pea aphids, the wing disc in wingless-destined aphids degenerated 30-hr postbirth and that this degeneration was due to autophagy rather than apoptosis. Activation of autophagy in first instar nymphs reduced the proportion of winged aphids, and suppression of autophagy increased the proportion. REPTOR2, associated with TOR signaling pathway, was identified by RNA-seq as a differentially expressed gene between the two morphs with higher expression in the thorax of wingless-destined aphids. Further genetic analysis indicated that REPTOR2 could be a novel gene derived from a gene duplication event that occurred exclusively in pea aphids on autosome A1 but translocated to the sex chromosome. Knockdown of REPTOR2 reduced autophagy in the wing disc and increased the proportion of winged aphids. In agreement with REPTOR\'s canonical negative regulatory role of TOR on autophagy, winged-destined aphids had higher TOR expression in the wing disc. Suppression of TOR activated autophagy of the wing disc and decreased the proportion of winged aphids, and vice versa. Co-suppression of TOR and REPTOR2 showed that dsREPTOR2 could mask the positive effect of dsTOR on autophagy, suggesting that REPTOR2 acted as a key regulator downstream of TOR in the signaling pathway. These results revealed that the TOR signaling pathway suppressed autophagic degradation of the wing disc in pea aphids by negatively regulating the expression of REPTOR2.

Aging is a process accompanied by widespread degenerative changes which are a major cause of human disease and disability. One goal of aging research is to develop interventions or drugs that can extend organism lifespan and treat age-related diseases. Here, we report the identification of a broad spectrum anti-viral agent, ribavirin, as a potential pharmacological aging intervention. Ribavirin extended the lifespan and healthspan of Caenorhabditis elegans by inhibiting Target of Rapamycin (TOR) signaling and activating AMP-activated protein kinase (AMPK). Moreover, our data indicate that ribavirin activated AMPK by reducing the levels of adenosine triphosphate (ATP) and lysosomal v-ATPase-Ragulator-AXIN Complex. Thus, our studies successfully identify ribavirin as a potential anti-aging drug, and indicate that its anti-aging effect is mediated via AMPK-TOR signaling.

In traditional Chinese medicine, soft-shelled turtle protein and peptides serve as a nutraceutical for prolonging the lifespan. However, their effects on anti-aging have not been clarified scientifically in vivo. This study aimed to determine whether soft-shelled turtle peptides (STP) could promote the lifespan and healthspan in Drosophila melanogaster and the underlying molecular mechanisms. Herein, STP supplementation prolonged the mean lifespan by 20.23% and 9.04% in males and females, respectively, delaying the aging accompanied by climbing ability decline, enhanced gut barrier integrity, and improved anti-oxidation, starvation, and heat stress abilities, while it did not change the daily food intake. Mechanistically, STP enhanced autophagy and decreased oxidative stress by downregulating the target of rapamycin (TOR) signaling pathway. In addition, 95.18% of peptides from the identified sequences in STP could exert potential inhibitory effects on TOR through hydrogen bonds, van der Walls, hydrophobic interactions, and electrostatic interactions. The current study could provide a theoretical basis for the full exploitation of soft-shelled turtle aging prevention.

The removal of drivers\' active engagement in driving tasks can lead to erratic gaze patterns in SAE Level 2 (L2) and Level 3 (L3) automation, which has been linked to their subsequential degraded take-over performance. To further address how changes in gaze patterns evolve during the take-over phase, and whether they are influenced by the take-over urgency and the location of the human-machine interface, this driving simulator study used a head-up display (HUD) to relay information about the automation status and conducted take-over driving experiments where the ego car was about to exit the highway with variations in the automation level (L2, L3) and time budget (2 s, 6 s). In L2 automation, drivers were required to monitor the environment, while in L3, they were engaged with a visual non-driving related task. Manual driving was also embodied in the experiments as the baseline. Results showed that, compared to manual driving, drivers in L2 automation focused more on the HUD and Far Road (roadway beyond 2 s time headway ahead), and less on the Near Road (roadway within 2 s time headway ahead); while in L3, drivers\' attention was predominantly allocated on the non-driving related task. After receiving take-over requests (TORs), there was a gradual diversion of attention from the Far Road to the Near Road in L2 take-overs. This trend changed nearly in proportion to the time within the time budget and it exaggerated given a shorter time budget of 2 s. While in L3, drivers\' gaze distribution was similar in the early stage of take-overs for both time budget conditions (2 s vs. 6 s), where they prioritized their early glances to Near Road with a gradual increase in attention towards Far Road. The HUD used in the present study showed the potential to maintain drivers\' attention around the road center during automation and to encourage drivers to glance the road earlier after TORs by reducing glances to the instrument cluster, which might be of significance to take-over safety. These findings were discussed based on an extended conceptual gaze control model, which advances our understanding of gaze patterns around control transitions and their underlying gaze control causations. Implications can be contributed to the design of autonomous vehicles to facilitate the transition of control by guiding drivers\' attention appropriately according to drivers\' attentional state and the take-over urgency.