Autophagy must be dynamically tuned to nutrient availability, yet how plants prevent excessive autophagy during nutrient starvation while preserving basal flux under replete conditions remains elusive. Here, we identify the redundant U-box E3 ligases PUB25 and PUB26 as critical negative regulators of nitrogen starvation-induced autophagy in Arabidopsis. Loss of PUB25/PUB26 enhances autophagy and improves plant growth under nitrogen deprivation, whereas their overexpression suppresses autophagy. Mechanistically, PUB25/PUB26 directly interacts with the autophagy-initiating kinase ATG1 and target it for proteasomal degradation. During nitrogen starvation, the receptor kinase FERONIA (FER) is transcriptionally upregulated and enhances PUB25/PUB26 activity by phosphorylating it at Thr95/Thr94, acting as a molecular brake on ATG1 to fine-tune autophagy to proper cellular levels during prolonged deprivation. Additionally, under nitrogen-rich conditions, FER directly phosphorylates ATG13 to repress ATG1-ATG13 complex assembly, thereby sustaining low basal autophagy. Together, our findings reveal a nutrient-responsive FER-PUB25/PUB26 regulatory module that integrates constitutive and inducible regulatory checkpoints to dynamically modulate autophagy activity across variable nitrogen status, optimizing plant fitness.

Background Plants have evolved multiple strategies to cope with the ever-changing external environment. Autophagy, as one of the crucial mechanisms involved, has been demonstrated to play a pivotal role in plant responses and adaptation to abiotic stresses. However, the precise molecular mechanisms underlying the role of autophagy in mediating cold stress remain to be fully elucidated. Results In this study, we demonstrated that autophagy mutants presented increased freezing tolerance under both non-acclimated and cold-acclimated conditions. Autophagy positively regulates the expression of anthocyanin biosynthesis-related genes, thereby influencing anthocyanin accumulation in Arabidopsis under low-temperature conditions. Moreover, we found that cold stress directly suppresses the expression of autophagy-related genes and reduces autophagic flux. The RNA-seq data revealed that cold-responsive genes were pre-activated in the autophagy mutant atg13ab even before cold treatment. Additionally, we observed constitutive accumulation of the dehydrin protein COR47 in atg13ab. Conclusions Taken together, these data suggest that autophagy is a negative regulator of freezing tolerance in Arabidopsis.

BACKGROUND:Plants have evolved multiple strategies to cope with the ever-changing external environment. Autophagy, as one of the crucial mechanisms involved, has been demonstrated to play a pivotal role in plant responses and adaptation to abiotic stresses. However, the precise molecular mechanisms underlying the role of autophagy in mediating cold stress remain to be fully elucidated. RESULTS:In this study, we demonstrated that autophagy mutants presented increased freezing tolerance under both non-acclimated and cold-acclimated conditions in Arabidopsis. Autophagy positively regulates the expression of anthocyanin biosynthesis-related genes, thereby influencing anthocyanin accumulation in Arabidopsis under low-temperature conditions. Moreover, we found that cold stress directly suppresses the expression of autophagy-related genes and reduces autophagic flux in Arabidopsis. The RNA-seq data revealed that cold-responsive genes were pre-activated in the autophagy mutant atg13ab even before cold treatment. Additionally, we observed constitutive accumulation of the dehydrin protein COR47 in atg13ab mutant. CONCLUSIONS:Taken together, these data suggest that autophagy is a negative regulator of freezing tolerance in Arabidopsis.

The forkhead-associated (FHA) domain, a well-characterized small protein module that mediates protein–protein interactions by targeting motifs containing phosphothreonine, is present in many regulatory molecules like protein kinase, phosphatases, transcription factors, and other functional proteins. FHA-domain containing proteins in yeast and human are involved in a large variety of cellular processes such as DNA repair, cell cycle arrest, or pre-mRNA processing. Since the first FHA-domain protein, kinase-associated protein phosphatase (KAPP) was found in plants, the interest in plant FHA-containing proteins has increased dramatically, mainly due to the important role of FHA domain-containing proteins in plant growth and development. In this review, we provide a comprehensive overview of the fundamental properties of FHA domain-containing proteins in plants, and systematically summarized and analyzed the research progress of proteins containing the FHA domain in plants. We also emphasized that AT5G47790 and its homologs may play an important role as the regulatory subunit of protein phosphatase 1 (PP1) in plants.

14 Autophagy, a conserved pathway which carries out the bulk degradation of 15 cytoplasmic material in eukaryotic cells, is critical in plant physiology and 16 development. It is tightly regulated by ATG13, a core component of ATG1 kinase 17 complex which initiates autophagy. Although it has been reported that ATG13 is 18 dephosphorylated immediately after nutrient starvation, the phosphatase regulating 19 this process is poorly understood. Here, we demonstrated that the septuple mutant 20 (topp-7m) and octuple mutant (topp-8m) of type one protein phosphatase (TOPP) 21 exhibited significantly reduced tolerance to fixed-carbon (C) starvation due to 22 compromised autophagy activity. Genetic analysis placed TOPP upstream of 23 autophagy. Interestingly, ATG13a was found to be an interactor of TOPP. And TOPP 24 directly dephosphorylated ATG13a in vitro and in vivo. Meanwhile, eighteen 25 phosphorylation sites of ATG13a were identified by LC-MS. Mimic 26 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which this version posted April 10, 2022. ; https://doi.org/10.1101/2022.04.10.487768 doi: bioRxiv preprint

Autophagy, a conserved pathway that carries out the bulk degradation of cytoplasmic material in eukaryotic cells, is critical for plant physiology and development. This process is tightly regulated by ATG13, a core component of the ATG1 kinase complex, which initiates autophagy. Although ATG13 is known to be dephosphorylated immediately after nutrient starvation, the phosphatase regulating this process is poorly understood. Here, we determined that the Arabidopsis (Arabidopsis thaliana) septuple mutant (topp-7m) and octuple mutant (topp-8m) of TYPE ONE PROTEIN PHOSPHATASE (TOPP) exhibited significantly reduced tolerance to fixed-carbon (C) starvation due to compromised autophagy activity. Genetic analysis placed TOPP upstream of autophagy. Interestingly, ATG13a was found to be an interactor of TOPP. TOPP directly dephosphorylated ATG13a in vitro and in vivo. We identified 18 phosphorylation sites in ATG13a by LC-MS. Phospho-dead ATG13a at these 18 sites significantly promoted autophagy and increased the tolerance of the atg13ab mutant to fixed-C starvation. The dephosphorylation of ATG13a facilitated ATG1a-ATG13a complex formation. Consistently, the recruitment of ATG13a for ATG1a was markedly inhibited in topp-7m-1. Finally, TOPP-controlled dephosphorylation of ATG13a boosted ATG1a phosphorylation. Taken together, our study reveals the crucial role of TOPP in regulating autophagy by stimulating the formation of the ATG1a-ATG13a complex by dephosphorylating ATG13a in Arabidopsis.

Brassinosteriods (BRs) are plant hormones essential for plant growth and development. The receptor-like kinase (RLK) BRI1 perceives BRs to initiate a well-known transduction pathway which finally activate the transcription factors BZR1/BES1 specifically regulating BR-mediated gene expression. The RLK EMS1 governs tapetum formation via the same signaling pathway shared with BRI1. BRI1 and EMS1 have a common signal output, but the gene structural specificity and the molecular response remain unclear. In this study, we identified that the transmembrane (TM), intracellular juxtamembrane (iJM), kinase, and leucin-rich repeats 1-13 (LRR1-13) domains of EMS1 could replace the corresponding BRI1 domain to maintain the BR receptor function, whereas the extracellular juxtamembrane (eJM) and LRR1-14 domains could not, indicating that the LRR14-EJM domain conferred functional specificity to BRI1. We compared the kinase domains of EMS1 and BRI1, and found that EMS1's kinase activity was weaker than BRI1's. Further investigation of the specific phosphorylation sites in BRI1 and EMS1 revealed that the Y1052 site in the kinase domain was essential for the BRI1 biological function, but the corresponding site in EMS1 showed no effect on the biological function of EMS1, suggesting a site regulation difference in the two receptors. Furthermore, we showed that EMS1 shared the substrate BSKs with BRI1. Our study provides insight into the structural specificity and molecular mechanism of BRI1 and EMS1, as well as the origin and divergence of BR receptors.

Autophagy is a conserved system from yeast to mammals that mediates the degradation and renovation of cellular components. This process is mainly driven by numerous autophagy-related (ATG) proteins. Among these components, the ATG1/ATG13 complex plays an essential role in initiating autophagy, sensing nutritional status signals, recruiting downstream ATG proteins to the autophagosome formation site, and governing autophagosome formation. In this review, we will focus on the ATG1/ATG13 kinase complex, summarizing and discussing the current views on the composition, structure, function, and regulation of this complex in plants.

ABSTRACT Autophagy, a conserved pathway which carries out the bulk degradation of cytoplasmic material in eukaryotic cells, is critical in plant physiology and development. It is tightly regulated by ATG13, a core component of ATG1 kinase complex which initiates autophagy. Although it has been reported that ATG13 is dephosphorylated immediately after nutrient starvation, the phosphatase regulating this process is poorly understood. Here, we demonstrated that the septuple mutant ( topp-7m ) and octuple mutant ( topp-8m ) of type one protein phosphatase (TOPP) exhibited significantly reduced tolerance to fixed-carbon (C) starvation due to compromised autophagy activity. Genetic analysis placed TOPP upstream of autophagy. Interestingly, ATG13a was found to be an interactor of TOPP. And TOPP directly dephosphorylated ATG13a in vitro and in vivo . Meanwhile, eighteen phosphorylation sites of ATG13a were identified by LC-MS. Mimic dephosphorylation of ATG13a at these 18 sites significantly promoted autophagy and increased the atg13ab mutant tolerance to fixed-C starvation. Further study showed that the dephosphorylation of ATG13a facilitated ATG1a-ATG13a complex formation. Consistently, the recruitment of ATG13a for ATG1a was markedly inhibited in topp-7m-1 . In addition, TOPP-controlled dephosphorylation of ATG13a boosted ATG1a phosphorylation. Taken together, our study reveals the crucial role of TOPP in regulating autophagy by stimulating the formation of ATG1a-ATG13a complex through dephosphorylating ATG13a in Arabidopsis .

Oral infection with Toxoplasma gondii results in dysbiosis and enteritis, both of which revert to normal during chronic infection. However, whether infection leaves a lasting impact on mucosal responses remains uncertain. Here we examined the effect of the chemical irritant dextran sodium sulfate (DSS) on intestinal damage and wound healing in chronically infected mice. Our findings indicate that prior infection with T. gondii exacerbates damage to the colon caused by DSS and impairs wound healing by suppressing stem cell regeneration of the epithelium. Enhanced tissue damage was attributable to inflammatory monocytes that emerge preactivated from bone marrow, migrate to the intestine, and release inflammatory mediators, including nitric oxide. Tissue damage was reversed by neutralization of inflammatory monocytes or nitric oxide, revealing a causal mechanism for tissue damage. Our findings suggest that chronic infection with T. gondii enhances monocyte activation to increase inflammation associated with a secondary environmental insult.

Toxoplasma is a widespread parasite of animals including many rodents that are a natural part of the transmission cycle between cats, which serve as the definitive host. Although wild rodents, including house mice, are relatively resistant, laboratory mice are highly susceptible to infection. As such, laboratory mice have been used to compare pathogenesis of natural variants and to evaluate the contributions of both host and parasite genes to infection. Protocols are provided here for evaluating acute and chronic infection with different parasite strains in laboratory mice. These protocols should provide uniform standards for evaluating natural variants and attenuated mutants and for comparing outcomes across different studies and between different laboratories.

Calcium dependent protein kinase 1 (CDPK1) is an essential Ser/Thr kinase that controls invasion and egress by the protozoan parasite Toxoplasma gondii. The Gly gatekeeper of CDPK1 makes it exquisitely sensitive to inhibition by small molecule 1H-pyrazolo[3,4-d]pyrimidine-4-amine (PP) compounds that are bulky ATP mimetics. Here we rationally designed, synthesized, and tested a series of novel PP analogs that were evaluated for inhibition of CDPK1 enzyme activity in vitro and parasite growth in cell culture. Optimal substitution on the PP scaffold included 2-pyridyl ethers directed into the hydrophobic pocket and small carbocyclic rings accessing the ribose-binding pocket. Further optimization of the series led to identification of the lead compound 3a that displayed excellent potency, selectivity, safety profile, and efficacy in vivo. The results of these studies provide a foundation for further work to optimize CDPK1 inhibitors for the treatment of acute and chronic toxoplasmosis.

The gastrointestinal tract hosts the largest collection of commensal microbes in the body. Infections at this site can cause significant perturbations in the microbiota, known as dysbiosis, that facilitate the expansion of pathobionts, and can elicit inappropriate immune responses that impair the intestinal barrier function. Dysbiosis typically occurs during intestinal infection with Toxoplasma gondii. Host resistance to T. gondii depends on a potent Th1 response. In addition, a Th17 response is also elicited. How Th17 cells contribute to the host response to T. gondii remains unclear. Here we show that class I-restricted T cell-associated molecule (CRTAM) expression on T cells is required for an optimal IL-17 production during T. gondii infection. Moreover, that the lack of IL-17, results in increased immunopathology caused by an impaired antimicrobial peptide production and bacterial translocation from the intestinal lumen to the mesenteric lymph nodes and spleen.

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile culture methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using “air-liquid interface” (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable complete development of the parasite life cycle in vitro. ALI cultures supported parasite expansion >100-fold and led to the production of viable oocysts that were transmissible in vitro and to mice. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating conventional meiosis and Mendelian segregation. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

Innate lymphoid cells (ILCs) were originally classified based on their cytokine profiles, placing natural killer (NK) cells and ILC1s together, but recent studies support their separation into different lineages at steady-state. However, tumors may induce NK cell conversion into ILC1-like cells that are limited to the tumor microenvironment and whether this conversion occurs beyond this environment remains unknown. Here, we describe Toxoplasma gondii infection converts NK cells into ILC1-like cells that are distinct from both steady-state NK cells and ILC1s in uninfected mice. These cells were Eomes-dependent, indicating that NK cells can give rise to Eomes- Tbet-dependent ILC1-like cells that circulate widely and persist independent of ongoing infection. Moreover, these changes appear permanent, as supported by epigenetic analyses. Thus, these studies markedly expand current concepts of NK cells, ILCs, and their potential conversion.

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using "air-liquid interface'' (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion > 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

In contrast to the importance of type II interferon-γ (IFN-γ) in control of toxoplasmosis, the role of type I IFN is less clear. We demonstrate here that TgIST, a secreted effector previously implicated in blocking type II IFN-γ signaling, also blocked IFN-β responses by inhibiting STAT1/STAT2-mediated transcription in infected cells. Consistent with a role for type I IFN in cell intrinsic control, ∆Tgist mutants were more susceptible to growth inhibition by murine and human macrophages activated with IFN-β. Additionally, type I IFN was important for production of IFN-γ by natural killer (NK) cells and recruitment of inflammatory monocytes at the site of infection. Mice lacking type I IFN receptors (Ifnar1−/−) showed increased mortality following infection with wild-type parasites and decreased virulence of ∆Tgist parasites was restored in Ifnar1−/−mice. The findings highlight the importance of type I IFN in control of toxoplasmosis and illuminate a parasite mechanism to counteract the effects of both type I and II IFN-mediated host defenses.

Current diagnosis of toxoplasmosis relies almost exclusively on antibody detection, and while detection of IgG provides a useful estimate of prior infection, it does not alone indicate immune status. In contrast, detection of IFN-γ responses toT. gondiiantigens has been used to monitor immune responsiveness in HIV-infected patients, thus providing valuable predictions about the potential for disease reactivation. However, specificT. gondiiantigens that can be used in assays to detect cellular immunity remain largely undefined. In this study, we examined the diagnostic potential of microneme antigens ofT. gondiiusing IFN-γ detection assays. Our findings demonstrate that MIC antigens (MIC1, MIC3, MIC4, and MIC6) elicit IFN-γ responses from memory T cells in chronically infected mice. Monitoring IFN-γ production by T cells stimulated with MIC antigens provided high sensitivity and specificity for detection ofT. gondiiinfection in mice. Taken together, these studies suggest that microneme antigens might be useful as an adjunct to serological testing to monitor immune status during infection.

Cross-presentation allows for the presentation of exogenous viral and tumor antigens to CD8+ T cells by XCR1+ classical dendritic cells (cDC1s). Previously, the role of cross-presentation in cytotoxic T cell responses in vivo was unclear due to the lack of mouse models specifically deficient in cross-presentation. We developed a CRISPR-Cas9 screen to find novel regulators of cross-presentation and found that the protein WDFY4 is essential for cross-presentation of cell-associated antigens but not for direct presentation or presentation on MHCII. WDFY4-deficient mice had normal cDC1 development and were able to survive infections with Toxoplasma gondii through production of IL-12. However, WDFY4-deficient mice failed to prime CD8+ T cells to viral antigens and were completely unable to control immunogenic tumors. WDFY4 interacts with proteins related to vesicular formation and trafficking such as clathrin and HSP90ab1 and localizes near the cell surface, suggesting its involvement in vesicular targeting after antigen uptake during cross-presentation.