Atg Proteins Research Articles

New tricks for the old autophagy protein Atg8.

New tricks for the old autophagy protein Atg8.

Neuronal VCP loss of function recapitulates FTLD-TDP pathology.

SUMMARYThe pathogenic mechanism by which dominant mutations in VCP cause multisystem proteinopathy (MSP), a rare neurodegenerative disease that presents as fronto-temporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), remains unclear. To explore this, we inactivate VCP in murine postnatal forebrain neurons (VCP conditional knockout [cKO]). VCP cKO mice have cortical brain atrophy, neuronal loss, autophago-lysosomal dysfunction, and TDP-43 inclusions resembling FTLD-TDP pathology. Conditional expression of a single disease-associated mutation, VCP-R155C, in a VCP null background similarly recapitulates features of VCP inactivation and FTLD-TDP, suggesting that this MSP mutation is hypomorphic. Comparison of transcriptomic and proteomic datasets from genetically defined patients with FTLD-TDP reveal that progranulin deficiency and VCP insufficiency result in similar profiles. These data identify a loss of VCP-dependent functions as a mediator of FTLD-TDP and reveal an unexpected biochemical similarity with progranulin deficiency.

Abstract 1942: Quantitative D2O proteomics reveals ATG9A-dependent autophagy substrates that are degraded independently of the LC3-lipidation machinery

Abstract Autophagy is an essential catabolic process that cancer cells use to survive metabolic stress and maintain basal cellular homeostasis via degradation of dysfunctional organelles and protein aggregates. One protein with an unclear, yet essential, role in autophagy is the multi-pass transmembrane protein ATG9A. ATG9A is an apical autophagy regulator that is hypothesized to help seed the formation of nascent autophagosomes by providing membrane through its own unique ATG9A vesicles. To understand the role ATG9A plays in autophagic protein turnover, we performed whole proteome deuterium exchange and quantitative mass spectrometry to measure protein degradation rates in Wild-Type (WT) and ATG9A Knock-Out (KO) cells. This experiment revealed that ATG9A KO cells display significantly decreased degradation rates of aggregation prone proteins and autophagy adapters, such as p62/SQSTM1. In an effort to understand the relationship between ATG9A and p62/SQSTM1, we found that ATG9A associates directly with p62/SQSTM1 and mediates the basal degradation of p62/SQSTM1-associated protein aggregates. This ATG9A-mediated degradation occurs in a non-canonical way, involving a subset of ATG proteins (e.g., ATG13, ATG101), but independent of the LC3-lipidation machinery ATG5 and ATG7. Taken together, our data suggests that ATG9A controls the degradation of p62 in an ATG5/ATG7-independent manner. Citation Format: Colten M. McEwan, David G. Broadbent, Ashari R. Kannangara, Daniel M. Poole, Bradley C. Naylor, John C. Price, Jens C. Schmidt, Josh L. Andersen. Quantitative D2O proteomics reveals ATG9A-dependent autophagy substrates that are degraded independently of the LC3-lipidation machinery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1942.

The evolution of autophagy proteins - diversification in eukaryotes and potential ancestors in prokaryotes.

Autophagy is a degradative pathway for cytoplasmic constituents, and is conserved across eukaryotes. Autophagy-related (ATG) genes have undergone extensive multiplications and losses in different eukaryotic lineages, resulting in functional diversification and specialization. Notably, even though bacteria and archaea do not possess an autophagy pathway, they do harbor some remote homologs of Atg proteins, suggesting that preexisting proteins were recruited when the autophagy pathway developed during eukaryogenesis. In this Review, we summarize our current knowledge on the distribution of Atg proteins within eukaryotes and outline the major multiplication and loss events within the eukaryotic tree. We also discuss the potential prokaryotic homologs of Atg proteins identified to date, emphasizing the evolutionary relationships and functional differences between prokaryotic and eukaryotic proteins.

Dicer promotes Atg8 expression through RNAi independent mechanism in Cryptococcus neoformans.

ATG8 is one of the critical genes that participate in several essential autophagic steps. The expression of ATG8 must be exquisitely regulated to avoid physiological disorder and even cell death. However, the mechanisms of regulating ATG8 expression remain to be fully uncovered. In this investigation, we found that Dicer homologs in Cryptococcus neoformans could activate the expression of ATG8 independent of RNAi. Deletion of two Dicer homologs (DCR1 and DCR2) from C. neoformans, especially DCR2, led to significantly reduced Atg8 protein level, but deletion of other RNAi components did not result in the same phenotype. The autophagic flux, the numbers of autophagic bodies and the tolerance to glucose starvation of dcr2∆ were also significantly reduced. Further investigation showed that Dcr2 activates the expression of ATG8 through the promoter region, not the Open Reading Frame or 3' Untranslated Region. We also found that a similar phenomenon exists in mammalian cells, as DCR1 instead of AGO2 knockdown also reduced the expression of LC3, indicating that this mechanism may be conservative in eukaryotic cells. Therefore, a novel transcription activation mechanism was revealed in this paper.

TOR and MAP kinase pathways synergistically regulate autophagy in response to nutrient depletion in fission yeast

ABSTRACT General autophagy is an evolutionarily conserved process in eukaryotes, by which intracellular materials are transported into and degraded inside lysosomes or vacuoles, with the main goal of recycling those materials during periods of starvation. The molecular bases of autophagy have been widely described in Saccharomyces cerevisiae, and the specific roles of Atg proteins in the process were first characterized in this model system. Important contributions have been made in Schizosaccharomyces pombe highlighting the evolutionary similarity and, at the same time, diversity of Atg components in autophagy. However, little is known regarding signals, pathways and role of autophagy in this distant yeast. Here, we undertake a global approach to investigate the signals, the pathways and the consequences of autophagy activation. We demonstrate that not only nitrogen but several nutritional deprivations including lack of carbon, sulfur, phosphorus or leucine sources, trigger autophagy, and that the TORC1, TORC2 and MAP kinase Sty1 pathways control the onset of autophagy. Furthermore, we identify an unexpected phenotype of autophagy-defective mutants, namely their inability to survive in the absence of leucine when biosynthesis of this amino acid is impaired. Abbreviations: ATG: autophagy-related; cAMP: cyclic adenosine monophosphate; cDNA: complementary deoxyribonucleic acid; GFP: green fluorescence protein; Gluc: glucose; Leu: leucine; MAP: mitogen-activated protein; MM: minimal medium; PI: propidium iodine; PKA: protein kinase A; RNA: ribonucleic acid; RT-qPCR: real time quantitative polymerase chain reaction; S. cerevisiae: Saccharomyces cerevisiae; S. pombe: Schizosaccharomyces pombe; TCA: trichloroacetic acid; TOR: target of rapamycin; TORC1: target of rapamycin complex 1; TORC2: target of rapamycin complex 2; YE5S: yeast extract 5 amino acid supplemented.

Inhibition of endoplasmic reticulum stress-related autophagy attenuates MCLR-induced apoptosis in zebrafish testis and mouse TM4 cells

Microcystin-leucine arginine (MCLR), a widespread environmental contaminant produced by cyanobacteria, poses a severe threat to the male reproductive system. However, the mechanisms of MCLR-induced testis injury accompanied by autophagy are still obscure. This study aimed to investigate the effects of MCLR on autophagy and apoptosis on the male reproductive system and its mechanism both in vitro and in vivo. MCLR caused damage to the testis of zebrafish, resulting in decreased hatching and growth retardation in the offspring. It also remarkably enhanced autophagic flux by elevating the expression of LC3BII, ATG5, and ATG12 proteins. The autophagic flux was also confirmed through the formation of autophagosomes in the ultrastructure of the zebrafish testis and the accumulation of LC3-positive puncta in zebrafish testis and mouse TM4 cells. Further evaluations revealed that inhibition of autophagy by 3-methyladenine (3-MA) significantly attenuated MCLR-induced apoptosis. This finding indicated that autophagy plays an essential role in cell death in the male reproductive system. Besides, inhibiting endoplasmic reticulum (ER) stress using 4-phenylbutyrate (4-PBA) remarkably blocked autophagy and partially suppressed apoptosis in TM4 cells induced by MCLR. This phenomenon suggested that ER stress-related autophagy was involved in MCLR-induced apoptosis. This study reveals crosstalk between ER stress and autophagy via the PERK/eIF2α/ATF4 signaling pathway. It further suggests that ER stress-related autophagy contributes to MCLR-induced apoptosis and injury in the male reproductive system. These findings provide a novel insight into MCLR-induced impairments of the testis.

Developmental Consequences of Defective ATG7-Mediated Autophagy in Humans

BackgroundAutophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare.MethodsWe performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle–biopsy specimens, mouse embryonic fibroblasts, and yeast.ResultsWe found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7.ConclusionsWe identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.)

Lipidation status of single membrane-associated ATG8 proteins

ATG8 are core autophagy proteins, the lipidated forms of which decorate double-membraned autophagosomes, as well as single-membraned organelles such as endolysosomes. Recent studies from the Florey and Münz laboratories delineate the status of single membrane-associated ATG8 proteins by indicating that their membrane anchoring can involve phosphatidylserine conjugation and their stabilization depends on ATG4 protease inhibition.

High intensity interval training induces dysregulation of mitochondrial respiratory complex and mitophagy in the hippocampus of middle-aged mice

Autophagy is involved in aging-related cognitive impairment. Aerobic exercise training can improve cognitive function in the elderly and this effect may be associated with autophagic mechanisms and mitochondrial respiratory function. High intensity interval training (HIIT) has beneficial effects on heart and skeletal muscles by activating autophagy and/or mitophagy, but the effects of HIIT on autophagy/mitophagy in the aging brain are unknown. This study investigated the effects of HIIT on the mitochondrial respiratory complex and autophagy/mitophagy, and its relation to brain function. Thirteen middle-aged male ICR mice underwent HIIT for 7 weeks. The exercise program reduced the spontaneous behavior and exploration activities of the mice. The phosphorylation level of cAMP response element binding protein (CREB) and the protein expression of brain-derived neurotrophic factor (BDNF) decreased after the 7-week HIIT. Exercise downregulated the protein expression of Complex Ⅰ and upregulated the protein expression of Complex Ⅲ, Complex Ⅳ and Complex Ⅴ. HIIT also decreased the expression of mitophagy-related proteins in the mitochondrial fractions of the hippocampus. However, HIIT did not change the expression of autophagy-related proteins LC3, P62, Atg5, Atg7, Beclin-1 and Lamp2 in the total lysate of the hippocampus. These data indicated that HIIT might have negative effects on the plasticity of the hippocampus in middle-aged mice. The effects may be related to the dysregulation of CREB-BDNF signaling, mitochondrial respiratory complex and mitophagy induced by HIIT.

Sphingosine-1-phosphate induces myocyte autophagy after myocardial infarction through mTOR inhibition

Sphingosine-1-phosphate (S1P)/S1P receptor 1 signaling exerts cardioprotective effects including inhibition of myocyte apoptosis. However, little is known about the effect of S1P treatment on myocyte autophagy after myocardial infarction (MI). In the present study, we tested the hypothesis that S1P induces myocyte autophagy through inhibition of the mammalian target of rapamycin (mTOR), leading to improvement of left ventricular (LV) function after MI. Sprague–Dawley rats underwent MI or sham operation. The animals were randomized to receive S1P (50 μg/kg/day, i.p.) or placebo for one week. H9C2 cardiomyocytes cultured in serum- and glucose-deficient medium were treated with or without S1P for 3 h. MI rats exhibited an increase in LV end-diastolic dimension (EDD) and decreases in LV fractional shortening (FS) and the maximal rate of LV pressure rise (+dP/dt). S1P treatment attenuated the increase in LV EDD and decreases in LV FS and +dP/dt. In the MI placebo group, the LC3 II/I ratio, a marker of autophagy, was increased, and increased further by S1P treatment. S1P also enhanced the autophagy-related proteins Atg4b and Atg5 after MI. Similarly, in cultured cardiomyocytes, autophagy was increased under glucose and serum deprivation, and increased further by S1P treatment. The effect of S1P on myocyte autophagy was associated with mTOR inhibition after MI or in cultured cardiomyocytes under glucose and serum deprivation. S1P treatment prevents LV remodeling, enhances myocyte autophagy and inhibits mTOR activity after MI. These findings suggest that S1P treatment induces myocyte autophagy through mTOR inhibition, leading to the attenuation of LV dysfunction after MI.

Post-translational regulation of autophagy is involved in intra-microbiome suppression of fungal pathogens

BackgroundMicrobiome interactions are important determinants for ecosystem functioning, stability, and health. In previous studies, it was often observed that bacteria suppress potentially pathogenic fungal species that are part of the same plant microbiota; however, the underlying microbe-microbe interplay remains mostly elusive. Here, we explored antagonistic interactions of the fungus Fusarium graminearum and bacterium Streptomyces hygroscopicus at the molecular level. Both are ubiquitous members of the healthy wheat microbiota; under dysbiosis, the fungus causes devastating diseases.ResultsIn co-cultures, we found that Streptomyces alters the fungal acetylome leading to substantial induction of fungal autophagy. The bacterium secrets rapamycin to inactivate the target of rapamycin (TOR), which subsequently promotes the degradation of the fungal histone acetyltransferase Gcn5 through the 26S proteasome. Gcn5 negatively regulates fungal autophagy by acetylating the autophagy-related protein Atg8 at the lysine site K13 and blocking cellular relocalization of Atg8. Thus, degradation of Gcn5 triggered by rapamycin was found to reduce Atg8 acetylation, resulting in autophagy induction in F. graminearum.ConclusionsAutophagy homeostasis plays an essential role in fungal growth and competition, as well as for virulence. Our work reveals a novel post-translational regulation of autophagy initiated by a bacterial antibiotic. Rapamycin was shown to be a powerful modulator of bacteria–fungi interactions with potential importance in explaining microbial homeostasis in healthy plant microbiomes. The autophagic process provides novel possibilities and targets to biologically control pathogens.3Zu6EQMv78RbwrwJHF2G1TVideo abstract

Evidence for Lysosomal Dysfunction within the Epidermis in Psoriasis and Atopic Dermatitis

Atopic dermatitis and psoriasis are frequent chronic inflammatory skin diseases. Autophagy plays a substantial role in the homeostasis of an organism. Loss or impairment of autophagy is associated with multiple diseases. To investigate the possibility that autophagy plays a role in atopic dermatitis and psoriasis, we investigated the levels of key ATG proteins in human skin specimens as well as in primary human epidermal keratinocytes exposed to inflammatory stimuli invitro. Although TNF-α facilitated the induction of autophagy in an initial phase, it reduced the levels and enzymatic activities of lysosomal cathepsins in later time periods, resulting in autophagy inhibition. Therefore, TNF-α appears to play a dual role in the regulation of autophagy. The relevance of these invitro findings was supported by the observation that the protein levels of cathepsins D and L are decreased in both psoriasis and atopic dermatitis skin specimens. Taken together, this study suggests that TNF-α blocks autophagy in keratinocytes after long-term exposure, a mechanism that may contribute to the chronicity of inflammatory diseases of the skin and, perhaps, of other organs.

Alternative careers at the autophagy factory.

During autophagy, proteins of the ATG8 family are conjugated to phosphatidylethanolamine (PE) in double-membrane structures called phagophores. Now, Durgan et al. have found that, during the non-canonical process of conjugating ATG8 proteins to single-membrane structures, ATG8 can be ligated to phosphatidylserine. Here, I discuss the potential consequences of their findings.

Atg32-dependent mitophagy sustains spermidine and nitric oxide required for heat-stress tolerance in Saccharomycescerevisiae.

In Saccharomyces cerevisiae, the selective autophagic degradation of mitochondria, termed mitophagy, is critically regulated by the adapter protein Atg32. Despite our knowledge about the molecular mechanisms by which Atg32 controls mitophagy, its physiological roles in yeast survival and fitness remains less clear. Here, we demonstrate a requirement for Atg32 in promoting spermidine production during respiratory growth and heat-induced mitochondrial stress. During respiratory growth, mitophagy-deficient yeast exhibit profound heat-stress induced defects in growth and viability due to impaired biosynthesis of spermidine and its biosynthetic precursor S-adenosyl methionine. Moreover, spermidine production is crucial for the induction of cytoprotective nitric oxide (NO) during heat stress. Hence, the re-addition of spermidine to Atg32 mutant yeast is sufficient to both enhance NO production and restore respiratory growth during heat stress. Our findings uncover a previously unrecognized physiological role for yeast mitophagy in spermidine metabolism and illuminate new interconnections between mitophagy, polyamine biosynthesis and NO signaling.

P300/HDAC1 regulates the acetylation/deacetylation and autophagic activities of LC3/Atg8\u2013PE ubiquitin-like system

Protein acetylation plays potential roles in regulating autophagy occurrence. However, it varies greatly between yeast and mammals, and has not been thoroughly investigated in other organisms. Here, we reported that the components of BmAtg8–PE ubiquitin-like system (BmAtg3, BmAtg4, BmAtg7, and BmAtg8) in Bombyx mori were localized in the nucleus under nutrient-rich conditions, whereas they were exported to the cytoplasm upon autophagy induction. RNAi of BmP300 and inhibition of BmP300 activity resulted in nucleo-cytoplasmic translocation of BmAtg3 and BmAtg8, as well as premature induction of autophagy in the absence of stimulus. Conversely, RNAi of BmHDAC1 and inhibition of class I/II HADCs activities led to the nuclear accumulation of BmAtg3 and BmAtg8. In addition, acetylation sites in Atg proteins of BmAtg8–PE ubiquitin-like system were identified by mass spectrometry, and acetylation-site mutations caused nucleo-cytoplasmic translocation of BmAtg3, BmAtg4, and BmAtg8 along with autophagy promotion. Similarly, the subcellular localization of human ATG4b is determined by acetylation modification. In general, BmP300-mediated acetylation sequesters the components of BmAtg8–PE ubiquitin-like system in the nucleus, thus leading to the autophagy inhibition. Oppositely, BmHDAC1-mediated deacetylation leads to the nucleo-cytoplasmic translocation of the components of BmAtg8–PE ubiquitin-like system and promotes autophagy. This process is evolutionarily conserved between insects and mammals.

Autophagy Modulators in Cancer Therapy.

Autophagy is a process of self-degradation that plays an important role in removing damaged proteins, organelles or cellular fragments from the cell. Under stressful conditions such as hypoxia, nutrient deficiency or chemotherapy, this process can also become the strategy for cell survival. Autophagy can be nonselective or selective in removing specific organelles, ribosomes, and protein aggregates, although the complete mechanisms that regulate aspects of selective autophagy are not fully understood. This review summarizes the most recent research into understanding the different types and mechanisms of autophagy. The relationship between apoptosis and autophagy on the level of molecular regulation of the expression of selected proteins such as p53, Bcl-2/Beclin 1, p62, Atg proteins, and caspases was discussed. Intensive studies have revealed a whole range of novel compounds with an anticancer activity that inhibit or activate regulatory pathways involved in autophagy. We focused on the presentation of compounds strongly affecting the autophagy process, with particular emphasis on those that are undergoing clinical and preclinical cancer research. Moreover, the target points, adverse effects and therapeutic schemes of autophagy inhibitors and activators are presented.

Cadmium Impairs Autophagy Leading to Apoptosis by Ca2+-Dependent Activation of JNK Signaling Pathway in Neuronal Cells.

Autophagy, a process for self-degradation of intracellular components and dysfunctional organelles, is closely related with neurodegenerative diseases. It has been shown that cadmium (Cd) induces neurotoxicity partly by impairing autophagy. However, the underlying mechanism is not fully elucidated. In this study, we show that Cd induced expansion of autophagosomes with a concomitant abnormal expression of autophagy-related (Atg) proteins in PC12 cells and primary murine neurons. 3-MA, a classical inhibitor of autophagy, attenuated Cd-induced expansion of autophagosomes and apoptosis in the cells. Further investigation demonstrated that Cd activated JNK pathway contributing to autophagosome expansion-dependent neuronal apoptosis. This is supported by the findings that pharmacological inhibition of JNK with SP600125 or expression of dominant negative c-Jun markedly attenuated Cd-induced expansion of autophagosomes and abnormal expression of Atg proteins, as well as apoptosis in PC12 cells and/or primary neurons. Furthermore, we noticed that chelating intracellular free Ca2+ ([Ca2+]i) with BAPTA/AM profoundly blocked Cd-elicited activation of JNK pathway and consequential expansion of autophagosomes, abnormal expression of Atg proteins, and apoptosis in the neuronal cells. Similar events were also seen following prevention of [Ca2+]i elevation with EGTA or 2-APB, implying a Ca2+-dependent mechanism involved. Taken together, the results indicate that Cd impairs autophagy leading to apoptosis by Ca2+-dependent activation of JNK signaling pathway in neuronal cells. Our findings highlight that manipulation of intracellular Ca2+ level and/or JNK activity to ameliorate autophagy may be a promising intervention against Cd-induced neurotoxicity and neurodegeneration.

Glucocappasalin Induces G2/M-Phase Arrest, Apoptosis, and Autophagy Pathways by Targeting CDK1 and PLK1 in Cervical Carcinoma Cells.

Glucocappasalin (GCP), a natural product derived from the seeds of Descurainia sophia (L.) Webb. ex Prantl, exhibits potential antitumor activity in HeLa cervical carcinoma cells. In this study, we investigated the anti-cervical cancer property of GCP through the induction of cell cycle arrest, apoptosis, and autophagy in vitro and in vivo, and elucidated the underlying molecular mechanisms. We demonstrated that treatment with GCP inhibited the growth of HeLa, Siha, and Ca Ski cell lines in a dose-dependent manner, with HeLa cells displaying particular sensitivity to the GCP treatment. Subsequently, the expression of cyclin-dependent kinase 1 (CDK1) and polo like kinase 1 (PLK1) were evaluated in HeLa cells using the CDK1 kinase assay kit, the fluorescence polarization assay, real-time quantitative PCR, and western blotting. Our results demonstrate that GCP could be employed to attenuate the expression of CDK1 and PLK1 in a dose- and time-dependent manner. The complementary results obtained by flow cytometry and western blotting allowed us to postulate that GCP may exhibit its antitumor effects by inducing G2/M cell cycle arrest. Moreover, HeLa cells treated with GCP exhibited a loss in mitochondrial membrane potential, together with the activation of caspases 3 and 9, and poly ADP-ribose polymerase (PARP). Additionally, we found that GCP could increase the formation of acidic vesicular organelles (AVOs), as well as the levels of Beclin1, LC3-II, p62, and Atg5 proteins in HeLa cells. Further studies indicated that GCP triggered autophagy via the suppression of the PI3K/AKT/mTOR signaling pathways. The autophagy inhibitor 3-methyladenine (3-MA) was used to determine whether autophagy affects the apoptosis induced by GCP. Interestingly, the inhibition of autophagy attenuated apoptosis. In vivo anti-tumor experiments indicated that GCP (60 mg/kg, i.p.) markedly reduced the growth of HeLa xenografts in nude mice without apparent toxicity. Taken together, we demonstrate that GCP induces cell cycle G2/M-phase arrest, apoptosis, and autophagy by acting on the PI3K/AKT/mTOR signaling pathways in cervical carcinoma cells. Thus, GCP may represent a promising agent in the eradication of cervical cancer.

AoATG5 plays pleiotropic roles in vegetative growth, cell nucleus development, conidiation, and virulence in the nematode-trapping fungus Arthrobotrys oligospora.

Autophagy is an evolutionarily conserved process in eukaryotes, which is regulated by autophagy-related genes (ATGs). Arthrobotrys oligospora is a representative species of nematode-trapping (NT) fungi that can produce special traps for nematode predation. To elucidate the biological roles of autophagy in NT fungi, we characterized an orthologous Atg protein, AoAtg5, in A. oligospora. We found that AoATG5 deletion causes a significant reduction in vegetative growth and conidiation, and that the transcript levels of several sporulation-related genes were significantly downregulated during sporulation stage. In addition, the cell nuclei were significantly reduced in the ΔAoATG5 mutant, and the transcripts of several genes involved in DNA biosynthesis, repair, and ligation were significantly upregulated. In ΔAoATG5 mutants, the autophagic process was significantly impaired, and trap formation and nematocidal activity were significantly decreased. Comparative transcriptome analysis results showed that AoAtg5 is involved in the regulation of multiple cellular processes, such as autophagy, nitrogen metabolism, DNA biosynthesis and repair, and vesicular transport. In summary, our results suggest that AoAtg5 is essential for autophagy and significantly contributes to vegetative growth, cell nucleus development, sporulation, trap formation, and pathogenicity in A. oligospora, thus providing a basis for future studies focusing on related mechanisms of autophagy in NT fungi.