BTB/POZ-MATH (BPM) proteins are key regulators to modulate the stability of their substrates via the 26S proteasome. However, their function in plant immunity, particularly in modulating the activity of nucleotide-binding, leucine-rich repeat (NLR) receptor, remains unknown. Here, we demonstrate that maize ZmBPM1, but not its close homolog ZmBPM2, negatively regulates the autoactive NLR protein Rp1-D21 by promoting its degradation through both the 26S proteasome and the autophagy pathways. ZmBPM1 overexpression inhibits Rp1-D21-mediated hypersensitive response (HR), whereas its mutation enhances the HR strength in maize. Notably, ZmBPM1 is primarily localized in the autophagosome-like punctate structures and facilitates relocation of Rp1-D21 from the nucleo-cytoplasmic compartment to these punctate structures. Furthermore, ZmBPM1 promotes the degradation of Rp1-D21 through autophagy pathway by interacting with the autophagy-related protein ZmATG6a/ZmATG6b, therefore inhibiting Rp1-D21-mediated HR. Strikingly, ZmBPM1 also acts as a negative regulator of resistance against southern corn rust caused by Puccinia polysora, a fungus that appears to enhance ZmBPM1-mediated autophagic activity. Our study thus identifies an autophagy-dependent mechanism by which a BPM protein finely controls NLR homeostasis and plant immunity, revealing a previously unrecognized immune regulation mechanism in plants.

Autophagy is a conserved eukaryotic intracellular degradation pathway that exhibits specialized forms, such as the biosynthetic cytoplasm-to-vacuole targeting (Cvt) pathway in yeast. This pathway selectively delivers several hydrolase precursors to the vacuole. While the core autophagy machinery is highly conserved between yeast and plants, the existence of a similar Cvt-like pathway for vacuolar hydrolase trafficking in plants remains unclear. Here, we found that Arabidopsis thaliana endo-β-mannosidase (AtEBM), a glycosidase involved in degrading vacuolar N -glycans released from glycoproteins, is selectively transported to the vacuole via autophagy. Further analyses revealed that AtEBM directly interacts with the core autophagy protein ATG8 through an ATG8-interacting motif (AIM). This interaction is essential for sequestering AtEBM into autophagosomes. Remarkably, the AIM is conserved among EBM orthologs across the green plant lineage. In addition, several phenotypic studies showed that atebm mutants generated through CRISPR-Cas9 editing did not exhibit autophagy defects, including early senescence and hypersensitivity to nutrient starvation, but rather had reduced fecundity. Thus, these findings reveal a Cvt-like pathway in plants that fulfills a biosynthetic role by selectively delivering the glycosidase AtEBM to the vacuole.

Light-harvesting efficiency is crucial for plant photosynthesis, and leaves must adjust their angles to maximize sunlight capture. Leguminous plants have evolved a specialized motor organ, the pulvinus, located between the leaf blades and petioles, enabling rapid leaflet reorientation toward sunlight. Although the role of pulvinus in orienting leaflets is well understood, the exact mechanisms behind this light response remain unclear. In this study, we identified a fast-neutron radiated mutant with the back-bent leaflet (bbl) phenotype in Medicago truncatula. The mutant leaves fail to orient properly toward light and excessively open under white light (WL) or blue light (BL). The adaxial motor cells of the bbl pulvinus contain a higher potassium concentration than those of wild-type plants, causing excessive cell swelling under WL. BBL encodes a Phototropin 2 homologue from Arabidopsis thaliana, and is highly expressed in the pulvinus. BBL/MtPHOT2 is associated with the plasma membrane in the dark, and translocates to the Golgi body upon BL exposure. Our findings demonstrate that BBL/MtPHOT2 regulates leaflet movement by modulating potassium concentration in the adaxial pulvinus motor cells, ensuring accurate leaf orientation toward the light. Our study provides genetic evidence that fills the gap between pulvinus-mediated leaflet movement and light direction.

Autophagy, a critical process for the vacuolar degradation of proteins and organelles, is governed by multiple conserved autophagy-related (ATG) proteins. The central component of the ATG machinery is the ubiquitin-like protein ATG8, which is essential for multiple steps of the autophagy process, including phagophore expansion, autophagosome closure, trafficking and fusion with the lysosome/vacuole, and selective cargo recruitment. Currently, our understanding of the roles of ATG8 in plant autophagy and the functional specialization of ATG8 family members is limited due to genetic redundancy. To assess the roles of ATG8 genes in plant autophagy, here we used CRISPR/Cas9 technology to systematically knockout the Arabidopsis ATG8 genes. By analyzing the atg8 mutants, we found that in contrast to mammalian ATG8s, in which the LC3s and GABARAP subfamilies play distinct roles in the autophagic process, Arabidopsis ATG8s perform an overlapping function in controlling autophagic flux. Combinatorial mutations of Clade I and Clade II ATG8s resulted in severely impaired autophagy under nutrient-starved conditions. Furthermore, we found that RABG3 proteins, members of the RAB7/RABG GTPase family, interact with ATG8s through AIM-LDS interfaces, and that such interaction is essential for the association of RABG3 proteins with the autophagosomal membrane and probably for the fusion of autophagosome with the vacuole, but is not required for endosomal trafficking. With the collection of multiple high-order atg8 mutants generated in this study, we now provide a venue to study the roles of ATG8 genes in canonical autophagy and non-canonical autophagy in Arabidopsis. ### Competing Interest Statement The authors have declared no competing interest.

Autophagy is a highly conserved intracellular degradation system that is crucial for nutrient recycling, thus regulating plant growth and development as well as in response to various stresses. Halophytic plant Lycium ruthenicum Murray (L. ruthenicum) is considered as a potential model plant for studying the physiological mechanisms of salt stress tolerance in plants. Although the genome sequence of L. ruthenicum is available, the characteristics and functions of the salt stress-related genes remain largely unknown. In the present study, a total of 36 AuTophaGy-related (ATG) genes were identified in L. ruthenicum and detailed characteristics of them were given. Quantitative real-time polymerase chain reaction analysis revealed that the expression of 25 LrATGs was significantly upregulated after salt stress treatments. Furthermore, the autophagic marker line pSuper:GFP-LrATG8g was generated and used to demonstrate the salt stress-induced autophagy, as revealed by measuring autophagic flux and observing autophagosome formation. The pSuper:LrATG5-GFP overexpression (OE) lines were also generated and further phenotypic analysis showed that OE-LrATG8g and OE-LrATG5 plants exhibited better salt tolerance than that of WT plants. To the best of our knowledge, this study firstly reports a detailed overview of LrATGs-mediated autophagy in L. ruthenicum response to salt stress. These findings contribute to a global understanding of the characteristics of ATG genes in L. ruthenicum and lay a foundation for future functional study.

While ATG8ylation, the lipidation of ATG8-family proteins, is canonically linked to double-membrane autophagosome formation, emerging studies demonstrate its non-canonical association with single-membrane organelles. The functional significance of ATG8ylation in these compartments, however, remains unclear. Here, we demonstrate that ionophores rapidly trigger ATG8 conjugation to the vacuolar membrane (tonoplast), a process reliant on the ATG conjugation system rather than the upstream autophagic regulators. Inhibiting reactive oxygen species (ROS) generation or V-ATPase function greatly impedes the targeting of ATG8 to the tonoplast. Intriguingly, the attachment of ATG8 to the tonoplast enhances its invagination and fosters the formation of intraluminal vesicles within vacuoles, which is achieved independently of the ESCRT machinery or cytoskeletal components. The emergence of ATG8-positive vesicles may facilitate the restoration of vacuolar acidification by redirecting proton flow from the vacuole-to-cytoplasm to an intravacuolar direction, which aids in the rapid recovery of plant growth after removal of monensin. Furthermore, under alkaline stress, ATG8 targets the tonoplast and induces vacuolar membrane invagination via a regulatory mechanism similar to that of monensin, indicating that ATG8ylation-mediated vacuolar remodeling represents an adaptive mechanism against environmental alkalinization in plants.

The ubiquitin-like protein ATG8 is a central component of the autophagy process and is required at multiple steps during both bulk and selective autophagy. Currently, our understanding of the roles of ATG8 in plants and the possible functional specialization of its family members is limited by genetic redundancy. Here, we employed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 targeting technology to systematically inactivate all nine Arabidopsis thaliana ATG8 loci. Subsequent analyses of the resulting mutants revealed that, unlike mammalian ATG8 family members, which have distinct roles, Arabidopsis isoforms largely overlap in their functions controlling autophagic flux. Notably, combinatorial mutations have similarly impaired autophagy and misregulated proteomes much like other autophagy mutants. We further examined the functional redundancy of Arabidopsis ATG8s in late autophagy stages by investigating their interactions with Rab GTPase (RABG)3/RAB7 proteins. We found that all ATG8 representatives could interact with RABG3 proteins via ATG8-interacting motif-LC3-interacting region-docking site interfaces. Such interactions are crucial for RABG3 binding to the autophagosome membrane and probably for the fusion of autophagosomes with the vacuole. However, they are not necessary for endosomal trafficking. With this collection of multiple high-order atg8 mutants, we provide a venue to selectively study the roles of individual ATG8 isoforms during both canonical and noncanonical autophagy in Arabidopsis.

Nitrogen (N) is a key macronutrient whose availability often determines maize growth and productivity. Improving nitrogen use efficiency (NUE) is critical to increase maize yield while reducing N input and, more importantly, to alleviate environmental pollution. However, only a few genes have been exploited for maize NUE improvement thus far. Here, we identified 44 candidate genes associated with NUE-related traits by performing a genome-wide association analysis in a maize natural population. We further found that the natural variations in ZmNRL1, encoding a DUF630 and DUF632 domains-containing protein, strongly associated with chlorophyll content under N starvation. Loss of function of ZmNRL1 reduced nitrogen content and weakened plant growth under hydroponic and soil conditions, whereas overexpression of ZmNRL1 conferred better tolerance to N stress and elevated yields in transgenic maize and Arabidopsis. Comparative transcriptome analysis further revealed that ZmNRL1 has a broad impact on the expression of many N utilization and signaling genes. Moreover, we showed that ZmNRL1 anchored to the plasma membrane, probably through the dual lipid modifications of myristoylation and palmitoylation. Thus, we propose that ZmNRL1 is a key regulator of the adaptation response to N limitation in maize and could be a potential target for breeding high-yield maize with enhanced NUE.

In eukaryotic cells, autophagosomes are double-membrane vesicles that are highly mobile and traffic along cytoskeletal tracks. While core autophagy-related proteins (ATGs) and other regulators involved in autophagosome biogenesis in plants have been extensively studied, the specific components regulating plant autophagosome motility remain elusive. In this study, using TurboID-based proximity labeling, we identify the retromer subcomplex comprising sorting nexin 1 (SNX1), SNX2a, and SNX2b as interacting partners of ATG8. Remarkably, SNX proteins decorate ATG8-labeled autophagosomes and facilitate their coordinated movement along microtubules. Depletion of SNX proteins restricts the motility of autophagosomes in the cytoplasm, resulting in decreased autophagic flux. Furthermore, we show that the microtubule-associated protein CLASP is a bridge, connecting the SNX-ATG8-decorated autophagosomes to the microtubules. Genetically, the clasp-1 mutant phenotype resembles that of plants with disrupted SNXs or microtubule networks, displaying diminished autophagosome motility and reduced autophagic flux. Collectively, our study unveils a hitherto unanticipated role of the SNXs subcomplex in connecting autophagosomes with microtubules to promote autophagosome mobility in Arabidopsis.

Plant immunity is suppressed in the symbiotic nodule cells, thereby facilitating rhizobial infection. Medicago truncatula NODULES WITH ACTIVATED DEFENSE1 (MtNAD1) is crucial for suppressing immunity in nodules; however, its molecular function is unclear. We explored the molecular basis of the role of MtNAD1 in suppressing innate immunity in M. truncatula nodules. Medicago truncatula mutants lacking MtATG7 produced defective nodules, sharing some similarities with the Mtnad1 mutant nodules. Furthermore, MtNAD1 interacted with several immunity-related proteins, including BAX-inhibitor1a (MtBI-1a), two Lysin-motif proteins (MtLYM1/2), Pathogenesis-related10 (MtPR10c/d), MtMPK3/6, and two Lysin-motif receptor kinases (MtLYK8/9). In addition, MtNAD1 and the autophagy pathway contributed to the reduction of MtBI-1, MtPR10c/d, and MtLYM1/2 protein levels in planta. Knocking out either the MtBI-1 or MtLYM1/2 gene in the M. truncatula nad1 mutant can partially restore the defective nodules of the nad1 mutant. Our results demonstrate that MtNAD1 associates with the autophagy pathway by interacting with MtATG8, contributing to the degradation of several immunity-related proteins in M. truncatula nodules during rhizobial colonization and thereby supporting the development of a successful symbiosis.

Above-optimal growth temperatures, usually referred to as heat stress (HS), pose a challenge to organisms’ survival as they interfere with essential physiological functions and disrupt cellular organization. Previous studies have elucidated the complex transcriptional regulatory networks involved in plant HS responses, but the mechanisms of organellar remodelling and homeostasis during plant HS adaptations remain elusive. Here we report a non-canonical function of ATG8 in regulating the restoration of plant Golgi damaged by HS. Short-term acute HS causes vacuolation of the Golgi apparatus and translocation of ATG8 to the dilated Golgi membrane. The inactivation of the ATG conjugation system, but not of the upstream autophagic initiators, abolishes the targeting of ATG8 to the swollen Golgi, causing a delay in Golgi recovery after HS. Using TurboID-based proximity labelling, we identified CLATHRIN LIGHT CHAIN 2 (CLC2) as an interacting partner of ATG8 via the AIM–LDS interface. CLC2 is recruited to the cisternal membrane by ATG8 to facilitate Golgi reassembly. Collectively, our study reveals a hitherto unanticipated process of Golgi stack recovery from HS in plant cells and uncovers a previously unknown mechanism of organelle resilience involving ATG8.

The selective turnover of macromolecules by autophagy provides a critical homeostatic mechanism for recycling cellular constituents and for removing superfluous and damaged organelles, membranes, and proteins. To better understand how autophagy impacts seed maturation and nutrient storage, we studied maize (Zea mays) endosperm in its early and middle developmental stages via an integrated multiomic approach using mutants impacting the core macroautophagy factor AUTOPHAGY (ATG)-12 required for autophagosome assembly. Surprisingly, the mutant endosperm in these developmental windows accumulated normal amounts of starch and Zein storage proteins. However, the tissue acquired a substantially altered metabolome, especially for compounds related to oxidative stress and sulfur metabolism, including increases in cystine, dehydroascorbate, cys-glutathione disulfide, glucarate, and galactarate, and decreases in peroxide and the antioxidant glutathione. While changes in the associated transcriptome were mild, the proteome was strongly altered in the atg12 endosperm, especially for increased levels of mitochondrial proteins without a concomitant increase in mRNA abundances. Although fewer mitochondria were seen cytologically, a heightened number appeared dysfunctional based on the accumulation of dilated cristae, consistent with attenuated mitophagy. Collectively, our results confirm that macroautophagy plays a minor role in the accumulation of starch and storage proteins during maize endosperm development but likely helps protect against oxidative stress and clears unneeded/dysfunctional mitochondria during tissue maturation.

Macroautophagy/autophagy has been recognized as a central antiviral defense mechanism in plant, which involves complex interactions between viral proteins and host factors. Rhabdoviruses are single-stranded RNA viruses, and the infection causes serious harm to public health, livestock, and crop production. However, little is known about the role of autophagy in the defense against rhabdovirus infection by plant. In this work, we showed that Rice stripe mosaic cytorhabdovirus(RSMV) activated autophagy in plants and that autophagy served as an indispensable defense mechanism during RSMV infection. We identified RSMV glycoprotein as an autophagy inducer that interacted with OsSnRK1B and promoted the kinase activity of OsSnRK1B on OsATG6b. RSMV glycoprotein was toxic to rice cells and its targeted degradation by OsATG6b-mediated autophagy was essential to restrict the viral titer in plants. Importantly, SnRK1-glycoprotein and ATG6-glycoprotein interactions were well-conserved between several other rhabdoviruses and plants. Together, our data support a model that SnRK1 senses rhabdovirus glycoprotein for autophagy initiation, while ATG6 mediates targeted degradation of viral glycoprotein. This conserved mechanism ensures compatible infection by limiting the toxicity of viral glycoprotein and restricting the infection of rhabdoviruses.Abbreviations: AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; ANOVA: analysis of variance; ATG: autophagy related; AZD: AZD8055; BiFC: bimolecular fluorescence complementation; BYSMV: barley yellow striate mosaic virus; Co-IP: co-immunoprecipitation; ConA: concanamycin A; CTD: C-terminal domain; DEX: dexamethasone; DMSO: dimethyl sulfoxide; G: glycoprotein; GFP: green fluorescent protein; MD: middle domain; MDC: monodansylcadaverine; NTD: N-terminal domain; OE: over expression; Os: Oryza sativa; PBS: phosphate-buffered saline; PtdIns3K: class III phosphatidylinositol-3-kinase; qRT-PCR: quantitative real-time reverse-transcription PCR; RFP: red fluorescent protein; RSMV: rice stripe mosaic virus; RSV: rice stripe virus; SGS3: suppressor of gene silencing 3; SnRK1: sucrose nonfermenting1-related protein kinase1; SYNV: sonchus yellow net virus; TEM: transmission electron microscopy; TM: transmembrane region; TOR: target of rapamycin; TRV: tobacco rattle virus; TYMaV: tomato yellow mottle-associated virus; VSV: vesicular stomatitis virus; WT: wild type; Y2H: yeast two-hybrid; YFP: yellow fluorescent protein.

In plants, autophagy is a conserved process by which intracellular materials, including damaged proteins, aggregates, and entire organelles, are trafficked to the vacuole for degradation, thus maintaining cellular homeostasis. The past few decades have seen extensive research into the core components of the central autophagy machinery and their physiological roles in plant growth and development as well as responses to biotic and abiotic stresses. Moreover, several methods have been established for monitoring autophagic activities in plants, and these have greatly facilitated plant autophagy research. However, some of the methodologies are prone to misuse or misinterpretation, sometimes casting doubt on the reliability of the conclusions being drawn about plant autophagy. Here, we summarize the methods that are widely used for monitoring plant autophagy at the physiological, microscopic, and biochemical levels, including discussions of their advantages and limitations, to provide a guide for studying this important process.

Protein-targeting technologies represent essential approaches in biological research. Protein knockdown tools developed recently in mammalian cells by exploiting natural degradation mechanisms allow for precise determination of protein function and discovery of degrader-type drugs. However, no method to directly target endogenous proteins for degradation is currently available in plants. Here, we describe a novel method for targeted protein clearance by engineering an autophagy receptor with a binder to provide target specificity and an ATG8-binding motif (AIM) to link the targets to nascent autophagosomes, thus harnessing the autophagy machinery for degradation. We demonstrate its specificity and broad potentials by degrading various fluorescence-tagged proteins, including cytosolic mCherry, the nucleus-localized bZIP transcription factor TGA5, and the plasma membrane-anchored brassinosteroid receptor BRI1, as well as fluorescence-coated peroxisomes, using a tobacco-based transient expression system. Stable expression of AIM-based autophagy receptors in Arabidopsis further confirms the feasibility of this approach in selective autophagy of endogenous proteins. With its wide substrate scope and its specificity, our concept of engineered AIM-based selective autophagy could provide a convenient and robust research tool for manipulating endogenous proteins in plants and may open an avenue toward degradation of cytoplasmic components other than proteins in plant research.

Light is arguably one of the most important environmental factors that determines virtually all aspects of plant growth and development, but the molecular link between light signaling and the autophagy pathway has not been elucidated in plants. In this study, we demonstrate that autophagy is activated during light-to-dark conversion though transcriptional upregulation of autophagy-related genes (ATGs). We showed that depletion of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to enhanced autophagy activity and resistance to extended darkness and nitrogen starvation treatments, contributing to higher expression of ATGs. HY5 interacts with and recruits HISTONE DEACETYLASE 9 (HDA9) to ATG5 and ATG8e loci to repress their expression by deacetylation of the Lys9 and Lys27 of histone 3. Furthermore, we found that both darkness and nitrogen depletion induce the degradation of HY5 via 26S proteasome and the concomitant disassociation of HDA9 from ATG5 and ATG8e loci, leading to their depression and thereby activated autophagy. Genetic analysis further confirmed that HY5 and HDA9 act synergistically and function upstream of the autophagy pathway. Collectively, our study unveils a previously unknown transcriptional and epigenetic network that regulates autophagy in response to light-to-dark conversion and nitrogen starvation in plants.

The plant membrane-trafficking system plays a crucial role in maintaining proper cellular functions and responding to various developmental and environmental cues. Thus far, our knowledge of the maize membrane-trafficking system is still limited. In this study, we systematically identified 479 membrane-trafficking genes from the maize genome using orthology search and studied their functions by integrating transcriptome and evolution analyses. These genes encode the components of coated vesicles, AP complexes, autophagy, ESCRTs, retromers, Rab GTPases, tethering factors, and SNAREs. The maize genes exhibited diverse but coordinated expression patterns, with 249 genes showing elevated expression in reproductive tissues. Further WGCNA analysis revealed that five COPII components and four Rab GTPases had high connectivity with protein biosynthesis during endosperm development and that eight components of autophagy, ESCRT, Rab, and SNARE were strongly co-upregulated with defense-related genes and/or with secondary metabolic processes to confer basal resistance to Fusarium graminearum. In addition, we identified 39 membrane-trafficking genes with strong selection signals during maize domestication and/or improvement. Among them, ZmSec23a and ZmVPS37A were selected for kernel oil production during improvement and pathogen resistance during domestication, respectively. In summary, these findings will provide important hints for future appreciation of the functions of membrane-trafficking genes in maize.

Seed storage proteins (SSPs) are of great importance in plant science and agriculture, particularly in cereal crops, due to their nutritional value and their impact on food properties. During seed maturation, massive amounts of SSPs are synthesized and deposited either within protein bodies derived from the endoplasmic reticulum, or into specialized protein storage vacuoles (PSVs). The processing and trafficking of SSPs vary among plant species, tissues, and even developmental stages, as well as being influenced by SSP composition. The different trafficking routes, which affect the amount of SSPs that seeds accumulate and their composition and modifications, rely on a highly dynamic and functionally specialized endomembrane system. Although the general steps in SSP trafficking have been studied in various plants, including cereals, the detailed underlying molecular and regulatory mechanisms are still elusive. In this review, we discuss the main endomembrane routes involved in SSP trafficking to the PSV in Arabidopsis and other eudicots, and compare and contrast the SSP trafficking pathways in major cereal crops, particularly in rice and maize. In addition, we explore the challenges and strategies for analyzing the endomembrane system in cereal crops.

Techniques for disrupting of protein function are essential for biological researches and therapeutics development. Though well-established, genetic perturbation strategies may have off-target effects and/or trigger compensatory mechanisms, and cannot efficiently eliminate existing protein variants or aggregates1, 2. Therefore, precise and direct protein-targeting methods are highly desired. Here we describe a novel method for targeted protein clearance by engineering an autophagy receptor with a binder to provide target specificity and an ATG8-binding motif (AIM) to link the targets to nascent autophagosomes, thus harnessing the autophagy machinery for degradation. We demonstrate its specificity and broad potentials by degrading various fluorescent-tagged proteins and peroxisome organelle, using a tobacco-based transient expression system, and by degrading endogenous proteins in transgenic Arabidopsis expressing engineered receptors. With the wide substrate scope and specificity of selective autophagy, our method provides a convenient and robust strategy for eliminating proteins and aggregates, and may enable developing new treatments for protein-related disorders.

Autophagy is an intracellular trafficking mechanism by which cytosolic macromolecules and organelles are sequestered into autophagosomes for degradation inside the vacuole. In various eukaryotes including yeast, metazoans, and plants, the precursor of the autophagosome, termed the phagophore, nucleates in the vicinity of the endoplasmic reticulum (ER) with the participation of phosphatidylinositol 3-phosphate (PI3P) and the coat protein complex II (COPII). Here we show that Arabidopsis thaliana FYVE2, a plant-specific PI3P-binding protein, provides a functional link between the COPII machinery and autophagy. FYVE2 interacts with the small GTPase Secretion-associated Ras-related GTPase 1 (SAR1), which is essential for the budding of COPII vesicles. FYVE2 also interacts with ATG18A, another PI3P effector on the phagophore membrane. Fluorescently tagged FYVE2 localized to autophagic membranes near the ER and was delivered to vacuoles. SAR1 fusion proteins were also targeted to the vacuole via FYVE2-dependent autophagy. Either mutations in FYVE2 or the expression of dominant-negative mutant SAR1B proteins resulted in reduced autophagic flux and the accumulation of autophagic organelles. We propose that FYVE2 regulates autophagosome biogenesis through its interaction with ATG18A and the COPII machinery, acting downstream of ATG2.